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Introduction
Identifying bacteria with MALDI-TOF (Figure 1.) a single isolated colony (A) or
simple cell extract is spotted onto a stainless steel target plate (B) and overlaid
with an ultra violet absorbing molecule. The target plate is inserted into the
MALDI-TOF (Bruker Microflex) instrument (C). Nitrogen pulsed laser ionization is
applied to the sample and the proteins are ionized. They are then separated
based on their mass/charge ratio (D). The resulting spectra, a protein finger print
(2,000 20,000 Dalton range) are compared to a database of known spectra
(Burker BioTyper) (E). Processing time is quicker than both genotypic and
biochemical technologies to obtain an identification, taking 10 minutes for the first
sample to be read with an additional 1 minute/sample thereafter. A match factor
for each sample is calculated based on the number and amplitude of the peaks
that match as well as the number of peaks that do not match the known spectra.
This study evaluates MALDI-TOF technology for its ability to correctly identify
environmental isolates.
A B
D
Ribosomal Spectra
Methods
A set of 31 known bacterial strains from culture collections and 146 different
strains representing 43 different species know to frequently occur in
Pharmaceutical and manufacturing environments were used for this study.
Identities of all organisms and strains were verified using 16S sequencing cross
referenced against the Accugenix library database.
Bacteria for each experiment were tested using both the direct spotting method
and extract method (ethanol-formic acid-TFA) per manufactures
recommendations.
Results
20.3%
26.1%
Secure Species ID
Probable Species ID
Probable Genus ID
No ID
22.7% 30.9%
30.2%
Species
No ID
69.8%
Figure 4. Performance of sample identifications with the match factor cut off set
at 1.75 for a species level identification using DNA sequencing as a reference.
Overall Performance of MALDI-TOF (n=1873)
1.8%
5.9%
Species
22.5%
No ID
MisIdentification
No Spectra
69.8%
Discussion
17.1%
Species
No ID
82.7%
The percentage of sample identifications increased from 69.8% to 82.7% with the
addition of 15 relevant species to the AccuPRO spectra database (match factor
cut off set at 1.75 for a species level identification).
Conclusions
The low cost, ease of use and rapid turnaround time of MALDI-TOF analysis are
strong advantages of this technology. A customized database optimized for
environmental monitoring of isolates commonly encountered in the
Pharmaceutical and related manufacturing industries are required for better
performance. The ability of shipping ethanol extract samples to a contract
service provider is an inexpensive proposition, as well as a technical benefit
because temperature and time dependencies are eliminated.
About Accugenix:
For over 20 years, Accugenix has been the worlds leading FDA-registered,
cGMP service laboratory for microbial identification and characterization. The
company has tested and identified more microorganisms than any other
industrial company or service laboratory, which has allowed Accugenix to
develop and refine a
validated proprietary library and method of interpretation unrivaled in
industry. More than 1000 facilities around the world rely on the companys
expertise to meet their microbial identification needs.
Try out AccuPRO-ID for free! Simply submit 5 bacterial samples and write the
redemption code: Pharmig-AD in the Comments column on the Identification
Request Form (AccuPRO-ID IRF Page 1). To access the form, please visit us at
http://accugenix.com/info_ref.php. For questions concerning sample submission
contact our Technical Support Organization at technicalsupport@accugenix.com
or call 1 302.292.8888.
Mark Calmann
Sr. Product Manager
Accugenix
223 Lake Drive
Newark, DE 19702
markcalmann@accugenix.com