Tim Sandle

Introduction
Topics:
Identification of environmental monitoring
contaminants.
What, why and when.
Questions of culture media and incubation for
environmental monitoring.
Other types of environmental monitoring.
Microbial identification systems
Range of different
systems available
Vary on:
Phenotypic
Genotypic
Databases
Cost
Reliability
Ease of qualification
Phenotypic systems
Typically incorporate
reactions to different
chemicals or different
biochemical markers.
Based on a small number
of tests.
Growth dependent
methods.
Require pure cultures.
Examples: API, VITEK,
OmniLog.


Phenotypic systems
More recent:
Mass spectrometry e.g.
MALDI-TOF
Analyses microbial
proteins.
Flow cytometry
Limited to the detection
and discrimination of
viable culturable, viable
nonculturable, and non-
viable organisms.
Genotypic systems
Genotypic methods utilise
one of two alternatives:
hybridization or
sequencing
Most commonly of the
gene coding for 16S rRNA.
With hybridisation, this is
DNA-DNA homology (or
how well two strands of
DNA from different
bacteria bind [hybridize]
together).

Genotypic systems
Pulsed-field gel electrophoresis (PFGE)
Multilocus sequence typing (MLST)
Ribotyping
Repetitive sequence-based PCR (rep-PCR)
DNA Microarrays
Why carry out identifications?
To look for changes from the norm.
Changes from the norm may signal cleaning or
disinfection concerns.
In the event of a sterility test failure.
Only genotypic comparison can invalidate.
To assist with investigations into out-of-limits results.
Grouping microorganisms into different categories.

Types of contamination
Human related:
Skin:
Staphylococcus
Micrococcus
PropionibacteriaCory
neforms.
Low levels of
Acinetobacter.
Oral:
Streptococcus

Environmental:
Bacillus and related genera.
Fungi.
Water:
Pseudomonads and related
genera.
Reference:
Sandle, T. (2011): A Review of
Cleanroom Microflora: Types,
Trends, and Patterns, PDA
Journal of Pharmaceutical
Science and Technology, Vol. 65,
No. 4, JulyAugust 2011, pp392-
403

How many identifications?
Grade A
Identify all microorganisms
These should be few
They raise concerns about a breakdown of control
Grade B
Identify when a Grade A event has happened
E.g. Was similar contamination found near an isolator port?;
Was something similar recovered from an operators gown?
When at action level:
Identify other times on the basis of sample location and risk.
How many identifications?
Lower grade cleanrooms:
Identify a selection over the course of a year to examine for
changes from the norm
Maybe 25%?
Which level to identify to?
Depends on the level, location and risk rating.
Helpful to risk assess environmental monitoring locations.
Depends on what is to be done with the data
Batch rejection decisions species level
Concerned about disinfectants species for GPSR and GNR only
Benchmarking - a selection only
Checking for staff aseptic practices Gram stain may be sufficient

Visual identification
Good for a quick check on
differences:
Size & colony shape (circular,
irregular, rhizoid)
Colony edge (smooth,
filamentous, undulating)
Elevation (flat, raised, convex,
crateriform)
Surface (wrinkled, rough, waxy,
glistening)
Opacity (transparent, translucent,
opaque)
Pigmentation
Colour (red, yellow, white)
Water solubility (water soluble -
colour tints surrounding media)

Disinfectant resistance
How many identifications?
Environmental monitoring
remains a spot check for
indicators of cleanroom
contamination.
Just as you cannot capture
everything, you cannot
identify everything.
However, it is useful to
know what your most
frequent isolates are.
Disinfectant efficacy studies
Are the challenge organisms used in disinfectant
effectiveness studies representative of the organisms
found in the environment?
Are the necessary log reductions achieved when the
organisms are exposed to disinfectants?
FDA have an expectation of environmental isolates.
Trending
Reviewing efficiency of cleaning and disinfection
regimes e.g. are spore formers or Gram-negatives
surviving?


Media growth promotion
There may also be implications for the test panel used
for media growth promotion.
The use of environmental isolates is a longstanding
debate:
Are environmental isolates representative of the stressed
or sublethally damaged microbial forms more
commonly found within the pharmaceutical
environment;?
Or do they resemble laboratory cultures, of a type
adjusted to grow readily on enriched laboratory culture
media ?

Environmental monitoring
What is the point of environmental monitoring?
Environmental control is more important than
monitoring.
Individual results do not tell us very much.
The methods of monitoring are variable and each carries
a degree of inaccuracy.
Rapid and alternative methods are promising but, as yet,
unproven.
Culture media.
Culture media
Between 70 and 90% of microorganisms in the
environment are viable but non-culturable
There is no single culture medium that will detect all
of the culturable microorganisms.
Does this matter?
Should two different media be used?
There are also the variants of:
Incubation time,
Incubation temperature.
Incubation time
Avoiding the chicken or the egg scenario:
Incubation time needs to be decided once the dual
media debate and incubation temperature questions
have been decided.
Maximum incubation time should be assessed for:
At what point do colony forming units stop appearing?
Is there a point when the media ceases to be able to support
slow-growing microorganisms?
Incubation time
Studying incubation time:

Sandle, T., Skinner, K. and
Yeandle, E. (2013). Optimal
conditions for the recovery of
bioburden from
pharmaceutical processes: a
case study, European Journal
of Parenteral and
Pharmaceutical Sciences, 18
(3): 84-91





Studying the effect of
desiccation of media
following UDAF exposure
and at the end of
incubation:

Sandle, T. (2011): 'Microbial
recovery on settle plates in
unidirectional airflow
cabinets', Clean Air and
Containment Review, Issue 6,
pp8-10

The one or two media debate
Should two different culture media be used?
One to recover bacteria
One to recover fungi
Raises two questions:
A) If it is necessary, which two media?
B) Is it really necessary?
Optimal fungal medium
It is generally accepted that TSA is a good, general medium
for the recovery of bacteria.
If two media are used, what is the appropriate fungal
medium?
Study:

Gebala, B. and Sandle, T. (2013). Comparison of
different fungal agar for the environmental
monitoring of pharmaceutical-grade cleanrooms, PDA
J Pharm Sci Technol.;67(6):621-33



Fungal media
Malt Extract Agar
Sabouraud Dextrose
Agar
Rose Bengal Agar
Potato Dextrose Agar
Fungal media study #1
Cleanrooms in south-east England
EU GMP Grade C and Grade D.
Media:
Malt Extract Agar (MEA),
Malt Extract Agar with Penicillin and Streptomycin
supplement (MEP),
Potato Dextrose Agar (PDA) containing no antibiotics,
Rose Bengal Agar (RBA) with chloramphenicol
Sabouraud Dextrose Agar (SDA) with chloramphenicol.

Fungal media study #2
Aims:
To assess if there is any significant variation in the
number of fungal isolates recovered by five selective
agars.
To assess any variation in recovery of different species or
genera by the selective agars.

Fungal media study #3
Outcomes:
Recovery of fungi was relatively low.
Mean counts varying between 0.1 and 7.8 colonies per sample
type.
Higher results from active air samplers.
Lowest results from surface contact plates.
More filamentous fungi in the environment than yeasts.

Fungal media study #4
Agars
Examined for significance using Students t-test
Yeats: MEA agar, followed by SDA, recovered the
greatest variety. PDA recovered the lowest variety.
Filamentous fungi: Largest variety recovered on RBA,
second SDA, and the smallest variety from MEA.
PDA was less selective, recovering higher numbers of
bacteria.
Optimal agar: SDA.

Fungal media study #5
Main types:
Top group: Cladosporium spp. and Penicillium spp.
Middle group: Aspergillus spp. and Bionectria sesquicillii
Samuels (anam. Clonostachys).

One medium
Regulators express an interest but no documents
produced by regulatory agencies mention types of
media required.
FDA guidance for environments used for aseptic filling
requires the agar to have undergone growth promotion
of bacteria and fungi.
Arguments against 2 media:
Unnecessary TSA will grow bacteria and fungi,
Increase the number of aseptic manipulations,
Costly.

One medium
Many sites use one general purpose culture medium
incubated at two temperatures:
30-35oC, to encourage the recovery of skin
commensurable bacteria;
Based on the skin microbiota.
Staphylococci, Micrococci & Corynebacteria.
20-25oC, designed to recover fungi.
Based on the growth characteristics of most fungi.
Alternaria, Trichophyton, Aspergillus & Cladosporium.

One medium: What is the appropriate order of
incubation?

Higher temperature:
Majority of
microorganisms recovered
are mesophilic bacteria.
Few fungi are recovered.
Filamentous fungi could
grow and obscure bacteria.
Destruction of lytic
cellular enzymes in fungi
if high temperature first.


Lower temperature:
Higher temperature may inhibit
fungal growth.
Higher temperature may
damage fungal enzymes, leading
to no recovery.
Avoids overgrowth of mycelia
obscuring bacterial colonies if
low temperature first.
Some bacteria may not grow at
this temperature.

One medium
Study:

Sandle, T. (2014) Examination of the Order of
Incubation for the Recovery of Bacteria and Fungi from
Pharmaceutical Cleanrooms, International Journal of
Pharmaceutical Compounding, 18 (3): 242 247



Order of incubation study #1
Medium:
Tryptone soya agar (aka = soya-bean casein digest
medium, tryptic soya agar)
Referenced in EP, USP & JP.
2 approaches:
In situ study: assessing microorganisms recovered from
a cleanroom environment.
In vitro study: plating out cultured microorganisms onto
TSA.

Order of incubation study #2
Two scenarios for a two-tiered incubation scheme:
Incubate at a higher temperature first, followed by the lower
temperature:
Regime A: 30-35
o
C for 2 days, followed by 20-25
o
C for 5 days
Incubate at a lower temperature first, followed by the higher
temperature:
Regime B: 20-25
o
C for 5 days, followed by 30-35
o
C for 2 days
One of two possible outcomes:
That there is a significant difference between the two
incubation regimes.
That there is not a significant difference between the two
incubation regimes.


Order of incubation study #3
Taking note of:
Time taken for the incubated plates to reach the
required temperature;
Time that the plates are placed into the incubator;
The day and the time that the plates are removed from
the incubator (either for temperature transfer or final
read).
Order of incubation study #4
Study aspects:
39 cleanrooms: EU GMP Grade C / ISO 14644 class 8 (in
operation) and EU GMP Grade D.
Changing rooms;
Wash bays;
Corridors;
Ambient processing areas;
Cleanrooms subject to a warmer temperature (such as an autoclave
preparation area);
Cold rooms (operating at 2-8C).
Surface samples taken (contact plates).
Two enable close to possible duplicate sampling.
136 samples.

Order of incubation study #5
Regime Number and
percentage of samples
recording higher
counts (n=136)
Mean total colony
count (CFU)
A
39 (28.7%) 8
B
54 (39.7%) 11
Data sets where results
were equivalent
43 (31.6%) <1
The data for the total count (bacterial and fungal colonies) was analysed using
an unpaired Students t-test (0.05 significance level, 95% confidence interval).
Order of incubation study #6
The total colony count results for the incubation
regime B gave a higher mean count than those from
incubation regime A
A = mean count of 8 CFU/plate
B = mean count of 11 CFU/plate.
This difference was shown not to be statistically
significant .
Therefore there is no optimum incubation regime for
total count.
If you do not find many fungi, then there is no need to
explore further.

Order of incubation study #7
What if fungi are recovered regularly?
Out of the 136 data sets, 15 sample results showed
fungal colony growth.
A greater number of fungal colonies were isolated by
incubation regime B incubation regime.
Regime A recovered ~ <1 fungal colony
Regime B recovered ~11 fungal colonies.
To determine whether this was significant or nor
required a second Student's t-test to be constructed.

Order of incubation study #8
This time, there the difference was shown to be
statistically significant .
Therefore incubation regime B produces a higher
fungal count.
Regime B: 20-25
o
C for 5 days, followed by 30-35
o
C for 2
days


Order of incubation study #9
Does this matter?
Perhaps if there is a lot of fungi recovered?
But should fungi be being recovered in high numbers?
The most important element is consistency of practice:
to use the same incubation parameters so that results
can be meaningfully compared over time.
Growth promotion testing
Growth promotion testing of media is important, to
demonstrate:
To ensure that monitoring results are not affected by the
quality of the media used.
That the collection and recovery of microorganisms will
not be affected by the presence of inhibitory substance
(such as disinfectant residues).
With a single medium, it is important to have a test
panel made up of bacteria and fungi.
There is a long-standing debate with the use of
environmental isolates.
Other types of environmental monitoring #1
Anaerobic:
Environmental monitoring may need to be adjusted for
anaerobic microorganisms, given the relatively high
levels of Propionibacterium spp. associated with hair
follicles.
Where nitrogen gas or compressed air lines are used as part of
the filling.

Other types of environmental monitoring #2
Thermophilic
No case at all
Pyschrophilic
Highly unlikely
Cold rooms may contain psychrotolerant organisms, but most
of these will grow under mesophilic conditions.
Few true psychrophiles expected.
See: Sandle, T. and Skinner, K. (2013). Study of psychrophilic
and psychrotolerant microorganisms isolated in cold rooms
used for pharmaceutical processing, Journal of Applied
Microbiology, 114 (4), 11661174
Summary
An identification strategy for cleanroom isolates.
Whether one or two culture media should be used for
environmental monitoring.
The optimal incubation regime, where one medium is
used.
The implications of the human microbiome project.
Pharmaceutical microbiology resources:
http://www.pharmamicroresources.com/

For copies of any papers, please email: tim.sandle@bpl.co.uk