chia Streptomyces Bartonella HafniaTerrimonas Pseudoxanthomonas Corynebacterium Streptococcus Aerococcus Saccharothrix Facklamia SchizosaccharomycesTetrageno

alieria Clavibacter Shimwellia Brachybacterium Leclercia Providencia Trabulsiella Xanthobacter Emericella Gardnerella Sporothrix Leuconostoc Pseudoclavibacter Alkali
omyces Turicella Roseomonas Ruminococcus Scedosporium Dysgonomonas Staphylococcus Peptostreptococcus Paenibacillus Balneatrix Solibacillus Prototheca Cupr
cillus Aspergillus Arthrobacter Mesorhizobium Acholeplasma Filobasidium Propioniferax Azohydromonas Chromobacterium Curtobacterium Kloeckera Austwickia Hyphomic
coccus Rummeliibacillus Budvicia Aquincola Enterobacter Sporobolomyces Brevundimonas Capnocytophaga Tatlockia Neisseria Salinivibrio Pullulanibacillus Arcanoba
ella Eggerthella Methanomonas Mucor Mobiluncus Caulobacter Helcococcus Psychrobacillus Campylobacter Blastomonas Wohlfahrtiimonas Thermoactinomyces Hermini
murella Mycobacterium Bordetella Pichia Vibrio Iodobacter Tenacibaculum Listeria Plesiomonas Haloarcula Shewanella Paecilomyces Thauera Viridibacillus Yokenella Ma
phingobium Ornithobacterium Epidermophyton Oligella Paracoccus Aureobasidium Eubacterium Dietzia Salimicrobium Klebsiella Mycoplasma Variovorax Sa
phyllum Scopulariopsis Odoribacter Anaerotruncus Abiotrophia Burkholderia Sodalis Empedobacter Sphingopyxis Lactococcus Sphingobium Microsporum Pepton
occus Beauveria Morganella Pasteurella Cedecea Bifidobacterium Micrococcus Propionimicrobium Starkeya Prevotella Histophilus Sphingomonas Acetobacter Fra
acterium Propionibacterium Aneurinibacillus Arthrographis Aromatoleum Pediococcus Phoma Xenorhabdus Methylobacillus Fusarium Wolinella Bacteroides Zygosacchar
simicrobium Helicobacter Rhizobium Terrabacter Ralstonia Butyricimonas Microsporum Castellaniella Borrelia Microbacterium Rheinheimera Wautersiella Saccharopo
la Nocardioides Gluconobacter
Kytococcus Chryseobacterium Alishewanella Gemella Methylobacterium Haemophilus Adlercreutzia
ochrobactrum Leminorella Candidatus Xanthomonas Pectobacterium Brevibacterium Arthroderma
imonas Actinomyces
obacter Lysinibacillus
MALDI Biotyper

bacterium Stenotrophomonas

Poster Hall 2016

ia Halomonas Rhodococcus Bergeyella Malikia
bacillus Chromohalobacter Yersinia Oerskovia Gallibacterium Erwinia Agromyces Filifactor Devosia Pragia Massilia Collinsella Finegoldia
a Pantoea Elizabethkingia Leifsonia Pseudozyma Streptosporangium Macrococcus
lla Delftia
coccus Janthinobacterium Kerstersia Luteibacter Photorhabdus Proteus Arcobacter Actinobaculum Alternaria Citrobacter Dichelobacter Achromobacter Candida Ew
hyton Granulicatella Leptothrix Suttonella Dermabacter Hydrogenophaga Cellulomonas AzoarcusTrichosporonTatumella Rhodospiridium Acidaminococcus Actinobacillus
hum Fusobacterium Lodderomyces Anaerococcus Colletotrichum Edwardsiella Comamonas Pigmentiphaga Moellerella Nesterenkonia Listonella Udeniomyces Erysip
chia Streptomyces Bartonella HafniaTerrimonas Pseudoxanthomonas Corynebacterium Streptococcus Aerococcus Saccharothrix Facklamia SchizosaccharomycesTetrageno
alieria Clavibacter Shimwellia Brachybacterium Leclercia Providencia Trabulsiella Wolinella Bacteroides Zygosaccharomyces Cellulosimicrobium Helicobacter Rhi
acter Ralstonia Butyricimonas Microsporum Castellaniella Borrelia Microbacterium Rheinheimera Wautersiella Saccharopolyspora Rahnella Nocardioides Glucon
obacterium Mannheimia Cohnella Aggregatibacter Cronobacter Lecythophora Riemerella Chaetomium Atopobium Rhizopus Acidovorax Rothia Kytococcus Chryseoba
wanella Gemella Methylobacterium Haemophilus Adlercreutzia Buttiauxella Weeksella Alloiococcus Bacillus Arxiozyma Halococcus Rhodotorula Pseudochrobactrum Lem
atus Xanthomonas Pectobacterium Brevibacterium Arthroderma Slackia Trueperella Inquilinus Brevibacillus Brachyspira Porphyromonas Aurantimonas Actin
la Kitasatospora Magnusiomyces Psychrobacter Acidiphilium Amycolatopsis Lactobacillus Marinibacillus Megamonas Dermatophilus Grimontia Acinetobacter Lysini
niaspora Parvimonas Moesziomyces Legionella Aliivibrio Dermacoccus Exiguobacterium Virgibacillus Raoultella Gordonia Dialister Parabacteroides Cardioba
rophomonas Sporobolomyces Coprobacillus Sporosarcina Brenneria Rathayibacter Arsenophonus Penicillium Pseudomonas Rubrivivax Bilophila Alloscardovia N
onas Rhodococcus Bergeyella Malikia Actinocorallia Aeromonas Micromonospora Alcaligenes Alistipes Pannonibacter Dickeya Kocuria Ochrobactrum Agrococcus Gracili
ohalobacter Yersinia Oerskovia Gallibacterium Erwinia Agromyces Filifactor Devosia Pragia Massilia Collinsella Finegoldia Phenylobacterium Methyloarcula Jonesia P
thkingia Leifsonia Pseudozyma Streptosporangium Macrococcus Veillonella Sporolactobacillus Moraxella Clostridium Pandoraea Flavobacterium Halobacterium Ta
Sinomonas Carnobacterium Myroides Exophiala Sporopachydermia Nocardiopsis Avibacterium Blautia Salmonella Weissella Herbaspirillum Ideonella Kingella K
coccus Janthinobacterium Kerstersia Luteibacter Photorhabdus Proteus Arcobacter Actinobaculum Alternaria Citrobacter Dichelobacter Achromobacter Candida Ew
hyton Granulicatella Leptothrix Suttonella Dermabacter Hydrogenophaga Cellulomonas AzoarcusTrichosporonTatumella Rhodospiridium Acidaminococcus Actinobacillus
hum Fusobacterium Lodderomyces Anaerococcus Colletotrichum Edwardsiella Comamonas Pigmentiphaga Moellerella Nesterenkonia Lis
myces Erysipelothrix Escherichia Streptomyces Bartonella Hafnia Terrimonas Pseudoxanthomonas Corynebacterium Streptococcus Aero
rothrix Facklamia Schizosaccharomyces Tetragenococcus Lechevalieria Clavibacter Shimwellia Brachybacterium Leclercia MALDI-TOF
Providencia Trab
Innovation with Integrity
Changing Microbiology
Dear customers
Welcome to the 5th edition of our MALDI Biotyper (MBT) Poster Hall, which showcases a selection of
scientific publications presented at recent conferences and meetings.
With over 2000 systems in operation, the MALDI Biotyper has become the gold standard for routine
identification of microorganisms in clinical laboratories, and its use is steadily increasing for routine identifi-
cation tasks in environmental labs, the pharmaceutical industry, and food and beverage markets.
Probably the most compelling reasons for the wide acceptance and implementation of the MALDI Biotyper
in microbiology labs are its ease of use, its robustness, and broad species coverage which we are con-
tinuously expanding.
Compared to 2010, the newest reference library contains double the number of reference spectra. One
focus of the additions to the library in the 2016 update is a broad coverage of anaerobe strains. Many of
these reference spectra were generated in collaboration with the European Network for the Rapid Identifi-
cation of Anaerobes (ENRIA).
Identification of mycobacteria has been further improved by updating the MBT Mycobacteria Library,
which now covers 159 of 169 known species, and adapted bioinformatics for high-sensitivity and
high-specificity species identification (as illustrated in Dr. Pranadas poster on page 32).
With sepsis being a potentially life-threatening clinical indication, rapid identification of blood stream
infections is a prerequisite for initiating adequate antibiotic therapy. The MALDI Biotyper Sepsityper
enables MALDI-TOF mass spectrometry-based proteomic profiling and rapid enrichment and identification
of bacteria and yeast directly from positive blood cultures. In Europe, the MALDI Sepsityper Kit is labeled
according to the IVD-Directive 98/79/EC. Compared to conventional workflows, the combination of the
MBT Sepsityper IVD Kit and the MBT Sepsityper IVD software module allows a much earlier microbial
identification from positive blood culture samples derived from patients with a potential sepsis. Following
the MBT Sepsityper IVD Kit workflow, the sample is submitted to the MALDI Biotyper for identification
less than 30 minutes after detection of a positive blood culture. The MBT Sepsityper IVD Module supports
result management for more complex blood culture samples by automatically adapting the peak-picking
mass range and the identification confidence levels, and by optionally allowing mixed culture detection.
To meet the demands of leading microbiology laboratories for even higher productivity and faster turn-
around times, we expanded the MALDI Biotyper product range recently by the introduction of the
MALDI Biotypersmart mass spectrometer system. The MALDI Biotypersmart system features Brukers
proprietary smartbeam solid-state laser technology, which is more than 3 times faster than conventional
nitrogen lasers. Additionally, the smartbeam laser combines the at least one order of magnitude higher
lifetime of solid-state lasers with the unmatched MALDI performance of nitrogen lasers used in microbial
identification.
To complement Brukers established reusable stainless steel MALDI target plates, we enlarged the
MALDI Biotyper sample preparation portfolio by introducing the disposable MBT Biotarget 96. These
new MALDI target plates provide increased workflow efficiency by offering 96 sample positions, and by
employing Brukers proprietary AnchorChip technology, they enable precise and homogeneous sample
preparations in liquid workflows.
The MBT Biotarget 96 is fully compatible with the paperless and traceable workflow using the MBT Pilot,
which supports guided target preparation based on micro-projection technology, and the MBT Galaxy
automated MALDI target preparation system, which frees laboratory personal from routine matrix and
formic acid pipetting while ensuring highest preparation quality.
The new MBT Subtyping software module enables the automated analysis of specific resistances that
are associated with specific bacterial subtypes. In this context, the MBT Subtyping Module supports the
detection of carbapenem resistance associated with cfiA-encoded class B metallo-beta-lactamase in
Bacteroides fragilis and the analysis of MRSA Staphylococcus aureus.
Bruker is committed to Innovation with Integrity. A great deal of this innovation for the MALDI Biotyper
product line is evidenced in this new edition of our Poster Hall.
As every year, my sincere thanks go to our customers who contributed to this edition and whose scientific
spirit encourages us to develop new applications for the MALDI Biotyper. I am looking forward to reading
your future posters and publications!
With best regards,
Dr. Wolfgang Pusch,
Executive Vice President
Clinical MALDI, Bruker Daltonik GmbH, Germany
2
Index of Posters

Primary ID Blood Culture and Resistance

20 Gram-Negative Bacilli Isolated from Bronchial Aspiration from Patients in the Intensive Care Unit and 40 Antimicrobial Stewardship Combined with MALDI-TOF and -Lacta Test Performed on Gram-Negative
Performance of MALDI-TOF MS for Routine Identification Bacilli Blood Culture is Effective for Sparing the Use of Carbapenems
21 Clinical Case of Brucellosis Identified by MALDI-TOF-MS
22 Identification of Highly Pathogenic Microorganisms Using MALDI-TOF Mass Spectrometry Results Resistance
of an Inter-Laboratory Ring Trial 41 Detection of OXA-48-Producing Enterobacteriaceae through MALDI-TOF Using Temocillin as
23 Evaluation of Matrix-Assisted-Laser-Desorption-Ionisation Time-of-Flight Mass Spectrometry for Diagnostic Marker
the Identification of Capnocytophaga canimorsus and of Capnocytophaga cynodegmi 42 Discriminating Carbapenemase Production in a UK Clinical Laboratory: The Role of the MALDI-TOF
24 Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) Hydrolysis Assay
and Prosthetic Joint Infection 43 Rapid Detection of Tobramycin Resistant Gram-Negative Bacteria by MALDI-TOF MS
25 It is Time for Accurate Identification of Raoultella spp. 44 Rapid Detection of OXA-48 Expressing Enterobacteriaceae by MALDI TOF-MS
26 Evaluation of the MALDI-TOF MS Method for Identication of Nocardia Species 45 Optimization of the MBT-ASTRA
27 Evaluation of MALDI-TOF as a High-Volume Enteric Pathogen Screening Method Compared to 46 Rapid Antibiotic Susceptibility Detection Using Mass Spectrometric-Antibiotic Susceptibility
an In-House PCR Based Algorithm at a Large Outpatient Laboratory Rapid Assay (MS-ASTRA) in Klebsiella spp.
28 The Role of MALDI-TOF to Discover New Pathogens: Propionibacterium kocii, a Human Pathogen 47 Detection of Extended Spectrum Beta-Lactamase among Enterobacteriaceae Using MALDI-TOF
Anaerobic Bacterium
29 Do You Know What is Growing in Your Hospital? Impact on the Trend of Yeasts and Molds Identification
in a Large Italian Hospital Using Mass Spectrometry Resistance and Subtyping
48 MALDI-TOF Mass Spectroscopy and blakpc Gene Phylogenetic Analysis: an Outbreak of Multidrug
Primary ID of Mycobacteria and Carbapenem Resistant K. pneumoniae Isolates?
30 Use of MALDI-TOF Mass Spectrometry for the Identification of Mycobacteria in Clinical Practice 49 Epidemiological Typing of Methicillin-Resistant Staphylococcus aureus (MRSA): Spa-typing
31 Mycobacteria Identification by MALDI Biotyper System: Evolution of Database Content versus MALDI-TOF
and Evaluation Criteria 50 Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass-Spectrometry (MALDI-TOF) Based
32 Looking at the Differences: First Report of Possibilities to Separate Mycobacterium chimaera from Typing of an ESBL Escherichia coli Outbreak in a Hospital Setting
Mycobacterium intracellulare by MALDI-TOF MS
33 Evaluation of the MALDI-TOF Biotyper for Identification of Mycobacterium spp. Isolates in Environmental and Food Microbiology
the Clinical Microbiological Laboratory 51 / 52 ESKAPE Pathogens - Understanding Nosocomial Risk Factors
34 Determination of Non Tuberculous Mycobacteria with Matrix Assisted Laser Desorption Ionization-Time 53 Rapid and Accurate Listeria Species Identification by MALDI Biotyper Database Improvement
of Flight Mass Spectrometry 54 Identification of Food Microorganisms by MALDI-TOF Mass Spectrometry
35 Performance of MALDI-TOF MS in Species Identification of Clinical Mycobacterial Isolates
Veterinary Microbiology
Primary ID in Urine 55 The Use of MALDI-TOF MS for the Identification of Mastitis Pathogens
36 Rapid Identification of Pathogens Directly from Urine by MALDI-TOF
37 Proposal of a New Protocol for Rapid Bacterial Identification and Susceptibility Testing Directly from New Applications
Urine Samples 56 MALDI-TOF Mass Spectrometry for the Identification of Trichomonas vaginalis: Preliminary Study
57 Bacterial Antibiotic Resistance Determination by Metal Oxide-Catalyzed MALDI MS Fatty Acid Profiling
Blood Culture 58 Identification of Animal Species from Meat Using MALDI-TOF MS
38 Fast Bacterial Identification by Mass Spectrometry in Blood Culture Broth of Bacteremic Patients
Allows Quick Adaptation of the Empirical Antibiotic Therapy Typing of Microorganisms by FT-IR
39 Validation and Implementation of MALDI-TOF: a Quick and Easy Method for Bacterial Identification 59 Differentiation of Klebsiella pneumoniae Strains by Fourier Transform Infrared Spectroscopy
from Clinical Samples

3
Bruker Analytical Excellence, Acknowledged Expertise and Global Presence

The Performance Leader in Life Science, Analytical and Clinical Systems

Right from the beginning, which is now more than fty years ago, Bruker has been driven by the idea: Mass Spectrometry and Separations
to provide the best technological solution for each analytical task. Today, worldwide, more than 6,000 em- Infrared and Raman Spectroscopy
ployees in over 90 locations on all continents are focusing their efforts on this permanent challenge. Bruker X-ray Diffraction and Elemental Analysis
systems cover a broad spectrum of applications in all elds of research and development and are used in all Magnetic Resonance
industrial production processes for the purpose of ensuring quality and process reliability. Bruker continues Surface Analysis
to build upon its extensive range of products and solutions, expand its broad base of installed systems and Preclinical Imaging
maintain a strong reputation amongst its customers. As one of the world's leading analytical instrumentation Mobile Detection
companies, Bruker remains focused on developing state-of-the-art technologies and innovative solutions for
todays ever complex analytical questions.

Bruker Offices
Additional information: www.bruker.com/map 4
Bruker Daltonics Facilities
Bruker Daltonik GmbH, Bremen, Germany
Bruker Corporation
The Bruker Group is a leading provider of high-performance scientific instruments and solutions for molecular
research, materials research, and also for industrial and applied analysis. Bruker Corporation (NASDAQ:BRKR),
headquartered in Billerica, Massachusetts, is the publicly traded parent company of Bruker AXS, Bruker Biospin,
Bruker Daltonics and Bruker Optics. We employ more than 6,000 people based in more than 90 production faci-
lities around the world.
For more than 50 years, Bruker has enabled scientists to make breakthrough discoveries and develop new applicati-
ons that improve the quality of human life. Brukers high-performance scientific research instruments and high-value
analytical solutions enable scientists to explore life and materials at molecular, cellular and microscopic levels. In
close cooperation with our customers, Bruker is enabling innovation, productivity and customer success in life sci-
ence molecular research, in applied and pharma applications, and in microscopy, nano-analysis and industrial applica-
tions, as well as in cell biology, preclinical imaging, clinical research, microbiology and molecular diagnostics.
Bruker Daltonics, Billerica, MA, USA
Bruker Daltonik GmbH,
Leipzig, Germany
Bruker Daltonics Bremen Facility
Bruker Daltonics has sold more than 6,000 MALDI-TOF systems and employs more than 1,000 people around the
world spread across three main sites; Billerica (USA), Leipzig & Bremen (Germany). The microbiological research
and development group started in 1998 with MALDI Biotyper development at our Bremen facility where we
employ more than 150 people in R&D.
The Bremen facility is spread over 250,000 sq ft. and houses three class II microbiology laboratories as well as the
largest demonstration center in Europe which carries out more than 550 demonstrations and 150 training courses
every year. Bruker Daltonics has more than 150 trained MALDI engineers representing almost one thousand years
of experience between them, most have more than 10 years of service. Additionally we can provide remote web-
based support around the clock anywhere in the world.
5
 MALDI Biotyper a Success Story
1985:
Establishment of the
mass spectrometry
business in Bremen,
Germany
2009:
Launch of the IVD MALDI
Biotyper which complies
with the European Directive
for In-Vitro Diagnostica
IVD 98/79/EC
2007: Major private laboratories
Bruker establishes a quality extend their accreditation
1998: system in compliance with according to DIN EN ISO
The microbiological ISO 13485 based on its 15189
research and develop- decade-long experience with MALDI Biotyper library
ment department quality management expands to include over
starts operation according to ISO 9001 3,000 strains
2013:
2011:
> 300 MALDI Biotyper
systems sold worldwide
MALDI Biotyper library
expanded to > 4,000
strains
2015:
FDA clearance of MALDI
Biotyper CA System
> 1000 MALDI Biotyper
systems sold worldwide
Bruker and Charles River
sign collaboration agree-
ment
FDA clearance for
expanded claim of the
MALDI Biotyper CA
System
IVD certification for
MALDI Sepsityper Kit
Introduction of MALDI
Biotyper smart, the
first microbial MALDI-
TOF system with life-
time laser
Expanded MBT Myco-
bacteria Library with
spectra for 149 of 165
known species
MALDI Biotyper library
expanded to > 5,900
strains

1960 1980 2000 2005 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016

1960: 1991: 2004: 2008: 2010: 2012: 2014: 2016:

Foundation of the
Bruker company by
Prof. Laukien, father
of the current CEO
Frank Laukien
Introduction of the
rst commercial
MALDI-TOF
mass spectrometer
Launch of MALDI First MALDI Biotyper
Biotyper 1.0 as de- systems at microbio-
dicated microbiological logical diagnostic labs
and taxonomical and industry sites
research tool First worldwide multi-
center studies validating
the outstanding identi-
cation performance of
MALDI Biotyper
Establishment of an
international group of
experts working on iden-
tication of mycobacte-
ria and lamentous fungi
with the MALDI-TOF
approach
Bruker and Kiestra sign
collaboration agreement
Launch of the MALDI
Sepsityper Kit for
direct identication of
pathogens from positive
blood culture bottles
Bruker and BD sign
collaboration agreement
> 700 MALDI Biotyper
Systems sold worldwide
Launch of Filamentous
Fungi and Mycobacteria
Libraries
Bruker signs OEM agree-
ments with both BD and
Siemens to distribute the
MALDI Biotyper system
Bruker completes patent
portfolio for Functional
Resistance Detection
and Strain Typing
Pharmaceutical
validation and support
for 21 CFR part11
compliance
1,500 MALDI Biotyper
systems sold
New MBT STAR-BL
product line for selec-
tive testing of antibiotic
resistance
Introduction of MBT
Galaxy and MBT Pilot
instruments for facilitated
target preparation
MALDI Biotyper library
expanded to > 5,600
strains
> 2,000 MALDI Biotyper
systems sold
Introduction of the
MBT Subtyping Module
enabling the automated
detection of strain spe-
cific characteristics
Launch of the MBT
Mycobacteria Module for
optimized data analysis
of mycobacteria spectra
MALDI Biotyper library
expanded to > 6,900
strains

6
MALDI Biotyper References
The MALDI Biotyper system uses a pattern-matching algorithm to compare extracted proteomic fingerprint peak lists with a dedicated library containing an extensive set of reference spectra (main spectra) from
various microorganisms. It has been used successfully for the identification of various groups of microorganisms, as reported innumerous publications.
2016
Andersen KM, Kristoffersen AK, Ingebretsen
A, Vikholt KJ, rtengren UT, Olsen I, Enersen
M, Gaustad P (2016). Diversity and antifungal
susceptibility of Norwegian Candida glabrata
clinical isolates. J Oral Microbiol 8:29849
Bernhard M, Zautner, AE, Steinmann J, Weig
M, Gro U, Bader O (2016). Towards proteomic
species barcoding of fungi - An example using
Scedosporium/ Pseudallescheria complex
isolates. Fungal Biol 120:162165
Blosser, SJ, Drake SK, Andrasko JL, Henderson
CM, Kamboj K, Antonara S, Mijares L, Conville
P, Frank KM, Harrington SM, Balada-Llasat JM,
Zelazny AM (2016). Multi-Center MALDI-TOF MS
Study for the Identication of Clinically-Relevant
Nocardia spp. J. Clin. Microbiol. doi:10.1128/
JCM.02942-15 [Epub ahead of print]
Buckwalter SP, Olson SL, Connelly BJ,
Lucas BC, Rodning AA, Walchak RC, Deml
SM, Wohlel SL, Wengenack NL (2016).
Evaluation of Matrix-Assisted Laser Desorption
Ionization-Time of Flight Mass Spectrometry
for Identication of Mycobacterium species,
Nocardia species, and Other Aerobic
Actinomycetes. J. Clin. Microbiol. 54:376384
Camoez M, Sierra JM, Dominguez MA,
Ferrer-Navarro M, Vila J, Roca I (2016).
Automated categorization of methicillin-resistant
Staphylococcus aureus clinical isolates into
different clonal complexes by MALDI-TOF mass
spectrometry. Clin. Microbiol. Infect. 22:161.
e1161.e7
Chen JHK, Cheng VCC, Wong OY, Wong
SCY, So SYC, Yam WC, Yuen KY (2016). The
importance of matrix-assisted laser desorption
ionization-time of ight mass spectrometry
for correct identication of Clostridium difcile
isolated from chromID C. difcile chromogenic
agar. J Microbiol Immunol Infect. doi:10.1016/j.
jmii.2015.12.002 [Epub ahead of print]
Emami K, Nelson A, Hack E, Zhang J, Green
DH, Caldwell GS, Mesbahi E (2016). MALDI-
TOF Mass Spectrometry Discriminates Known
Species and Marine Environmental Isolates of
Pseudoalteromonas. Front Microbiol 7:104
Flores-Gonzlez JC, Guerrero-Lozano I,
Prez-Guerrero JJ, Hernndez-Gonzlez A,
Galn-Snchez F, Quintero-Otero S, Rubio-
Quiones F, Pantoja-Rosso S (2016). First report
of invasive fungal disease by Candida fabianii in
a non-neonatal paediatric patient. Rev Iberoam
Micol 33:4850
Fraser M, Brown Z, Houldsworth M, Borman
AM, Johnson EM (2016). Rapid identication
of 6328 isolates of pathogenic yeasts using
MALDI-ToF MS and a simplied, rapid extraction
procedure that is compatible with the Bruker
Biotyper platform and database. Med. Mycol. 54:
8088.
Kajiwara H (2016). Direct detection of the plant
pathogens Burkholderia glumae, Burkholderia
gladioli pv. gladioli, and Erwinia chrysanthemi
pv. zeae in infected rice seedlings using matrix
assisted laser desorption/ionization time-of-ight
mass spectrometry. J. Microbiol. Methods 120:
15
Karatuna O, Celebi B, Can S, Akyar I, Kilic
S (2016). The use of Matrix-assisted laser
desorption ionization-time of ight mass
spectrometry in the identication of Francisella
tularensis. Bosn J Basic Med Sci. doi:10.17305/
bjbms.2016.894 [Epub ahead of print]
Kodana M, Tarumoto N, Kawamura T, Saito
T, Ohno H, Maesaki S, Ikebuchi K (2016).
Utility of the MALDI-TOF MS method to
identify nontuberculous mycobacteria. J. Infect.
Chemother. 22:3235
Lee WS, Ou TY, Chen FL, Hsu CW, Jean SS,
(2016). Shewanella putrefaciens bacteremia
in a uremic patient receiving hemodialysis. J
Microbiol Immunol Infect 49:159160
Leroy AG, Malandain D, Duchalais , Meurette
G, Corvec S, (2016). Accurate MALDI-TOF mass
spectrometry identication of a colistin-resistant
Moellerella wisconsensis strain. Med Mal Infect.
doi:10.1016/j.medmal.2016.01.009 [Epub ahead of
print]
Lin WH, Hwang JC, Tseng CC, Chang YT, Wu
AB, Yan JJ, Wu JJ, Wang MC (2016). MALDI-
TOF MS Accelerates Pathogen Identication and
May Confer Benet in the Outcome of Peritoneal
Dialysis-Related Peritonitis. J. Clin. Microbiol.
doi:10.1128/JCM.03378-15 [Epub ahead of print]
Lotte R, Lotte L, Ruimy R (2016). Actinotignum
schaalii (formerly Actinobaculum schaalii): a newly
recognized pathogen-review of the literature.
Clin. Microbiol. Infect. 22:2836
Magnette A, Huang TD, Renzi F, Bogaerts P,
Cornelis GR, Glupczynski Y (2016). Improvement
of identication of Capnocytophaga canimorsus by
matrix-assisted laser desorption ionization-time of
ight mass spectrometry using enriched database.
Diagn. Microbiol. Infect. Dis. 84:1215
Marekovi I, Bonjak Z, Jakopovi M, Boras
Z, Jankovi M, Popovi-Grle S (2016).
Evaluation of Matrix-Assisted Laser Desorption/
Ionization Time-of-Flight Mass Spectrometry in
Identication of Nontuberculous Mycobacteria.
Chemotherapy 61:167170
Ponce-Alonso M, Rodrguez-Rojas L, Del
Campo R, Cantn R, Morosini MI (2016).
Comparison of different methods for identication
of species of the genus Raoultella: report of 11
cases of Raoultella causing bacteraemia and
literature review. Clin. Microbiol. Infect. 22:252257
Rams TE, Sautter JD, Getreu A, van Winkelhoff
AJ (2016). Phenotypic identication of
Porphyromonas gingivalis validated with matrix-
assisted laser desorption/ionization time-of-ight
mass spectrometry. Microb. Pathog. doi:10.1016/j.
micpath.2016.01.021 [Epub ahead of print]
Rodrguez-Snchez B, Ruiz-Serrano MJ, Ruiz
A, Timke M, Kostrzewa M, Bouza E (2016).
Evaluation of MALDI Biotyper mycobacteria
library v3.0 for the identication of non-
tuberculous mycobacteria. J. Clin. Microbiol.
doi:10.1128/JCM.02760-15 [Epub ahead of print]
7
MALDI Biotyper References

Sala-Comorera L, Vilar C, Galofr B, Blanch

AR, Garca-Aljaro C (2016). Use of matrix-
assisted laser desorption/ionization-time of ight
(MALDI-TOF) mass spectrometry for bacterial
monitoring in routine analysis at a drinking
water treatment plant. Int J Hyg Environ Health.
doi:10.1016/j.ijheh.2016.01.001 [Epub ahead of print]

Salzer HJF, Rolling T, Schmiedel S, Klupp

EM, Lange C, Seifert H (2016). Severe
Community-Acquired Bloodstream Infection
with Acinetobacter ursingii in Person who Injects
Drugs. Emerg Infect Dis 22:134137

Schulthess B, Bloemberg GV, Zbinden A,

Mouttet F, Zbinden R, Bttger EC, Hombach M
(2016). Evaluation of the Bruker MALDI Biotyper
for Identication of Fastidious Gram-Negative
Rods. J. Clin. Microbiol. 54:543548

Schweitzer VA, van Dam AP, Hananta IPY,

Schuurman R, Kusters JG, Rentenaar RJ
(2016). Bruker matrix-assisted laser desorption/
ionization time-of-ight mass spectrometry
identication of Neisseria gonorrhoeae is
improved by a database extension. J. Clin.
Microbiol. doi:10.1128/JCM.00016-16 [Epub
ahead of print]

Sparbier K, Schubert S, Kostrzewa M (2016).

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MALDI Biotyper Poster Hall 2016 Primary ID

Gram-negative bacilli isolated from bronchial aspiration from patients in the Intensive Care
Unit and performance of MALDI-TOF MS for routine identification.
Mara Dolores Guerrero1, Luz Balsalobre1, Cristina Santa Olalla1, Teresa Alarcn1.
1U.H. La Princesa, Madrid, Spain.

RESULTS

The rapid identification of Gram-negative bacilli in ventilator-

295 isolates from 98 patients were obtained. Type and frequency of gram negative bacilli is shown in table 1.
associated pneumonia is crucial to the therapeutic management of
124 out of 295 isolates (42.0%) underwent no identification by MALDI-TOF MS because there was no clinical
intensive care unit (ICU) patients.
need or because the patients had the same bacteria in a previous culture or because the morphology was
suggestive of a particular species. Of the remaining 171 isolates, the identification between MALDI-TOF MS
and the traditional methods coincided to species level in 95.9% (n=164) and to genus level in 4.1% (n=7)
(Acinetobacter, Enterobacter, Flavobacterium, Klebsiella and Serratia). MALDI-TOF MS identification was
OBJECTIVES obtained 1 to 6 days before MScan identification (Graphic.1). 95.3% of the gram negative rods were identified
by MALDI-TOF with a score 2.0 and the remaining 4.7% with scores of 1.700 to 1.999.

Isolates
Microorganisms
To study the frequency of isolates of gram negative bacilli from Number Percentage (%)

bronchial aspirations (BAS) in ICU patients and to analyze the Pseudomonas aeruginosa
71 24.1
breakthrough time of the identification by Matrix-Assisted Laser Non-fermenting Stenotrophomonas maltophilia 5 days 6 days
29 9.8 4 days
Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF Gram-negative bacteria 1,7 % 1,2 %
Acinetobacter 6,4 %
MS) compared with traditional methods. baumannii/haemolyticus 23 7.8

Delftia acidovorans 1 0.3

Flavobacterium spp. 1 0.3
3 days
Klebsiella pneumoniae
63 21.4 21,6 % 1 day
Serratia marcescens 20 6.8
48 %
Enterobacter cloacae 19 6.4 2 days
METHODS Enterobacteriaceae Proteus mirabilis 17 5.8
21,1 %
Escherichia coli 15 5.1
Klebsiella oxytoca 15 5.1
Morganella morganii 10 3.4
Enterobacter aerogenes
5 1.7
Proteus vulgaris 3 1.0
Retrospective study from 09/01/2013 to 30/09/2014 of gram- Hafnia alvei 2 0.7
negative bacilli isolated from BAS in ICU patients at the U.H. La Cronobacter sakazakii
1 0.3
Princesa. MALDI-TOF MS (Bruker) was compared with the Graphic 1. Time gained in the
Total 295 100
identification by MALDI TOF MS in
conventional phenotypic methods used routinely in our laboratory relation to Mscan.
for the identification of microorganisms, Mscan WalkAway Table 1. Frequency of isolates of Gram-negative bacilli from ICU patients.
(Siemens). MALDI-TOF MS mass spectra were acquired in the
range 2-20 KDa, using a Microflex LT mass CONCLUSIONS
spectrometer, processed through the flexControl 3.4 software and
analyzed through flexAnalysis 3.4 provided by the manufacturer.
Scores 2.0 were considered reliable for species level Pseudomonas aeruginosa, Klebsiella pneumoniae and Stenotrophomonas maltophilia were the microorganisms
identification, scores of 1.700 to 1.999 were considered reliable for more frequently isolated from BAS in ICU patients. MALDI-TOF MS technology proves to be a fast and reliable
genus level, and scores lower than 1.700 were not considered identification method, improving the routine identification of ICU isolates. In most cases it was between 1 to 3
reliable. The difference in the identification time between the two days ahead of conventional methods. This is of vital importance to direct appropriate therapy to critically ill
systems was also analyzed. patients. In view of these facts, it could replace conventional identification methods.

20
MALDI Biotyper Poster Hall 2016 Primary ID

Clinical case of Brucellosis identified by MALDI-TOF-MS

S.Horridge1, W. Shinwari1 and P.G. Munthali1,2.
1University Hospitals Coventry and Warwickshire NHS Trust, Coventry
2Warwick University, Warwickshire

AIM: The aim of this was to use MALDI Biotypers Security-Related (SR) database to identify a possible Brucella species from blood culture growth in a patient with indicative clinical presentation.
BACKGROUND: Brucella spp. are a zoonosis that can cause human infections and can be considered a bioterrorism threat 1. The organism is associated with travel to endemic areas 2 and contact with animals and unpasteurised dairy products 1. MALDI-TOF is a rapid Identification (ID) system 3 used for routine diagnostics in bacteriology at
Coventry and Warwickshire Pathology Services (CWPS) since June 2015 and has a security database which can be used to identify containment level 3 organisms following extraction to render them safe for handling outside of CL3 laboratory.
METHOD: Patient presented with history of high fevers for four weeks since return from Somalia. Blood cultures were taken on admission. A gram stain was performed and showed gram negative cocco-bacilli (Figure 1). Sample was cultured on chocolate and blood agar, incubated at 37oC in CL3 incubator and growth examined at 24 and 48 hrs.
At 24hours, colonies from the plate were used for carrying out the full formic acid extraction method for MALDI-TOF 4,5 and target plate run through the SR database.
RESULTS: MALDI-TOF produced an ID of Brucella melitensis (score 2.423) (Figure 2a). Cultures and serology were sent to the reference laboratory. The patients antibiotic treatment was optimised for brucellosis and they were discharged.
CONCLUSION: Previously, the isolate would have required biochemical testing for provisional ID 1 and sending to reference laboratory for full identification.
MALDI-TOF provided a rapid ID confirming diagnosis of Brucellosis six days before the reference laboratory confirmatory report was received. This resulted in more effective patient management in this case of Brucellosis, as well as efficient management of potentially exposed laboratory staff.

Table 1: Comparison of methods for diagnosing Brucellosis

Introduction Method: MALDI TOF extraction and database use Gram stain and Serology Reference laboratory MALDI-TOF
Direct ID of CL3 organisms can not be performed as they require inactivation using extraction to culture testing
Brucella spp. are zoonotic organisms consisting of six species of which four have been
associated with human infections: B.melitensis, B.abortus, B. canis and B.suis 4. It has render them safe for handling outside of CL3 and analysis using MALDI-TOF MS 8. Method Blood culture bottles IgM, IgG, Total antibody Culture is sent off to the Colonies of organism
flag positive on the (Brucella Capt) are reference laboratory are extracted using
worldwide distribution but is endemic in the Arabian peninsula, Indian subcontinent, instrument. measured and where growth formic acid full
Extraction method was performed as recommended by manufacturer. Briefly 5 small colonies
Mediterranean basin and parts of South and Central America, Central Asia and Africa 2. Gram stain is Complement Fixation characteristics related extraction, spotted
were added to 300 l deionized water in a 1.5 ml tube (Eppendorf, Germany) and mixed with a
Human acquisition is usually via direct contact with infected animals, consumption of pipette. 900 l ethanol was added. Mixture was centrifuged at 13,000 rpm for 2 minutes.
performed. Organism Test (CFT) performed to urea, H2s, CO2, basic onto a MALDI-TOF
unpasteurised dairy products or inhalation of aerosols 1. Goats are the most common will appear as gram from serum at fuchsin and thionin are target plate and run
Supernatant was decanted, centrifuge step repeated, residual alcohol removed and pellet left to negative pleomorphic presentation, 2, 6, 12 assessed. Reactions through the routine
host of B.melitensis, along with camels and sheep and this particular species has the air-dry at room temperature. 40 l 70% formic acid was added to the pellet, mixed with a pipette cocco-bacilli 1 ; Figure and 24 weeks. with monospecific sera and SR database for
widest distribution 1. and 40 l acetonitrile added. Mixture was centrifuged as before, 1 l of supernatant spotted 1). Titres used to make are performed as well genus and species ID.
onto steel target plate. Spot was air-dried, overlaid with 1 l matrix (-cyano-4-hydroxy-cinnamic Blood culture is diagnosis. as phages looking for
The most common presentation is high and persistent fever possibly with headaches, acid in organic solvent of 50% acetonitrile and 2.5% trifluoroacetic acid) and air dried 4,5. inoculated onto blood, lysis.
MALDI Biotyper version 3.1 software was used to analyse the organism on a microflex LT mass chocolate and
back pain, malaise and anorexia 1. anaerobic agar and
Due to low infectious dose and aerosolisation being one of its transmission routes 6, it is spectrometer 6 using the Biotyper reference library and subsequently the SR database 7.
incubated at 37oC in
a potential bioterrorism agent and poses an infection risk to laboratory staff if not CO2 incubator.
handled in Containment Level 3 (CL3) laboratory 4,7. Organism will appear
as grey colonies.
Time to Gram stain - 5mins Assay performed at Cultures- Minimum Cultures Minimum
MALDI-TOF-MS Biotyper is a rapid ID system that uses mass spectrometry and an
result Cultures - Minimum reference laboratory - 18hours of growth * 18hours of growth
extensive database to identify organisms to genus and species level 3. It uses a laser to 18hours of growth. weekly. Report received - 5-7 Full extraction- 20-
ionize proteins from a bacteria or yeast, which are accelerated through an electric field Reports back to days 30mins
and flight tube. It separates them based on their mass-to-charge ratio, detects them and laboratory- variable (2- *Can send blood culture Running isolate
4weeks) bottle if cultures do not through database
produces a spectrum 6 (Figure 2b), which is compared to an existing database of known
grow <5mins
spectra. As of June 2015, Coventry and Warwickshire Pathology Services (CWPS)
bacteriology department at UHCW NHS Trust have used it for routine diagnosis of (Sepsityper can be used
clinical isolates. It has an additional Security-Related (SR) database which can be used direct from blood
Figure 2: a) MALDI-TOF results following extraction of organism with a score confirming ID to species level. B) Example of culture bottle reducing
for identifying CL3 organisms. spectrum for B.melitensis produced by MALDI Biotyper 4 the ID time to <1hour.)
Result Presumptive ID only Presumptive diagnosis 1 Confirmed ID to species Confirmed ID to species
The aim of this was to use MALDI Biotyper to identify possible Brucella species from
blood culture growth in a patient with indicative clinical presentation.
Case presentation and timeline level level
Twelve year old boy was admitted to hospital following four weeks of ~40oC fevers two to three
times a day. Patient had a history of recent travel to Somalia with his father and exposure to
handling goats whilst there. Potentially drank unpasteurised goats milk. Conclusions
On admission (Day 1): 1st malaria screen negative, urine negative, normal chest X-ray. MALDI Biotyper provided a rapid, accurate identification of a CL3 organism, B.
melitensis in this case.
Day 2: Blood cultures taken, normal abdominal ultrasound. 2nd malaria screen negative. It allowed for effective targeted treatment of the patient for Brucellosis and
Day 3: Virology & parastiology screens negative, Brucella serology sent to reference laboratory. subsequent discharge.
Clinical suspicion and so swift movement of the samples and cultures into CL3
Day 4: 3rd malaria screen negative. laboratory, along with confirmed ID by MALDI-TOF ensured efficient management of
Day 6: Stool, ova and cyst parasite investigations negative. Aerobic blood culture bottle became laboratory staff potentially exposed.
positive, gram stain performed and gram-negative cocco-bacilli seen. Brucellosis suspected so
plates and cultures taken into CL3 laboratory.
Repeat blood cultures requested.
Patient started on ceftriaxone and doxycycline while awaiting ID.
Acknowledgements
Staff from the Bacteriology Department at UHCW NHS Trust for performing the extraction and MALDI-TOF identification of the isolates
Microbiology clinical team for their quick handling of the situation and good liaison with paediatric department
Day 7: Growth on chocolate and blood agar. Full extraction method performed, isolate run Dr Peter Hoffmann, Consultant Clinical Scientist Public Health England
through MALDI Biotyper and ID of B. melitensis given (score of 2.423) (Figure 2). Patient put on Dr Pankaj Lal Consultant Medical Microbiologist
Lorraine Perrett, Brucella Reference Laboratory of the Animal Health and Veterinary Laboratory Agency
Brucellosis treatment; doxycycline and rifampicin1, and discharged. UHCW Occupational Health Department for efficient follow-up and reassurance for exposed staff members
Incident meeting held. Specimen sent to reference laboratory.

Day 8: Occupational health started prophylaxis for staff considered high-risk of exposure.
Day 13: (6 days post ID by MALDI Biotyper)- reference laboratory
confirmation of B.melitensis serovar 1; sensitive to tetracycline, rifampicin, ciprofloxacin,
streptomycin, gentamicin.
References
1 Young, E.J. (2010). Brucella Species. In: Mandell, G.L. Bennett J.E. and Dollin,R. eds. Mandell, Douglas and Bennetts Principles and Practice of
Infectious Diseases (7th Edition). Philadelphia: Churchill Livingstone Elsevier, 2921-2925
2 Pappas, G. Papadimitriou, P. Akritidis, N.et al. (2006). The Lancet Infectious Diseases, 6(2): 91-99
3 Seng, P. Drancourt, M. Gouriet, F. et al. (2009). Clin. Infect. Dis., 49(4): 543-51
4 Ferreira,L. Vega Castao, S. Snchez-Juanes, F. et al. (2010). PLoS ONE, 5(12): e14235
5 Marklein, G. Josten, M. Klanke, U. et al. (2009). J. Clin. Microbiol., 47(9): 2912-2917

Day 29: Serology results back from reference laboratory confirming acute brucellosis 6 Hartmeyer, G.N. Jensen, A.K. Bcher, S. et al. (2010). Scandinavian Journal of Infectious Diseases, 42(9): 716-718

Figure 1: Gram stain from patients blood culture showing pleomorphic gram negative cocco-bacilli (IgM >2560, IgG 80, CFT 128) 6 Cunningham, S.A. and Patel, R. (2013).J Clinc.Microbiol, 51(5): 1639-1640
7 Drevinek, M. Dresler, J. Klimentova, J. et al. (2012). Letters in Applied Microbiology, 55: 40-46

21
 22
Primary ID

Identification of Highly Pathogenic Microorganisms

using MALDI-TOF Mass Spectrometry
Results of an Inter-Laboratory Ring Trial
Peter Lasch1, Tara Wahab 2, Sandra Weil 3, Bernadett Plyi 4, Herbert Tomaso 5, Sabine Zange 6,
Beathe Kiland Granerud 7, Michal Drevinek 8, Branko Kokotovic 9, Matthias Wittwer 10, Valentin Pflger 11,
Antonino Di Caro 12, Maren Stmmler 1, Roland Grunow 13 and Daniela Jacob 13
Motivation: In the case of a release of highly pathogenic bacteria (HPB) there is an urgent need for rapid, accurate and reliable
diagnostics. MALDI-TOF mass spectrometry is a rapid, accurate and cost effective technique which is becoming increasingly important in
microbiological diagnostics to complement classical microbiology, PCR and genotyping of HPB. In the present study, the results of a joint
exercise with eleven partner institutions from nine European countries are presented. In this exercise ten distinct microbial samples, among
them five HPB, Bacillus anthracis, Brucella canis, Burkholderia mallei, Burkholderia pseudomallei and Yersinia pestis were characterized
under blinded conditions.
Time line
Second meeting
in Solna (SWE): evaluation
Pilot tests on non-
of pilot tests, exchange of
First meeting pathogenic strains Inactivation of HPB:
data base spectra
at RKI (GER): standardization & -inactivation (30 kGy)
optimization of sample prep/meas
conditions
European Ring Trial Shipment of the samples
to the partners (UN3373)
Final MALDI measurements,
analysis, preparation Third meeting in Spectra analysis at evaluation on site,
of a publication Brussels (BEL): analysis the study centre (RKI), approach A
and discussion approaches B&C
Samples Results
# Genus / Species / Strain Concentration (cfu/mL) Identification Approach A Identification Approach B Identification Approach C
No. Sample Identity Partly Partly Partly
1 Burkholderia pseudomallei A101-10 1.1 109 Correct Incorrect Correct Incorrect Correct Incorrect
correct correct correct
10
2 Francisella tularensis ssp. holarctica Ft 32 1.7 10 1
9 1 1 9 1 0 10 1 0
Burkholderia pseudomallei A101-10
3 Brucella canis A183-5 10 9.5 (86%) 9.5 (95%) 10.5 (95%)
1.9 10
4.5 6.5 0 9 1 0 11 0 0
2 Francisella tularensis ssp. holarctica Ft 32
4 Bacillus anthracis AMES 6.4 107 7.75 (70%) 9.5 (95%) 11 (100%)
5 Ochrobactrum anthropi A148-11 2.0 1010 3 Brucella canis A183-5
3 8 0 10 0 0 11 0 0
7 (64%) 10 (100%) 11 (100%)
6 Yersinia pseudotuberculosis type III 1.3 109
9 0 2 9 1 0 11 0 0
7 Burkholderia mallei A106-3 9 4 Bacillus anthracis AMES
1.0 10 9 (82%) 9.5 (95%) 11 (100%)
8 Burkholderia thailandensis E125 5.6 1010 5 Ochrobactrum anthropi A148-11
10 1 0 10 0 0 11 0 0
10.5 (95%) 10 (100%) 11 (100%)
9 Yersinia pestis A106-2 1.3 107 8 0 3 6 3 1 9 1 1
10 Bacillus thuringiensis DSM350 8 6 Yersinia pseudotuberculosis type III
8.6 10 8 (73%) 7.5 (75%) 9.5 (86%)
11* Escherichia coli RKI A139 9 0 2 9 1 0 8 3 0
7 Burkholderia mallei A106-3
9 (82%) 9.5 (95%) 9.5 (86%)
12* Bacillus cereus BW-B
10 1 0 10 0 0 11 0 0
8 Burkholderia thailandensis E125
13 Bacillus cereus ATCC 10987 10.5 (95%) 10 (100%) 11 (100%)
14 Bacillus thuringiensis DSM 5815 7 3 1 6 4 0 6 5 0
9 Yersinia pestis A106-2
8.5 (77%) 8 (80%) 8.5 (77%)
15 Burkholderia thailandensis DSM 13276 4 2 5 10 0 0 9 2 0
10 Bacillus thuringiensis DSM350
16 Yersinia enterocolitica DSM 4780 5 (45%) 10 (100%) 10 (91%)
Summary of the different identification results of the MALDI-TOF MS ring trial with the number of correct, partly correct and incorrect identifications. The
Overview on microbial strains and species used in the inter-laboratory ring trial (samples 1-10) cells contain furthermore a point sum (correct identification: one point, partly correct: half point and incorrect: zero points) and the corresponding
* Strains utilized for -inactivation test measurements in advance of the ring trial identification accuracy (in %). Color scheme, green: the identification accuracy of the given microbial strain is equal or larger than 90%, yellow:
Strains used for pilot tests on non-HPB accuracy is equal or larger than 75% and below 90% and red: accuracy below 75%.
Partner Institutions Conclusions
1
Robert Koch Institute, Proteomics and Spectroscopy, ZBS6, Berlin, An inter-laboratory external quality assurance exercise (EQAE) has been
Germany conducted by eleven partner institutions from nine European countries
Public Health Agency of Sweden, Solna, Sweden
2
3
Austrian Agency for Health and Food Safety, Vienna, Austria MALDI-TOF MS was used as a tool for rapid, reliable and cost-effective
4
National Center for Epidemiology, Department of Bacteriology, identification of highly pathogenic microorganisms
Budapest, Hungary Preparatory phase: pilot tests on non-pathogenic strains to optimize and
5
Friedrich-Loeffler-Institut, Institute of Bacterial Infections and Zoonoses, standardize the experimental procedures
Jena, Germany
6
Institute for Microbiology of the Bundeswehr, Munich, Germany Irradiation by -rays proved to be a MALDI-TOF MS compatible and
Norwegian Institute of Public Health, Oslo, Norway induced only subtle spectral changes
7
MALDI Biotyper Poster Hall 2016

National Institute for Nuclear, Chemical and Biological Protection,

8
Milin, Czech Republic The average identification accuracy equaled 77% when using non-standard
9
National Veterinary Institute, Technical University of Denmark, mass spectral databases
Frederiksberg, Denmark The accuracy could be improved to > 93% when spectral diagnoses were
10
Spiez Laboratory, Federal Office for Civil Protection, Spiez, Switzerland attained on the basis of an optimized spectral database with a better
MABRITEC AG, Riehen, Switzerland
11
coverage of highly pathogenic and related species
12
Microbiology Laboratory and Infectious Diseases Biorepository,
L. Spallanzani National Institute for Infectious Diseases, Rome, Italy The compilation of comprehensive databases is considered to be of
13
Robert Koch Institute, Highly Pathogenic Microorganisms, ZBS2, Berlin, paramount importance for reaching accurate spectral diagnoses
Germany
Future efforts to improve the diagnostic capabilities should focus on the
exchange of validated reference spectra
Acknowledgments
The authors wish to thank Dr. T. M. Fuchs (ZIEL, Technical University Munich, Germany), Dr. J. Rau (CVUA, Stuttgart, Germany),
Dr. W. Beyer (University of Hohenheim, Stuttgart, Germany), Dr. A. Paauw (TNO, Rijswijk, Netherlands), M. Dybwad (NDRE, Kjeller, Publications
Norway), and Dr. N. Schrch (Labor Spiez, BABS, Spiez, Switzerland) for providing strains, samples, or spectra of important Lasch P. et al., Identification of Highly Pathogenic Microorganisms using MALDI-TOF Mass
microbial pathogens. S. Weil, Dr. S. Zange and Dr. B. Plyi are grateful to Dr. P. Hufnagl (AGES, Vienna, Austria), Dr. B. Thoma Spectrometry Results of an Inter-Laboratory Ring Trial, J Clin Microbiol 2015. 53(8):2632-40
(InstMikroBioBw, Munich, Germany) and Dr. M. Ivn (Semmelweis University, Budapest, Hungary), respectively. In addition, we like
to thank S. Becker, P. Lochau, A. Schneider, S. Howaldt, and R. Andrich (all RKI, Berlin, Germany) for excellent technical Wittwer M. et al., First Report: Application of MALDI-TOF MS within an External Quality
assistance. Moreover, we are very grateful to the European Commission and CHAFEA for financially and technically supporting the Assurance Exercise for the Discrimination of Highly Pathogenic Bacteria from
QUANDHIP Joint Action (CHAFEA Grant Agreement n 2010 21 02). Contaminant Flora. Applied Biosafety. 2012. 17(2): 59-63.
MALDI Biotyper Poster Hall 2016 Primary ID

A. Magnette1, T.-D. Huang1, F. Renzi, P. Bogaerts1, G. Cornelis, Y. Glupczynski1 Poster

1Laboratory
of Clinical Microbiology, CHU Dinant-Godinne UCL Namur, B-5530 Yvoir, Belgium
Research Unit in Biology of Microorganisms, Namur Institute for Life Sciences (NARILIS), University of Namur, B-5000 Namur, Belgium eP224
Introduction Objectives Results
Capnocytophaga canimorsus (CC) and Capnocytophaga The aim of this study was to evaluate the MALDI-TOF MS identification of CC and CD isolates with the original MALDI BioTyper database
cynodegmi (CD) are gram-negative bacteria that belong ability of MALDI-TOF MS to identify these two For the CC strains (n=94):
to the commensal oropharyngeal flora and that can be Capnocytophaga species and to assess the Identification to the species level (log score 2) for 1 (1%) strain
transmitted to humans from dogs or cats and cause added value of a home-made built reference Identification to the genus level (log score between <2 and 1.7) for 15 (16%) strains
serious infections. Routine bacteriological methods are database of mass spectra to improve their Unreliable identification (log score <1.7) obtained for the 78 (82,9%) remaining strains
not optimal for their accurate and timely identification. identification. For the CD strains (n=10):
Identification to the species level (log score 2) for the 10 (100%) strains
Constitution of an enriched CC and CD database
Materials and Methods Generation of a dendrogram with species-specific cluster patterns with the mass spectra of the extracted strains.
94 CC and 10 CD isolates including animal and clinical strains were included in the study (Table 1). This dendrogram shows:
All strains were subcultured twice on 5% sheep blood Schaedler agar supplemented with vitamin K1 1 cluster of the 8 CD strains
(Becton-Dickinson) and incubated for 2-3 days at 35C with 5% CO2. 2 clusters (clusters I and II) among the isolates of CC : cluster I included 9 isolates all from dogs only and cluster II
First step: Identification of all isolates by MALDI-TOF MS using Microflex LT (Bruker Daltonics) based on the contained 23 and 19 isolates from dogs and humans, respectively (Figure 1).
MALDI BioTyper database (version 3.0; mean spectra [MSP] of 5627 cellular organisms). A first score was
obtained after submitting the downloaded raw spectra to the original MALDI BioTyper database.
Second step: Establishment of a complementary homemade reference database by the analysis of 51 CC and 8
CD isolates in the MALDI BioTyper software. Species Human/animals Clinical sites Country No. of isolates
Belgium 13
Dog mouth
Extraction procedure and creation of MSP: Dogs
(n = 75)
Italy 1
After 2-3 days of incubation, colonies were suspended in distilled Switzerland 61

water + ethanol solution, centrifuged and suspended in formic acid Capnocytophaga

Belgium 10
Germany 1
solution + acetonitrile. canimorsus
Blood cultures Netherlands 1
1 L of the resulting supernatant, spotted ten times on the steel Human
(n = 19) Switzerland 3
surface of a target plate, was overlaid with 1 L of matrix and Sweden 1
subjected to MALDI-TOF MS analysis. USA 3
Each spot was measured three times by the MBT_FC.par flexControl Belgium 1
Dog mouth
method and the MBT-autoX.axe autoExecute method. Dogs
(n = 7)
Switzerland 5

30 spectra obtained per strain were closely analyzed in the flexAnalysis Capnocytophaga
USA 1
Hand wound
program and a minimum of 20 accurate spectra were downloaded to cynodegmi Human
(n = 2)
USA 2

the MALDI BioTyper software in order to generate a single MSP Unknown

Unknown origin 1
accounting for the extracted Capnocytophaga strain with the BioTyper (n = 1)

MSP creation standard method. Table 1: CC and CD isolates included in the study (n=104)

Third step: Challenge of the MALDI BioTyper database and the generated database with 45 blind-coded isolates
of CC and CD. Figure 1: Dendrogram of 59 Capnocytophaga strains analyzed, including 8 CD strains and 51 CC strains.

Conclusions
The enrichment with our own database by including additional Capnocytophaga canimorsus
isolates significantly improved the performance of identification of CC to the species level and
may represent a useful complement to Brukers MALDI BioTyper database for the diagnosis of
these difficult to identify organisms.
Correspondance: Dr. Te-Din Daniel Huang. Laboratory of Microbiology CHU Dinant-Godinne UCL Namur, 1 Avenue Dr. G. Therasse, 5530 Yvoir, Belgium.
te-din.huang@uclouvain.be
MALDI-TOF MS identification of CC and CD challenge strains using the enriched database
After including the spectra of 59 extracted CC and CD isolates, the newly constituted database combined with the
MALDI BioTyper database were challenged with 43 randomly selected CC strains not included in the new
database
Following addition of the homemade database in the BioTyper software, MALDI-TOF MS provided reliable
identification to the species level for all 43 CC strains tested with an average score of 2.33 (range 2.012-2.543).

23
MALDI Biotyper Poster Hall 2016 Primary ID

24
MALDI Biotyper Poster Hall 2016 Primary ID

It is time for accurate identification of Raoultella spp.

L. Rodrguez-Rojas, P.M. Ponce-Alonso, R. del Campo, R. Cantn, M.I. Morosini
Microbiology and Parasitology Laboratory. Ramn y Cajal University Hospital, Madrid, Spain
Introduction and purpose. Raoultella is an
Primers
Antibiotic Age
enterobacterial genus recently split from the genus Case Year MicroScan Api 20E MALDI-TOF (score)
resistance
Sex
(years)
Diagnosis
blaorn blapla
Klebsiella that may be misidentified at routine
Ampicillin,
1 2011 K. oxytoca K. oxytoca R. ornithinolytica (2.50) + - Female 63 Acute cholangitis
laboratories if only phenotypic identification systems are Fosfomycin

used. It has been recently described that mass 2 2012 K. oxytoca K. oxytoca R. ornithinolytica (2.52) + - Ampicillin Male 78 Acute cholangitis

spectrometry has an adequate resolution power to 3 2012 K. oxytoca K. oxytoca R. ornithinolytica (2.53) + -
Ampicillin,
Male 60
Renal and hepatic cyst infection
Fosfomycin (hepatorenal polycystosis)
differentiate both genera.
4 2012 K. oxytoca K. oxytoca R. ornithinolytica (2.38) + - Ampicillin Male 77 Acute cholangitis
The aim of this work was to retrospectively re-identify
K. K.
the putative Raoultella isolates that had been reported as 5 2012 R. planticola (2.51) - + Ampicillin Male 67 Fever of unknown origin
pneumoniae pneumoniae
Klebsiella spp. in positive blood cultures of our 6 2013 K. oxytoca K. oxytoca R. ornithinolytica (2.42) + - Ampicillin Male 77 Acute cholangitis
Institution. K. K. Acute cholecystitis with
7 2014 R. ornithinolytica (2.32) + - Ampicillin Male 74
pneumoniae pneumoniae gallblader perforation. Sepsis
Material and Methods. A retrospective search among the
K. K.
8 2014 R. planticola (2.38) - + Ampicillin Male 87 Short febrile illness with unknown focus
blood culture isolates collected in our Microbiology pneumoniae pneumoniae
Post-endoscopic retrograde
Department (2011-2014) was performed, selecting Ampicillin, Cefalotin,
9 2014 K. oxytoca K. oxytoca R. ornithinolytica (1.76) + - Male 33 Cholangiopancreatography, acute
Amoxicillin/Clavulanic
cholangitis. Sepsis
samples initially identified as K. oxytoca or K.
10 2014 K. oxytoca K. oxytoca R. ornithinolytica (2.5) + - Ampicillin Female 81 Acute lithiasic pancreatitis
pneumoniae. A total of 80 isolates were recovered and
submitted to MALDI-TOF re-identification. A subset of 10 Results. Discrepancies between results from the identification methods are shown in the Table. Considering the blaorn and blapla gene sequences as
isolates (12.5%), consistently identified as Raoultella (8 R. the gold-standard method, MALDI-TOF accurately differentiated 8 as R. ornithinolytica and 2 as R. planticola with high scores (median, 2.45). As
ornithinolytica and 2 R. planticola), was further studied expected, all isolates exhibited resistance to ampicillin, with variable susceptibility to other antibiotics.
by MicroScan (EUCAST criteria), API 20E (bioMrieux,
Conclusions. At least 10 isolates of Raoultella spp. have been previously misidentified as Klebsiella spp. in our laboratory in the last 4 years.
France) and 16S rRNA, blaorn and blapla PCR amplifications
MALDI-TOF seems to be a suitable alternative to properly identify these still infrequent isolates. The strong association between bacteraemia
and further nucleotide sequencing. Clinical data were
due to Raoultella and biliary tract-related pathologies justifies the need of a correct identification as it can help clinicians to guide clinical
obtained after patients chart revision.
diagnosis, particularly in those patients with fever of unknown origin.

25
MALDI Biotyper Poster Hall 2016 Primary ID

Evaluation of the MALDI-TOF MS method for identification of Nocardia species

Segura C, Plasencia V, Clotet A, Salvad M, Sez-Nieto JA. Laboratorio de Referencia de Catalua. Barcelona

OBJECTIVE MALDI-TOF MS data interpretation. RESULTS C O N C L U S I O N S

To evaluate a rapid and simple Nocardia species Scores of >2.0 were accepted for species Fifty-one Nocardia strains were identified by DTF method is a good procedure to
identification method using Matrix-assisted laser assignment and scores between 1.7 and 2.0 for Bruker MALDI Biotyper system, discarding those identify Nocardia species. The implementation of
desorption ionization-Time of flight Mass identification to the genus level, and scores below that are not found in BruKer Daltonik database MALDI-TOF in the workflow of a microbiology
spectrometry (MALDI-TOF MS). For this purpose 1.7 for not reliable interpretation. (N. flavorosea, N. coubleae, N. levis and N. mexicana) laboratory would reduce the turnaround time
two different sample preparation protocols were the identification rate rose at 93.48% (DTF) and for the identification of Nocardia. 16S rRNA gene
compared. 84,78 % (PEM). Correct identification at species sequencing would only be necessary in those
level was achieved for 37 (80,43%) (DTF) and 21 cases when MALDI-TOF does not give us re l i a
M E T H O D S Figure 1. Correct Nocardia identification (n=51). (45,65%) (PEM). All N. farcinica and 90 % of N. b l e re s u l t s . U n f o r t u n at e l y,
Bacterial strains and culture conditions. cyaricigeorgica were identified at species level. 3 some Nocardia species are still not found in
Fifty-one Nocardia were collected, all isolates from strains (Nocardia abscessus, N. otitidiscaviarium, Biotyper database, therefore it is necessary to
clinical specimens between 2007 and 2013 in Nocardia brasilensi) were not identified. amplify this database in order to improve these
Laboratorio de Referencia de Catalua results.
(Barcelona). These bacterial strains had been
previously identified by 16S rRNA gene
sequencing. Six N. farcinica, 4 N. abscessus, 2 N.
brasilensis, 1 N. coubleae, 29 N.cyaricigeorgica, 2 N.
flavoroseae, 1 N. levis, 1 N. mexicana, 3 N. nova, 2 N.
otitidiscaviarium. The clinical origins of the strains
were from respiratory tract. Organisms were
cultured in blood agar with 5% sheep blood, for
48 to 72 hours at 37 C in CO2.
Sample preparation protocols.
Direct transfer-formic acid method (DTF). 1 l of
formic acid (70%) was added to the bacterial Figure 2. Correct Nocardia identification, without
spot; air dried and overlaid 1 l of matrix Nocardia species not found on Biotyper database
solution. (n=46)
Protein extraction method (PEM). 1) 10 colonies
were resuspended in 500 l distilled water and
boiled. 2) 2 min. centrifugation at 13,000 rpm. 3)
300 l distilled water and 900 l of ethanol were
added to the pellet. 4) two series of
centrifugation of 2 min at 13,000 rpm. 5) The
dried pellet was resuspended in 50 l of formic
acid and incubated for 15 min at room
temperature. 6) 50 l of acetronitrile was added
and it was centrifuged for 2 min at 13,000 rpm.
Finally, 1 l supernatant was analysed by MALDI-
TOF.

This could be a place for your sources.

26
MALDI Biotyper Poster Hall 2016 Primary ID

Evaluation of MALDI-TOF as a high volume enteric pathogen screening method compared to an

in-house PCR based algorithm at a large outpatient laboratory
V. Singh1, J. Davidson1, R. Reyes1,2, M. Imperial1,2, MT Kelly1,2
Lifelabs Medical Laboratory Services, Burnaby, B.C, Canada
Department of Pathology and Laboratory Medicine, University of British Columbia, Canada

Abstract Methods Results

Background A two phase study was conducted to compare the incumbent method (in-house PCR with supplemental Of the 214 enteric pathogens listed in table 2:
biochemicals/sequencing ) to an algorithm incorporating the MALDI-TOF as the primary identification During Phase 1, a total of 2132 suspected isolates were tested by both algorithms. A combined 209
The detection and identification of enteric pathogens for many laboratories remains a complex method. enteric pathogens were then isolated during both Phase 1 and 2 of the study. Accuracy of identification to genus level was 67% for MALDI-TOF and 57% for PCR. While
algorithm of using differential and selective media followed by various identification methods for accuracy to species level was 50% for MALDI-TOF and 12% for PCR. Unidentified organisms were
suspected enteric pathogens. This can be both time consuming and expensive. MALDI-TOF The incumbent identification method consisted of a previously validated conventional gel-based multiplex Compared to the incumbent method, the MALDI-TOF based algorithm: 14% for MALDI-TOF and 31% for PCR. These results are summarized in Table 2.
has the ability to improve the turn around time and reduce the complexity of the identification of PCR method with primers targeting specific loci of the following enteric pathogens: Salmonella spp,
suspect enteric pathogens, despite its well described limitations such as being unable to Aeromonas spp, Yersinia spp. Shigella spp., Enteroinvasive E.coli, Shiga
Shiga-toxin
toxin producing E.coli (via Correctly identified: C. jejuni, C.coli , E.tarda, P.shigelloides, Aeromonas hydrophila group spp. Of the remaining 1918 non-enteric
non enteric pathogens tested:
differentiate Shigella spp from Eschericia coli. This study explores the use of MALD-TOF as a detection of stx1/2 genes), Yersinia spp, Vibrio spp. Supplemental biochemical testing (API and Vitek) and V.parahaemolyticus 8.8% of resutls did not correlate between MALDI-TOF and PCR. Additional testing methods were
high volume stool screening method at a large outpatient laboratory and explores the limitations and serology are used for more specific identification of Salmonella spp, Shigella spp. and Vibrio spp. A Correctly identified Salmonella only to genus level, secondary testing required to serotype via implemented to determine identficiation. PCR displayed 3.3% as non-amplified. MALDI-TOF testing
inherent with the method as compared to an in-house developed PCR identification algorithm. supplemental multiplex PCR assay is used for speciating organisms identified as Yersinia spp. serology or Vitek (same as incumbent PCR method) resulted in an initial 0.4% with no peaks obtained and 0.7% with no reliable identification.
Campylobacter spp. are identified by isolation of suspicious colonies from Campylobacter selective media Misidentified 4 Y.frederiksenii as Y.enterocolitica. Discrepants resolved by a combination of PCR,
Methods at 42 Celsius, morphology (gram stain) and oxidase. Suspected Campylobacter isolates were then sent biochemical testing (rhamnose) and reference lab sequencing.
to a reference laboratory which performed sequencing for speciation. Could not distinguish between Shigella, EIEC and E.coli. All organisms identified as E.coli by
An in-house developed conventional multiplex PCR assay for the identification of suspect MALDI-TOF were tested by the in-house PCR for the detection of stx1/2 (STEC) and the Shigella
Shigella spp, Entero-invasice E.coli, Shiga-toxin producing E.coli, Yersinia spp, Aeromonas The MALDI-TOF incorporating algorithm consisted of using MALDI-TOF as the preliminary identification invasion plasmid genes (EIEC, Shigella spp).
spp, Vibrio spp, and Salmonella spp. from conventional enteric culture was evaluated against
an algorithm incorporating MALDI-TOF (Bruker, Daltronics). The study occurred in two phases.
Phase one was a month long prospective comparison of all suspect enteric isolates by both
method to identify all suspected enteric isolates using the same media and protocols for selecting
suspected isolates as the incumbent method. Because of previously known limitations of the current
MALDI-TOF method, any organisms identified by MALDI-TOF as E.coli/Shigella spp were then tested by
Conclusions
methodologies, while phase two was a comparison of any positive identifications by the current the existing PCR method to rule out Shigella, EIEC and STEC. Organisms identified by MALDI-TOF as
Table1: Identification summary of MALDI-TOF compared to multiplex PCR
methodology against the MALDI-TOF identification, with a target of at least 200 positives Salmonella spp. were tested by Vitek/API to rule out Salmonella typhi/paratyphi and also sent to a
pathogens over the course of the two phases. Discrepant results were tested by alternative reference lab for confirmation of serotype. Species # isolates # isolates identified Total MALDI-TOF has proven to be fast and reliable in identifying most enteric pathogens. There are
methods such as biochemical identification or 16S sequencing at a reference laboratory. identified by by MALDI
MALDI-TOF
TOF some notable
t bl exceptions
ti (S l
(Salmonella
ll serotype,
t Shi ll EIEC,
Shigella, EIEC STEC,
STEC some Yersinia
Y i i species)
i ) that
th t
For MALDI-TOF testing, a sterile stick is used to pick a single isolated colony from freshly grown bacteria, PCR
are mitigated by implementing secondary testing methods, which in our case is using a subset of
Results which is directly smeared onto a 96 well target plate, covered with 1L HCCA Matrix solution, air dried
Aeromonas species 34 36 37 our pre-existing PCR assays. This algorithm results in a shorter time-to-result of anywhere from 1 to
and loaded into Bruker Auto-flex. Mass spectrum was analyzed via FlexControl, MALDI Biotyper RTC
24 hours and requires only a small amount of bacterial growth for testing.
A total of 2132 suspect enteric isolates were compared during phase 1 of the study. A total of and MALDI Biotyper 3.0 software. Manufacturer recommended score cut-offs were used to determine Campylobacter species 0 45 45
214 enteric pathogens were isolated in the course of 2 phases. Previously described limitations genus-level (1.700 to 1.999) or species level (>= 2.000) identification
PCR has many benefits but can be expensive, time consuming and requires more specialized
of MALDI-TOF with respect to the discrimination of Shigella spp, EIEC and from Eschericia coli Edwardsiella tarda 0 1 1
personnel to develop, run, troubleshoot and interpret results. For instance if a specimen required
were resolved through the use of the PCR method as a secondary step after identification of Discrepant results between identification algorithms were resolved through either additional biochemical
Plesiomonas shigelloides 0 9 9 repeat testing, by PCR this would require a further 24 hour delay in turn around time due to
any E. coli species. Resolution of Salmonella serotype could not be reliably performed by testing and/or sent to a reference laboratory which performed 16S sequencing.
workflow constraints (only 1 PCR run per day) compared to MALDI TOF which typically would take
MALDI-TOF. There were also several misidentifications of Yersinia frederiksensii as Yersinia Salmonella species 51 52 52 no longer than 1 hour due to the ease of either re-acquiring spectra from the same spot or preparing
enterocolitica by MALDI-TOF. However, MALDI-TOF performed well for the identification of Phase 1 of the study consisted of a month long period of prospective parallel testing where all suspect
a fresh-spot.
Aeromonas spp, Plesiomonas spp , Vibrio spp. and Campylobacter spp. as a solitary method. enteric isolates identified by the incumbent PCR method were also simultaneously tested by MALDI-TOF. Vibrio parahaemolyticus 9 9 9

Yersinia entercolitica 26 27 27 In comparison to PCR the MALDI-TOF algorithm is faster, cheaper and easy to use with improved
Conclusion Phase 2 of the study consisted of prospectively comparing enteric pathogens identified by the incumbent
turn-around-time for patient results, particularly for negative samples and for those organisms which
method to the identification provided by the MALDI-TOF algorithm. The end point of this phase was the Shigella species 20 0 20 MALDI-TOF can reliably identify without supplemental testing. Aside from E.coli, MALDI-TOF is an
MALDI TOF can reliably identify most enteric pathogens with some notable exceptions.
MALDI-TOF accumulation of at least 200 enteric pathogens for comparison accumulated over both phases.
excellent
ll t stool
t l pathogen
th screening
i method.
th d
Limitations can be mitigated through the use of a supplemental PCR based identification EIEC (Enteroinvasive E.coli) 5 0 5
algorithm.
STEC (Shiga-toxin producing E.coli) 5 0 5 With advances in technology and addition of sufficient spectra to the Bruker database it would be no
surprise to see MALDI-TOF be the primary research and diagnostic tool in high volume laboratories
in the near future due to its accuracy, precision and ability to process high volumes of isolates.3
These characteristics allow for accurate, timely and cost-effective results.
Table 2: Accuracy of the MALDI-TOF and PCR
No. (%) correctly identified

Introduction References
MALDI-TOF PCR

Isolate ID No. of Species Genus NO ID Species Genus NO ID

strains level level level level

A. caviae 10 10 (100) 10(100) 7(70) 3(30) 1. Murray. R.P. What Is New in Clinical MicrobiologyMicrobial Identification by MALDI-TOF Mass
Enteric culture has been a very important tool for understanding gastrointestinal infections.
Spectrometry. A Paper from the 2011 William Beaumont Hospital Symposium on Molecular
I l ti off pathogenic
Isolation th i b
bacteria
t i from
f stool
t l directs
di t antimicrobial
ti i bi l treatment
t t t when
h indicated,
i di t d A. hydrophilia 7 7 (100) 7 (100) 7 (100) Pathology. J Mol Diagn. 2012 Sep; 14(5): 419423.
helps prevent secondary complications and allows for public health agencies to track and
manage gastroenteritis outbreaks to prevent transmission of illness. A. jandaei 3 2 (67) 3 (100) 3 (100)
2. Ying He,Haijing Li, Xuedong Lu,Charles W. Stratton,and Yi-Wei Tang. Spectrometry Biotyper
System Identifies Enteric Bacterial Pathogens Directly from Colonies Grown on Selective Stool
Laboratory identification and isolation of enteric pathogens has traditionally been done via A.media 3 3 (100) 3 (100) 3 (100) Culture Media.,J Clin Microbiol. 2010 Nov; 48(11): 38883892. Published online 2010 Sep 15.
selective media, culture and isolation followed by biochemical identification and serology. doi: 10.1128/JCM.01290-10.
These methods can be costly and time consuming, taking 2 to 4 days to obtain a result. This A. ichthiosmia 1 1 (100) 1 (100) 1 (100) 3. J. Kathleen Lewis, Jing Wei, and Gary Siuzdak. Matrix-assisted Laser Desorption/Ionization
longer turn-around-time can have negative consequences for patient care and treatment. Mass Spectrometry in Peptide and Protein Analysis. Encyclopedia of Analytical Chemistry R.A.
A. salmonicida 1 1 (100) 1 (100) 1 (100)
Providing rapid, accurate, and specific identification of enteric bacterial pathogens is Meyers (Ed.) pp. 5880 5894, John Wiley & Sons Ltd, Chichester, 2000.
considered essential for directing the antimicrobial therapy of diarrheal illnesses.2
A. veronii 12 12 (100) 12(100) 12(100)
The current methodology employed at Lifelabs Medical Laboratory Services (Burnaby site)
C. campy 6 5 (83) 1 (17) 6(100)
uses traditional culture methods to isolate suspected bacteria but instead of biochemical
identification methods, a previously validated in-house multiplex gel-based PCR method is
C. coli 2 2 (100) 2 (100) 2(100)
used for definitive identification of enteric pathogens. Turn-around time for PCR is between 4-
8 hours and is an improvement over traditional biochemical identification.
identification However
However, there C. jejuni 36 19(53) 17(47) 36(100)
exist opportunities for further improvements.
C. upsaliensis 1 1 (100) 1 (100) 1(100)
Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) is a technology that has
been developed that enables rapid identification of microorganisms. Isolated colonies are E. tarda 1 1 (100) 1 (100) 1(100)
spotted on a target plate, covered with matrix, dried and then subjected to a laser pulse. The
laser induces desorption and ionization of the bacteria-matrix layer, which is then separated P. shigelloides 9 8 (89) 1 (11) 9(100)
based on particulate mass/charge ratio1. A characteristic mass spectra is obtained which is
compared to a database of known spectra to definitively identify the microorganism2, Salmonella species 52 52(100) 51(98) 1(2)
Results are typically available within a few minutes of loading a target plate into the mass
V. parahaemolyticus 9 8 (89) 1 (11) 9(100)
spectrometer.
Y. enterocolitica 27 26 (96) 27(100) 26(96) 26(96) 1(4)
MALDI-TOF implementation as a high volume enteric pathogen screening method has the
potential to improve patient result turn-around-time at a high volume community outpatient lab Y. frederiksenii (a) 4 4(100) 4(100)
such as Lifelabs which processes several thousand stool cultures a month.
Shigella species (b) 20 20(100) 20(100)

EIEC (c) 5 5(100) 5(100)

STEC (d) 5 5(100) 5(100)

Totals 214 106(50) 140(67) 35(14) 26(12) 125(57) 64(31)

a ) Y.frederiksenii: identified by API20E and reference laboratory

b ) Shigella species identified by PCR by detecting virulance factors that were then confirmed via serology
c ) Enteroinvasive E.coli (EIEC) and Shigella identified by PCR by detection of invasion plasmid gene
d) Shigatoxin producing E.coli (STEC) identified by PCR detection of stx1/stx2 genes

27
MALDI Biotyper Poster Hall 2016 Primary ID

The role of MALDI-TOF to discover new pathogens: Propionibacterium kocii,

a human pathogen anaerobic bacterium
Edit Urbn1, Judit Hunyadkrti2, Pl Barz, Istvn Nagy2
Institute of Clinical Microbiology1 Faculty of Medicine, University of Szeged, Biological Research Centre of the Hungarian Academy of Sciences2, Hungary

Background Methods Results Conclusion

Propionibacterium species are nonsporulating, Gram-positive anaerobic
bacilli that are considered as commensal bacteria on the skin. They are
usually non-pathogenic and are common contaminants of blood and body-
fluid cultures. Sometimes, it can be difficult to determine whether positive
culture results for propionibacteria reflect contamination or true infection.
The role of P. acnes in the pathogenesis of acne has been debated for
decades, but never adequately proven. Serious infections due to
propionibacteria are rarely reported, but this bacterium is increasingly
recognized as a cause of serious infections, such as endocarditis, prosthetic
joint infection, endophthalmitis, osteomyelitis and central nervous system
infections.
Case 1
Case 1 was a 15-year-old female patient with chronic inflammatory acne
receiving systemic lymecycline treatment. Only a Propionibacterium sp.
strain was isolated from her acne sample in high CFU. The strain was
tetracycline sensitive, sorbitol and protease positive.
Three clinical isolates from these cases were identified in routine MALDI-TOF gave only a genus level identification compared with the Propionibacterium infections are usually characterized by a paucity
laboratories as non-identified Propionibacterium sp. by the commonly reference strains in the case of 3 isolates. MALDI-TOF analysis gave of classical symptoms of infection or inflammation. Invasive
used phenotypic methods, such as presumptive identifications, classical only Propionibacterium sp. results, because this species was not included propionibacterium infection typically occurs in the setting of after
biochemical tests, Rapid ID 32A (ATB) and API 20A (bioMrieux, Fr.). until this time in the database. Genome sequencing was performed by surgery. Given the low virulence of propionibacteria, infections
MALDI-TOF MS and the dedicated BioTyper software (BRUKER combining the cycled ligation sequencing on SOLiD V4 System (Life with these organisms are usually indolent. In our cases we could
Daltonik Gmbh. Gr.) using ATCC reference strains as references were Technologies) with 454 FLX pyrosequencing (Roche). It has a single detect this species by molecular methods and we found a new
circular chromosome of 2,410,997 bps, with a GC content of ~60%; there species when clinical manifestations were connected to this
used to compare identification in species level and to broaden the
are 2205 putative coding sequences, 49 tRNAs, and 9 rRNA loci. bacterium. Using to a new molecular techniques, new or earlier
database for the most frequent clinical isolates of Propionibacterium.
non-pathogenic bacteria can be indentified in various clinical
All strains were cultured on Columbia-based anaerobic blood agar for Newly accepted species as Propionibacterium kocii could be identified
pictures or we can elucidate the pathogenic role in these settings.
24 hours in an anaerobic chamber. Because isolates gave discrepant by the MALDI-TOF. Clear differentiation could be achieved for less
identifications with the biochemical tests and only low scores (1.406; frequent clinical isolates the taxonomic place of which was recently
1.02; 1.203) of the MALDI-TOF method, the 16S rRNA sequencing established. Figure 5. API 20A result of the isolated the isolated Propionibacterium kocii
was used to confirm the identification. 75% ethanol extract of the cells
were stored in -20C till sending to the BRUKER laboratory for
verification the MALDI-TOF analysis again and to compare the strains
with their database.
Table 1. Identification scheme in API 20A

Case 2
ATCC Strains IND URE GLU MAN LAC SAC MAL SAL XYL ARA GEL ESC GLY CEL MNE MLZ RAF SOR RHA TRE CAT

P. acnes 90 0 99 36 0 1 0 0 0 0 74 0 99 0 100 0 0 18 0 1 95
Figure 6. CT scan of case 3
P. granulosum 0 0 99 43 0 93 31 0 0 0 4 0 99 0 99 12 56 0 4 75 90
Case 2 was a 55-year-old female patient with a known benign brain
P. avidum 0 0 99 32 45 99 72 62 0 0 98 99 99 0 99 60 40 0 0 90 90
tumour had a tumour resection. About 1 month before the operation, she
P. propionicus 0 10 99 96 50 90 64 0 0 14 50 0 60 0 40 0 40 0 0 42 0
had meningeal symptoms: high fever, nausea, vomiting, acute headache.
Strain 1./Case 1. + - + - - - - - - - + - + - + - - - - - -
Intracranial CT showed a 20 mm right-sided parieto-occipital ring-form
Strain 2./Case 2. + - + - - - - - - - + + +/- - - - - - -
plexus with extensive gloves-finger like oedema. This space occupying - -

Strain 3./Case 3. + - + - - - - - - - + + + - - - - - -
lesion might be a metastasis. The pathological opinion was glioblastoma - -

multiforme, WHO grade IV. In the postoperative period, her clinical

condition improved, but 1 day before discharging the patient from the
Figure 1. Typical mass spectra of Propionibacterium acnes strain Figure 2. Mass spectra of Propionibacterium kocii strains
hospital, her wound became infected. In spite of regular wound toilet at
the surgical site, discharge could be detected. Thus new surgical
intervention could be performed again, when cranial bone-lobe was
totally removed. From this site, microbial sample was collected. We
could isolate only a Propionibacterium sp. strain as a pure culture in
high CFU. After this, the surgical site was completely healed.

Figure 7. Principal Component Analysis (PCA) using whole

Case 3 genomes of the genus Propionibacterium

In April, 2011 a 60-year-old male patient had an urgent emergency

admission to the ER, because of hemiparesis and speech disorder. 4 days
before his admission, he had a teeth extraction, his symptoms manifested
after this intervention. He had no fever, no meningeal symptoms at the
time on admission. CT scan showed a large space-occupying lesion with
Figure 3. Presumptive identification of the Propionibacterium kocii strains after
oedema. On the basis of radiological and neurosurgical examinations, the
48 hours of anaerobic incubation on anaerobic blood agar. Figure 4. ATB ID 32 A result of the isolated Propionibacterium kocii
possibility of malignant brain tumour, glioblastoma has been arisen. Vancomycin 5 g, Kanamycin 1000 g, Colistin 10 g and Metronidazole 5 g disks
Because of this, he underwent a neurosurgical operation. After three
months of the first hospital treatment (the patient was treated on the basis
of the general oncology protocol), the patient was readmitted to the
hospital, because of deteriorations in his general and neurological
conditions. On the basis of the new CT scan, the previous surgical site
was inflamed, thus new neurosurgical intervention was carried out
urgently. Large abscess could be found and sample collection from the
wall of this abscess was performed. Only a Propionibacterium sp. strain
was isolated in very high CFU. 2 years after the first hospital admission,
the patient died because of his basic illness.

28
MALDI Biotyper Poster Hall 2016 Primary ID

29
MALDI Biotyper Poster Hall 2016 Primary ID of Mycobacteria

30
MALDI Biotyper Poster Hall 2016 Primary ID of Mycobacteria

berrtliche Berufsausbungsgemeinschaft
MVZ Dr. Eberhard & Partner Dortmund

Mycobacteria Identification by MALDI Biotyper System: Evolution of Database Content and Evaluation Criteria
A. B. PrAnAdA1, M. TiMke2, e. WiTT1, M. kosTrzeWA2
ECCMID 2015, Copenhagen
1
MVZ Dr. Eberhard & Partner Dortmund, Department of Medical Microbiology, Dortmund, Germany Poster 1190
2
Bruker Daltonik GmbH, Bremen, Germany apranada@labmed.de

Introduction Materials and Methods Results Comparison Database V2.0 vs. V3.0 and Evaluation of New Threshold Values Conclusion
Identification of mycobacteria by matrix-assisted laser Mycobacterial isolates (n = 1045) were inoculated on Out of 1045 analyses of pure cultures log(score) values Mycobacteria Identifications with Current Threshold Values Mycobacteria Identifications with Proposed New Threshold Values
Every MALDI Biotyper database reference is attributable
desorption/ionisation time-of-flight (MALDI-TOF) mass solid Lwenstein-Jensen medium or in liquid BD BAC- were 2.0 for 82.9 % and between 1.7 and < 2.0 for Library
1.2 %
Library
0.8 % to a strain or an isolate. A higher number of references
spectrometry (MS) requires an optimised preparation TEC MGIT tubes (BD, Heidelberg). 12.1 %, representing the current high and low confidence 94.1 % 4.5 %
2.000
97.3 % 1.9 %
2.000 per species covers potential natural variabilities of a spe-
V3.0 V3.0
method and a corresponding database. In addition, patient material was inoculated in MGIT identification results with database version 2.0. 1.900-1.999
1.800-1.899
1.900-1.999
1.800-1.899 cies.
tubes. Positive cultures (n = 93) were further analysed. Using the adapted values and database version 3.0, Besides this, there can be other influences like human,
1.700-1.799 1.700-1.799
1.600-1.699 1.600-1.699

97.3 % and 1.9 % of high and low confidence level iden- instrument or medium based variability due to practical
1.500-1.599 1.500-1.599

Evolution of databases for mycobacteria ID

1.400-1.499 1.400-1.499

tification were obtained, respectively (Figures 1a and 1b). knowledge, instrument settings or liquid / solid medium,
1.300-1.399 1.300-1.399

Preparation of positive cultures and isolates V2.0 V2.0

1.200-1.299 1.200-1.299

Mycobacteria Library 1.0 (Bruker Daltonik, Bremen, Ger- Very few discrepancies were observed (n = 8) using da- High Confidence: 82.9 % Low Confidence: 12.1 % High Confidence: 91.6 % Low Confidence: 5.4 % respectively. All these minor variations have been bal-
many) was released in 2012 and version 2.0 in 2014 with Biomass was collected from solid and liquid media and tabase version 2.0 (Table 2), but only for very closely re- anced by several references per species by the presented
Unreliable: 5.1 % Unreliable: 3.1 %

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

173 and 313 references, respectively. processed using the mycobacteria extraction protocol. lated species and of log(score) values below 2.0. Figure 1a: Identification results for 1045 analyses using database V2.0 and V3.0 Figure 1b: Identification results for 1045 analyses using database V2.0 and V3.0 database extension.
with current threshold values. with proposed new threshold values.
An extension by 542 additional references mainly clin- The update of the database eliminated 7 of these dis- As a result, the proportion of high confidence level iden-
ical isolates leads to version 3.0 in this year. Optimised extraction protocol for mycobacteria crepancies. Isolate
Mycobacteria Library 2.0 Mycobacteria Library 3.0 Table 2: Divergences out of 1045 identifications. tifications increased with database version 2.0 and a fur-
The remaining misidentification is likely due to a mix- ID Result log(score) OK? ID Result log(score) OK? ther gain could be observed with version 3.0.
Collect biomass from solid medium or 1.2ml from MGIT M. immunogenum 8608 M. abscessus 1.944 NO M. immunogenum 2.389 OK For identification with database version 2.0
Here we present results obtained for mass spectra com- medium, centrifuge, discard supernatant up of samples: M. nebraskense and M. gordonae usually M. engbaekii 026468 M. hiberniae 1.795 NO M. engbaekii 2.449 OK
n = 8 divergences were observed for closely
Add water, heat inactivation (boiling for 30 min) related species with log(score) values < 2.0.
pared to these three database versions with focus on can be differentiated unambigously from each other by M. kansasii IV 11649 M. gastri 1.753 NO M. kansasii 2.437 OK
Add 900 l ethanol, mix, centrifuge, discard supernatant Increased sensitivity without loss in specificity
sensitivity and specificity. Centrifuge again, discard supernatant MALDI-TOF MS. A comparison of the mass spectra yields M. elephantis 457 M. pulveris 1.743 NO M. elephantis 2.812 OK Database version 3.0 enables the correct identi-
Dry pellet at room temperature M. elephantis 44368 M. pulveris 1.743 NO M. elephantis 2.443 OK fication in these cases.
Add zirconia/silica beads (0.5 mm) to a maximum log(score) value of only 1.24. M. elephantis 125 M. pulveris 1.669 NO M. elephantis 2.828 OK One remaining discrepancy is likely due to a Reliability of identification results is based on log(score)
Resuspend in 10-50 l acetonitrile, vortex for 1 min M. elephantis 101 M. pulveris 1.665 NO M. elephantis 2.853 OK mix-up of samples. values. It was possible to lower threshold values without
Evaluation of adapted threshold values Addition of 70 % formic acid, vortex for 5sec,
centrifuge Improved identification for clinical specimens with M. nebraskense J605 M. gordonae 1.619 NO M. gordonae 1.749 NO risking false species identification results using Myco-
In addition to the investigation of the database versions Transfer 1 l of supernatant to MALDI target newer database versions and adapted thresholds bacteria Library 3.0.
we also have evaluated current threshold values and pro- Clinical Specimens Database Comparison and Evaluation of Threshold Values Both improvements have led to an increased sensitivi-
pose adapted ones for Mycobacterium spp. (Table 1). Out of the 93 enrichment cultures of directly inoculated
ty without a decrease of specificity thus avoiding time
clinical specimens 78.5 % resulted in log(score) values Clinical Specimens and Current Threshold Values Clinical Specimens and Proposed New Threshold Values
Mass spectrometer, databases and software Mycobacteria
Library
Mycobacteria
Library consuming repetitions and additional tests.
2.0 using the current database version 2.0. The new
2.2 %

Table 1: Current and proposed log(score) thresholds for mycobacteria

V3.0 1.1 % V3.0
Current log(score) thresholds for mycobacteria Proposal Mass spectra were recorded with a microflex LT instru- extended database version 3.0 led to an improvement 89.2 % 9.7 % 97.8 %
This benefit was demonstrated in a routine laboratory
2.000 3.000 identification at high confidence level 1.800 3.000 ment and compared to Mycobacteria Library 1.0, 2.0 and resulting in 89.2 % identifications at high confidence 2.400-2.499 2.400-2.499
2.300-2.399 2.300-2.399 with patient inoculated cultures.
1.700 1.999 identification at low confidence level 1.600 1.799 the extended database version 3.0 using MALDI Biotyper level. 2.200-2.299
2.100-2.199
2.200-2.299
2.100-2.199
0.000 1.699 no reliable identification 0.000 1.599
3.1 software (Bruker Daltonik, Germany). With adapted threshold values, 94.6 % were considered V2.0 2.000-2.099
1.900-1.999
V2.0 2.000-2.099
1.900-1.999

as high confidence and 5.4 % as low confidence iden- 78.5 % 18.3 % 3.2 % 1.800-1.899
1.700-1.799
94.6 % 5.4 % 1.800-1.899
1.700-1.799
Conflicts of Interest / Disclosures:
The reliability of identifications obtained by MALDI-TOF tifications using database version 2.0. All these species 1.600-1.699 1.600-1.699 A. B. Pranada presents his data in an ECCMID symposium organised by Bruker Dal-
Reference methods for identification 1.500-1.599 1.500-1.599 tonik GmbH and receives speaker fees.
MS is indicated by log(score) values. identifications were correct. E. Witt: None.
V1.0 V1.0
The reference method for study isolates was GenoType The extended database version 3.0 further improved these High Confidence: 62.4 % Low Confidence: 32.3 % High Confidence: 90.3 % Low Confidence: 8.6 %
M. Timke and M. Kostrzewa are employees of Bruker Daltonik GmbH.

In comparison to usual bacterial isolates some myco- Mycobacterium CM (Hain Lifescience, Nehren, Germa- results to 97.8 % for high confidence and 2.2 % at low Unreliable: 5.4 % Unreliable: 1.1 %
Contact:
bacteria show lower log(score) values. Nevertheless, the ny). Sequencing of 16S rRNA gene or ITS sequence was confidence level without any identification in the unre- 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Dr. med. Arthur B. Pranada
Figure 2a: Identification results from clinical specimens (n = 93) in comparison to Figure 2b: Identification results from clinical specimens (n = 93) in comparison to MVZ Dr. Eberhard & Partner Dortmund (BAG)
identification results still seem to be reliable. performed for a few isolates. liable category (Figures 2a and 2b). the different database versions and applying current thresholds. the different database versions and applying adapted thresholds. Balkenstr. 17-19, 44137 Dortmund, Germany apranada@labmed.de

For research use only. Not for use in diagnostic procedures.

31
MALDI Biotyper Poster Hall 2016 Primary ID of Mycobacteria

D-223
Looking at the Differences: First Report of Possibilities to
Separate Mycobacterium chimaera from Mycobacterium intracellulare by MALDI-TOF MS
A. B. PrAnAdA1, M. TiMke2, e. WiTT1, M. kosTrzeWA2
1
MVZ Dr. Eberhard & Partner Dortmund, Department of Medical Microbiology, Dortmund, Germany
Poster D-223
2
Bruker Daltonik GmbH, Bremen, Germany
apranada@labmed.de

correct for 30 of our strains it failed for six M. chimaera

Introduction and Background Optimised extraction protocol for mycobacteria
strains (Table 1).
Results Differentiation of Mycobacterium chimaera and Mycobacterium intracellulare by MALDI-TOF MS
Collect biomass from solid medium or 1.2ml from MGIT
medium, centrifuge, discard supernatant
Table 1: MALDI Biotyper results for six M. chimaera/intracellulare group members Table 2: Characteristic peaks and log(IQ) values of some reference strains
M. chimaera and M. intracellulare Add water, heat inactivation (boiling for 30 min)
MALDI Biotyper
Correct manual ID at species level for 97 % ID by MALDI-TOF MS analysis
Add 900 l ethanol, mix, centrifuge, discard super- Definite ID by
natant Isolate Mycobacteria Library V3.0 Strain Definite ID by ITS sequencing Characteristic peaks Software algorithm
Mycobacterium chimaera and M. intracellulare are close- Centrifuge again, discard supernatant
ITS sequencing
Best match log(score) OK? To allow a definite identification by MALDI-TOF MS, three 6446 m/z 6476 m/z 7359 m/z log(IQ) value
Resulting species ID OK?
ly related species within the Mycobacterium avium com- Dry pellet at room temperature M. chimaera DSM 44623(T) M. chimaera M. intracellulare DSM 44161 2.358 NO peaks have to be checked manually. Using this model, 35 M. chimaera DSM 44623(T) Mycobacterium chimaera + - + 0.618 M. chimaera Yes
Add zirconia/silica beads (0.5 mm)
plex (MAC). Resuspend in 10-50 l acetonitrile, vortex for 1 min
Mycobacterium sp. 12136335 M. chimaera M. intracellulare 3887 2.271 NO isolates (97 %) were assigned to the correct species. Ex- M. chimaera DSM 44622 Mycobacterium chimaera + - + 0.947 M. chimaera Yes
Mycobacterium sp. 13001113 M. chimaera M. intracellulare 3887 2.378 NO M. intracellulare DSM 43223T Mycobacterium intracellulare - + - -0.803 M. intracellulare Yes
Addition of 70 % formic acid, vortex for 5sec, Mycobacterium sp. 14099066 M. chimaera M. intracellulare DSM 44161 2.099 NO emplary results of this identification approach can be seen M. intracellulare DSM 44161 Mycobacterium intracellulare - + - -0.421 M. intracellulare Yes
centrifuge
Mycobacterium sp. 12030617 M. chimaera M. intracellulare 3887 2.601 NO in Tables 2 and 3. Only one isolate could not be assigned M. intracellulare 27852 Mycobacterium intracellulare - + - -0.728 M. intracellulare Yes
Pathogenicity and clinical relevance Transfer 1 l of supernatant to MALDI target
Mycobacterium sp. 15000561 M. chimaera M. intracellulare DSM 44161 2.270 NO manually to any species (Table 3). M. intracellulare CCUG 2800 Mycobacterium intracellulare - + - -0.713 M. intracellulare Yes
M. intracellulare was often associated with pulmonary Table 3: Characteristic peaks and log(IQ) values of the six strains that were incorrectly classified by the standard Biotyper algorithm
disease while for M. chimaera, newly described in 2004, From these six M. chimaera strains three matched with a ID by MALDI-TOF MS analysis
Software identifies all isolates (100 %) correctly Strain Definite ID by ITS sequencing Characteristic peaks Software algorithm
a lower pathogenicity was assumed. ence spectra using MALDI Biotyper 4.0 software (Bruker reference stored in the database as M. intracellulare 3887. 6446 m/z 6476 m/z 7359 m/z log(IQ) value
Resulting species ID OK?
Daltonik, Germany). Identification of the corresponding reference isolate re- For a more convenient approach, a software for real-time M. chimaera DSM 44623(T) Mycobacterium chimaera + - + 0.618 M. chimaera Yes
vealed that it was erroneously labelled as M. intracellulare identification was developed and tested. Mycobacterium sp. 12136335 Mycobacterium chimaera + - + 0.779 M. chimaera Yes
Invasive M. chimaera infections in cardiac surgery Mycobacterium sp. 13001113 Mycobacterium chimaera + - + 0.788 M. chimaera Yes
as ITS sequencing resulted in M. chimaera. This demands In short, the software algorithm is based on the intensity
Development of algorithm for differentiation Mycobacterium sp. 14099066 Mycobacterium chimaera - - - 0.279 (?) / M. chimaera Yes
However, heater cooler devices used in cardiac surgery for a correction in the database in future versions. of several characteristic peaks for M. chimaera and M.in- Mycobacterium sp. 12030617 Mycobacterium chimaera + - + 0.836 M. chimaera Yes
were recently supposed to be the source of invasive M.chi- Spectra were checked manually with FlexAnalysis soft- tracellulare. Mycobacterium sp. 15000561 Mycobacterium chimaera + - + 0.778 M. chimaera Yes
maera infections reported from Europe. ware (Bruker Daltonik, Germany) for characteristic peaks The three remaining strains matched with the reference
for the two species. Based on these peaks a (manual) iden- M.
M. intracellulare DSM 44161. As in this case the ITS se- As a result a so called log(IQ) value indicates with positive The calculated log(IQ) values allowed correct classifica- reliable classification thresholds defining a zone of indif-
tification algorithm was developed. quence confirmed the database entry it remains that three values the identification as M. chimaera or with negative tion of all isolates (100 %). ferent results might be appropriate.
Differentiation of both species is difficult
out of 36 strains could not be identified correctly at spe- values as M. intracellulare (Figure 2). As some values were quite close to zero for reasons of a So currently log(IQ) values between -0.15 and +0.15 could
A reliable differentiation of these two species in daily cies level by the MALDI Biotyper standard software. be regarded as no reliable identification, while -0.15
Software tool for real-time identification
routine is challenging. Test systems based on DNA hy- would indicate M. intracellulare and 0.15 M. chimaera,
bridization, e.g. like GenoType Mycobacterium CM (Hain For real-time differentiation of mass spectra a software Identification of M. chimaera and M. intracellulare by software algorithm respectively.
Characteristic peaks of M. chimaera / M. intracellulare
Lifescience, Nehren, Germany) identify both species as tool was developed. For this purpose a peak detection al- 1.200

M. intracellulare. MALDI Biotyper (Bruker Daltonik GmbH,
Bremen, Germany) groups both species into a complex.
Even the complete 16S rRNA gene differs in only onebase.
Materials and Methods
gorithm was implemented. The result of this algorithm is
a log(IQ) value that indicates which of the two species
has been detected.
Results Identification at Group Level
Therefore a more reliable method for differentiation of
1.000
M. chimaera and M. intracellulare by MALDI-TOF MS was Conclusions
investigated. As a first basic approach the mass spectra
0.800
of the two species were screened for characteristic peaks. Differentiation of M. chimaera and M. intracellulare is dif-
Indeed, several peaks allowing a reliable differentiation 0.600 ficult in routine diagnostics but may be clinically relevant.
could be found (Figure 1).
0.400 Here, we present two easy to perform and reliable MALDI-
Intens. [a.u.]

m/z 6446 Mycobacterium chimaera DSM 44623T

3000
TOF MS based methods for definite identification.
Isolates of M. chimaera and M. intracellulare Correct ID at group level with standard software 0.200
2500
First, characteristic peaks were identified for a manual ap-
Isolates (n = 36) of M. chimaera (n = 23) and M. intracel- All Mycobacterium chimaera (n = 23) and M. intracellu- 2000 M. chimaera proach. Second, a software tool for automated real-time
log(IQ) 0.000
lulare (n = 13) were cultured on solid Lwenstein-Jensen lare (n = 13) strains were correctly identified by the stan- 1500 m/z 7359 M. intracellulare identification was developed. All differentiation results
medium or in liquid BD BACTEC MGIT tubes (BD, Hei- dard MALDI Biotyper algorithm as M. chimaera/intracel- 1000
-0.200 were correct.
delberg) according to standard procedures. lulare group (data not shown). 500

0 -0.400 To our knowledge these are the first methods suitable for
routine to differentiate M. chimaera/intracellulare.
Intens. [a.u.]

Mycobacterium intracellulare DSM 43223T

Reference identification method Differentiation M. chimaera / M. intracellulare unrelia- 1250 m/z 6476
-0.600
ble with standard software
All study isolates were identified by ITS sequence analysis
1000

Conflicts of interest / Disclosures:

-0.800 A. B. Pranada presented his data in a symposium organized by Bruker Daltonik GmbH
as reference method. In addition to the identification at group level the Biotyp- 750

and received speaker fees.

er software also shows the exact reference spectrum that 500
E. Witt: None.
-1.000 M. Timke and M. Kostrzewa are employees of Bruker Daltonik GmbH.
matched in parentheses, e.g. Mycobacterium chimaera_
MALDI-TOF MS 250

intracellulare_group (MM chimaera DSM 44622 DSM b). 0 -1.200

Contact:
Dr. med. Arthur B. Pranada
Mass spectra were recorded with a microflex LT instru- This can be used as a hint, which species might have been 6400 6600 6800 7000 7200 7400
m/z
Figure 2: From the 36 strains of this study 465 MALDI-TOF mass spectra were acquired. The newly developed software algorithm could separate spectra of M. chimaera MVZ Dr. Eberhard & Partner Dortmund (BAG)
ment and compared to Mycobacteria Library 3.0 refer- detected but so far this is not reliable. Although it was Figure 1: Characteristic MALDI-TOF MS peaks for M. chimaera and M. intracellulare (positive log(IQ) values) from spectra of M. intracellulare (negative log(IQ) values). Balkenstr. 17-19, 44137 Dortmund, Germany apranada@labmed.de

32
MALDI Biotyper Poster Hall 2016 Primary ID of Mycobacteria

33
 34
Primary ID of Mycobacteria

Determination of Non Tuberculous Mycobacteria with

Matrix Assisted Laser Desorption Ionization-Time Of
Flight Mass Spectrometry
Maartje van den Boomgaard, Niels Koehorst, Renate
Leussenkamp-Hummelink, Maria de Boer, Adri van der
Zanden, Bert Mulder
Non tuberculous mycobacteria (NTM) differ in their capacity to cause clinical disease. Quick and
accurate identification is therefore important. Current determination, based on biochemical
phenotyping and DNA sequencing, is time-consuming and expensive. We investigated the
determination of NTM with Matrix Assisted Laser Desorption Ionization-Time of Flight Mass
Spectrometry (MALDI-TOF MS) on accuracy, speed and cost.
Strains tested Influence of databases
Retrospectively, 122 isolates of 27 different Databases with a larger number of
species were determined with MALDI-TOF MS. mycobacterial spectra (2,0) show significantly
Sequence analysis were considered as gold better results than databases with fewer
standard. spectra (BDAL). However, the score of some
Species Nr Species Nr strains worsened using a larger database. It is
M. abcessus 4 M. llatzerense 1
worth to use the BDAL database combined
M. africanum 3 M. malmoense 8
with other Mycobacteria Libraries, that
M. avium 10 M. marinum 9
apparently contain other spectra.
Influence databases on reliability
M. avium complex 10 M. neoaurum 1 scores
M. bovis 2 M. palustre 1 100%
Percentage strains

M. bovis BCG 6 M. peregrinum 6 80%

M. celatum 2 M. scrofulaceum 2
60%
M. chelonae 8 M. simiae 2
M. chimaera 5 M. smegmatis 1 40% <1,699
1,700-1,999
M. engbaekii 1 M. szulgai 2 20% >2,000
M. fortuitum 5 M. terrae 2
0%
M. gordonae 5 M. tuberculosis 6 BDAL 1.0 2.0 BDAL 1.0 2.0
M. intracellulare 5 M. xenopi 5 MGIT LJP
M. kansasii 10 Database and medium
Influence of colony age Conclusion
MALDI-TOF MS analysis correctly identified
Both in the group rapid growers as in the group
92% of the isolates on liquid media and 91%
slow growers, the number of scores above
of the isolates on solid media. Of those 75%
2,000 decreases as the colonies grow older.
(liquid media) and 67% (solid media) had a
score >2,000, indicating accurate species
Influence of age Influence of age
for slow growers
identification. This combined with the low
for rapid
cost of 47,12 (MALDI-TOF MS) versus
growers
MALDI Biotyper Poster Hall 2016

100%
100%
90% 376,54 (sequencing)) and quick results
80%
80% 70% (daily versus weekly) makes MALDI-TOF MS
60%
60%
50%
a promising first identification method for
40%
40% NTM. Prospective studies on patients must
30%
20% 20% show its practical feasibility. In addition, it is
0%
10% important to use an extensive database and
0%
day 4 day 7 day 10 day 10 day 14 day 21 to carry out the MALDI-TOF MS analysis
rapidly after the initial colony growth.
Laboratorium Microbiologie Twente Achterhoek, Hengelo, Nederland
www.labmicta.nl / info@labmicta.nl
MALDI Biotyper Poster Hall 2016 Primary ID of Mycobacteria

Performance of MALDI-TOF MS in Species Identification of Clinical Mycobacterial Isolates

Dzen Maltepe
Grkem Yaman1, Elcin A. Alasehir2 Laboratories University
1Dzen Laboratories Group, Department of Microbiology and Tuberculosis, Istanbul Group Faculty of
2Maltepe University, Faculty of Medicine, Department of Medical Microbiology Medicine

Introduction: Results: Table 4:

Matrix-assisted laser desorption/ionization time-of-flight (MALDI- From a total of 210 mycobacteria strains 167 (79.5%) were Mycobacteria correctly identified MALDI-TOF Scores
TOF MS) instruments are being extensively used for accurate and identified as M. tuberculosis complex (MTBC) and 43 (20.5%) were with MALDI-TOF MS
<1.3 1.3-1.7 >1.7 >2.0
rapid identification of bacteria and yeasts in clinical microbiology identified as non tuberculosis mycobacteria (NTM) (Table 1). MTBC (144/167) 2 19 39 84
laboratories. Identification of mycobacterial hspecies still usually 144 (86.2%) of MTBC strains were accurately identified with M.abscessus (10/10) 0 1 2 7
depends on molecular methods which demand ighly technical MALDI-TOF MS (Table 2). In 127 (88.2%) of MTBC strains a score M.fortuitum (4/7) 0 0 1 3
environment and are relatively expensive. We have evaluated the higher than 1,7 and above was obtained which is accepted to be M.intracellulare (3/4) 0 0 0 3
performance of MALDI-TOF MS directly from the positive MGIT correct to the genus level according to manufacturer guidelines.
M.porcinum (4/4) 0 0 0 4
bottles for identification of mycobacterial strains which were From 23 (13.8%) misidentified strains only 1 had a score above 1,7
M.simiae (4/4) 0 0 4 0
isolated from routine clinical patient specimens. whereas in 21 (91.3%) the score was lower than 1,3 which is very
M.avium (3/3) 0 0 0 3
low. From 43 NTM, 38 (88.4%) were correctly identified with
Method: M.kansasii (2/2) 0 0 0 2
MALDI-TOF MS. From 34 (89.5%) of correctly identified NTM a
Various clinical specimens obtained between April 2013- M.xenopi (2/2) 0 0 1 1
score more than 1.7 was obtained.
September 2014 were cultivated both in BD BACTEC MGIT 960 and M.gordonae (1/1) 0 1 0 0
182 (86.7%) of all mycobacteria strains were accurately identified
LJ solid media. A total of 210 mycobacterium isolates from positive with MALDI-TOF MS (Table 3 and 4). In 163 (77.6%) strains, a score M.haemophilum (1/1) 0 0 0 1
MGIT bottles were identified routinely with MPT64 1,7 or above was obtained with MALDI-TOF MS and 160 (98.2%) of M.gastri (1/1) 0 1 0 0
immunochromatographic test (SDTB Ag MPT64 Rapid Kit, Republic these results were determined to be accurate M.lentiflavum (1/1) 0 1 0 0
of Korea) and PCR-RFLP assay (PRA). Biomass from MGIT bottles M.marinum (1/1) 0 0 0 1
were processed using a zirconia/silica bead based method and M.mucogenicum (1/1) 0 0 0 1
Table 2
identification was done with Mycobacteria Library v2.0 using
:
M.smegmatis (0/1) 0 0 0 0
Bruker Microflex LT (Bruker Daltonics, Germany) instrument. Method MTBC (167) TDM (43)
TOTAL (182/210) 2 23 47 110
Concordant results obtained with MPT64, PRA and MALDI-TOF MS MPT64 161 (+) 43 (-)
5 (faintly +)
were determined to be accurate. Discordant strains were identified
1 (-)
molecularly with HAIN Geno Type Mycobacterium CM/AS (HAIN
PRA 163 MTBC 29 correct Conclusion:
Lifescience, Germany) line probe assay kits.
3 MTBC? 5 correct (group) When a score of 1,7 or above is obtained with MALDI-TOF
1 no ID 9 no ID MS using zirconia/silica bead based method and
Table 1:
MALDI-TOF 144 (86.2%) MTBC 38 (88.4%) correct Mycobacteria Library v2.0 both MTBC and NTM strains
Strains No Strains No could be identified rapidly and accurately isolated from
23 mis-ID (13.8%) 5 mis-ID (11.6%)
M.tuberculosis complex 167 M.xenopi 2 routine clinical BD MGIT culture bottles.
MALDI-TOF MS alone may not be sufficient to serve as a
M.abscessus 10 M.gordonae 1 Table 3:
mycobacteria identification tool directly from positive MGIT
M.fortuitum 7 M.haemophilum 1 MALDI Result Number (%) Score >1.7 Score <1.3 bottles mainly because of contamination however a simple
M.intracellulare 4 M.gastri 1 Correct 182 (%86,7) 157 (%86,2) 2 (%1) combination with rapid MPT64 antigen test increases the
False 28 (%13,3) 3 (%10,7) 22 (%78,6) correct identification rate to 97.6%.
M.porcinum 4 M.lentiflavum 1
M.simiae 4 M.marinum 1
M.avium 3 M.mucogenicum 1
M.kansasii 2 M.smegmatis 1 Copyright 2015 Dr. Gorkem Yaman, Duzen Laboratories Group Cemal Sahir Sok. No:14 Mecidiyekoy Istanbul, Turkey; gorkemyaman@dzen.com.tr

35
 36
Primary ID in Urine

Rapid identification of pathogens

directly from urine by MALDI-TOF
Katarzyna Schmidt1, Helen Williams2, David M. Livermore1
1
Medical Microbiology, University of East Anglia (UEA), Norwich, 2 Norfolk and Norwich University Hospital (NNUH)
INTRODUCTION METHODS I Urine samples
Out- and in- patients
(n=150)
Care Quality Commission data show UK hospital
1
Triage
admissions for UTIs increasing progressively.
Automated iQ 200 96 culture positive
Early recognition of the pathogens and their resistances (Iris Diagnostics)
System
27 culture negative BUT microscopy positive
27 insignificant
may help to tailor treatment, preventing morbidity and
- in elderly urosepsis patients - mortality2; it should also Sample preparation
(see Methods II)
support antibiotic stewardship. If >40 WBC/l
>5 bacteria/l
= MALDI-TOF Culture, CLED agar
Quantitative Culture confirm I/D re-I/D
Current identification takes c. 24-48h3. To achieve rapid Chrom agar
results we sought to use MALDI-TOF directly on urine,
without culture.
Results agreed Negative or
with culture discrepant
= =
Accepted Results repeated
Figure 1. Flow chart for pathogen identification from urine samples
METHODS II
Washed &
1%SDS/ Formic acid/
Human cells
removed
Collected
supernatant
Collected
bacterial pellet
Lysozome
treatment
Acetonitrile
extraction
E. coli 2.170
2000rpm - 2min 13500rpm - 5min
Human cells
Bacteria Score:
0-1.699 no reliable identification
Urine sample 1.7-1.999 probable genus identification
2.0 species identification
Figure 2. Sample preparation for the MALDI-TOF
RESULTS Pathogens Culture MALDI-TOF I/D
total total agreement
Escherichia coli 22 25 17
MALDI-TOF identified pathogens in 81/96 culture-positive urines (Table 1). Pseudomonas aeruginosa 16 14 13
In 69/81 the same pathogen was found by culture and MALDI-TOF (Table 2). Enterococcus faecalis/faecium 15 14 12
10/15 failures to find pathogens largely related to low bacterial counts (Table 3). Other Coliform species*
Citrobacter freundii
13 10
1
9
For 12/81 culture-positive urines identifications disagreed, with little clear pathern. Citrobacter koseri
Enterobacter cloacae
1
3
Klebsiella pneumoniae 4
Klebsiella oxytoca 1
MALDI-TOF also detected organisms by MALDI-TOF in 8/27 culture-negative urines Proteeae spp** 11 8 8
(Table 3) including organisms (e.g. Gardnerella vaginalis) unable to grow in routine Proteus mirabilis
Providencia stuartii
6
1
media, and in 16/27 "insignificant" urines. Morganella morganii 1
Streptococcus spp (Grp B) 8 5 5
Staphylococcus aureus 4 5 3
Sensitivity was 84.4%; specificity, 70%, but increased to 100% if the 8 organisms Staphylococcus saprophiticus 2 2 1
identified by MALDI-TOF from culture-negative urines were taken as true positives. Candida albicans
CoagNegStaph
1
3
1
1
1
0
Streptococcus spp (Grp A) 1 2 0
Overall, MALDI-TOF allowed identification for 89 urine samples (59%) with Acinobacillus schaalii
Clostridium spp
0
0
4
1
0
0
monomicrobial infection and 16 (10%) with polymicrobial infection (Table 1). Gardnerella vaginalis 0 2 0
Lactobacillus spp 0 2 0
Peptoniphilus harei 0 1 0
SUM 96 97 69
*As based on Chrom ID culture: split as Citrobacter spp., Enterobacter spp., Klebsiella spp. by MALDI-TOF
**As based on Chrom ID culture: split as Morganella morganii, Proteus mirabilis, Providencia stuartii by MALDI-TOF
Table 2. Identification agreement between culture and MALDI-TOF
Culture -ve Culture +ve Totals Culture -ve Culture +ve Totals MALDI TOF ve results from +ve culture (n=15*)
MALDI+ve 8* 81 89 0 81 81 Detection limit <105 /ml 10
MALDI Biotyper Poster Hall 2016

Mixed culture 3
CONCLUSION
MALDI -ve 19 15 34 27 15*(8 GNB 42
7 GPB) Unknown reason 2
Totals 27 96 123 27 96 123
MALDI TOF disagreement from +ve culture (n=12)
Sensitivity 84.4% 84.4%
Specificity 70% 100%
Aerobic GNB bacteria able to grow on standard medium
Aerobic GPB bacteria able to grow on standard medium
6
3
Direct use of MALDI-TOF allowed
Monoinfection Polyinfection MALDI Anaerobic GPB bacteria difficult to cultivate 3
bacterial identification from urines
with >10 cfu/ml within 1.5 hours.
found by found by +ve/-ve 5
MALDI MALDI results MALDI TOF +ve results from ve culture (n=8*)
+ve(n=96) 73 8 81/15
-ve culture (n=27) 8 0 8/19
Aerobic bacteria able to grow on standard medium 6
This significantly shorten ed
Anaerobic bacteria difficult to cultivate 2
Mixed culture (n=27) 8 8 16/11
identification time and may aid
Table 1. Positivity/negativity agreement by culture and MALDI-TOF Table 3. Summary of MALDI-TOF: culture disagreement
selection of appropriate therapy.
ACKNOWLEDGEMENT
REFERENCES
1. http://www.cqc.org.uk/file/4346 We gratefully acknowledge all the laboratory staff for support
2. Foxman B. Epidemiology of Urinary Tract Infections Infections: Incidence, Morbidity and Economic Costs. Am J Med 2002;113:5-13 in this research and help in collecting urine samples; also Brker
3. Schmiemann G et al. The Diagnosis of Urinary Tract Infection. Dtsch Arztebl Int 2010;107:361-367 for loan of the MALDI-TOF.
MALDI Biotyper Poster Hall 2016 Primary ID in Urine

37
MALDI Biotyper Poster Hall 2016 Blood Culture

S-901 Fast Bacterial Identification by Mass Spectrometry in Blood Culture Broth of

Bacteremic Patients Allows Quick Adaptation of the Empirical Antibiotic Therapy
F. Gourmelen1, C. Piau1, C.Leyer1-2, G.Auger1, P. Hisberg1, C. Le Blanc1, P. Vincent1-2, S. Kayal1-2.
1CHU Rennes, Deparment of Microbiology; 2University of Rennes-1, Medical school; Rennes- France
# Frederic.gourmelen@chu-rennes.fr

ABSTRACT
RESULTS
Delayed or inappropriate treatment of bloodstream infec3ons is detrimental
for the prognosis of sep3c pa3ents. In addi3on to Gram staining of growing
bacteria, we tested whether a rapid bacterial iden3ca3on in blood culture
broths (RBI) based on matrix-assisted laser desorp3on/ioniza3on 3me-of- Demographic and clinical characteris2c of
Bacterial species directly iden2ed in the BCBs Global view of the changement of empirical an2bio2c treatment (EAT1
ight mass spectrometry (MALDI-TOF) could improve empirical an3bio3c
treatment (EAT).
pa2ents N= 180 and EAT2) occurred in the studied popula2on
During a 6-month period, we prospec3vely evaluated for 180 pa3ents with
monomicrobial bloodstream infec3on and analyzed empirical treatments
started before an3bio3c suscep3bility tes3ng (AST). For each pa3ent we
considered the treatment decisions made before (EAT1) and aSer (EAT2) RBI,

and analyzed their respec3ve appropriateness based on the nal AST of the
isolate.

Onset of a unique blood culture growing with a coagulase nega3ve Microbiological
Staphylococcus (CNS) was nally considered as a contamina3on (n=40). diagnosis procedure
Pa3ents experiencing a bloodstream infec3on (140), Gram nega3ve isolates
(56%) were mostly Enterobacteriaceae (65) and non-fermen3ng bacteria
(11), and Gram posi3ve isolates (44%) consisted in S.aureus (28), CNS (11),
Streptococcus and Enterococcus (22). RBI in blood culture broths was achieved
at species level for 94% of isolates. Before RBI, EAT1 was not adapted for 21
BCBs sampling
not treated pa3ents, and 37 receiving inadequate EAT. ASer RBI, EAT2 was de
novo introduced for 23 pa3ents and modied in 24 others. This resulted in a
nal op3mal treatment for 89% of pa3ents compared to 57% before RBI. This

improvement was mainly achieved by adap3ng EAT regimen against Rapid ID
Rapid
Pseudomonas species and other non fermen3g bacteria. Among the 140
pa3ents 26 pa3ents were documented with severe sepsis or sep3c shock and

for 5 of them EAT1 was not adapted whereas EAT2 could be quickly modied
according to bacterial iden3ca3on: P. aeruginosa (2), E. faecium, E.
aerogenes, K. pneumoniae. Conv ID
Conv
Gram staining alone is no appropriate to predict the natural or the expected All the iden3ca3ons obtained directly from BCBS were
resistance prole to an3bio3cs. Taking into account natural resistance and
knowledge of local ecology, RBI with MALDI-TOF is accurate enough to give a
conrmed by conven3onal iden3ca3on of sub-cultured
one day prior to the AST results recommenda3ons to improve EAT. colonies.
We did not observe any misiden3ca3on Adequacy of the adapta2on of the rst of second line of EAT according to
iden2ed bacterial species

Direct iden2ca2ons of bacterial species are at
OBJECTIVES least 24h more rapid than conven2onal Anaerobic

Delayed or inappropriate treatment of bloodstream iden2ca2ons.

Enterococcus
infec3ons is detrimental for the prognosis of sep3c 120

pa3ents.
100 Streptococcus
In addi3on to Gram staining of growing bacteria, we EAT1 adapted
tested whether a rapid bacterial iden3ca3on in blood 80
S.aureus
EAT2 adapted
culture broths (RBI) based on matrix-assisted laser
desorp3on/ioniza3on 3me-of-ight mass spectrometry 60
Enterobacteriace
(MALDI-TOF) could improve empirical an3bio3c treatment
40
(EAT). Non fermenJng

20
(ICU=Intensive Care Unit, ESBL=Extended spectrum beta lactamase, 0% 20% 40% 60% 80% 100%
MRSA=Methicilin Resistant S.aureus, VRE= Vancomycin resistant

METHODS
enterococcus) 0
ConvID RapID Of pa3ents

Kaplan-Meier es3mate survival likelihood of the studied 1. No-one of the bacteremia with non-fermen2ng microorganism ( mainly P. aeruginosa) was adapted.
RapdID= Iden3ca3on performed by mass
popula3on at 30 day was as expected from other RBI allows quick adapta2on of EAT for 73% of included pa2ents
During a 6-month period, we prospec3vely collected 203 posi3ve blood spectrometry directly on BCBs when detected 2. When the species S. aureus was iden:ed and according to the global resistance epidemiology of the
culture. When Gram staining was polymorphic and when RBI by mass survey :
posi3ve care department, adapted EAT enhances from 54% to 96%
spectrometry failed the pa3ent has not been taken into account n=23 . Overall 86,5%
ConvID = conven3onal iden3ca3on of isolated 3. Dieren:a:on between E. faecalis and E. faecium by RBI signicantly increased the adequacy of EAT
Sepsis 88% from 43% to 86%
Finally we evaluated 180 pa3ents with a unique posi3ve blood culture bacteria
Sep3c shock 63,7%
growing with a single microorganism and analysed empirical treatments
started before an3bio3c suscep3bility tes3ng (AST). For each pa3ent we
considered the treatment decisions made before (EAT1) and aSer (EAT2)
bacterial iden3ca3on in BCBs.
CONCLUSION
The appropriateness of EAT was based on its adequacy with the nal References
an3bio3c suscep3bility tes3ng (AST) the isolate
EAT is considered as adequate the infec3ng microorganism was
! Gram staining alone is not appropriate to predict the natural or the expected resistance prole to an2bio2cs. Kumar A, Ellis P, Arabi Y, Roberts D, Light B, Parrillo JE, et al. Ini3a3on of
inappropriate an3microbial therapy results in a vefold reduc3on of survival in
subsequently found to be suscep3ble to the given an3bio3c. ! Taking into account natural resistance and knowledge of local ecology, species iden2ca2on by mass spectrometry human sep3c shock. Chest. nov 2009;136(5):123748
Ibrahim EH, Sherman G, Ward S, Fraser VJ, Kollef MH. The inuence of
RBI is considered to have an impact if the an3bio3c therapy was either directly in BCBs with MALDI-TOF is accurate enough to give a one day prior to the AST results recommenda2ons to inadequate an3microbial treatment of bloodstream infec3ons on pa3ent
started or changed (spectrum broadening, spectrum streamlining, outcomes in the ICU sejng. Chest. juill 2000;118(1):14655.
introduc3on of empirical an3bio3c therapy or discon3nua3on) following improve EAT. Seifert H. The clinical importance of microbiological ndings in the diagnosis
the iden3ca3on and before obtaining an3biogram. and management of bloodstream infec3ons. Clin Infect Dis O Publ Infect Dis
! Pseudomonas aeruginosa iden2ca2on and Enterococcus species dieren2a2on allow a quick adapta2on of the empirical Soc Am. 15 mai 2009;48 Suppl 4:S23845

an2bio2c chemotherapy and facilitate severe infec2on management

38
 39
Blood Culture

Validation and implementation of MALDI-TOF: a quick and easy method for bacterial identification from clinical samples
Manar Najim Mashhadani M B ChB, MSc, PhD MED MICRO, PGCert Infec Sciences
Department of Microbiology, Northampton General Hospital, United Kingdom
Aims To validate the use of the Bruker Biotyper MALDI-TOF MS system for the routine identification of bacterial and yeast isolates from cultures. To
evaluate whether the conventional identification methods, taken from clinical samples sent to the Department of Microbiology at Northampton
General Hospital from hospital wards and GPs surgeries, warrants replacing.
Background METHODS
The use of the direct examination of both macroscopic and microscopic colony morphology is a v All isolates were recovered from routine examination of clinical specimens submitted to the
traditional approach for the identification of bacterial and yeast isolates from clinical specimens. microbiological laboratory, such as blood, urine, pus, biopsy, and swabs from any site of the
These tests are laborious, involve long incubation periods, require a significant amount of hands- body, cerebrospinal fluid, respiratory tract, and wound specimens. Retrospective isolates
on time, and are subject to human error. Automation tests and molecular-based DNA techniques including previously identified isolates, reference isolates, and quality control isolates, are also
can improve turnaround time and provide results with high sensitivity and specificity, but they used in the evaluation to test a wide range of different pathogens and to cover varieties of
have disadvantages. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass genera and species. The isolates were recovered after aerobic, microaerophilic, or anaerobic
spectrometry, however, is a molecular platform method for automated, rapid, easy to use, and less incubation at 35C on 5% sheep blood agar, chocolate agar, cysteine-lactose electrolyte-
expensive identification of pathogens that provides a viable alternative to the direct examination deficient (CLED) agar and Sabouraud's agar media, Oxford (Perth, UK). A total of 134 isolates
of macroscopic and microscopic colonies and PCR tests. were used validate MALDI -TOF for routine use in Northampton General Hospital (NGH) and 44
positive blood culture bottles used to evaluate MALDI SepsityperTM Kit .
v Conventional Methods used for identification purposes included Gram staining, catalase and
oxidase activities, latex and DNase for staphylococci, serotyping for Streptococci spp.
Results & Summary v Further identification using automated system, vitek2 (bioMrieux) and appropriate cards and
Gram negative species - 98% showed the same genus and species identification. Only one appropriate Phoenix identification panels and Phoenix apparatus (BD)
isolate had discrepancies: the first occurred at genus level with Acientobacter spp being
identified as Lactobacillus johnsonii by MALDI-TOF and Acinetobacter ursingii by Vitek2. v MALDI-TOF (BD-Bruker) and MALDI SepsityperTM Kit were evaluated against the conventional
methods and were used according to the manufacture instructions.
Staphylococci Species - 100% results compatibility of MALDI-TOF to Phoenix and Vitek 2.
Streptococci Species - all tested species were identified correctly by MALDI-TOF, compared
to either Phoenix or Vitek2 in addition to Lancefield grouping results, except one isolate
being identified as Lactococcus greavieae by Vitek2 and Streptococcus angiosus by
MALDI-TOF.
Yeast Isolates - All yeast isolates except one had the same identification compared to
Vitek2. Stephanoascus ciferrii 89% was identified using Vitek2, MALDI gave an excellent
identification of Candida albicans. Further extraction was required for some isolates to
gain high MALDI-TOF scores.
One isolate which was GPB failed to be identified using Phoenix and Vitek2, but API testing
gave a positive identification of Brevibacterium with 83%, while MALDI identified it as
Bacillus pumilus with high score. Colony morphology gave similar classical morphology of
Bacillus spp. on agar plate.
Sepsityper Kit results showed 41/44 isolates had the same identification when comparing
direct extraction form positive blood culture bottles and MALDI-TOF identification from
agar culture method. One sample had no reliable identification by both methods and
thought to be a contamination. Two flagged positive culture bottled failed to give an
identification by the extraction method, but there was no organism seen by Gram staining
and no growth after 48 hour on the culture plates. Both samples had lots of white blood
cells which triggered the flagging. Fig 1 : MALDI-TOF Instrument at NGH and MALDI SepsityperTM Kit.
No. Test/ Phenotypically ID Gram Oxidase Phoenix/ Vitek2 Maldi
GRAM NEGATIVE BACTERIA

1 NLF-CLED Coliform GNB Neg Acientobacter lwoffii Acientobacter lwoffii

2 NLF-CLED Coliform GNB Neg Acientobacter baumannii Acientobacter baumannii
1 NLF-CLED Coliform GNB Neg Acientobacter ursingii Acientobacter ursingii
1 NLF-CLED Coliform GNB Neg Acientobacter ursingii Acientobacter johnsnii
1 NLF-CLED Coliform GNB Neg Citrobacter Koseri Citrobacter Koseri
1 NLF-CLED Coliform GNB Neg Citrobacter freundii Citrobacter freundii
19 LF-CLED Coliform GNB Neg E. coli E. coli
4 NLF-CLED Coliform GNB Neg Enterobacter cloacae Enterobacter cloacae
1 NLF-CLED Coliform GNB Neg Enterobacter aerogenes Enterobacter aerogenes
2 LF-CLED Coliform GNB Neg Klebsilla Oxytoca Klebsilla Oxytoca
3 LF-CLED Coliform GNB Neg Klebsilla pneumoniae Klebsilla pneumoniae
13 NLF-CLED Coliform GNB Pos P. auerogenosa P. auerogenosa
1 NLF-CLED Coliform GNB Pos P. putida P. putida
3 NLF-CLED Coliform GNB Neg Serratia marcescens Serratia marcescens
1 NLF-CLED Coliform GNB Neg Serratia liquefaciens Serratia liquefaciens
1 NLF-CLED Coliform GNB Neg Ptoteus mirablis Ptoteus mirablis
1 NLF-CLED Coliform GNB Neg Strernotro. Maltophilia Strernotro. Maltophilia
No. Test/ Phenotypically ID Gram Grouping Phoenix/ Vitek2 Maldi
Streptococci Spp.

1 Alpha haemolysis Streptococci Spp. GPC
2 Gamma (no) haemolysis Streptococci Spp. GPC
3 Gamma (no) haemolysis Streptococci Spp. GPC
9 Beta haemolysis Streptococci Spp. GPC
5 Beta haemolysis Streptococci Spp. GPC
1 Beta haemolysis Streptococci Spp. GPC
1 Alpha haemolysis Streptococci Spp. GPC
2 Alpha haemolysis Streptococci Spp. GPC
7 Beta haemolysis Streptococci Spp. GPC
1 Gamma (no) haemolysis Streptococci Spp. GPC
1 Alpha haemolysis Streptococci Spp. GPC
No. Test/ Phenotypically ID Gram
NoT Aerococcus urinae
Group D Eenterococcus faecalis
Group D Eenterococcus faecium
Group B Streptococcus agalactiae
Group G Sterptococcus dysgalactiae
Group C Sterptococcus equisiminis
NoT Streptococcus lutetiensis
NoT Streptococcus pneumoniae
Group A Streptococcus pyogenes
Group D Streptococcus salivarius
NoT Lactococcus garvieae
sts/Latex & Dna Phoenix/ Vitek2
Aerococcus urinae
Eenterococcus. faecalis
Eenterococcus. faecium
Streptococcus. agalactiae
Sterptococcus. dysgalactiae
Sterptococcus. equisiminis
Streptococcus. lutetiensis
Streptococcus. pneumoniae
Streptococcus. pyogenes Table 2: Comparable isolate identification using MALDI-TOF ID/Kit and MALDI-TOF ID/Culture
Streptococcus. salivarius
Streptococcus. angiosus
Maldi

Spp.
Staphylococci
20
2
White- gray coolonies Staphylococci Spp.
White- gray coolonies Staphylococci Spp.
GPCL
GPCL
Positive
Negative
Staphylococcus aureus Staphylococcus aureus
Staphylococcus. epidermidis Staphylococcua. epidermidis
Conclusion
2 White- gray coolonies Staphylococci Spp. GPCL Negative Staphylococcus. simulans Staphylococcus. simulans Turnaround times reduced with the use of MALDI-TOF and Phoenix, and
1 White- gray coolonies Staphylococci Spp. GPCL Negative Staphylococcus. saprophyticus Staphylococcus. saprophyticus laboratory workflow adapted.
1 White- gray coolonies Staphylococci Spp. GPCL Positive Staphylococcus.intermedius Staphylococcus.intermedius
MALDI Biotyper Poster Hall 2016

1 White- gray coolonies Staphylococci Spp. GPCL Negative Staphylococcus Lugdunensis Staphylococcus Lugdunensis MALDI-TOF identification and Phoenix AST replaced all Gram staining,
No. Test/ Phenotypically ID Gram Wet prep Vitek2 Maldi
supplementary tests for identification and the use of combi ID/AST Phoenix

Spp.
Candida
13 White- creamy colonies Candida spp. Yeast Yeast Candida albicans Candida albicans
1 White- creamy colonies Candida spp. Yeast Yeast Stephanoascus ciferrii 89% Candida albicans panels.
1 White- creamy colonies Candida spp. Yeast Yeast Candida parapsilosis Candida parapsilosis
1 White- creamy colonies Candida spp. Yeast Yeast Candida krusei Candida krusei MALDI-TOF provide reliable, quick and reproducible results. The current
1 White- creamy colonies Candida spp. Yeast Yeast Candida lusitaniae Candida lusitaniae workflow in the NGH microbiology laboratory has significantly improved the
2 White- creamy colonies Candida spp. Yeast Yeast Candida galbrata Candida galbrata
turnaround times, flexibility and reduced human errors results from multiple
tests and possibility of contamination.
Table 1: The results of 134 isolates identification using conventional methods, Phoenix or
Vitek2 and MALDI-TOF
MALDI-TOF cost around 0.02 ($0.03) to 0.06 ($0.10)
Turnaround times were reduced with the use of MALDI-TOF.
compared to around 3.23 ($5.50) for conventional
methods.
Identification time was cut from 7hrs to >24hrs using cultures, as opposed to
between only 14mins to 16mins using the Sepsityper Kit.
Sepsityper Kit for blood culture samples cost 3.30
($5.57) per sample.
MALDI Biotyper Poster Hall 2016 Blood Culture and Resistance

Antimicrobial Stewardship Combined With Maldi-tof and -Lacta Test

Dr Marie-Paule Fernandez-Gerlinger
2157 Unit Mobile de Microbiologie Clinique, Service de Microbiologie
Hpital europen Georges Pompidou
20, rue Leblanc, 75015 Paris, France

Performed on Gram-Negative Bacilli Blood Culture is Effective for Sparing the use of Carbapenems
marie-paule.gerlinger@egp.aphp.fr

A. Aubry1, A. Fournier1, H. Pereira2, S. Katsahian2,3, H. Bensekhri1, J-L. Mainardi1,3 and M-P. Fernandez-Gerlinger1,3
1 Unit Mobile de Microbiologie Clinique, Service de Microbiologie, Hpital europen Georges Pompidou, AP-HP, Paris, France; 2 Ple Biostatistique et Sant Publique, Hpital europen Georges Pompidou, AP-HP, Paris, France ;3 Facult de Mdecine Paris Descartes, Universit Paris Descartes, Paris, France

Introduction and purpose Results

Table 1: Characteristics of the 128 cases of Gram-negative bacteremia analyzed Antimicrobial Stewardship with and without Maldi-tof and -Lacta
Deal with: Female n (%) 60 (47) Test: Strat. B compared to Strat. C
1) Emerging antibiotic resistance in Gram-negative bacilli (GNB), e.g. extended- N= 340 Bacteremia due to GNB during the Age mean years 69 50
study period (168 days) Nosocomial* n (%) 77 (60) p=0.03
spectrum beta-lactamase (ESBL) and carbapenemase-producing Previous ESBL carriage n (%) 14 (10)
Enterobacteriaceae Recent antimicrobial therapy n (%) 69 (54) 45
Severe sepsis/septic shock n (%) 29 (23)
2) Critical issue in patients with GNB bloodstream infections (BSI) and high ICU admission n (%) 19 (15%) 40
N= 209 Bacteremia due to GNB during the
morbidity and mortality in patients with BSI caused by ESBL (1) study period excluded (week-end and Polymicrobial bacteremia n (%) 9 (7)
Source of infection
3) And exposure to broad-spectrum antimicrobials, as carbapenems. positive in the afternoon)
Urinary tract n (%) 50 (39)
35
Catheter infection n (%) 45 (35)
- Catheter 22 30
Thus, rapid antibiotic optimization is a key of importance in the context of - PAC 17
BSI caused by GNB. N= 131 Bacteremia due to GNB during the - lymphangitis 6

Days
analyzed period (120 days) 25 Strat B
Digestive tract n (%) 11 (9) p=0.04
- Peritonitis 3 Strat C
Antimicrobial optimization is improved by: - Translocation 8 20
1) Rapid identification of the microorganism with Maldi-tof Mass-spectrometry N= 3 Bacteremia due to GNB during the
- Angiocholitis 9
- Cholecystitis 1 15
(MT) analyzed period (120 days) excluded - Liver abscess 1 p=0.6
(anaerobic bacteria and Stenotrophomonas
2) Combined with antimicrobial stewardship (AMS) intervention spp)
- Other 1
10
In terms of length of stay, morbidity, mortality, total hospital costs, in the context Pneumonia n (%) 6 (5)
of GNB bacteremia (2,3), and for patients with antibiotic-resistant Gram-negative Others n (%) 4 (3) 5
N= 128 Bacteremia due to GNB during the - OSI 2
bacteremia (4). analyzed period (120 days) included - Unknown 2
*
Nosocomial: hospital acquired and healthcare associated; ICU: intensive care unit; PAC : Port-a-cath
0
Spare carbapenem Narrow spectrum Avoid inadequat
The purpose of this study was to assess the combination of AMS with rapid implantable central venous access device ;OSI operative site infection. antibiotherapy

identification by MT, and a new rapid biochemical test able to detect ESBL: -
Lacta-test (BLT) (Bio-Rad, Marnes-la-Coquette, France). Table 2: Organism distribution

Pathogens n (%) Agreement Strat.B and -Lacta Test or Maldi-tof MS estimated through Cohens
Patients and methods Enterobacteriaceae
Escherichia coli
100 (84)
62 (50) ESBL carriage and bacteremia kappa
Proteus mirabilis 1
Klebsiella pneumoniae 17 (14) ESBL
All Non ESBL -Lacta Test Maldi-tof MS
Klebsiella oxytoca 3 (2) bacteremia
Bacteremia bacteremia
Enterobacter cloacae 8 (7) N (%)
N (%) N(%)
Enterobacter aerogenes 2 (1)
Prospective observational study (168 days- 24 weeks): Enterobacter sakasakii 1 Spare carbapenem Strat. B
= -0,0465 = -0,0483 not
ESBL known p = 2 .10-13 p=0
-All patients with GNB positive blood cultures during 168 days Serratia marcescens 3 (2) agreement
Pantoea agglomerans 1 carriage
14 (10) 6 (43) 8 (57)
-Analyzed was performed from Monday to Friday morning (120 days). Morganella morganii 1 N (%)
p = 0.001 = 0,85 = 0,82
Citrobacter freundii 1 Avoid inadequate
p=0 p=0
MT and BLT were performed simultaneously on 3h incubated solid medium Nonfermentative 17 (12)
ESBL not OR = 9.65 antibiotherapy Strat.B
Pseudomonas aeruginosa 15 (12)
subcultures Acinetobacter pittii 1
known agreement
carriage 114 (90) 8 (7) 106 (93) = 0,95 = 0,885
Three strategies were compared: Chryseobacterium spp. 1
N (%) Narrow spectrum Strat. B p=0 p=0
Other aerobic 2 (1)
(A) empiric antibiotic therapy initiated by the physician in charge of the patient Salmonella spp. 2 (1)
knowing GNB bacteremia without MT and BLT results Polymicrobial 9 (7)
(B) empiric antibiotic therapy recommended by AMS without MT and BLT results
(C) AMS advice with MT and BLT results.

Distribution between different tests were compared using a Mac Nemar test for Conclusion
paired data. Agreement was estimated through the Cohen's Kappa. We have also
tested whether Kappa coefficient is different from 0.
References 1) Rapid identification with Maldi-tof MS and EBLSE rapid test -Lacta Test associated with antimicrobial stewardship (Strat C) is more efficient than AMS alone to narrow spectrum and avoid inadequate antibiotherapy.
(1) Schwaber et al. JAC 2007 2) Apart, BLT and MT are not in agreement with AMS in term of sparing carbapenem. BLT and MT are in agreement with AMS in terms of narrowing spectrum and avoiding inadequate antibiotherapy.
(2) Clerc et al. CID 2013
(3) Huang et al. CID 2013
3) In this study, ESBL carriage is associated with ESBL bacteremia.
(4) Perez et al. J Infect 2014

40
MALDI Biotyper Poster Hall 2016 Resistance

Detection of OXA-48-producing Enterobacteriaceae through MALDI-TOF using temocillin as diagnostic

marker
Marina Oviao Garca1, Mara Jos Barba Miramontes1, Begoa Fernndez Prez1, Jess Oteo Iglesias2, Jos Campos Marqus2, Sebastien Van de Velde3, Germn Bou1.
1Complejo Hospitalario Universitario A Corua, A Corua, Spain. 2Instituto de Salud Carlos III, Madrid, Spain. 3Eumedia, Manage, Belgium

Introduction and Results Diameter of distribution of temocillin inhibition (30 g, Rosco) for the carbapenemase
enzymes tested in the assay.
Purpose CO2 Structural analysis and experimental
data over temocillin [M], MS spectrum
40
OXA
Group D carbapenemases (especially

Number of isolates
reveal the following mass peaks: IMP
blaOXA-48 isolates) detection represents [M + H]+ ...415 Da 30
a challenge as these enzymes are an NDM
[M + Na]+ ....437 Da
emerging healthcare concern. As the [Mdecarb. + H]+ ......371 Da 20
VIM
carbapenems MICS are low, [Mhydrol/decarb. + H]+.....389 Da KPC
carbapenemases often go undetected H2O

and are overloooked by most

10 negative
Mass peaks of the decarboxilated species are the most
phenotypic methods, besides the fact intense. Temocillin resistance is considered either for the
that there is no specific inhibitor for disappearance of temocillin mass peaks or for the appearance
0
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
group D detection. Most if not all of the hydrolyzed form. Diameter zones (mm)
classical ESBL and AmpC enzymes 4
Temocillin disk diffusion MALDI-TOF MS
are sensitive to temocillin. However, (x10) A
isolates harbouring OXA-48 Isolates per assay
-Lactamase
carbapenemases show very high -lactamase Average zone % Average time (min) of
0.8 class Sensitivity
levels of resistance. We propose to (n) diameter (mm) Resistances temocillin hydrolysis
perform temocillin structural analysis (<12mm) detection %
and asses a MS method for detecting 370.951
none 5 24,6 0 - -
carbapenemases enzymes, focused 0.4
on OXA-48-type. AmpC 11 18 0 - -
436.929 ESBL 5 24 0 - -
414.996
KPC 15 18,8 0 - -
Material and methods
0.0

B VIM 23 10,5 70 (16/23) 36 88 (14/16)

Temocillin behaviour was evaluated by 8000
MALDI-TOF and by disk diffusion NDM 3 11 67 (2/3) 75 100 (2/2)
method, Rosco, in a series of 94 IMP 12 17,5 25 (3/12) 45 100 (3/3)
characterized isoltes, , 73 of them OXA 20 9,2 100 (20/20) 15 100 (20/20)
carrying a carbapenemase enzyme: 15
4000
blaKPC, 23blaVIM, 3blaNDM, 12blaIMP and Bacterial isolates with no antimicrobial resistance, AmpC, ESBL and KPC-type enzymes
20blaOXA.For the disk diffusion method, remain susceptible to temocillin. On the other hand, and as previously reported, isolates
bacteria were overnight cultured on carrying a carbapenemase enzyme, show different levels of resistance, being VIM-type and
MH agar with a temocillin disk. The 388.407 * specially OXA-type enzymes the most resistant. Global sensitivity for detecting temocillin
susceptibility cut-off used for the study resistance by MALDI-TOF is 95%, being 100% for OXA-48-type enzymes. OXA-48-type
0
was the one proposed by Huang et al. 370 380 390 400 410 420 430 440 450 460 enzymes hydrolyze temocillin in 15 min, and the average hydrolysis time for the rest of
m/z
(<12mm). For the MALDI-TOF (A) Mass spectrum of temocillin and (B) its hydrolyzed form at 15 min enzymes is higher. This involve that a hydrolized temocillin spectrum at 15 min has a
analysis, the reaction between 10 l of after exposure to a blaOXA-48 K. pneumoniae strain. The indicated mass positive predictive value of 87% (20/23) for owning an OXA-48-type enzyme. The negative
temocillin (Negaban, Eumedica, peaks are representative of temocillin and its hydrolyzed form (*). predictive value at the same time is 100%.
1500 mg/L, 20 mM Tris-HCl; 50mM
NH4HCO3, pH 7.0) and the amount of
bacteria filling 1l inoculation loop, Conclusions
incubated at 37C, was monitored. For Results show that temocillin is a good marker for OXA-48 isolates, but cant be used as a discriminatory agent, because other isolates carrying carbapenemases
the measuring 1l of the analyte was enzymes, with the exception of KPCs, may also be resistant. To achieve an exact class carbapenemase detection, a carbapenem plus inhibitors should be add
applied on a target and allowed to dry, to the assay. However, MALDI-TOF allow a more accurate identification of OXA-48-type isolates than phenotypical methods, as they are the fastest enzymes in
then it was covered by the matrix hydrolyzing temocillin. Besides, in areas where OXA-48 producers are predominant, testing temocillin alone in the MALDI-TOF assay, rather than by disk
solution (HCCA), and measures were diffusion, can be a highly sensitive marker. The method also helps to discount the presence of CPE, because of its high negative predictive value. This is the first
taken after drying. time temocillin is described by MS and proved to be a good method for detecting OXA-48-type enzymes.

41
MALDI Biotyper Poster Hall 2016 Resistance

Discriminating Carbapenemase Production in a UK Clinical Laboratory:

The Role Of The MALDI-ToF Hydrolysis Assay.
Pitty LM1, Moore LSP1,2, Rebec M1, Holmes AH2, Donaldson H1,2.
1Department of Microbiology, Imperial College Healthcare NHS Trust London (UK), 2Division of Infectious Diseases, Imperial College London (UK).

Introduction Methods Results Discussion

Carbapenemase producing Gram negative
bacteria pose a significant public health
threat, yet current methods to clinically
discriminate them from organisms with
other mechanisms of carbapenem-
resistance frequently fail to balance
acceptable expense, turn-around-time and
predictive values.
Seventy-eight Gram negative isolates derived from clinical Investigation of the variables identified a reproducible and This study has provided proof of concept
isolates from patients attending primary, secondary and tertiary optimally discriminable spectra for carbapenem/hydrolysate that discrimination of carbapenemase-
care across West London were prepared as Macfarland 7 peaks was best obtained with: production from other mechanisms of
innocula from overnight cultures. Sixty one of these isolates (i) an ertapenem concentration of 1mg/ml carbapenem-resistance can be achieved in
where referred to the Antimicrobial Resistance and Healthcare (ii) incubated for 4 hours the clinical setting with MALDI-ToF MS.
Associated Infections Reference Unit (Public Health England, (iii) in a buffer of ammonium citrate
Colindale) to confirm the mechanism of carbapenem resistance. (iv)a matrix diluent of 0.5% trifluoracetic acid
Isolate Types / Strain /
Carbapenemase Enzyme Number of Isolates As was found in a recent study3 it was
determined that ammonium citrate buffer
Carbapenem Resistant Isolates 61 Isolate Types / Strain / Ertapenem Hydrolysis Assay (No of
Carbapenem Sensitive Isolates 17 Carbapenemase Enzyme Number of Isolates positives detecteddefined as >0.4 logRQ)

was determined as the most appropriate

Carbapenemase Producing Isolates 37 MIC Break Point (mg/l) Carbapenemase Producing Isolates 37 Run 1 Run 2
Lactose Fermenting Isolates Ertapenem Imipenem Meropenem
K. pneumonia 14

Recent evidence suggests that Gram buffer for use in this assay as the 20mM
K. pneumonia 14 >1.0
bla-NDM 5 (1 reference strain) bla-NDM 5 (1reference strain) 5 4
bla-KPC 4 (1 reference strain)

negative organism carbapenemase- bla-OXA-48

bla-VIM
3 (1 reference strain)
2 (2 reference strains)
bla-KPC (1 reference strain)
bla-OXA-48 (1 reference strain)
4 (1 reference strain)
3 (1 reference strain)
3
0
3
0 When applied to TRIS-HCl buffer provided poor peak
production can be demonstrated through E. coli
bla-OXA-48
7
4
>1.0
bla-VIM 2 (2 reference strain) 2 0
all 78 isolates, resolution. Interestingly, Hrabk et al2
detection of carbapenem molecules and determined TRIS HCl to be the optimum
bla-NDM 2
E. coli 7

this method was

bla-IMP 1 (1 reference strain)
bla-KPC 0 bla-OXA-48 4 0 0

their hydrolysates using matrix-assisted K. oxytoca bla-VIM

Klebsiella sp.bla-NDM
1
1
>1.0
>1.0
bla-NDM 2 2 1
able to provide buffer for their study detecting meropenem
laser desorption/ ionisation time-of-flight Non-Lactose Fermenting Isolates
P. aeruginosa bla-VIM 8 (1 reference strain) >8.0 >8.0
bla-IMP
bla-KPC
1 (1 reference strain)
0
1
0
1
0 results to hydrolysis, whilst Kempf et al4 selected
mass spectrometry (MALDI-ToF),1,2 A. baumanii
bla-OXA 51
4
2
>8.0 >8.0
K. oxytoca bla-VIM 1 1 1
delineate 0.45% NaCl for detection of imipenem. This
potentially providing a low cost, rapid suggests that the selection of optimum
bla-OXA 58 1 Klebsiella sp.bla-NDM 1 0 0
bla-NDM 1
P. aeruginosa bla-VIM 8 (1 reference strain) 1 4 carbapenemase-
method. buffer type may be specific to the type of
A. indicus bla-OXA 58 1 >8.0 >8.0

producing from
P. mirabilis bla-NDM 1 >8.0 >8.0 A. baumanii 4

carbapenem from which hydrolysis is

Non-Carbapenemase Producing Isolates 24
bla-OXA 51 2 1 0

clinical isolates
K. pneumonia 11 >1.0
E. cloacae 5 >1.0 bla-OXA 58 1 0 0
P. aeruginosa 4 >8.0 >8.0
bla-NDM 1 0 0
in 5 hour testing being detected.
This study analyses how these described
E. coli 1 >1.0
Klebsiella sp 1 >1.0 A. indicus bla-OXA 58 1 0 0

research methods translate into a large UK

S. marcescens
Enterobacter sp
1
1
>1.0
>1.0
P. mirabilis bla-NDM 1 0 0
time for batched
Non-Carbapenemase Producing Isolates 24

clinical laboratory, validated against An ertapenem hydrolysis assay was performed using a MALDI- K. pneumonia 11 0 0 samples. A positive cut-off value for logRQ was set
clinical isolates. It is hoped that ToF mass spectrometer (Bruker Daltronics ). Four variables E. cloacae 5 0 0
at 0.4 for this evaluation, but almost all
P. aeruginosa 4 0 0

Implementation of this methodology will were investigated to optimise methods for translation to a E. coli 1 0 0 non-carbapenemase producing isolates
significantly improve antimicrobial clinical laboratory including: Klebsiella sp 1 0 0
produced logRQ values below 0.25. If
S. marcescens 1 0 0

stewardship and in turn assist in the (i) concentration of ertapenem (1 - 3mg/ml), Enterobacter sp 1 0 0 implemented into a routine microbiology
reduction in the transmission of Multi-Drug (ii) incubation time (3 - 4 hours) Carbapenem Sensitive Isolates 17 0 0
laboratory the positive cut-off value could
Total 16 14

Resistant Organisms (MDROs) causing (iii) buffer (ammonium citrate vs TRIS-HCl) potentially be lowered, resulting in
Healthcare Associated Infections (HCAIs). (iv) -cyano-4-hydroxy-cinnamic acid matrix diluent increased sensitivity without reducing
(0.5 2.5% trifluoroacetic acid). specificity.
Figure 1 Non-carbapenemase producer

Spectra peaks associated

References
1. Hrabak J et al. Detection of NDM-1, VIM-1, KPC, OXA-48,
and OXA-162 carbapenemases by matrix-assisted laser
desorption ionization-time of flight mass spectrometry. J
Clin Micro. 2012; 50(7):2441-3.
2. Sparbier K et al. Matrix-assisted laser desorption ionization-
time of flight mass spectrometry-based functional assay for
rapid detection of resistance against -lactam antibiotics. J
Clin Micro. 2012;50(3):927-37.
3. Papagiannitsis C et al. MALDI-TOF MS meropenem
hydrolysis assay with NH4HCO3, a reliable tool for the direct
detection of carbapenemase activity. J Clin Micro. 2015;
pii=JCM.03094-14.
4. Kempf M et al. Rapid detection of carbapenem resistance in
Acinetobacter baumannii using matrix-assisted laser
desorption ionization-time of flight mass spectrometry. Plos
ONE. 2012;7(2):e31676
5. Tijet N et al. Evaluation of the Carba NP test for rapid
detection of carbapenemase-producing Enterobacteriaceae
and Pseudomonas aeruginosa. Antimicrob Agents
Chemother. 2013;57(9): 4578-80.
with intact ertapenem and
Image 2 Conclusions
the ionic salts were: 475Da,
498Da, 542Da and 520Da This low cost method can detect
(Figure 1). The hydrolysed Figure 2 Carbapenemase producer
carbapenemase enzymes at a sensitivity
and decarboylated and specificity comparable to other
ertapenem spectra peaks methods currently available such as the
CarbaNP test5; more cheaply than multi-
were 450Da, 472Da and
538Da (Figure 2). plex PCR platforms; and with a faster turn-
Analysis of repeat runs (quadruplet) enable logQR values for around-time than the 24 hours required for
Mass spectra derived after clinical isolate incubation with the relative peak intensity to be plotted. Using a threshold value of the Modified Hodge Test.
ertapenem were analysed with Flexanalysis 3.3 to identify intact 0.4 obtained optimal discrimination of carbapenemase
vs hydrolysed ertapenem peaks, and relative peak intensity was producing organisms from non-carbapenemase producing However the absolute sensitivity refutes
compared using MBT STAR-BL software (Bruker DaltronicsImage ). organisms (Figure 2 demonstrates) yet still the sensitivity of this its utility as a stand-alone test for
assay was only 51%, with a specificity of 100%. Sub-group
LogQR values for relative peak intensity were calculated production of carbapenemase-production
1 by: analysis excluding OXA-type isolates improved sensitivity to in its current form; further optimisation of
logRQ = log(sum[hydrolyzed peak intensities])
(sum[non-hydrolyzed peak intensities]) 69% whilst maintaining specificity at 100%. this assay is warranted.

Author contact: lee.pitty@imperial.nhs.uk. Acknowledgements: NIHR Health Protection Research Unit in Healthcare Associated Infections and Antimicrobial Resistance at Imperial College London, and NIHR Imperial Biomedical Research Centre.

42
MALDI Biotyper Poster Hall 2016 Resistance

Rapid Detection of Tobramycin resistant Gram-negative Bacteria by MALDI-TOF MS

M. Rammler1, C. Lange1, M. Kostrzewa1, K. Sparbier1
D-1144 1 Bruker Daltonik GmbH, Fahrenheitstrae 4, 28359 Bremen, Germany
ICAAC 2015 katrin.sparbier@bruker.com

Introduction 2,5 Figure 4: Plotting of Results

BHI K.pneumoniae, 2 h
2
relative growth
The constantly growing number of resistant values derived from Optimization of the incubation time revealed
1,5
bacteria is an increasing health care problem. BHI MALDI mea- individual requirements for each species. To
+Antibiotic
1 surements against
Quick and cost-efficient methods are required Cell lysis with
lysis reagent the Tobramycin con-
achieve a separation between resistant and
to facilitate rapid resistance evaluation for Cell suspension
sensitive strains K. pneumoniae had to be
Incubation at 37C 0,5
Overnight culture containing internal
BHI McF 0.5 Species dependent time standard centration after
hygienic and therapeutic measures. MALDI-TOF 0
2 4 8 16 32 64 128 256 incubation of the incubated 2 h, P. aeruginosa 3 h, and A.
MS provides quick analysis methods for respective species baumannii 3.5 h The relative growth cutoff
2
for the indicated
identification and meanwhile also for resistance P. aeruginosa, 3 h
time. Green bars
value had to be set to 0.4 (data not shown).

Relative growth
detection. Quantitative MALDI-TOF MS was 1,5
represent sensitive Titration of Tobramycin revealed a minimum
recently applied to detect resistance of 1 strains and red bars inhibitory concentration (ASTRA MIC) for each
Meropenem-resistant K. pneumoniae based on Acquisition Target represent resistant strain. A breakpoint concentration of 32 to 64
Evaluation of MS preparation 0,5
strains. Values > 0.4
bacterial growth. Here, we apply this approach profile
spectra represent growth
g/ml for K. pneumoniae, 16 to 32 g/ml for P.
for detection of Tobramycin-resistant K. 0
2 4 8 16 32 64 128 256 and < 0.4 growth aeruginosa, and 8 g/ml for A. baumannii
pneumoniae, P. aeruginosa, and A. baumannii. Figure 1: Workflow of the MALDI-TOF MS-based resistance inhibition. Red boxes resulted in a separation equivalent to the Etest
test MBT-ASTRA
2
A.baumannii, 3.5 h indicate the results between sensitive and resistant strains
Tobramycin concen-
(Fig. 4).
Methods
1,5
tration(s) resulting
A Susceptible strain
B
Resistant strain
1 in the correct Plotting the ASTRA MIC against the Etest MIC
Fresh overnight cultures of 20 K. pneumoniae, 0,5
classification of showed a direct correlation of both MIC values.
strains (ASTRA Compared to the Etest MIC the ASTRA MIC was
20 P. aeruginosa, and 20 A. baumannii strains breakpoint
0
increased due to the short incubation time of
Spectra number

Spectra number

were analyzed by E-Test and an MS-based 2 4 8 16 32 64 128 256 concentration) with

resistance test (MBT-ASTRA). MBT-ASTRA
employs quantitative MALDI-TOF MS to
compare the growth of bacteria in BHI medium
containing different concentrations
Tobramycin concentration [g/ml]
respect to the Etest. few hours (Fig. 5A). Alternatively to the time
consuming titration experiments, the relative
growth at the ASTRA breakpoint concentration
of A 1000 B 2 of the respective species could be plotted
1,8
against the Etest MIC. This resulted in a clear

Relative growth at breakpoint conc.

Tobramycin to the growth of the same strain in Figure 2: MALDI-TOF mass spectra of a susceptible (A) and a
1,6
separation of sensitive and resistant strains

ASTRA MIC [g/mL]

medium without antibiotic after a respective resistant K. pneumoniae strain (B) after 2 h incubation with 100
1,4

incubation at 37C (Fig. 1). After growing, the Tobramycin (upper panel) or without Tobramycin (lower panel),
1,2
1
which is in concordance to the Etest MIC
cells were lysed in the presence of an internal
respectively. 0,8 results (Fig. 5B).
10
0,6
standard. 1 l of the lysates is spotted on a
Conclusions
0,4
A
MALDI target and overlaid with HCCA matrix B 0,2
1
after drying. MS profile spectra are acquired on
0
0,01 0,1 1 10 100 1000 0,01 0,1 1 10 100 1000
MBT ASTRA results are available after
a microflex LT/SH benchtop mass spectrometer Etest MIC Tobramycin [mg/mL] Etest MIC Tobramycin [g/mL]
a few hours
(Bruker Daltonik GmbH)(Fig. 2). The relative Figure 5: ASTRA MIC plotted against the respective Etest MIC of each strain (A). Relative growth MBT ASTRA results are concordant to
protein amount was calculated using the values derived at the breakpoint concentration of the MBT ASTRA plotted against the Tobramycin Etest results
internal standard (Fig. 3A). The ratio of the Etest MIC values (B). K.pneumoniae; red dots, P. aeruginosa; green dots, A. baumannii; blue
Species specific assay conditions
dots. The horizontal dotted red, green, and blue lines represent the MBT ASTRA breakpoint
protein content for the BHI plus Tobramycin Figure 3: Evaluation of MBT-ASTRA spectra. Area under the (antibiotic concentration, incubation
concentration of the respective species. The horizontal orange line indicates the threshold of the
setup and for the BHI only setup was curve (AUC) of four different strains each without and with RG of the MBT-ASTRA. The vertical red line indicates the threshold of the MIC for K.pneumoniae, time) have to be optimized and
calculated (relative growth, RG, Fig. 3B). Tobramycin (A). Normalization of growth of antibiotic setups to the vertical green line for P. aeruginosa, and vertical blue line for A. baumannii. applied
the growth in BHI resulted in the Relative Growth(B).

MALDI BiotyperTM
For research use only. Not for use in diagnostic procedures.

43
MALDI Biotyper Poster Hall 2016 Resistance

Rapid Detection of OXA-48 expressing

Enterobacteriaceae by MALDI TOF-MS
ASM 2015 Poster 555

K. Sparbier, M. Peer, M. Schneider, Methods

C. Lange, M. Kostrzewa 0.4 as positive, and between 0.2 and
Bruker Daltonik GmbH, Bremen, DE 20 genetically confirmed OXA-48 0.4 as intermediate. A IMI 30 min B ETP - 2 h
positive K.pneumoniae strains, 72 Classical MIC determination (Etest) was
1,4 1,4

negative K.pneumoniae isolates, and performed applying EUCAST guidelines

1,2 1,2

respective control strains were incubated 1,0 1,0

Introduction with 0.25 mg/ml IMI for 30 min or with
for evaluation. 0,8 0,8

Norm LogRQ

Norm LogRQ
0.1 mg/mL Ertapenem (ETP) for 2 h at 0,6 0,6

The increasing number of carbapenem 37C in incubation buffer. Stability of Results 0,4 0,4

resistant Gram-negative bacteria is a dissolved IMI without bacteria was 0,2 0,2

challenging health care problem. Well- analyzed by LC-ESI-MS(n). After co- LC-ESI-MS(n) analysis revealed an 0,0 0,0

directed handling of this challenge incubation of antibiotics and bacteria, acceptable in-use stability of dissolved -0,2 -0,2

requires reliable, rapid, and cost efficient
assays for detection of carbapenem
resistance. The detection of OXA-48
positive strains using routine assays
such as Etest or disc diffusion often fails.
MALDI-TOF MS is an established method
in microbiological laboratories.
Recently, this technique has also been
employed for detection of
carbapenemase activity in
Enterobacteriaceae. Here, we
demonstrate a reliable, rapid, and cost
efficient MALDI-TOF MS-based approach
for the detection of OXA-48 expressing
Enterobacteriaceae isolates employing
Imipenem (IMI) as reporter substance.
cell-free supernatants were spotted onto IMI without bacteria. Within 24 h, only -0,4
0,01 0,1 1 10 100
-0,4
0,01 0,1 1 10 100
a MALDI target. Dried spots were 1.3 % of the IMI dissolved in incubation MIC [g/ml IMI]
MIC [g/ml IMI]
overlaid with matrix containing an buffer was hydrolyzed (Fig. 2).
internal standard. Spectra were acquired The MALDI analysis using IMI as Fig. 3: Comparison of the MALDI-TOF MS derived results
on a microflex LT/SH mass spectrometer benchmark antibiotic correctly detected normalized values of the OXA-48 strains (normalized LogRQ values) and the Etest derived MIC
(Bruker Daltonik). Fully-automated data all OXA-48 strains after 30 min revealed no difference in the hydrolytic values for IMI (A) and ETP (B). OXA-48 strains are
marked red and susceptible strains are marked green.
processing based on an intensity ratio incubation. After normalization towards capacity between those strains correctly Above dashed red line: positive; below green dashed
calculation of internal standard and non- a known negative and a respective detected and those strains which had line: negative.
hydrolyzed IMI or of hydrolyzed and positive control (kpc positive strain), the been misclassified by Etest (Fig. 3A).
non-hydrolyzed ETP was performed normalized values range between 0.43 Using ETP as benchmark antibiotic
using a dedicated software tool and and 1.29. All susceptible strains were required a prolonged incubation time. Conclusions
included normalization to respective correctly classified with normalized Nevertheless, 4 of the OXA-48 strains
negative and positive control strains (= values below 0.2. Sensitivity and were tested negative and additional
NormLogRQ value). Strains with a value specificity were 100% each. In contrast, three strains revealed only an MALDI-TOF MS and
below 0.2 were considered as negative, Etest (IMI) detected 6/20 OXA-48 intermediate hydrolysis of ETP even after
Imipenem provide a
above 0.4 as positive, and between 0.2 and isolates as positive. Comparison of the 2 h. The corresponding Etest with ETP
detected all OXA-48 strains. reliable approach for the
Table 1: Summary of the numbers of isolates correctly detected as resistant and detection of OXA-48 strains
susceptible using IMI as reporter substance, respectively. Total number of resistant and
susceptible strains and specificity and sensitivity of the respecive approach 1,20E+08
IMI_hydro IMI Accelerated procedure
MIC STAR-BL MIC STAR-BL
Gene
IMI IMI (0.5h) ETP ETP (2h)
1,00E+08
(30 min incubation time;
OXA-48 correctly
20 9 20 20 16
8,00E+07
60 min overall time) com-

Area [AU]
found
Susceptible correctly
6,00E+07
pared to the established
72 71 72 72 72 4,00E+07 standard procedures
found
Total resistant 20 10 20 20 16 2,00E+07

Total susceptible 72 82 72 72 76 0,00E+00

Cost-efficient in contrast
0 1 4 6 8 24 to other fast approaches
False negative 0 10 0 0 4 Time [h]
like PCR
False positive 0 1 0 0 0
Specificity 100 0.99 1.00 100 1.00 Fig. 2: Stability of IMI dissolved in buffer for 24 h at 4C.
Fig. 1: Workflow for the analysis of carbapenem resistance with the MBT Only a slight increase of the hydrolysis products (red
STAR-BL system (Bruker Daltonik GmbH). Sensitivity 100 0.50 1.00 100 0.80 bars) is detected. MALDI-Biotyper
For research use only not for use in diagnostic procedures

44
MALDI Biotyper Poster Hall 2016 Resistance

Bruker Daltonics

Optimization of the MBT-ASTRA

Katrin Sparbier, Beatrix Wegemann, Christoph Lange, Markus Kostrzewa
Bruker Daltonik GmbH, Germany
Objective: Methods: For comparison, Etest analysis was Best results were found at an antibiotic
performed in parallel applying EUCAST concentration of 8 g/ml MEM using a cut off
Fast and reliable methods for the detection of 38 K. pneumoniae isolates (10 KPC, 10
guideline for evaluation. of 0.5 for the relative growth value. Under
bacterial resistance are mandatory for a well- NDM, 10 Oxa-48, and 8 susceptible) were
these conditions two susceptible strains were
directed handling of resistant bacteria. incubated at McF 0.5 in BHI and BHI with
Results: found in the intermediate range, only.
MALDI-TOF MS is an established method in decreasing meropenem (MEM)
microbiological laboratories providing quick concentrations (256 g/ml to 0.25 g/ml) for Applying different antibiotic concentrations
1000 A
species identification. Recent developments 1h and for 2h at 37C. Subsequently, cells and different incubation times require an

ASTRA Breakpoint [g/ml]

100
provided methods for MALDI-TOF MS based were washed and lysed with formic acid and adaptation of the cutoff values for the
resistance detection, e.g. of -lactam acetonitrile supplemented with an internal decision susceptible or resistant. One hour 10

resistance or more general resistance standard. Lysates were spotted onto a incubation and application of 4 g/ml to 16
1
detection using heavy amino acids. Very MALDI target and dried. Spots were overlaid g/ml MEM lead to two to four miss-classified
recently, we published a quantitative MALDI- with HCCA matrix. MALDI-TOF MS profile strains and two strains showing an 0,1
0,01 0,1 1 10 100
TOF MS approach facilitating the rapid spectra were acquired on a microflex LT/SH insufficient growth preventing any evaluation. MIC [g/ml]
detection of meropenem resistant K. mass spectrometer (Bruker Daltonik) in the Depending on the MEM concentration and 1000 B
pneumoniae. The principle of this assay is mass range from 2 to 20 kDa. Spectra were the applied cut off value, either some of the

ASTRA Breakpoint [g/ml]

based on the comparison of protein profile normalized to the standard and the AUC was resistant strains were classified negative or 100

spectra derived either from cells after in calculated for each setup. The ratio of the some of the susceptible strains were
10
incubation with BHI medium or from the AUC for the BHI plus meropenem to the AUC classified as positive strains, respectively.
same cells after incubation in BHI with of the BHI only setup was calculated (relative Increasing the incubation time up to 2 h one 1

antibiotics. Here, we demonstrate the effects growth). Different relative growth cut offs to six misclassifications were observed.
of different assay parameters on the were tested to detect resistant strains (low Table 1: Outcome for the different strains at 4, 8, and 16 g/ml 0,1
0,01 0,1 1 10 100
MEM after 1 and 2 hours incubation (green: correctclassification,
outcome. growth corresponds to a low relative growth red: mis-classification, yellow: invalid due to poor growth of the
MIC [g/ml]

value). Experiments were repeated once and control) Fig. 3: ASTRA breakpoint concentration versus MIC at a
the median was considered for evaluation. Cut off 0.5 4 g/ml 8 g/ml 16 g/ml cut off threshold of 0.5 after 1(A) or 2 (B) hours (Green:
Resistant 10 10 10 susceptible, red: KPC, blue: NDM, and purple: OXA-48)

KPC
Antibiotic
Suceptible 0 0 0 Dashed lines indicate breakpoint concentrations
Invalid 0 0 0 between susceptible and resistant.
BHI
Susceptible strain Resistant strain Resistant 10 8 8

NDM
1 h incubation
Incubation 37C
McF 0.5 x104 x104
Suceptible 0 2 2
Conclusion
1068PEG_CTX 0:D10 MS, BaselineSubtracted
Intens. [a.u.]

20028S_BHI 0:A8 MS, BaselineSubtracted

Intens. [a.u.]

Species dependent time

1.2

BHI only
2.0

1.0 Invalid 0 0 0
1.5
Resistant 9 8 7

Oxa-48
0.8

Suceptible 0 1 2
Lysis reagent
An incubation time of 2 h, a meropenem
1.0 0.6

with Cell lysis invalid 1 1 1

0.4

internal
Resistant 1 0 0
concentration of 8 g/ml, and a relative

ceptible
0.5

standard 0.2

Sus-
Target preparation Suceptible 7 8 8
growth cut off of 0.5 led to the most reliable
0.0 0.0
x104 x104 1068PEG_CTX 0:D10 MS, BaselineSubtracted

2 2 2
20028S_CTX 0:A10 MS, BaselineSubtracted

Invalid
Intens. [a.u.]

Intens. [a.u.]

BHI MEM
1.5

Resistant 10 10 10
detection of meropenem resistant K.
1.5

KPC
1.0
Suceptible 0 0 0
pneumoniae strains. Further validation with
1.0

Invalid 0 0 0
Resistant 10 10 9
additional strains will be necessary.
0.5
NDM
2 h incubation

0.5

Suceptible 0 0 1
Invalid 0 0 0
Optimization of the protocol for other species
0.0
Acquisition of 4000 5000 6000 7000 8000 9000 10000 11000 12000 13000 14000
0.0
4000 5000 6000 7000 8000 9000 10000 11000 12000 13000 14000

MS profile spectra
m/z m/z

Resistant 10 10 10
Oxa-48

Suceptible 0 0 0 according to incubation time, antibiotic

invalid 0 0 0
Fig. 2: MALDI-TOF spectra of a susceptible and a Resistant 6 2 0 concentration, and relative growth cut off will
ceptible

Fig. 1: Preparation scheme for the analysis of antibiotic resistant strain after incubation in BHI medium and BHI
Sus-

resistance by quantitative MALDI-TOF MS. with 8g/ml MEM for 1 h, respectively

Suceptible 4 8 10
be required.
Invalid 0 0 0

Lange C, Schubert S, Jung J, Kostrzewa M, Sparbier K. ;Quantitative matrix-assisted laser desorption ionization-time Copyright 2015 Katrin Sparbier, katrin.sparbier@bruker.com
of flight mass spectrometry for rapid resistance detection. J Clin Microbiol. 2014 Dec;52(12):4155-62.

45
MALDI Biotyper Poster Hall 2016 Resistance

Rapid antibiotic susceptibility detection using Mass Spectrometric-Antibiotic Susceptibility Rapid

Assay (MS-ASTRA) in Klebsiella spp.
Lawrence Tse1, Chistopher Coon2, Katherine Riebe2, Nathan A. Ledeboer2,3
1Medical College of Wisconsin, Milwaukee, WI USA; 2Dynacare Laboratories, Milwaukee, WI USA; 3Department of Pathology, Medical College of Wisconsin, USA

Background Results
In 2013, the CDC classified the growing CRE (carbapenem-resistant enterobacteriaceae) threat as Meropenem: 8g/ml Ceftriaxone: 64g/ml Ceftriaxone: 128g/ml Cefepime: 64g/ml Cefepime: 128g/ml
urgent, the highest threat level1 1.6 1.6 1.6 1.6 1.6
- considered an immediate public health threat that needs urgent and progressive action
- estimated that Klebsiella spp. made up 11% of CRE healthcare-associated infections1 1.4 1.4 1.4 1.4 1.4
- approximately 7900 cases in the US leading to 520 attributable deaths annually1
Almost half of all patients with carbapenem-resistant enterobacteriaceae bacteremia will die1 1.2 1.2 1.2 1.2 1.2
- up to 40% of bacteremia pt receive inadequate initial antibiotic treatment2
- mortality increases by 7.6% for each hour that appropriate treatment is delayed3

Relative Growth
Relative Growth

Relative Growth

Relative Growth

Relative Growth
1 1 1 1 1

0.8 0.8 0.8 0.8 0.8

Current susceptibility testing times:
- conventional methods such as E-tests require 18-24 hour incubations
0.6 0.6 0.6 0.6 0.6
- automated systems such as BD Phoenix and bioMerieux Vitek 2 require 8-18 hours
and 4-18 hours, respectively
0.4 0.4 0.4 0.4 0.4

Thus there is an ever growing need to determine antibiotic susceptibility to improve patient care and for 0.2 0.2 0.2 0.2 0.2
infection control to limit its spread.
0 0 0 0 0
0.1 1 10 0.1 1 10 100 0.1 1 10 100 0.1 1 10 100 0.1 1 10 100

Introduction MIC [g/ml] MIC [g/ml] MIC [g/ml] MIC [g/ml] MIC [g/ml]

Relative Growth vs. MIC (as determined by BD Phoenix). Vertical light green lines denote the MIC based definition of resistance according to CLSI. Horizontal dark green lines
While current methods commonly require overnight incubations, the recently developed Mass denote the Relative Growth cutoffs used.
Spectrometric-Antibiotic Susceptibility Test Rapid Assay (MS-ASTRA) can determine an organisms
susceptibility profile in less than 2 hours depending on the organisms doubling time. In this study, we
have adapted the MS-ASTRA technique to determine meropenem, ceftriaxone, and cefepime resistance Meropenem Ceftriaxone Cefepime
in Klebsiella spp. clinical isolates. Concentration (ug/ml) 8 64 64 128 64 64 128
RG cutoff 0.4 0.4 0.5 0.4 0.4 0.5 0.4
Methods Category Agreement
matching results
98.3% 89.8% 93.2% 98.2% 91.5% 94.9% 94.2%

- 59 Klebsiella spp. clinical isolates

Very Major Error 0.0% 1.7% 1.7% 0.0% 0.0% 0.0% 0.0%
false susceptible by MS-ASTRA
- 18 are resistant to meropenem, ceftriaxone, and cefepime: KPC (klebsiella producing carbapenamase) or OXA (oxacillinase)
- 41 are sensitive Major Error 1.7% 8.5% 5.1% 1.8% 8.5% 5.1% 5.8%
false resistant by MS-ASTRA

Table 1: Category agreement of MS-ASTRA with BD Phoenix and E-test

Meropenem Ceftriaxone Cefepime

+ internal standard Concentration (ug/ml) 8 64 64 128 64 64 128
Incubation Protein
(allows for RG cutoff 0.4 0.4 0.5 0.4 0.4 0.5 0.4
1 hour at 35C Extraction
+ Antibiotic spectral comparison)
2 Tail McNemar Test: p-value 1.00 0.22 0.63 1.00 0.06 0.25 0.25
BHI inoculation Table 2: Test of significance. Since all values are greater than 0.05, there is no statistically significant difference between the results
Fresh overnight Acquire spectra via from MS-ATRA with BD Phoenix and E-test.
MALDI-TOF
Mass Spectrometry
Susceptible Resistant Conclusions Acknowledgements
BHI
standard standard We have adapted the MS-ASTRA protocol for use with Klebsiella spp., resulting in a Department of Pathology
significant decrease in time needed to determine antibiotic susceptibility to under 2 hours Medical College of Wisconsin
standard standard
Milwaukee, WI, USA
Current limitations
-not yet practical in the clinical setting until can implement further automation
-current data set only has data regarding Gram negative organisms
Meropenem 8ug/ml -concentrations used are considerably larger than current CLSI antibiotic
susceptibility breakpoints References
-not yet able to determine MIC 1AntibioticResistance Threats in the United States, 2013 (CDC)
Future directions 2Sogaard M, Norgaard M, Schonheyder HC., First notification of positive

-reproducibility studies to confirm precision blood cultures and the high accuracy of the gram stain report. J. Clin.
Microbiol. 45 (2007)
-increase sample set to include more Klebsiella spp. isolates 3Kumar A, et al., Duration of hypotension before initiation of effective

-will need to increase study to other organisms and antibiotics antimicrobial therapy is the critical determinant of survival in human
septic shock. Crit Care Med. 34 (2006)

46
MALDI Biotyper Poster Hall 2016 Resistance

Detection of extended spectrum beta-lactamase among Enterobacteriaceae using MALDI-TOF

D-1145, ICAAC 2015

M. Ziegler1, M. Peer2, K. Sparbier2, S. Wirth1, H. Seifert3, H. Wisplinghoff1,3

ABSTRACT RESULTS MATERIALS AND METHODS

Objectives: MALDI-TOF MS, a common tool in routine laboratories, was Data acquisition and processing. Fully automated data acquisition and
1.0
evaluated for ESBL detection and characterization in Enterobacteriaceae Hydrolyzation Inhibition data pre-processing was performed using a benchtop MALDI-TOF MS and
utilizing a research approach. 0.8 the MBT STAR-BL Module (both Bruker Daltonik GmbH, Bremen,
0.6 CTX > 0.3 > 0.1 Germany) by calculating the ratio of hydrolyzed and intact antibiotic peak
Results intensities (normalized to controls).

logRQ
0.4 CPO > 0.3 > 0.1
MALDI-TOF MS sensitivity and specificity of individual antibiotics was The obtained hydrolysis results were subsequently processed and
comparable to the combined Disc Diffusion (DD) test. 0.2 evaluated manually according to the CA inhibition to detect an ESBL
Results varied depending on bacterial species and antibiotic/inhibitor CPM > 0.3 > 0.14 production. Cut-off values for inhibition results were estimated on an
0.0
used in the research assay setup empirical basis.
Negative Positive CAZ > 0.04 > 0.05
The simple and easy to use (semi-) automated workflow allows robust + CA - CA + CA - CA + CA - CA Results were directly compared to the disk diffusion test using the same
Control Control
and very rapid ESBL determinations. Isolate No. 1 Isolate No. 2 Process Control antimicrobial agents, except CPO. In order to determine the accuracy of
ESBL detection, results were also compared to a variety of phenotypical
Figure 1 Example of MBT-STAR BL-Assay results for CTX as presented in Table 1 Estimated normalized logRQ cut-off values
Conclusion used for subsequent manual data analysis and molecular methods that had been used in a previous study [1] including
the software after automated data acquisition and processing
MALDI-TOF MS is a promising and fast tool for ESBL detection isoelectric focusing, targeted PCR and sequencing.
In future, optimized assay conditions, fully automated and dedicated data
Bacterial strains. A total of 146 previously characterized
acquisition and data processing workflows will aid the fast, lean and MALDI-TOF MS DD test MALDI-TOF MS DD test MALDI-TOF MS DD test
CPM CPM CTX CTX CAZ CAZ Enterobacteriaceae isolates - including 96 ESBL producers and 50 non-
accurate detection of ESBLs in routine laboratories
pos neg pos neg pos neg pos neg pos neg pos neg
ESBL produces such as hyper-producers of chromosomal AmpC, Koxy, or
SHV enzymes, and wild-type strains (Escherichia coli (n=61), Klebsiella
agreement agreement agreement agreement agreement agreement
INTRODUCTION MALDI- MALDI- MALDI-
pneumoniae (29), K. oxytoca (16), Enterobacter cloacae (16), E. aerogenes
TOF MS 100% 100% 55.9% 86.4% TOF MS 100% 100% 83.9% 75.5% TOF MS 100% 100% 64.2% 78.5% (5), Citrobacter freundii (5), C. farmeri (1), Morganella morganii (5), Serratia
In recent years, multidrug resistance has emerged among the Gram- CPM CTX CAZ
marcescens (1), Proteus mirabilis (6) and P. vulgaris (1)) - were included in
negative bacteria. Rapid and accurate detection of prevalent resistance DD test
90.5% 45.8% 100% 100%
DD test
85.7% 72.7% 100% 100%
DD test
78.8% 63.8% 100% 100%
CPM CTX CAZ the study.
mechanisms, such as extended-spectrum beta-lactamase (ESBL) activity in
E. coli ATCC 25922, K. pneumoniae ATCC 700603, and Acinetobacter
Enterobacteriaceae, is crucial for timely guidance and application of
Tables 2 4 Correlations between MALDI-TOF and DD test for all bacterial strains (n=146). CPO was not available as DD test baumanii (laboratory strain) were used as controls.
appropriate antimicrobial therapies.
Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass Disk Diffusion Test. The disk diffusion test was conducted according to
Sensitivity Specificity
spectrometry (MS), so far primarily used for species identification in routine 100% CLSI guidelines using disks with 30g of CTX, CPM or CAZ with and
laboratories, has been proposed for the detection of resistance without 10g of clavulanic acid. CPO was not available as DD test.
80%
mechanisms. Only recently, it has been commercialized for fully-automated
SUMMARY & CONCLUSION
detection of a present -lactamase activity. This study was conducted to 60%
evaluate the potential of MALDI-TOF MS to detect and characterize ESBL MALDI-TOF MS results of this research study were in good agreement
in Enterobacteriaceae. 40% with results obtained with the Disc Diffusion (DD) test. Accuracy was also
in good correlation with other methods applied in routine (data not
20%
shown).
MATERIALS AND METHODS
0% A high correlation of results was found for individual bacterial strains,
Cefepime Cefpodoxime Ceftazidime Cefotaxime Disc Diffusion Test
Study design. A modified MBT STAR-BL assay, a dedicated workflow to dependent on individual antibiotics used.
detect a present -lactamase activity in Enterobacteriaceae by analyzing Figure 2 Specificity and Sensitivity of ESBL detection for all bacterial strains (n=146) based on cut off values from Table 1 Appropriate benchmark antibiotics (e.g. a combination CPO and CTX) in
the enzymatic hydrolyzation of the antibiotics -lactam ring, was developed combination with optimized assay conditions implementing fully
and evaluated for ESBL detection [2]. Briefly, bacterial strains were automated MALDI-TOF MS data acquisition and data processing may
CPM CTX CAZ CPM CTX CAZ CPM CTX CAZ CPM CTX CAZ CPM CTX CAZ
incubated 1 4 h with different cephalosporins (cefotaxime (CTX), finally lead to rapid and enhanced detection of ESBL production in
ceftazidime (CAZ), cefepime (CPM), or cefpodoxime (CPO)) with and Escherichia coli Klebsiella spp. Enterobacter spp. Proteus spp. (n=12) Citrobacter sp. (n=6) Enterobacteriaceae superior to currently applied conventional methods
(n=61) (n=45) (n=21) M. morganii (n=5) S.marescens (n=1)
without the presence of the -lactamase inhibitor clavulanic acid (CA), like DD or molecular methods.
respectively. Suitable incubation times and CA concentrations were MALDI / DD pos. agreement 85.4 88.6 74.4 48.5 76.5 75 60 100 36.4 37.5 66.7 25 0 100 0
evaluated in preliminary studies on an individual basis. REFERENCES
MALDI / DD neg. agreement 69.2 82.4 77.3 83.3 54.5 66.7 100 84.6 90 100 100 85.7 100 50 100
1: Wiegand, I., H. K. Geiss, D. Mack, E. Strenburg, H. Seifert. 2007. Detection of
DD / MALDI pos. agreement 88.6 92.9 85.3 89.5 83.9 72 100 80 80 100 100 50 57.1 25 71.4 Extended-Spectrum Beta-Lactamases among Enterobacteriaceae by Use of
Semiautomated Microbiology Systems and Manual Detection Procedures. J Clin Microbiol.
ACKNOWLEDGEMENTS 45(4): 1167-1174.
DD / MALDI neg. agreement 34.6 73.7 55.6 38.5 57.1 70 73.3 100 56.3 44.4 75 66.7 nd 100 nd
2: Bruker Corporation. March 2015. Standard operational procedure for MBT STAR-BL
This study was supported in part by Bruker Daltonics.
Table 5 Correlations between MALDI-TOF and DD test for selected Enterobacteriaceae (n=146). CPO was not available as DD test. Assay for MBT Compass (RUO).

Wisplinghoff laboratories, Cologne1, Bruker Daltonics, Bremen 2 , Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Cologne3, Germany

47
MALDI Biotyper Poster Hall 2016 Resistance and Subtyping

MALDI-TOF mass spectroscopy and blakpc gene phylogenetic analysis: an outbreak of multidrug and carbapenem
resistant K. pneumoniae isolates ?
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To% conrm% these% data,% the% gene;c% distance% was%
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to% the% cluster% II% of% the% dendrogram% resulted%

worldwide% clone,% as% reported% in% two% recent% studies%
(Deleo%et%al.,%2014;%Chen%et%al.%2014a)%is%the%ST258%strain%
and% it% can% be% divided% in% two% groups,% with% dierent% Figure%1%
capsule%polysaccharide%gene%(cps)%regions.%
The%aim%of%the%present%study%was%to%evaluate%a%rapid%and%
Results)and)Discussion)
inexpensive% method,% based% on% MALDI=TOF% technology,%
By% MALDI=TOF% analysis,% the% 25% KPCs% isolated% were%
able% to% characterize% KPC% isolates% according% to% their%
divided%in%two%clusters%(Cluster%I%and%Cluster%II).%More%
dierent%pa[ern%of%TOF%peaks.%%
than%90%%of%both%the%cross=valida;on%and%recogni;on%
Samples,)materials)and)methods)
capability% was% found% in% the% models% used% for% the%
Between% January% 2012% and% February% 2013% a% total% of% 25%
discrimina;on%of%the%Clusters.%The%MALDI%TOF%analysis%
consecu;ve% non=replicate% clinical% KPCs% were% isolated%
were% performed% using% PCA% sta;s;cal% analysis% (that%
from% inpa;ents% at% the% University% Hospital% Campus% Bio=
provides%informa;on%on%the%hetero/homogeneity%of%a%
Medico% in% Rome.% All% isolates% were% characterized% with%
data%set)%and%the%MPS%debdrogram%crea;on.%%
phenotyping% method% (An;microbial% suscep;bility% test:%
In% the% PCA% analysis,% the% rst% three% principle%
AST);% genotyping% method% (MLST,% Real=;me% PCR% cps%
components% (PC1,% PC2,% PC3)% dieren;ated% the%
gene,% PCR% and% sequencing% of% blakpc% resistance% gene);%
samples% remarkably.% With% the% default% seAngs% in%
phylogen;c% method% (CLUSTAL% X% sodware,% Bioedit%
ClinproTools,% two% clusters% have% been% dened% (Figure%
sodware% version% 7.0.9% and% MEGA6% sodware)% and%
1).))
proteomic%method%(class%dendrogram%construc;on%using%
the% ClinproTool% sodware,% Bruker).% Results% from% MALDI= Than% we% obtained% an% MSP% dendrogramm% and%
TOF% and% genotypic% analysis% were% compared% to% the% clustering% that% help% us% to% understand% the% rela;onship%
epidemiological%data.% between% our% 25% KPCs.% We% have% pu[ed% an% arbitrary%
% distance%level%cut=o%of%10%(logarithmic%transforma;on%
% of%the%value%500)%(Figure%2).%In%the%Cluster%I%the%isolates%
were%from%clinical%specimens%collected%from%January%to%
December% 2012;% in% the% Cluster% II% the% KPCs% were% from%
clinical% samples% collected% from% the% end% of% the% year%
2012% (October=November)% to% February% 2013.% This%
temporal% spliAng% conrms% the% ability% of% MALDI=TOF%
clustering% to% dieren;ate% those% isolates% presumably%
not%related.%%
Figure%2%
%%
A% further% analysis% of% the% KPC% isolates,% revealed% that%
strains% grouped% by% MALDI=TOF% in% Cluster% I% were%
p h e n o t y p i c a l l y% c h a r a c t e r i z e d% b y% a% m o r e%
heterogeneous%AST%proles%than%those%in%cluster%II%(all%
fosfomicin%resistant%and%colis;n%suscep;ble).%This%may%
suggests% the% ability% of% MALDI=TOF% to% Cluster% isolates%
phenotypically%dierent.%%
The%MLST%analysis,)as%other%authors%reported%(Maiden%
et( al.,% 2013;% Snitkin% et( al.,% 2012),% was% not% able% to%
evidence% substan;al% dierence% between% the% isolates.%
In% fact,% all% isolates% resulted% ST% 512% except% two% in%
Cluster%I%(ST%650)%and%one%in%Cluster%II%(ST%37).%The%cps%
gene% resulted% type% II% in% all% isolates% screened% and% it%
seems% not% useful% to% follow% isolates% spread% in% a%
reduced% temporal% frame,% alike% MLST% it% is% based% on% a%
conserved% gene.% Conversely,% the% % blakpc% gene% located%
on%the%transposon%Tn4401,%resulted%type%3%except%in%4%
cases%(type%2,%one%in%Cluster%I%and%three%in%Cluster%II).%
Phylogene;c% analysis% was% performed% to% conrm% if%
blakpc%genotyping%should%be%really%able%to%dieren;ate%
isolates%on%the%basis%of%their%KPC%type%and%to%evaluate%
the% performance% of% MALDI=TOF% in% isolates% clustering.%
The% results% of% this% further% analysis% eec;vely%
conrmed% the% rela;onship% between% the% three% KPC=2%
isolates%belonging%to%the%Cluster%II%of%the%dendrogram%
and% the% lack% of% rela;onship% with% the% KPC=2% isolate% in%
Cluster% I;% in% fact% it% is% located% in% the% Clade% I% of% the%
phylogene;c%tree%(Figure%3).%
related.% Noteworthy,% a% very% strong% rela;onship%
between%the%two%KPC=2%isolates%of%clade%II%(Gp=2)%
was% demonstrated% by% the% absence% of% gene;c%
distance.% From% the% epidemiological% analysis%
resulted% that% the% two% KPC=2% isolates% of% clade% II%
were% from% pa;ents% that% have% shared% a% portable%
RX% instrument.% This% instrument% could% be% the%
cause%of%the%clonal%diusion%of%these%two%strongly%
related%KPC=2.%%
% conclusion,% the% data% from% this% study% have%
In%
demonstrated% that% MALDI=TOF% technique% is% a%
rapid% and% cost% saving% method% for% KPC% isolates%
iden;ca;on% and% clustering.% Its% associa;on% with%
blakpc% genotyping% could% represent% a% reliable%
method% to% iden;fy% the% possible% clonal% spread% of%
these% isolates% in% nosocomial% seAng% and%
consequently% a% method% for% preven;ng% their%
further%diusion.%
GIANI%T.,%D'ANDREA%M.M.,%PECILE%P.,%BORGIANNI%L.,%NICOLETTI%P.,%TONELLI%F.,%
BARTOLONI% A.,% ROSSOLINI% G.M.% (2009).% Emergence% in% Italy% of% Klebsiella(
pneumoniae% sequence% type% 258% producing% KPC=3% Carbapenemase.%
J.(Clin.(Microbiol.%47,%3793=3794.%
DELEO% F.R.,% CHEN% L.,% PORCELLA% S.F.,% MARTENS% C.A.,% KOBAYASHI% S.D.,%
PORTER% A.R.,% CHAVDA% K.D.,% JACOBS% M.R.,% MATHEMA% B.,% OLSEN% R.J.,%
BONOMO%R.A.,%MUSSER%J.M.,%KREISWIRTH%B.N.%(2014).%Molecular%dissec;on%
of% the% evolu;on% of% carbapenem=resistant% mul;locus% sequence% type% 258%
Klebsiella%pneumoniae.%Proc.(Natl.(Acad.(Sci.%111,%4988=4993.%%
CHEN% L.,% MATHEMA% B.,% PITOUT% J.D.,% DELEO% F.R.,% KREISWIRTH% B.N.% (2014a).%
Epidemic%Klebsiella(pneumoniae%ST258%is%a%hybrid%strain.%MBio%5,%e01355=14.%
MAIDEN%M.C.,%JANSEN%VAN%RENSBURG%M.J.,%BRAY%J.E.,%EARLE%S.G.,%FORD%S.A.,%
JOLLEY% K.A.,% MCCARTHY% N.D.% (2013).% MLST% revisited:% the% gene=by=gene%
approach%to%bacterial%genomics.%Nat.(Rev.(Microbiol.%11,728=736.%
SNITKIN% E.S.,% ZELAZNY% A.M.,% THOMAS% P.J.,% STOCK% F.% % (2012).%
NISC% Compara;ve% Sequencing% Program% Group,% Henderson% DK,% Palmore% TN,%
Segre% JA.% Tracking% a% hospital% outbreak% of% carbapenem=resistant% Klebsiella(
pneumoniae%with%whole=genome%sequencing.%Sci.(Transl.(Med.%4,%148ra116.%

%
%

48
MALDI Biotyper Poster Hall 2016 Resistance and Subtyping

Epidemiological Typing of Methicillin-Resistant Staphylococcus

aureus (MRSA): Spa-typing versus MALDI-TOF

Klaus Biehler1, Elisabeth Niederfeilner1, Uwe Frank1,2

1 Freiburg University Hospital, Institute for Environmental Health Sciences, Freiburg, Germany
2 Heidelberg University Hospital, Centre of Infectious Diseases, Heidelberg, Germany

BACKGROUND: Early detection of epidemic strains of Representative peaks of the nosocomial MRSA complex Berlin
m/z [Da] 3210 3444 4306 4815 5034 5526 6819 6863 6889 9627
methicillin-resistant Staphylococcus aureus (MRSA) is CONCLUSIONS:
essential for preventing MRSA outbreaks in the healthcare MRSA 706 + + + + + + + + + +
MALDI-TOF may be a useful tool to facilitate
setting. Spa-typing and pulsed-field gel electrophoresis
microbiological typing and can be used for efficient MRSA 454 + + 0 + + 0 0 + + +

(PFGE) are the methods of choice for identifying epidemic

and rapid identification of epidemic MRSA complexes MRSA 243 + + + + 0 + + + + +

MRSA strains.
MRSA 1156 + + + + + + 0 0 + +

OBJECTIVE: To evaluate the reliability of detecting epidemic MRSA 1169 + + + + 0 + 0 0 + +

Figure 2: MRSA strains (n=21) concordance in spa-typing and MALDI-TOF

MRSA strains by matrix-assisted laser desorption ionization- MSP peaks characteristic of the major nosocomial MRSA
MRSA score (Maldi) result spa typ result concordance
time-of-flight mass spectrometry (MALDI-TOF MS) compared strain (Maldi) (Ridom) complex Berlin
to the accredited method of spa typing 1006 2,698 South German t110 No result
4303 2,406 Barnim t032 Barnim yes
Representative peaks of the nosocomial MRSA complex Rhine Hessen
METHODS: Spa typing was performed on a total of 1,018 4312 2,762 Barnim t032 Barnim yes
m/z [Da] 3209 3444 4046 4306 4447 4591 4814 5056 5244 5525 6423 6848 6889 8090 9625
4275 2,494 Barnim t032 Barnim yes
clinical MRSA isolates. Of these, 33 representative isolates 4313 2,73 Barnim t032 Barnim yes MRSA 901 + + + + + + + + + + + + + + +

belonging to the five major hospital acquired MRSA clonal 4236 2.56 Berlin t002 South German no MRSA 902 + + + + + + + + + + + + + + +
1026 2.596 Berlin t110 No result
complexes, i.e. Barnim (5), Rhine Hessen (9), Berlin (5), 2307 2.368 Rhine Hessen t003 Rhine Hessen yes
MRSA 903 + 0 0 0 0 0 + 0 0 0 + 0 0 + +

Southern German (7), Clonal Class III (7) provided the 4242 2.474 South German t1345 No result
MRSA 904 + + + + + + + + 0 + + + + + +

reference mass spectra for the analysis by MALDI-TOF. 2382 2.38 Barnim t032 Barnim yes MRSA 907 + + 0 + + + + 0 0 + + + + + +

1004 2.675 Rhine Hessen T037 Vienna no

Extracted proteins obtained by the Bruker method were
MRSA 151 + + + + + + + + + + + + + + +
4297 2.721 Berlin T032 Barnim no
MRSA 153 + + 0 + + 0 + 0 0 + + 0 + 0 +
analyzed. 1244 2.669 Rhine Hessen t002 Rhine Hessen yes
MRSA 193 + + + + + + + + + + + + + + +
4339 2.481 South German t045 No result

RESULTS: Characteristic MALDI-TOF peaks of the major 1005

0210
2.687
2.572
South German
Berlin
t892
t001
No result
South German no
MRSA 488 + + 0 + 0 0 + 0 0 + 0 0 + 0 +

MRSA lineages were detected and analyzed. A total of 370 2347 2.39 South German t041 No result MSP peaks characteristic of the major nosocomial MRSA
complex Rhine Hessen
MRSA isolates with different reproducible scores of MALDI- 4325 2.469 Rhine Hessen t002 Rhine Hessen yes
4338 2.501 South German t045 No result
TOF were tested and compared to the results of spa-typing. 4308 2.617 South German t012 No result
Representative peaks of the nosocomial MRSA complex Barnim
Among the five clonal complexes, the results of MALDI-TOF 1014 2.615 Berlin t001 South German no
m/z [Da] 3209 3444 4306 4816 5004 5527 6819 6865 6890 9627

showed satisfactory concordance with spa-typing. MRSA 707 + + + + + + + + + +

MRSA 232 + + + + + + 0 + + +

MRSA 235 + + + + + + 0 + + +

MRSA 229 + + + + + + 0 + + +

MRSA 952 + + + + 0 + + + + +

MRSA 954 0 + + + + + + + + +

MSP peaks characteristic of the major nosocomial MRSA

complex Barnim
Figure 1: Representative MRSA strain typed by spa sequencing and MALDI-TOF analysis

49
MALDI Biotyper Poster Hall 2016 Resistance and Subtyping

Matrix-assisted laser desorption/ionization-time of flight mass-spectrometry (MALDI-TOF)

based typing of an ESBL Escherichia coli outbreak in a hospital setting
A. Egli1, S. Tschudin-Sutter2, D. Goldenberger1, A. Widmer2, R. Frei1
1 Clinical Microbiology, University Hospital of Basel, Switzerland; 2 Division of InfecAous Diseases and Hospital Epidemiology, University Hospital of Basel, Switzerland

Purpose Results
q Rapid and specic idenAcaAon to subspecies level and determinaAon of strain idenAty are integral components of q Outbreak on neonatal intensive care unit (Tschudin-Su\er S et al. Emerging Infect Dis 2010)
outbreak invesAgaAon and control.
q ConvenAonal methods such as pulsed-eld gel electrophoresis (PFGE) are labour intensive and require up to one week
from sample collecAon to result.
q MALDI-TOF is able to idenAfy bacterial species and subspecies rapidly.
q We aimed to assess the validity of MALDI-TOF for detecAon of stain idenAty as compared to PFGE.

MALDI-TOF technology
Target plate Detector
Pulsed laser beam
Figure 3. PFGE of ESBL-producing E.coli outbreak
q Eight isolates collected during an outbreak investigation in the neonatal care unit, identifying transmission of
ESBL-producing E.coli from a mother to her newborn twins and subsequent spread to two other neonates and
one healthcare worker, were analyzed by PFGE and MALDI-TOF.
Target plate
Bacterial colony q In comparison to PFGE, PCA-based typing with MALDI-TOF spectra reproduced a very similar dendrogram
- 4 single cultures
- 3 times repeated
Electric field for acceleration of ions with identical clustering of six outbreak isolates. Two isolates of ESBL-producing E. coli not associated with the
Figure 1. Work-ow for MALDI-TOF typing. The protein signature is highly specic for bacterial species outbreak were correctly separated.

Figure 4. Principal component analysis of ESBL-producing E.coli isolates.

mz q PCA-based dendrogram and frameshift analysis of quadruplicates at three independent experiments showed a
Figure 2. FrameshiV analysis. Spectra were magnied and strain specic protein peaks were idenAed. high reproducibility of the results.

Methods Summary and Conclusions

q ESBL-producing E.coli isolates from a previously investigated and published report from our institution were q These results demonstrate that MALDI-TOF can provide a rapid means of typing of ESBL-producing E. coli
analysed using MALDI-TOF after a full protein extraction protocol. isolates reliably separating outbreak-related from unrelated isolates.
q The spectra profiling was done in quadruplicates and repeated three times and analyzed using principal q Our findings have important implications for outbreak investigations, which are hampered by the slow
component analysis (PCA) to generate a dendrogram. turnaround time of conventional typing techniques.
q Analysis was performed with Biotyper 3.0 software (Bruker Daltonic, Leipzig, Germany).

Corresponding author

Adrian Egli MD PhD, Clinical Microbiology, University Hospital Basel, Petersgraben 4, 4031 Basel, Switzerland, E-mail: a.egli@usb.ch

50
MALDI Biotyper Poster Hall 2016 Environmental and Food Microbiology

ESKAPE Pathogens
Understanding Nosocomial Risk Factors STENOTROPHOMONAS
The monitoring of water used in care homes and Acinetobacter baumannii ALS Environmental are one of the first UK laboratories to validate and have ALS Environmental are proud to be one of the few
healthcare facilities is covered by several pieces of Health Acinetobacter baumannii is a rapidly emerging pathogen in accredited to ISO 17025:2005 and the Drinking Water Testing Standard (DWTS), laboratories in the UK and Ireland to offer an analytical
and Safety Guidance (HSG). The majority of the guidance the health care system. A. baumannii is usually introduced accredit the rapid identification of positive Microbiological samples by Matrix speciation service for Stenotrophomonas species, which
is on the monitoring of Legionella and is supported by the into a hospital by a colonised patient. Due to its ability to Assisted Laser Disportion and Ionistation by Time of Flight Mass Spectrometry are of particular concern to healthcare facilities as they are a
Approved Code of Practice for Legionella (ACoPL8) and survive on artificial surfaces and resist desiccation it can (MALDI-ToF MS). The ground breaking identification technique employed by common water-borne organism. Although infection by this
Health Technical Memorandums (HTM); however, there are survive and potentially infect new patients for some time. It ALS Environmental, known as MALDI-ToF, allows us to remove the need for opportunistic bacteria is rare, cases of Stenotrophomonas
a range of other risk factors that need to be considered is suspected that A. baumannii growth favours nosocomial presumptive data for bacteriological analysis, with all data reported as Colony maltophila (S. maltophila) are potentially lethal. Following
in nosocomial scenarios. The ESKAPE pathogens are settings due to the constant use of antibiotics by patients Forming Units (CFU). The impact of MALDI-ToF confirmation on the ESKAPE a request from one of our established clients we have
emerging pathogens of concern. ALS Environmental are in the hospital and causes a wide range of infection pathogens is highlighted in the table below. developed a rapid technique to confirm and identify the
able to offer rapid identification of these bacteria using including bacteremia, pneumonia, meningitis, urinary tract colonies of Stenotrophomonas.
our revolutionary MALDI-ToF confirmation technique. The infection, and wound infection. The organisms ability to Bacteria Incubation Confirmation: Confirmation:
MALDI-ToF Saving
ESKAPE Pathogens are: survive under a wide range of environmental conditions, Time Traditional MALDI-ToF An aquatic, ubiquitous, opportunistic organism, S. maltophila
and to persist for extended periods of time on surfaces, Enterococci 2 days 1 day Minutes 1 day may be a particular burden to healthcare facilities due
Enterococcus faecium make it a frequent cause of outbreaks of infection and an Staphylococcus aureus 2 days 1 day Minutes 1 day to the susceptibility of immuno-compromised patients. ALS Environmental are employing our latest technology
Enterococcus faecium; formerly known as Streptococcus Although it has no natural infection route to humans S. in order to produce rapid species level identifications,
endemic, health careassociated pathogen. Klebsiella pneumoniae 1 day 1 day Minutes 1 day
faecium until its re-categorization in 1984, is a human maltophila infection can be facilitated by contaminated this means that we do not report presumptive results,
Acinetobacter baumannii 1 day 1 day Minutes 1 day
pathogen that causes nosocomial bacteremia, surgical Pseudomonas aeruginosa prosthetics such as catheters and intravenous lines. S. but instead provide a full list of the Stenotrophomonas
Pseudomonas aeruginosa 2 day 1 day Minutes 1 day
wound infection, endocarditis, and urinary tract infections. The monitoring for Pseudomonas is covered in HTM04- maltophilas resitance to most antibiotics has proved fatal species present in the sample. The full species breakdown
The bacteria can survive for long periods of time inside 01 Addendum. Serious infections of P. aeruginosa usually Enterobacteriaceae 1 day 1 day Minutes 1 day
to severely immunocompromised hosts. for Stenotrophomonas is important as not all species are
hospitals on a variety of surfaces as well as in soil and occur in the immunocompromised. Infections of the Stenotrophomonas 1 day 1 day Minutes 1 day
pathogenic to humans.
sewage. Growth temperatures range from 10oC to 45oC in blood, pneumonia, and infections following surgery can
basic or acidic environments, and in environments which lead to severe illness and death in these people. The Legionella 10 days 2 days Minutes 2 days
ALS Environmentals Microbiology Operations Manager,
are isotonic or hypertonic. Ent. faecium can be highly highly susceptible nosocomial patients include those on E-coli 1 day 1 day Minutes 1 day Pervinder Johal comments
drug resistant. The spread of the disease occurs between breathing machines, premature babies and patients with Coliforms 1 day 1 day Minutes 1 day
patients in hospitals due to transfer of the pathogen by wounds from surgery or from burns. Additionally, healthy Clostridium Perfringens 1 day 1 day Minutes 1 day When our customers first approached us to analyse
hands or medical instruments. Also, antibiotic use can people can also develop mild illnesses with Pseudomonas Salmonella 4 days 2 days Minutes 2 days for Stenotrophomonas we began our research and
decrease the number of other intestinal bacteria that are aeruginosa, especially after exposure to water. Ear development into validating a new method for this genus
Listeria 4 days 2 days Minutes 2 days
susceptible to the antibiotic and decrease competition for infections, especially in children, and more generalised group. By utilising the latest technology we were able to
the drug resistant Ent. faecium. skin rashes may occur after exposure to inadequately The instant confirmation of the ESKAPE bacteria allows infection control and provide a full speciation of Stenotrophomonas in under a
chlorinated hot tubs or swimming pools. water treatment to make rapid decisions on any potential remedial works that month.
Staphylococcus aureus may need to be undertaken. The MALDIToF can be used to identify any positive
The carriage of Staphylococcus aureus is an important Enterobacter species
bacteria, including Legionella. Species of the Stenotrophomonas genus are common in
source of nosocomial infection and community-acquired The genus Enterobacter is a member of the coliform
methicillin-resistant Staph. aureus (MRSA). Staph. aureus is group of bacteria. Enterobacter species, particularly E. Legionella the environment and human infection is rare. However,
Traditional testing for Stenotrophomonas involves a
common and often found in the nose or on the skin. Most cloacae and E. aerogenes, are important nosocomial The MALDI-ToF confirmation of Legionella removes the presumptive stage; general screen that produces presumptive results for infection risk of opportunistic pathogens such as the
of the time these bacteria do not cause any symptoms. pathogens responsible for various infections; including meaning that ALS Environmental report positive confirmed Legionella on the the prescence of members of the genus. This is usually maltophila species of Stenotrophomonas can be elevated
The ability of the nasal passages to harbour Staph. aureus bacteremia, lower respiratory tract infections, skin original read days (currently days 3, 7 and 10). This is 40% quicker than the followed by biochemical tests which indicate whether S. in healthcare settings and other environments where
results from a combination of a compromised host immune and softtissue infections. Risk factors for nosocomial traditional approach and is fully ISO 17025:2005 accredited. ALS Environmental maltophila is present. immuno-compromised patients, such as in care homes
system combined with the bacterias ability to evade a Enterobacter infections include hospitalisation of greater have one of the worlds largest Legionella species libraries held within our MALDI- may be exposed. Despite the low infection rate, nomo-
hosts innate immunity. The spectrum of Staphylococcus than 2 weeks, invasive procedures in the past 72 hours, ToF. With only 3 known species unidentifiable, two of these are Viable But Not social outbreaks of S. maltophila are of increasing concern
infections can range from skin abscess to life-threatening treatment with antibiotics in the past 30 days, and the Culturable (VBNC) and the final species is being sourced by our laboratory. in modern infection control because it can be difficult to
infections such as septicaemia or endocarditis. presence of a central venous catheter. Specific risk factors treat effectively as it is resistant to most broad-spectrum
for infection with nosocomial multidrug-resistant strains antibiotics.
Klebsiella pneumoniae of Enterobacter species include the recent use of broad-
In nosocomial settings, Klebsiella bacteria can be spread spectrum cephalosporins or aminoglycosides and ICU care. LEGIONELLA SPECIES TABLE
through person-to-person contact or by contamination of
the environment; it is important to note that the bacteria Stenotrophomonas Maltophilia Name of species Number of serotypes Linked to humaninfection Geographic origin Common Matrix Type
are not spread through the air. Patients in healthcare Stenotrophomonas maltophilia is an organism of low Legionella anisa YES USA Process, Drinking, Surface and Recreational
settings may be exposed to Klebsiella when they are virulence which can frequently colonise fluids used in the
Legionella adelaidensis Unknown Adelaide in South Australia Process Water
on ventilators, or have intravenous catheters or wounds. hospital setting (eg, irrigation solutions, intravenous fluids)
Unfortunately, these medical tools and conditions may and patient secretions (eg, respiratory secretions, urine, Legionella beliardensis Unknown Montbeliard in France Process Water
allow Klebsiella to enter the body and cause infection; wound exudates). S. maltophilia usually bypasses normal Legionella birminghamensis YES Birmingham, Alabama, USA Process Water
which can be fatal in the immuno-compromised. hosts defenses to cause human infection. The growth of Legionella bozemanii (bozemanae) 2 YES Named after F.Marilyn Bozeman Clinical Isolation
S. maltophilia from sites which would normally be sterile
Legionella brunensis Unknown Brno, Czech Republic Process Water
(e.g., blood) usually represents true infection; growth of
S. maltophilia in microbiological cultures of respiratory Legionella busanensis Unknown Busan in Korea Process Water
or urinary specimens is therefore sometimes difficult to Legionella cardiaca YES Northwestern University, Chicago, USA Clinical Isolation
interpret and not always a proof of infection. Legionella cherrii Unknown Minnesota, USA Process Water
Legionella cincinnatiensis YES Cincinnati, Ohio, USA Clinical Isolation
Legionella donaldsonii Unknown Process, Drinking, Surface and Recreational

MALDI-TOF - Microbiological Confirmations Legionella drancourtii

Legionella dresdenensis
Unknown
Unknown Dresden, Germany
Amoebae
Recreational and Surface Water
Matrix Assisted Laser Desorption/Ionisation Time of Flight Mass Legionella drozanskii Unknown Leeds, England, UK Process and Drinking Water
The technique is used to analyse the protein fingerprint Legionella dumoffii YES Clinical Isolation
of a micro-organism, producing an identification using a Legionella erythra 2 YES Seattle, WA,USA Process Water
validated reference library. Legionella fairfieldensis YES Faifield,Victoria, Australia Process Water

The ability to reduce confirmation times is as a result of Legionella fallonii Unknown Cruise ship in international waters Process Water ESKAPE Pathogens Understanding
MALDI-TOF being able to be performed on colonies from Legionella feeleii YES Process Water and Clinical Isolation Nosocomial Risk Factors Part 1
selective agar. This removes the need for sub culturing and
incubation, which is the stage that incurs additional time.
Depending on the organism being tested, this additional
time can be anything from 4 hours to 5 or more days.

The method was developed in consultation with both

the United Kingdom Accreditation Service (UKAS) and
the Drinking Water Inspectorate (DWI). As a result, we
have full ISO 17025 UKAS accreditation, since December 51
2013. This was extended to include Drinking Water Testing
Specification (DWTS) in February 2014. Although the
technology has been used extensively in the clinical sector
for a number of years, ALS is the first UK Environmental
laboratory to use and accredit this technology.
 for Stenotrophomonas we began our research and
decrease the number of other intestinal bacteria that are aeruginosa, especially after exposure to water. Ear development into validating a new method for this genus
susceptible to the antibiotic and decrease competition for infections, especially in children, and more generalised group. By utilising the latest technology we were able to
the drug resistant Ent. faecium. skin rashes may occur after exposure to inadequately The instant confirmation of the ESKAPE bacteria allows infection control and provide a full speciation of Stenotrophomonas in under a
chlorinated hot tubs or swimming pools. water treatment to make rapid decisions on any potential remedial works that month.
Staphylococcus aureus may need to be undertaken. The MALDIToF can be used to identify any positive
The carriage of Staphylococcus aureus is an important Enterobacter species
bacteria, including Legionella. Species of the Stenotrophomonas genus are common in
source of nosocomial infection and community-acquired The genus Enterobacter is a member of the coliform
methicillin-resistant Staph. aureus (MRSA). Staph. aureus is group of bacteria. Enterobacter species, particularly E. Legionella the environment and human infection is rare. However,
Traditional testing for Stenotrophomonas involves a
common and often found in the nose or on the skin. Most cloacae and E. aerogenes, are important nosocomial The MALDI-ToF confirmation of Legionella removes the presumptive stage; general screen that produces presumptive results for infection risk of opportunistic pathogens such as the
of the time these bacteria do not cause any symptoms. pathogens responsible for various infections; including meaning that ALS Environmental report positive confirmed Legionella on the the prescence of members of the genus. This is usually maltophila species of Stenotrophomonas can be elevated
The ability of the nasal passages to harbour Staph. aureus bacteremia, lower respiratory tract infections, skin original read days (currently days 3, 7 and 10). This is 40% quicker than the followed by biochemical tests which indicate whether S. in healthcare settings and other environments where
results from a combination of a compromised host immune and softtissue infections. Risk factors for nosocomial traditional approach and is fully ISO 17025:2005 accredited. ALS Environmental maltophila is present. immuno-compromised patients, such as in care homes
system combined with the bacterias ability to evade a Enterobacter infections include hospitalisation of greater have one of the worlds largest Legionella species libraries held within our MALDI- may be exposed. Despite the low infection rate, nomo-
hosts innate immunity. The spectrum of Staphylococcus than 2 weeks, invasive procedures in the past 72 hours, ToF. With only 3 known species unidentifiable, two of these are Viable But Not social outbreaks of S. maltophila are of increasing concern
infections can range from skin abscess to life-threatening treatment with antibiotics in the past 30 days, and the Culturable (VBNC) and the final species is being sourced by our laboratory. in modern infection control because it can be difficult to
infections such as septicaemia or endocarditis. presence of a central venous catheter. Specific risk factors treat effectively as it is resistant to most broad-spectrum
antibiotics.

MALDI Biotyper Poster Hall 2016 Environmental and Food Microbiology

for infection with nosocomial multidrug-resistant strains
Klebsiella pneumoniae of Enterobacter species include the recent use of broad-
In nosocomial settings, Klebsiella bacteria can be spread spectrum cephalosporins or aminoglycosides and ICU care. LEGIONELLA SPECIES TABLE
through person-to-person contact or by contamination of
the environment; it is important to note that the bacteria Stenotrophomonas Maltophilia
are not spread through the air. Patients in healthcare Stenotrophomonas maltophilia is an organism of low
settings may be exposed to Klebsiella when they are virulence which can frequently colonise fluids used in the
on ventilators, or have intravenous catheters or wounds. hospital setting (eg, irrigation solutions, intravenous fluids)
Unfortunately, these medical tools and conditions may and patient secretions (eg, respiratory secretions, urine,
allow Klebsiella to enter the body and cause infection; wound exudates). S. maltophilia usually bypasses normal
which can be fatal in the immuno-compromised. hosts defenses to cause human infection. The growth of
S. maltophilia from sites which would normally be sterile
Legionella brunensis Brno, Czech Republic
(e.g., blood) usually represents true infection; growth of
S. maltophilia in microbiological cultures of respiratory Legionella busanensis Unknown Busan in Korea Process Water
or urinary specimens is therefore sometimes difficult to Legionella cardiaca YES Northwestern University, Chicago, USA Clinical Isolation
interpret and not always a proof of infection. Legionella cherrii Unknown Minnesota, USA Process Water
Legionella cincinnatiensis YES Cincinnati, Ohio, USA Clinical Isolation
Legionella donaldsonii Unknown Process, Drinking, Surface and Recreational

MALDI-TOF - Microbiological Confirmations Legionella drancourtii

Legionella dresdenensis
Unknown
Unknown Dresden, Germany
Amoebae
Recreational and Surface Water
Matrix Assisted Laser Desorption/Ionisation Time of Flight Mass Legionella drozanskii Unknown Leeds, England, UK Process and Drinking Water
The technique is used to analyse the protein fingerprint Legionella dumoffii YES Clinical Isolation
of a micro-organism, producing an identification using a Legionella erythra 2 YES Seattle, WA,USA Process Water
validated reference library. Legionella fairfieldensis YES Faifield,Victoria, Australia Process Water
Legionella fallonii Unknown Cruise ship in international waters Process Water
The ability to reduce confirmation times is as a result of
MALDI-TOF being able to be performed on colonies from Legionella feeleii YES Process Water and Clinical Isolation
selective agar. This removes the need for sub culturing and Legionella geestiana Unknown Geest office building, London , England Drinking and Process Water
incubation, which is the stage that incurs additional time. Legionella genomspecies 1 Unknown Unknown
Depending on the organism being tested, this additional
Legionella gormanii YES Atlanta, USA Soil
time can be anything from 4 hours to 5 or more days.
Legionella gratiana Unknown Savoy region in France Recreational and Process Water
The method was developed in consultation with both Legionella gresilensis Unknown In France from the city Groux-les-Bains Recreational, Drinking and Process Water
the United Kingdom Accreditation Service (UKAS) and Legionella hackeliae YES USA, Pennsylvania Clinical Isolation
the Drinking Water Inspectorate (DWI). As a result, we Legionella impletisoli Unknown Japan Soils near Trade Effluent Waters
have full ISO 17025 UKAS accreditation, since December
Legionella israelensis YES In Gaash in Israel Surface Water and Recreational Water
2013. This was extended to include Drinking Water Testing
Specification (DWTS) in February 2014. Although the Legionella jamestowniensis Unknown Jamestown,New York Soil
technology has been used extensively in the clinical sector Legionella jeonii Unknown Amoebae
for a number of years, ALS is the first UK Environmental Legionella jordanis YES Jordan River, Indiana , USA Process, Trade Effluent and Untreated Sewage
laboratory to use and accredit this technology. Legionella lansingensis YES Lansing, Michigan , USA Clinical Isolation
Legionella londiniensis 2 Unknown London , UK Process, Recreational, Surface and Drinking waters
As traditional culture-based methods are still used for the
isolation, compliance with standards is maintained. We Legionella longbeachae 2 YES Long Beach, California , USA Soil and Compost
are therefore fully compliant with ACoP L8 (and HSG274), Legionella lytica YES Poland Soil and Amoebae
The Water Supply (Water Quality) Regulations, The Private Legionella maceachernii YES Drinking Water
Water Supply Regulations and HTM guidance.
Legionella massiliensis Unknown Marseille, France, Process Water

The reduction in confirmation times removes the risk Legionella micdadei -L. pittsburghensis YES USA Clinical Isolation and Drinking Water
associated with acting (or not acting) on presumptive Legionella monrovica YES USA Unknown
data by providing an accredited, confirmed result within Legionella moravica Unknown Moravia, Czech Republic Process Water
minutes of the presumptive result being generated. The Legionella nagasakiensis YES Aomori, Japan Clinical Isolation, Recreational and Drinking Water
added benefit is that in addition to a rapid confirmation
Legionella nautarum Unknown London , UK Drinking and Process Water
result, in the majority of cases, species-level identification
will also be available. ALS will provide this additional Legionella oakridgensis YES Oak Ridge, Tennessee , USA Clinical Isolation and Process Water
information as part of the standard service. Legionella parisiensis YES Paris, France Clinical Isolation, Process and Surface Water
Legionella pittsburghensis Unknown USA Clinical Isolation
If you would like more information on specific organisms Legionella pneumophila 15 YES USA Clinical Isolation, Surface and Recreational Water
that we are able to identify, please contact our customer
Legionella pneumophila ssp fraseri YES Los Angeles, USA Clinical Isolation
services department.
Legionella pneumophila ssp pascullei YES Clinical Isolation
Legionella pneumophila ssp pneumophila YES Philadelphia , USA Clinical Isolation
ALS SERVICE OVERVIEW Legionella quateirensis Unknown Quarteira, Portugal Process and Drinking Water

UK and Ireland Locations Legionella quinlivanii 2 Unknown Australia Process Water

Legionella rowbothamii Unknown Process Water and Sludge
Legionella rubrilucens YES Los Angeles,California, USA Drinking and Recreational Water
Quality
Providing customers with UKAS ISO 17025:2005, MCERTS Legionella sainthelensi 2 YES Mount St. Helens, Washington, USA Clinical Isolation, Surface, Recreational and Ground Water
and DWTS accredited data from our laboratories across the Legionella santicrucis Unknown St. Croix, Virgin Islands Drinking and Process Water
UK. We participate in a broad range of Proficiency Testing Legionella shakespearei Unknown Stratford-upon-Avon , England, UK Process Water
schemes and hold a DEFRA import licence for soils. Legionella spiritensis 2 Unknown St. Helens, Oregon, USA Recreational and Surface Water
Did you know that? Legionella steelei YES California, USA and South Australia Clinical Isolation
We are able to provide a broad range of additional services Legionella steigerwaltii Unknown St. Croix, Virgin Islands Drinking and Process Water
to help with your sampling including: Legionella taurinensis Unknown Turin, Italy Process Water
Internal refrigerated and tracked courier network
Legionella tucsonensis YES Tucson, Arizona , USA Clinical Isolation
National portfolio of drop-off locations
Pre-Registration of samples via our Pre-Reg system Legionella tunisiensis Unknown Lake Sabkha ,Tunisia Recreational and Surface Water
Dedicated customer service advisor Legionella wadsworthii YES Los Angeles, California, USA Clinical Isolation
Online reporting via our WebTrieve system Legionella waltersii 2 Unknown Adelaide , Australia Drinking and Process Water
Legionella worsleiensis Unknown Worsley, England, UK Process Water
Legionella yabuuchiae Unknown Japan Soils

Contaminated Land
We understand the time pressures of large scale
Remediation and Brownfield projects and are a member
of the AGS. Our Coventry laboratory utilises state of the
art analytical equipment with the backup of our sister
laboratories across Europe to ensure that we deliver your
projects on time every time
Legionella and Microbiology
Being members of the Legionella Control Association (LCA)
we understand the emphasis placed on laboratory analysis
for the Control of Legionella. With 3 methods for testing
Legionella (including rapid PCR) and an understanding and
appreciation the implications of ACoP L8, HSG 274 and
HTM04-01 we are your ideal analytical partner for all of
your water hygiene monitoring requirements.
Waste Management Drinking Water If you have any questions on our services or would like more information on
By working closely with some of the largest companies We are one of only a handful of commercial laboratories ALS Environmental please call the Coventry laboratory on 02476 42 12 13 or
in this sector we are able to offer unrivalled analytical to have a dedicated Drinking Water Testing Specification visit our website.
and administration services to ensure that your samples (DWTS) accredited laboratory, based in Wakefield, Yorkshire.
are processed swiftly and in line with the UKAS Deviating We are able to supply analysis to the Public and Private ALS Environmental Limited
Sample Guidance. Drinking Water Regulations T +44 (0)24 7642 1213
F +44 (0)24 7685 6575
E info.ukenviro@alsglobal.com

www.alsenvironmental.co.uk ESKAPE Pathogens Understanding

Nosocomial Risk Factors Part 2

52
 53
Environmental and Food Microbiology

Rapid and Accurate Listeria species

Identification by MALDI Biotyper
Database Improvement
FEMS 2015, P 0604
Marian Awad, Markus Timke, Markus Mass spectra were acquired by using microflex Listeria species can be correctly identified by
Kostrzewa LT (Bruker Daltonik, Bremen, Germany) and using MALDI Biotyper improved database with
identified with MALDI Biotyper Compass high log(score) value and constant ranking of
Bruker Daltonik GmbH, Bremen, DE.
software. the correct species (Table 1,2).
Species identity of all strains was confirmed by
Introduction characteristic peaks for each species
(Barbuddhe et al. 2008) (Fig.3) and16S rRNA
Table 1. Correct and constant ranking of species identification of
50 Listeria spp. strains using actual and extended database.
gene sequencing (Czajka et al. 1993). Where
Organism second best
Genus Listeria is phylogenetically divided into Analyte Name
Organism best match
database 5627
Score
Value
Organism second best Organism best match Score
match database 5627 new database 5989 Value
match new database
5989
four groups: L. fleischmannii, L. grayi, only three distinct single pair differences L. fleischmannii 25391T
DSM
L. monocytogenes 1.578 Listeria welshimeri Listeria fleischmannii 2.63 Listeria fleischmannii
L. weihenstephanensis, and L. monocytogenes between L.monocytogenes and L.innocua in the L. fleischmannii 24998T
DSM
Pseudomonas
lundensis
1.416 L. monocytogenes Listeria fleischmannii 2.6 Listeria fleischmannii
group most related members L. V9 region were considered as a signature L. grayi 24933 CCUG
L. grayi 24938 CCUG
Listeria grayi
Listeria grayi
2.427
2.121
Listeria grayi
Listeria grayi
Listeria grayi
Listeria grayi
2.855
2.738
Listeria grayi
Listeria grayi
monocytogenes, L. ivanovii, L. innocua, L. between them (Fig.4). L. grayi 24939 CCUG Listeria grayi 2.287 Listeria grayi Listeria grayi 2.756 Listeria grayi
welshimeri and L. seeligeri (Fig.1). L. grayi 4984 CCUG
L. grayi 20596 DSM
Listeria grayi
Listeria grayi
2.392
2.241
Listeria grayi
Listeria grayi
Listeria grayi
Listeria grayi
2.762
2.769
Listeria grayi
Listeria grayi
L. grayi 20601T DSM Listeria grayi 2.103 Listeria grayi Listeria grayi 2.683 Listeria grayi
L. weihenstephanesis
Listeria ivanovii 1.781 Listeria ivanovii L. weihenstephanesis 2.796 L. weihenstephanesis
24698T DSM
L. weihenstephanesis
Listeria ivanovii 1.716 Listeria innocua L. weihenstephanesis 2.867 L. weihenstephanesis
24699T DSM
L. monocytogenes
L. monocytogenes 2.472 L. monocytogenes L. monocytogenes 2.721 L. monocytogenes
32843 CCUG
L. monocytogenes
L. monocytogenes 2.549 L. monocytogenes L. monocytogenes 2.764 L. monocytogenes
32964A CCUG
L. monocytogenes
L. monocytogenes 2.32 L. monocytogenes L. monocytogenes 2.572 L. monocytogenes
33548 CCUG
L. monocytogenes
L. monocytogenes 2.515 L. monocytogenes L. monocytogenes 2.784 L. monocytogenes
35751 CCUG
L. monocytogenes
L. monocytogenes 2.445 L. monocytogenes L. monocytogenes 2.744 L. monocytogenes
49753 CCUG
L. monocytogenes
L. monocytogenes 2.366 L. monocytogenes L. monocytogenes 2.668 L. monocytogenes
53269 CCUG
L. monocytogenes
L. monocytogenes 2.447 L. monocytogenes L. monocytogenes 2.716 L. monocytogenes
59664 CCUG
Fig.3 Characteristic Listeria Peaks taken from B. Barbuddhe et al. L. monocytogenes
61052 CCUG
L. monocytogenes 2.473 L. monocytogenes L. monocytogenes 2.781 L. monocytogenes
2008 ASM. L. monocytogenes
L. monocytogenes 2.421 L. monocytogenes L. monocytogenes 2.802 L. monocytogenes
31527 CCUG
L. ivanovii 24940 CCUG Listeria ivanovii 2.418 L. monocytogenes Listeria ivanovii 2.86 Listeria ivanovii
L. ivanovii 24941 CCUG Listeria ivanovii 2.336 L. monocytogenes Listeria ivanovii 2.794 Listeria ivanovii
L. ivanovii 24942 CCUG Listeria ivanovii 2.39 Listeria ivanovii Listeria ivanovii 2.756 Listeria ivanovii
L. ivanovii 24943 CCUG Listeria ivanovii 2.354 Listeria ivanovii Listeria ivanovii 2.735 Listeria ivanovii
L. ivanovii 24944 CCUG Listeria ivanovii 2.442 Listeria ivanovii Listeria ivanovii 2.845 Listeria ivanovii
L. ivanovii 37344 CCUG Listeria ivanovii 2.378 Listeria ivanovii Listeria ivanovii 2.855 Listeria ivanovii
Fig.1 A dendogram showing the relation of the four Listeria spp. L. ivanovii 12491T DSM Listeria ivanovii 2.41 Listeria ivanovii Listeria ivanovii 2.824 Listeria ivanovii
groups based on mass spectra. L. ivanovii 19119T
L. monocytogenes 2.423 Listeria ivanovii Listeria ivanovii 2.498 Listeria ivanovii
AGES
L. innocua 18949 CCUG Listeria innocua 2.517 L. monocytogenes Listeria innocua 2.684 Listeria innocua
L. monocytogenes group members are closely L. innocua 21830 CCUG
L. innocua 22268 CCUG
Listeria innocua
Listeria innocua
2.501
2.512
L. monocytogenes
L. monocytogenes
Listeria innocua
Listeria innocua
2.815
2.765
Listeria innocua
Listeria innocua
related and show slight differences in L. innocua 22270 CCUG Listeria innocua 2.454 L. monocytogenes Listeria innocua 2.701 Listeria innocua
phenotypic and chemical reactions as well as in L. innocua 24924 CCUG Listeria innocua 2.539 L. monocytogenes Listeria innocua 2.757 Listeria innocua
16S rRNA gene sequencing. Also, they have a
L. innocua 24928 CCUG Listeria innocua 2.604 L. monocytogenes Listeria innocua 2.752 Listeria innocua
L. innocua 27612 CCUG Listeria innocua 2.598 L. monocytogenes Listeria innocua 2.811 Listeria innocua
very similar profile mass spectra with small L. innocua 35613 CCUG Listeria innocua 2.483 L. monocytogenes Listeria innocua 2.599 Listeria innocua
differences (Fig.2) but also with a few very
L. innocua 44511 CCUG Listeria innocua 2.435 L. monocytogenes Listeria innocua 2.72 Listeria innocua
Fig.4 Three distinct single base pair differences between L. innocua 44813 CCUG Listeria innocua 2.535 L. monocytogenes Listeria innocua 2.679 Listeria innocua
characteristic peaks. L. monocytogenes and L. innocua in the V9 region. L. seeligeri 24929 CCUG Listeria seeligeri 2.306 Listeria ivanovii Listeria seeligeri 2.689 Listeria seeligeri
L. seeligeri 24930
Listeria innocua 2.323 L. monocytogenes Listeria seeligeri 2.865 Listeria seeligeri
CCUG
L. seeligeri 24932
Results
L. monocytogenes 2.164 Listeria innocua Listeria seeligeri 2.592 Listeria seeligeri
CCUG
L. seeligeri 27609
Listeria seeligeri 2.454 Listeria ivanovii Listeria seeligeri 2.849 Listeria seeligeri
CCUG
L. seeligeri 27801
A set of mass spectral Listeria fingerprints from
Listeria ivanovii 2.318 Listeria ivanovii Listeria seeligeri 2.653 Listeria seeligeri
CCUG
L. seeligeri 27802
Listeria ivanovii 2.229 Listeria ivanovii Listeria seeligeri 2.722 Listeria seeligeri
50 reference strains of 8 species covering all CCUG
L. seeligeri 45639
Listeria seeligeri 2.376 Listeria innocua Listeria seeligeri 2.847 Listeria seeligeri
Listeria fingerprints and models (Fig.5) were
CCUG
L. seeligeri 20751T
Listeria seeligeri 2.307 Listeria ivanovii Listeria seeligeri 2.769 Listeria seeligeri
DSM
established in the improved database. i.e. L. welshimeri 24934
CCUG
Listeria welshimeri 2.371 L. monocytogenes Listeria welshimeri 2.839 Listeria welshimeri
L. fleischmannii (n = 2) , L. grayi (n = 6), L. welshimeri 24935
CCUG
Listeria welshimeri 2.476 L. monocytogenes Listeria welshimeri 2.858 Listeria welshimeri
L. weihenstephanensis (n = 2),
L. welshimeri 24936
Listeria welshimeri 2.293 L. monocytogenes Listeria welshimeri 2.875 Listeria welshimeri
CCUG
L. welshimeri 20650T
L. monocytogenes (n = 9), L. ivanovii (n = 8),
Listeria welshimeri 2.451 L. monocytogenes Listeria welshimeri 2.84 Listeria welshimeri
DSM
L. welshimeri 15452
Listeria welshimeri 2.593 L. monocytogenes Listeria welshimeri 2.593 Listeria welshimeri
L. innocua (n = 10), L. welshimeri (n = 5) and DSM
L. seeligeri (n= 5).
Table 2. Constant results with high log(score) value.
Fig.2 Listeria spp. showing very similar profile mass spectra but few
characteristic peaks.
Only L. monocytogenes and L. ivanovii exhibit
pathogenic features. Therefore rapid and
accurate identification of Listeria strains is then
essential for appropriate management and
control.
The accuracy and speed of data acquisition by
MALDI-TOF MS makes it an important tool for
microorganisms identification in food
MALDI Biotyper Poster Hall 2016

processing, quality control and disease

diagnosis, and therefore it is important to
Fig.5.a L. monocytogenes and L. innocua different Listeria model
reliably identify the different Listeria species by fingerprints with characteristically peaks are covered in the improved
MALDI Biotyper. MALDI Biotyper database.
Conclusions
Listeria spp. are so closely related
Methods and very important to acquire correct
Listera strains were grown on Columbia 5 % rapid identification.
Sheep Blood agar (BD, Heidelberg, Germany) at
37 C. Biomass was collected after one day, and Extended improved MALDI Biotyper
then processed by ethanol / formic acid database can rapidly and correctly
standard extraction method. identify Listeria species.
Fig.5.b L. Innocua and L. seeligeri characteristical peaks and slight
differences accurately and rapidly identified by the improved MALDI
Biotyper database.
MALDI Biotyper
For research use only. Not for use in diagnostic procedures.
 54
Environmental and Food Microbiology

Identification of food microorganisms

by MALDI-TOF mass spectrometry
Ondrej edoa,b, Marta Dukovc, Ines Laaninc, Josef Kamenkd, Renta Karpkovc,e, and Zbynk Zdrhala,b
aResearch Group Proteomics, CEITEC - Central European Institute of Technology, Masaryk University, Brno, Czech Republic contact e-mail: sedo@sci.muni.cz
bNational Centre for Biomolecular Research, Faculty of Science, Masaryk University, Brno, Czech Republic
cDepartment of Milk Hygiene and Technology, Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic
dDepartment of Meat Hygiene and Technology, Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic
eDepartment of Bacteriology, Veterinary Research Institute, Brno, Czech Republic
INTRODUCTION: Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass EXPERIMENTAL: After sampling and homogenization of the food material in a
Spectrometry (MALDI-TOF MS) is currently becoming the first method of choice for rapid, MRS broth (Oxoid), all colonies showing different morphological characteristics were
simple, and reliable bacterial identification. Apart from clinical diagnostics, the method is selected and purified on MRS agar plates. The bacterial cells were treated by using an
applicable also in other fields, including food microbiology. The aim of this work was to extraction protocol recommended by Bruker Daltonics. The MALDI-TOF MS analyses
examine the usefulness of MALDI-TOF MS for identification of bacterial isolates from dairy were performed with an Ultraflex III instrument (Bruker Daltonics) and the resulting
and meat products, and to assess the discriminatory power of the method. mass spectra were treated by Biotyper software (database MBT-BDAL-5627).
DAIRY PRODUCTS: 148 isolates from cheese, milk and fermented dairy products MEAT: 1453 isolates from fermented, hot smoked dry and cooked meat products
identification at the species level: 130 isolates (87,8 %) and meat processing plant environment
identification at the species level with ambiguity: 8 isolates (5,4 %) identification at the species level: 892 isolates (61,4 %)
identification at the genus level only: 7 isolates (4,7 %) identification at the species level with ambiguity: 109 isolates (7,5 %)
no identification: 3 isolates (2,0 %) identification at the genus level only: 268 isolates (18,4 %)
BIO II 67
M I 15
BIO I 49 Pediococcus spp.
no identification: 184 isolates (12,7 %)
BIO IV 31
BIO III 53
BIO IV 32
BIO I 48
US 34
US 30
Lactobacillus paracasei
O III 7 KAS 1500
US 22 KAS 1487
S II 19 KAS 519
S II 37 KAS 281
O VI 11 KAS 992
US 28 KAS 1463
US 5 KAS 1462
BIO III 69 KAS 1433
S II 69 KAS 1430
BIO II 65 KAS 1418
BIO I 5 KAS 1432
BIO III 49 KAS 1429
BIO III 24 KAS 1421
BIO III 39 KAS 1405
BIO III 37 KAS 1010
BIO III 22 KAS 987
BIO III 17 KAS 918
BIO III 12 KAS 935
Extension of commercially available
BIO IV 11 KAS 900
BIO IV 3
BIO I 11
KAS 582
KAS 255
BIO IV 4 KAS 253
BIO IV 2 KAS 248
BIO III 27 KAS 1333
BIO I 6 KAS 1362
database of reference mass spectra
BIO III 21 KAS 1325
BIO IV 12 KAS 947
BIO III 23 KAS 1372
KAS 202
Lactobacillus rhamnosus
BIO II 23
BIO III 28 KAS 1000
BIO III 26 KAS 492
BIO II 22 KAS 349
with Weissella confusa
BIO I 17 KAS 396
BIO III 25 KAS 495
BIO I 15 KAS 341
BIO I 28 KAS 1285
BIO II 24 KAS 1282
S II 20 KAS 775
BIO III 18 KAS 845
and Weissella hellenica entries
BIO III 15 KAS 846
BIO II 16 KAS 844
BIO II 15 KAS 850
BIO II 13 KAS 853
BIO II 25 KAS 737
BIO II 7 KAS 700
BIO III 16 KAS 331
resulted in identification
BIO II 8 KAS 408
BIO III 13 KAS 301
BIO II 5 KAS 1039
M I 19 KAS 978
Lactobacillus sakei
MI9 KAS 1360
M I 16 KAS 373
KAS 372
of additional 34 isolates.
M I 13
D 16 KAS 299
B 25 KAS 851
16 KAS 265
BIO I 45 KAS 266
US 27 KAS 264
Lactobacillus curvatus/fructivorans
B 10 KAS 921
BIO IV 21 KAS 204
BIO IV 33 KAS 1431
BIO IV 18 KAS 832
BIO IV 22 KAS 472
KAS 201
(ambiguous identification results)
BIO IV 30
BIO IV 23 KAS 198
BIO III 70 KAS 197
BIO III 67 KAS 189
BIO III 68 KAS 1329
BIO III 73 KAS 585
BIO III 66 KAS 185
BIO II 71 KAS 184
BIO I 46 KAS 193
O VI 9 KAS 183
BIO III 58 KAS 114
Lactobacillus plantarum
D 42 KAS 111
C 33 KAS 1222
BIO II 59 KAS 1172
BIO II 17 KAS 838
BIO I 16 KAS 1161
Lactobacillus fermentum
BIO II 57 KAS 1223
BIO IV 16 KAS 1135
KAS 797
Weissella confusa
BIO IV 14
Enterococcus spp.
BIO I 50 KAS 203
S II 38 KAS 1400
BIO I 22 KAS 1311
D 33 KAS 525
D 20 KAS 1257
(new reference)
BIO I 43 KAS 1259
Lactobacillus brevis
67 KAS 555
BIO I 44 KAS 483
O VI 8 KAS 1196
BIO III 62 KAS 990
BIO II 72 KAS 135
KAS 130
Weissella viridescens
BIO II 60 KAS 1296
MI6
MI4 KAS 584
D 51 KAS 387
BIO II 58 KAS 312
Lactobacillus delbrueckii
BIO IV 48 KAS 163
BIO IV 39 KAS 674
BIO II 69 KAS 650
BIO I 41 KAS 574
BIO III 38 KAS 1287
BIO I 37 KAS 1291
BIO II 6 KAS 1286
KAS 1079
Lactobacillus johnsonii
BIO II 32
BIO II 19 KAS 1019
BIO III 31 KAS 1077
BIO III 29 KAS 1049
BIO III 36 KAS 1076
Weissella hellenica
BIO I 39 KAS 1018
BIO I 34 KAS 40
BIO I 33 KAS 389
O III 1 KAS 1348
O VIII 1 KAS 1292
O VII 3 KAS 968
(new reference)
O VIII 7 KAS 967
O VIII 14 KAS 476
O VIII 2 KAS 335
Leuconostoc lactis
O VIII 10 KAS 334
O VIII 9 KAS 336
O VIII 4 KAS 333
O VIII 3 KAS 390
O VII 4 KAS 388
M I 14
KAS 386
KAS 385
Leuconostoc mesenteroides
M I 10 KAS 322
MI5 KAS 318
D 44 KAS 317
BIO II 18
BIO II 9 KAS 316
BIO III 11 KAS 315
BIO III 2 KAS 314
KAS 313
Leuconostoc pseudomesenteroides
BIO III 1 KAS 162
BIO I 4 KAS 554
BIO II 10 KAS 323
BIO I 9 KAS 325
BIO IV 38 KAS 324
BIO IV 36 KAS 327
BIO I 14 KAS 326
1000 900 800 700 600 500 400 300 200 100 0 1000 900 800 700 600 500 400 300 200 100 0
Distance Level Distance Level
Fig. 1. Dendrogram calculated on the basis of MALDI-TOF mass spectra of all 148 isolates from dairy products. Fig. 2. Dendrogram calculated on the basis of MALDI-TOF mass spectra of 184 unidentified isolates from meat.
DISCRIMINATORY POWER: 17 strains of the Lactobacillus acidophilus group were cultivated in triplicates to examine differentiation between closely related strains.
Lactobacillus jensenii CCM 7778
Lactobacillus jensenii CCM 7563 Lactobacillus helveticus CCM 7193 III Lactobacillus crispatus CCM 7777 II
Lactobacillus jensenii CCM 7560
Lactobacillus delbrueckii CCM 7190 Lactobacillus helveticus CCM 7193 II Lactobacillus crispatus CCM 7777 III
Lactobacillus delbrueckii CCM 4290 Lactobacillus helveticus CCM 7193 I Lactobacillus crispatus CCM 7777 I
Lactobacillus delbrueckii CCM 4289
Lactobacillus amylovorus CCM 4382 Lactobacillus helveticus CCM 3806 II Lactobacillus crispatus CCM 7776 III
Lactobacillus amylolovorus CCM4381 Lactobacillus helveticus CCM 3806 III Lactobacillus crispatus CCM 7776 II
Lactobacillus amylovorus CCM 4380
Lactobacillus acidophilus CCM 4833 Lactobacillus helveticus CCM 3806 I Lactobacillus crispatus CCM 7776 I
Lactobacillus acidophilus BCCM 8151 Lactobacillus helveticus CCM 4287 II Lactobacillus crispatus CCM 7010 II
Lactobacillus helveticus CCM 4287
Lactobacillus helveticus CCM 7193 Lactobacillus helveticus CCM 4287 III Lactobacillus crispatus CCM 7010 III
Lactobacillus helveticus CCM 3806
Lactobacillus helveticus CCM 4287 I Lactobacillus crispatus CCM 7010 I
Lactobacillus crispatus CCM 7010
Lactobacillus crispatus CCM 7777
1000 900 800 700 600 500 400 300 200 100 0 1000 900 800 700 600 500 400 300 200 100 0
Lactobacillus crispatus CCM 7776
Distance Level Distance Level
1000 900 800 700 600 500 400 300 200 100 0
Distance Level
MALDI Biotyper Poster Hall 2016

Fig. 3. Dendrogram separated closely related Lactobacillus spp. correctly.
CONCLUSIONS:
MALDI-TOF MS was found as a suitable tool for identification
of bacterial isolates from dairy products.
Extension of the database of reference spectra is needed for improved
performance of the method for identification of isolates from meat products.
The method can serve to discriminate strains of the same species,
however, in the standard arrangement, some strains remain indistinguishable.
Fig. 4. Three strains of L. helveticus were differentiated successfully. Fig. 5. Strain CCM 7010 was discriminated from the remaining two indistinguishable
Equivalent results were obtained also for L. jensenii and L. delbrueckii strains. strains. Equivalent results were obtained also for L. amylovorus strains.
REFERENCES:
edo et al., Mass Spectrom. Rev. 2011: 30(3), 417-434.
Dukov et al., Int. J. Food Microbiol. 2012: 159(2), 107-114.
Kamenk et al., Acta Vet. Brno 2013: 82(2), 181-186.
edo et al., Rapid Commun. Mass Spectrom. 2013: 27(24), 2729-2736.
ACKNOWLEDGEMENTS: This work was supported by the project
CEITEC (Central European Institute of Technology, CZ.1.05/1.1.00/02.0068)
funded from the European Regional Development Fund and by the
European Social Fund (CZ.1.07/2.3.00/20.0189).
 55
Veterinary Microbiology

THE USE OF MALDI-TOF MS FOR THE

IDENTIFICATION OF MASTITIS PATHOGENS
Andrew J Bradley1,2, Caroline Hunt1 and Barbara Payne1
1 Quality Milk Management Services Ltd, Cedar Barn, Easton Hill, Easton, Wells, Somerset, BA5 1DU, UK.
2 School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, Sutton Bonington, LE12 5RD, UK.
Introduction Figure 1: MALDI-TOF MS for bacterial identification
Bovine mastitis has a complex aetiology, with over 150
different causal organisms identified. Historically, the Organism
cultured using
A small amount of the
colony is applied to a
industry standard for identification of organisms has conventional target plate and overlaid
techniques
been bacteriology supplemented with biochemical with HCCA Matrix
tests; these methods are slow, labour intensive and
prone to error in interpretation. PCR techniques offer
more rapid screening, though at the expense of being
able to determine complex aetiologies. MALDI-TOF MS
has shown promise in the field of human bacteriology(1)
and potentially offers a more rapid and robust
Sample subject to
alternative to conventional techniques for the Identification offered with
log score reflecting MALDI-TOF MS
identification of bovine mastitis organisms. likelihood of a reliable ID
MALDI Biotyper
Materials and Methods
Organisms were collated from routine laboratory
submissions and existing isolate catalogues and were
typed using routine laboratory techniques. In addition
some Staphylococcal isolates had been identified by
sequencing of the rpoB gene(2) and some of Spectra compared to
Corynebacterium spp had been typed by restriction a known database of
Spectra
in excess of 3,000
analysis of a 16S rRNA gene fragment(3). MALDI-TOF organisms
generated
MS (MALDI Biotyper (Bruker UK Ltd)) was compared to A laser is used to desorb proteins from the surface
of the material applied, these are accelerated
conventional techniques. When DNA based typing was
before drifting between electrodes. The time of
flight is used to determine the mass of the protein
available MALDI-TOF MS and conventional approaches
were both assessed. Analytical capability was assessed Figure 2: A comparison of MALDI-TOF MS with conventional
typing methods
within the genera Staphylococcus, Streptococcus,
Corynebacterium and family Enterobacteriacae.
Results
A total of 954 isolates and 101 different species were
identified by conventional means and subjected to
MALDI-TOF MS. The sensitivity and specificity of
species level identification by MALDI-TOF MS compared
to conventional and DNA based methods is summarised
in Figures 2 and 3 respectively.
Discussion and Conclusions
The most significant constraint to this study was the
need to compare to conventional methods, which have
their own inherent inaccuracies. For example, S. uberis
and Enterococcus spp are often difficult to type Figure 3: An illustration of the performance of MALDI-TOF MS
definitively using conventional methods and this may in and conventional techniques compared to DNA based typing.
part explain the apparently poorer agreement of
MALDI-TOF MS with respect to these pathogens. These
results should therefore be interpreted in this light.
Where DNA based techniques were available as a gold
MALDI Biotyper Poster Hall 2016

standard MALDI-TOF MS appeared to offer clear

benefits, outperforming conventional methods.
This study has clearly demonstrated the utility of
MALDI-TOF MS for the identification of organisms from
bovine milk, offering the opportunity to increase both
speed and accuracy of routine bovine mastitis
diagnostics.
References
(1) T.C. Dingle and S.M. Butler-Wu. (2013) Clin. Lab. Med. 33:589609.
(2) C.D. Hudson et al. (2009) Cattle Practice 17:(3)190-195.
(3) J.N. Huxley et al. (2004). J. Dairy Sci. 87(1): 38-45.
School of Veterinary Medicine and Science
MALDI Biotyper Poster Hall 2016 New Applications

56
MALDI Biotyper Poster Hall 2016
D-1146
Bacterial Antibiotic Resistance Determination by Metal Oxide-Catalyzed MALDI MS Fatty Acid Profiling
ABSTRACT
Background
MALDI-TOF MS has emerged as a rapid approach for bacterial diagnostics. However, current protein-based
methods do not address the increasing demand for antibiotic resistance profiling. As a result, additional
culture-based testing is typically required, which adds significant time and expense and further delays patient
treatment.
New approaches that combine ID and resistance profiling into a single test would drastically reduce turnaround
and improve treatment of drug resistant infections. We investigated MALDI MS fatty acid analysis as a means
of simultaneous ID and antibiotic resistance profiling. The energy inherent to the MALDI laser was used to
drive in situ metal oxide-catalyzed lipid fragmentation into taxonomically viable fatty acids using conventional
MALDI instruments already widely in clinical use. With a CeO2 catalyst in place of a traditional matrix, we
achieved strain-level ID and where able to rapidly and accurately differentiate antibiotic resistance among a
diverse collection of methicillin resistant and susceptible Staphylococcus aureus (MRSA/MSSA).
Experimental Design
Lipids from five replicates each of six MRSA and eight MSSA were extracted and fatty acid profiles obtained by
CeO2-catalyzed metal oxide laser ionization (MOLI) MS. Differentiation of resistant strains was achieved by
assembly of a database of spectral profiles followed by principal component analysis and K-Nearest Neighbor
pattern recognition. Classifications were confirmed by leave-one-out cross validation and verified by Biotyper
protein profiling. Resistance was confirmed by disc diffusion.
Results
MOLI MS fatty acid profiling resulted in 100% correct differentiation of MRSA/MSSA. Odd-numbered fatty
acids (C15:0, C17:0) were more prevalent in sensitive isolates, while a shift to even-numbered fatty acids
(C14:0, C14:1, C16:0, C16:1) was observed in resistant strains. A separate study of 160 samples
encompassing 32 strains from 14 Staphylococcus species yielded accuracies of 98% and 96% at the species
and strain level, respectively.
Conclusions
MOLI MS lipid fragmentation readily produced unique species and strain-level staphylococcal fatty acid
profiles. Results from a 160-sample study of 32 strains resulted in highly accurate differentiation of resistant
isolates. Importantly, this allowed for simultaneous ID and resistance determination with a single test without
the need for secondary methods or additional culturing.
INTRODUCTION
Diagnostic bacterial ID by MALDI-TOF MS protein analysis has gained acceptance by the clinical and
research communities following U.S. FDA and European Commission CE Mark approval of commercial
systems.
However, a significant drawback exists when using protein profiling to differentiate closely related bacterial
phylotypes. Moreover, this approach does not allow for determination of antimicrobial resistance.
To address the problem of accurate ID of closely-related isolates, we previously reported the use of rapid,
fatty acid (FA) profiling by CeO2-catalyzed MALDI-TOF (MOLI-MS).1-3
By focusing on bacterial lipids as diagnostic biomarkers rather than proteins, and by exploiting the unusual
catalytic propensity of the rare-earth lanthanide CeO2 to cleave lipids to FAs, we obtained highly accurate and
reproducible species- and strain-specific bacterial FA profiles.
We hypothesize that this was achieved through rapid, in situ conversion of bacterial lipids into FAs by the 4+
reactive state of CeO2 using the laser energy inherent to MALDI-TOF MS.
While cerium has been used in a wide range of biomedical applications,4 to our knowledge, its capacity as a
biocatalyst for in situ conversion of bacterial lipids into taxonomically viable FAs using MALDI-TOF laser
energy is novel.
By exploiting the strain-level classification capabilities of MOLI-MS, here we demonstrate simultaneous
bacterial ID and antimicrobial resistance determination in a single rapid test using conventional MALDI-TOF
MS instrumentation, which is increasingly utilized in clinical diagnostics worldwide.
GENERALIZED WORKFLOW
Figure 1. Overview of MOLI MS for diagnostic bacterial ID. A) Bacterial sampling followed by enrichment on nutrient
agar and lipid extraction. B) In situ CeO2-catalyzed lipid conversion to profilable FAs by laser irradiation in contact with
the oxide surface using a conventional MALDI laser. C) Generation of strain-specific FA profiles of each colony type.
D) Automated comparison of spectra to database for rapid ID and differentiation of drug resistant isolates. Results are
generated in tabular form in descending rank order. E) Taxonomic classification and statistical analysis allow for
automated determination of phylotypic relatedness at strain level.
C.R. Cox, N.R. Stambach, N.R. Saichek, K.J. Voorhees
Colorado School of Mines, Department of Chemistry and Geochemistry, Golden, CO.
DIFFERENTIATION OF METHICILLIN RESISTANCE IN S. AUREUS
A.
Figure 2. A) Differentiation of MRSA/MSSA by MOLI-MS fatty acid profiling. B) Fatty acid classification by FuRES
discriminant weights analysis; 95% confidence interval. Negative weights correspond to larger features in MRSA; positive
weights to larger features in MSSA.
STRAIN-LEVEL MOLI-MS FATTY ACID PROFILING
Figure 3. MOLI-MS fatty acid-based differentiation of staphylococci at (A) species and (B) strain level. Species
represented by color; strains by shape.
Figure 4. Phylotypic classification of staphylococci by MOLI-MS fatty acid profiling. A) Species- and B) Strain-level
dendrograms constructed using average linkages and Euclidean distances.
New Applications
1310 Maple St., Golden, CO 80401
303-384-2493
crcox@mines.edu
CeO2-CATALYZED MALDI-TOF MS FATTY ACID PROFILING
B.
Figure 5. Representative CeO2-catalyzed fatty acid profiles. Chain length and degree of unsaturation
indicated for all peaks.
CONCLUSIONS
Novel CeO2-catalyzed MALDI-TOF MS provides a more accurate alternative to protein-based methods.
By coupling the catalytic activity of CeO2 with MALDI laser energy, we demonstrated in situ decomposition
of staphylococcal lipids into taxonomically useful FA constituents. This allowed for highly accurate ID and
differentiation of MRSA from MSSA.
Supervised and unsupervised learning techniques yielded accuracies of 98% and 96% at the species and
strain level, respectively.
MRSA and MSSA were differentiated with 100% accuracy.
The primary advantages of this new diagnostic technique are the avoidance of misidentification of closely
related phylotypes, greatly improved accuracy, and simultaneous ID and determination of antimicrobial
resistance in a single rapid test, which can improve therapeutic management and infection control.
METHODS
Bacterial strains and growth conditions.
Staphylococci were streaked to isolation on BHI agar and incubated for 18 hr at 37o C.
Lipid extraction.
Individual colonies were suspended in 100 L of 2:1 (Vol/Vol) chloroform/methanol followed by addition of an equal volume PBS as previously
described.1
Metal oxide-catalyzed mass spectrometry.
100 mg of CeO2 was added to one mL of 2:1 (Vol/Vol) chloroform/methanol. One L was then removed from the bottom of the resulting slurry
and spotted on a stainless steel MALDI plate. Two L aliquots of bacterial lipid extracts were spotted directly onto CeO2. Negative controls
were run on SBA-15 to ensure FA spectra were the result of CeO2 catalysis and not thermal lipid desorption. All data was obtained as
described.1-3 Briefly, biological and technical replicate FA spectra were obtained in negative-ion reflectron mode with a grid voltage of 50.3%,
delayed extraction of 120 ns, and a sampling frequency of 1kHz using a 355 nm Nd:YAG laser.
Data analysis.
23 FA spectral peaks were selected, centroided, assigned nominal masses, and compiled using software written in-house. Data were imported
into the R Statistics package (Ver. 3.0.2) for PCA and leave-one-out cross-validation (CV). The prcomp function was used to calculate PCA
scores. PCA scores were plotted using the plot function. CV was conducted using the lda function from the Modern Applied Statistics with S
(MASS) package.5 Results from CV are reported as percentages of correct assignments divided by total measurements.
Processed FA profiles were analyzed with MATLAB 2014a. Generalized prediction rates were measured using three Latin partitions and 100
bootstraps.6 Two classifiers were evaluated: a fuzzy rule-building expert system (FuRES)6 and partial least squares discriminant analysis
(PLS-DA).7 The PLS-DA algorithm used two Latin partitions and ten bootstraps to calculate average pooled prediction errors.7 The number of
components (latent variables) that minimized error was selected and used to build a model from the training data, which was then used as a
prediction set. Training data consisted of a set of profiles used to build the classifiers; the test data were the set of profiles used to evaluate the
performance of these classifiers. Hierarchical cluster analysis was used to generate dendrograms and graphically illustrate Euclidean linkage
distances obtained from an agglomerative algorithm. Distances were between pairs of profiles or between averages of profiles from
subclusters.
ACKNOWLEDGEMENTS
This work was supported in part by an NIH Career Development Award through Rocky Mountain Regional
Center of Excellence for Biodefense and Emerging Infectious Diseases Research NIAID grant U54 AI065357,
and NSF MRI GRANT CHE-1229156.
LITERATURE CITED
1. Cox, C.R et al. 2015. Strain-level bacterial ID by CeO2-catalyzed MALDI-TOF MS fatty acid analysis and comparison to commercial protein-based methods. Nature
Scientific Reports. 5:10470.
2. Voorhees, K.J. et al. 2015. Comparison of metal oxide catalysts for pyrolytic MOLI-MS bacterial ID. J. Anal. Appl. Pyrolysis. 113:78-83.
3. Voorhees, K.J. et al. 2013. Modified MALDI MS fatty acid profiling for bacterial identification. J. Mass Spectrom. 48, 850-855
4. Xu, C. & Qu, X. Cerium oxide nanoparticle: a remarkably versatile rare earth nanomaterial for biological applications. Nature Publishing Group Asia Mater 6, e90 (2014).
5. Venables, W.N. & Ripley, B.D. 2002. Modern Applied Statistics with S. Springer.
6. Harrington P.B. 1991. Fuzzy multivariate rulebuilding expert systems: Minimal neural networks. Journal of Chemometrics. 5: 467-486.
7. Harrington P.B. et al. 2009. Automated principal component-based orthogonal signal correction applied to fused near infrared-mid-infrared spectra of french olive oils. Anal.
Chem. 81: 7160-7169.
8. Harrington P.B. 2006. Statistical validation of classification and calibration models using bootstrapped Latin partitions. Trends Anal. Chem. 25: 1112-1124.
All metal oxide-based MALDI-TOF technology presented herein is protected by U.S. patent # 61985919.
57
 58
New Applications

Identification of animal species from

meat using MALDI-TOF MS
Stoll, P., Rau, J.
Joerg.Rau@cvuas.bwl.de
Chemisches und Veterinruntersuchungsamt Stuttgart, Schaflandstrae 3/2, 70736 Fellbach
Introduction A B C
Consumer protection against incorrectly labeled foods is an important goal
of official food control. The declaration of the animal species is especially (pig)
crucial for meat. Until now mostly molecular and immunological methods wild boar
have been used for the analytical confirmation.
pig
Mass spectrometry (MS) [1,2,3] offers a new approach to the species iden-
tification of animals. MALDI-TOF MS combines a matrix-assisted laser de- onager
sorption/ionisation (MALDI) with a time of flight (TOF) analyzer and MS horse
(cattle)
(Fig. 1). Hereby, ionizable large biopolymers, such as proteins from mi-
sheep
croorganisms, fish or meat, can be analyzed softly. The prevalence of this
technology is rapidly increasing and MALDI-TOF MS is especially applied goat
for the differentiation of bacteria. roe deer
The identification of an unknown cattle
sample is achieved by compa- (chicken)
turkey
ring the resulting mass spect-
rum with the references in a da- chicken
tabase. The database therefore
is central for success. For iden-
tification of animal species from
meat, however, no commercial
database is available. Fig. 4: A Dendrogram of MSPs from meat of different animal species
B Comparison of MSPs. Above: sample; below: reference-MSP from the database
C Hitlist of samples, depicting the identity with database entries, by descending
Fig. 1: MALDI-TOF MS score value
Reference Samples/ Sample Preparation Meat Database
As a reference for creating the database, raw muscle meat from livestock, So far, 51 independent data entries of 29 different animal species were
zoo, pet and wild animals was provided by our veterinary post mortem pa- added to the CVUAS-database. These generated MSPs can be trans-
thology. ferred to other equipment within the same system. A selection of existing
database entries with additional information to the current status is listed
Based on Post & Dikler 2010 [1] a sample preparation protocol
on http://www.maldi-up.ua-bw.de/ [4].
optimized for meat was developed:
In 1.5 ml safe-lock tubes, around 5 mg Validation
musculature were crushed with a pestle
and 5 mg zirconia/silica beads 0.1 mm
For the validation of the system the specificity and selectivity of each para-
(BioSpec), meter were determined using independent reference samples consisting
in a mixture of 50% acetonitrile, 47.5% of muscle meat. As an example the summary of the results for the para-
H2O and 2.5% trifluoroacetic acid.
15 s extraction on a laboratory mixer,
meters Sus scrofa (pig), Bos taurus (cattle), and Gallus gallus (chicken)
centrifugation for 120 s at 14000 rpm. are depicted (Fig. 5).
1 l supernatant was transferred onto a
steel target, dried at room temperature
(ca. 60 s), overlaid with 1 l matrix-solu-
tion (-Cyano-4-hydroxycinnamic acid,
HCCA, Bruker). Fig. 2
The HCCA was dried and gently crystallized at room temperature (ca. 120 s).
Processing the Spectra / Creation of the Database
The loaded target can be directly measured in the MALDI-TOF MS (Bio-
typer LT-microflex, Bruker Daltonik, Bremen, Germany) (Fig. 2). The raw
spectra collected in the range of 2-20 kDa are reduced to MSPs (Main
Spectra Projections) (Biotyper 3.0, Bruker) (Fig. 3). These can be included Fig. 5: Result of validation: left pig, right cattle and chicken.
as a reference in an own database. The resulting MSPs of different animal top left sensitivity; middle right specificity; # = not
species vary considerably (Fig. 4). Conclusion / Future Prospects
The result for a sample is given as a score-value by the Biotyper system, A transferable method, fit for the routine identification of a first selection of
which reflects the correspondence of the sample MSPs with the database animal species from raw muscle meat using MALDI-TOF MS, is establis-
entries. hed.
It consists of an optimized sample preparation, own database entries and
the first parameter validations.
The speed (<1h), ease of use and low cost of consumables already enable
MALDI Biotyper Poster Hall 2016

Fig. 3: Mass spectrum from beef (above)

and resulting MSP (main spectra
projection; below)
the screening of animal species.
Further additions to the database (animal species, technological proces-
sing steps) and the continued formal validation of parameters will increase
the acceptance of this method.
This work has been carried in full by P. Stoll during his internship in his 3rd year of studies of bio-
technology at the University of Applied Sciences Esslingen.
This is a translation of a poster presented on the 44. Deutschen Lebensmittelchemikertag,
14.09.-16.09.2015 Karlsruhe, Germany.
References
[1] Post A, Dikler S (2010) FACSS 2010, 583
[2] Stephan R et al. (2014) Food Control 46: 69
[3] Flaudrops C et al. (2015), J. Food Comp. Anal. 41: 104-112
[4] http://www.maldi-up.ua-bw.de
MALDI Biotyper Poster Hall 2016 Typing of microorganisms by FT-IR

Differentiation of Klebsiella pneumoniae Strains

Bruker Corporation
+49 421 2205-1258

by Fourier Transform Infrared Spectroscopy

Markus.Kostrzewa@bruker.com

N. Mauder1, T. Eisenberg2, A. Fawzy2, J. Rau3, and M. Kostrzewa1

1) Bruker Corp., Bremen, Germany [BR], 2) Hessian State Lab., Gieen, Germany [HL], 3) Chemical and Vet. Investigations Office, Stuttgart, Germany [CV]

D-716, ICAAC 2015

1800 1500 1200 900 600 300 Distance

Introduction
HL_00357

New groups were formed out of data preprocessing:

BR_00066
2 HL_00311
HL_00215
Many strains produced a pattern
HL_00320

confused strains and the new group 2nd derivative 800-1300 cm-1 vector
normalization PCA (95% variance)
CV_04888.2
HL_00310
CV_00283 with very few common bands which
Fourier transform infrared
HL_00297

composition was validated as just LDA squared Mahalanobis distance

clustered to a big group not allowing
CV_04890.2
BR_00024 exceptional broad distribution of
matrix from 6 LDA axes BR_00068
BR_00045 spectra measured at lab HL
spectroscopy (FT-IR) is a well-
BR_00070

described. further differentiation.

BR_00071 overlap with BR_00078
CV_01701
3 HL_00263

established technology that has also

BR_00078
HL_00275
HL_00357b

In parallel the strains were checked HL_01168

HL_00600

been used for the identification of 1 CV_10109

BR_00046

by randomly amplified polymorphic 3 BR_00047

Summary
BR_00111

microorganisms at species level and

BR_00008
BR_00125

DNA (RAPD) analysis.

3 BR_00045
BR_00106

even below. It has been applied to (1) BR_00064

BR_00037

FT-IR proved to be suitable for

BR_00121
HL_00243

many species, is fast and

BR_00069
HL_00620
HL_00273 spectra of
HL_01386
differentiating K. pneumonia strains
economical. This multi-center study
Results
HL_01717
BR_00021 BR
3 BR_30104
CV_09644
1 BR_00044 CV with a discriminatory power
evaluated the power to discriminate CV_04886.2
3 CV_00353

outperforming RAPD-PCR. The

CV_04872.2
HL
Comparability of grouping. The
HL_01088

bacterial strains of Klebsiella (K.) 3 BR_00101

BR_00077

system was suitable to be used

BR_00116

three labs could discriminate 46

3 HL_00338

pneumoniae, a clinically relevant

CV_09653.2
CV_04889.2

across labs for the species under

CV_04887.2
BR_00065

(BR), 43 (CV), and 45 (HL) very

3

pathogen. 1 BR_00032
BR_00038
HL_00251

similar groups composed of 60

2 HL_00967
BR_00079
HL_00003
study. It may enable fast and better
strains. 14 of the 180 (360) group tracking of outbreak strains or to
Hierarchical cluster analysis (HCA; UPGMA) of 60 isolates, measured at three
Methods assignments differed from the labs. Consensus groups are separated by alteration of black and grey letters. follow contamination routes in the
consensus grouping. The positive Numerals at the nodes indicate the number of labs that found this grouping. future. Thereby, FT-IR may be a
60 K. pneumoniae strains were Coloration indicates the lab where an exception occurs (BR, CV, HL). valuable tool to monitor isolates in
controls (two identical strains
measured in transmission mode at Principal component analysis of spectra of seven differently confused strains clinical, pharmaceutical or food
stocked in separate labs for years)
three different labs equipped with is shown on the left side. processing environments and as a
were always grouped together as
the same infrared spectrometer Ellipses represent the 95% concentration assuming bivariate normal pre-scanning method to reduce
expected. The identification of group
hardware, using the same distribution. workload for reference laboratories
spectra had mean intra-lab recall
incubation and measurement in outbreak scenarios.
rates of at least 99.8%.
parameters. Each lab conducted at Different groupings were the main Molecular typing. RAPD-PCR
least three independent cause for bad single recall rates. analysis could differentiate 42
60 strains
measurements in duplicate. Spectra With a broader definition of 39 strains into 19 groups. Comparison
of each strain were used to train and common groups these values of the grouping by these two
validate linear discriminant analysis BR CV HL
increased to 99.7%, 99.8% and methods gave somewhat different Conclusions
(LDA) models with stratified 97.8%. but mostly plausible results.
Good discriminatory power
three labs
measured
randomized sets 200 times while 32 spectra
per strain
adding up the confusion matrices.
Reproducibility between labs is
positive control DSM 30104:
grouping & BR_30104 & CV_09644
46 groups 43 groups 45 groups must be indistinguishable
internal cross
99.8% 99.8% 99.97%
validation mean recall mean recall mean recall
60 strains
6 (32) spectra per strain 200 times outgroup: HL_00357 high
initially one strain per group model model model
inter-lab
stratified, random
50:50 split
cross
validation
98.7% 96.9% 94.2%
Consistency of data is
paramount
mean recall mean recall mean recall
training validation (82.4% worst) (23.1% worst) (31.3% worst)
merge groups according
to confusion matrix
model
until all recall rates
satisfactory (e.g.>95%)
add to
Soft group assignment like HCA
Cross lab validation. Training of
confusion might be preferable
matrix an LDA model with the spectra and
group assignment of one lab and Strict grouping into spectrovars
group 07

group 11

group 03

group 14

group 36

group 60

group 59

group 48

group 13

group 07 31%
group 11 50%
0%
95%
0%
20%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0% validation with the spectra of the
group 03 19%
group 14 0%
5%
0%
80%
0%
0%
64%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
other labs resulted in mean recall is mainly necessary to quantify
group 36 0% 0% 0% 36% 100% 0% 0% 0% 0% 0% partially confused at HL
data preprocessing:
rates of 98.7%, 96.9% and
group 60 0% 0% 0% 0% 0% 70% 0% 0% 0% 0% due to high variance:
group 59 0%
group 48 0%
0%
0%
0%
0%
0%
0%
0%
0%
30%
0%
100%
0%
0%
100%
0%
0%
0%
0%
2nd derivative 800-1300 cm-1 vector normalization BR_00045 & BR_00078 discriminatory power.
94.2%.
group 13 0% 0% 0% 0% 0% 0% 0% 0% 100% 0%
100% 100% 100% 100% 100% 100% 100% 100% 100% 100%

Original data: IR spectra of five strains measured at three labs.

FT-IR spectroscopy
For research use only. Not for use in diagnostic procedures.

59
 chia Streptomyces Bartonella HafniaTerrimonas Pseudoxanthomonas Corynebacterium Streptococcus Aerococcus Saccharothrix Facklamia SchizosaccharomycesTetrageno
alieria Clavibacter Shimwellia Brachybacterium Leclercia Providencia Trabulsiella Xanthobacter Emericella Gardnerella Sporothrix Leuconostoc Pseudoclavibacter Alkali
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cillus Aspergillus Arthrobacter Mesorhizobium Acholeplasma Filobasidium Propioniferax Azohydromonas Chromobacterium Curtobacterium Kloeckera Austwickia Hyphomic
coccus Rummeliibacillus Budvicia Aquincola Enterobacter Sporobolomyces Brevundimonas Capnocytophaga Tatlockia Neisseria Salinivibrio Pullulanibacillus Arcanoba
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onas Rhodococcus Bergeyella Malikia Actinocorallia Aeromonas Micromonospora Alcaligenes Alistipes Pannonibacter Dickeya Kocuria Ochrobactrum Agrococcus Gracili
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hyton Granulicatella Leptothrix Suttonella Dermabacter Hydrogenophaga Cellulomonas AzoarcusTrichosporonTatumella Rhodospiridium Acidaminococcus Actinobacillus
hum Fusobacterium Lodderomyces Anaerococcus Colletotrichum Edwardsiella Comamonas Pigmentiphaga Moellerella Nesterenkonia Lis
myces Erysipelothrix Escherichia Streptomyces Bartonella Hafnia Terrimonas Pseudoxanthomonas Corynebacterium Streptococcus Aero
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