Contemporary issues in Pharmaceutical Microbiology

Contemporary issues in Pharmaceutical MicrobiologyTim SandleTitle page1IntroductionTopics:Identification of environmental monitoring contaminants.What, why and when.Questions of culture media and incubation for environmental monitoring.Other types of environmental monitoring.Topics:

Identification of environmental monitoring contaminants.What, why and when.Questions of culture media and incubation for environmental monitoring.Other types of environmental monitoring.

2Microbial identificationMicrobial identification3Microbial identification systemsRange of different systems availableVary on:PhenotypicGenotypicDatabasesCostReliabilityEase of qualification

Range of different microbial identification systems availableVary on:

PhenotypicGenotypicDatabasesCostReliabilityEase of qualification

4Phenotypic systemsTypically 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

Typically incorporate reactions to different chemicals or different biochemical markers.

These systems are:

Based on a small number of tests.Growth dependent methods.Require pure cultures.Examples: API, VITEK, OmniLog.

5Phenotypic systemsMore recent:Mass spectrometry e.g. MALDI-TOFAnalyses microbial proteins.Flow cytometryLimited to the detection and discrimination of viable culturable, viable nonculturable, and non-viable organisms.

Phenotypic systems

More recent:

Mass spectrometry e.g. MALDI-TOFAnalyses microbial proteins.

Flow cytometryLimited to the detection and discrimination of viable culturable, viable nonculturable, and non-viable organisms.

6Genotypic systemsGenotypic 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

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).

7Genotypic systemsPulsed-field gel electrophoresis (PFGE)Multilocus sequence typing (MLST)RibotypingRepetitive sequence-based PCR (rep-PCR)DNA MicroarraysPulsed-field gel electrophoresis (PFGE) a technique that allows the electrophoretic separation of low numbers of large DNA restriction fragments produced using restriction enzymesMultilocus sequence typing (MLST) sequencing 400-500 base pair fragments of DNARibotyping this technique relies on the relative stability of the 16S and 23S rRNA genes coding for ribosomal RNA. The genes are cut using restriction enzymes and resulting DNA fragments separated by electrophoresis. The resulting fingerprint is visualised using fluorescent probes.Repetitive sequence-based PCR (rep-PCR) bacterial and fungal genomes contain numerous non-coding, repetitive DNA sequences separating longer, single copy, sequences and their arrangement varies between strains.DNA Microarrays a microarray is a collection of DNA probes attached in an ordered pattern onto a solid surface. These probes can be used to detect the presence of complementary sequences in bacterial isolates, and thus can detect marker genes in specific bacterial strains.8Why 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.

Why identify?

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.

9Types of contaminationHuman related:Skin:StaphylococcusMicrococcusPropionibacteriaCoryneforms.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

Types of contamination

Human related:Skin:StaphylococcusMicrococcusPropionibacteriaCoryneforms.Low levels of Acinetobacter.Oral:Streptococcus

Environmental:Bacillus and related genera.Fungi.Water:Pseudomonads and related genera.

Paper ref.10How many identifications?Grade A Identify all microorganismsThese should be fewThey raise concerns about a breakdown of controlGrade BIdentify when a Grade A event has happenedE.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 Ids?

Grade A Identify all microorganismsThese should be fewThey raise concerns about a breakdown of controlGrade BIdentify when a Grade A event has happenedE.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.

11How many identifications?Lower grade cleanrooms:Identify a selection over the course of a year to examine for changes from the normMaybe 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 dataBatch rejection decisions species levelConcerned about disinfectants species for GPSR and GNR onlyBenchmarking - a selection onlyChecking for staff aseptic practices Gram stain may be sufficient

How many Ids?

Lower grade cleanrooms:Identify a selection over the course of a year to examine for changes from the normMaybe 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 dataBatch rejection decisions species levelConcerned about disinfectants species for GPSR and GNR onlyBenchmarking - a selection onlyChecking for staff aseptic practices Gram stain may be sufficient12Visual identificationGood 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)PigmentationColour (red, yellow, white)Water solubility (water soluble - colour tints surrounding media)

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)PigmentationColour (red, yellow, white)Water solubility (water soluble - colour tints surrounding media)

But GMP?13Disinfectant resistance

Hierarchy of disinfectant resistance14How 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.

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.

15Disinfectant efficacy studiesAre 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.TrendingReviewing efficiency of cleaning and disinfection regimes e.g. are spore formers or Gram-negatives surviving?

What about disinfectant efficacy? Are we using the appropriate panel of microorganisms? Do we need to review the test panel?

I think the answer here is yes we need to review.

The most frequently isolated microorganisms from an environmental monitoring programme could be periodically examined against our in-use disinfectants.

For example, we could subject the main isolates to microbroth dilution testing with cleanroom disinfectant agents, to confirm the sensitivity profile or alternatively, to conduct a surface coupon test.

Or work out the Minimum Inhibitory Concentration (lowest concentration of disinfectant that can inhibit microbial growth).

We can also trend isolates to see what might be surviving and therefore be resistant. If Gram-negatives or endospore forming microorganisms are regularly surviving this may suggest weaknesses with our cleaning and disinfection regimes.

16Media growth promotionThere 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 ?

There may also be implications for broadening the test panel used for media growth promotion.

If so, should these be environmental isolates and, if so, should these be introduced to support the release of microbiological culture media? Similar set to disinfectant efficacy panel.

The use of environmental isolates as an alternative type cultures is a longstanding debate.

The issue is, once subcultured in the lab:

Whether environmental isolates remain representative of the stressed or sublethally damaged microbial forms more commonly found within the pharmaceutical environment;

Or whether they resemble laboratory cultures, of a type adjusted to grow readily on enriched laboratory culture media (cf Brown and Gilbert, 1995).

Interesting debatenot answered here!

17Environmental monitoring & culture mediaEM & culture media18Environmental monitoringWhat 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.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.

19Culture mediaBetween 70 and 90% of microorganisms in the environment are viable but non-culturableThere 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.Culture media limitations

Between 70 and 90% of microorganisms in the environment are viable but non-culturableThere 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.20Incubation timeAvoiding 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

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?

21Incubation timeStudying 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

Some references:

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): 84-91Studying 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

22The one or two media debateShould two different culture media be used?One to recover bacteriaOne to recover fungiRaises two questions:A) If it is necessary, which two media?B) Is it really necessary?

One or two media?

Should two different culture media be used?One to recover bacteriaOne to recover fungiRaises two questions:A) If it is necessary, which two media?B) Is it really necessary?

23Optimal fungal mediumIt 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

What is the best 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

24Fungal media

Malt Extract Agar

Sabouraud Dextrose Agar

Rose Bengal Agar

Potato Dextrose AgarThese are selective agar designed, through pH or the presence of antibiotics to inhibit bacteria and promote the growth of fungi.25Fungal media study #1Cleanrooms in south-east EnglandEU 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 chloramphenicolSabouraud Dextrose Agar (SDA) with chloramphenicol.

My facility undertook a study, looking at different fungal agar

Cleanrooms in south-east EnglandEU 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 chloramphenicolSabouraud Dextrose Agar (SDA) with chloramphenicol.

We had limited resources, although plan to do more.

26Fungal media study #2Aims: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.

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.

27Fungal media study #3Outcomes: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.

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.

28Fungal media study #4AgarsExamined for significance using Students t-testYeats: 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.

Agars

Examined for significance using Students t-test

Yeasts: MEA agar, followed by SDA, recovered the greatest variety. PDA recovered the lowest variety.b: 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.

29Fungal media study #5Main types:Top group: Cladosporium spp. and Penicillium spp. Middle group: Aspergillus spp. and Bionectria sesquicillii Samuels (anam. Clonostachys).

In terms of what was recovered.

Main types:

Top group: Cladosporium spp. and Penicillium spp.

Middle group: Aspergillus spp. and Bionectria sesquicillii Samuels

30But are two media really necessary?But are two media really necessary?31One mediumRegulators 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.

Regulators express an interest in fungi and environmental monitoring, but no documents produced by regulatory agencies mention types of media required.

One exception is the Japanese Pharm.

However, FDA guidance for environments used for aseptic filling (2004), requires the agar to have undergone growth promotion of bacteria and fungi.

Many sites use one general purpose culture medium incubated at two temperatures: Higher one: 30-35oC, to encourage the recovery of skin commensurable bacteria;Lower one: 20-25oC, designed to recover fungi

32One mediumMany 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 considerations

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.33One 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.

If the temp wrong and either:

Temp too high = destruction of lytic cellular enzymes leading to underestimation of the amount of fungi.Temp too low = overgrowth of mycelia obscuring bacterial colonies.

34One mediumStudy:

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

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 24735Order of incubation study #1Medium: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.

With in situ do not use clinical isolates, ensure they are representative

Decided on in situ because the morgs would be representative of the cleanroom environment.36Order of incubation study #2Two scenarios for a two-tiered incubation scheme:Incubate at a higher temperature first, followed by the lower temperature:Regime A: 30-35oC for 2 days, followed by 20-25oC for 5 daysIncubate at a lower temperature first, followed by the higher temperature: Regime B: 20-25oC for 5 days, followed by 30-35oC for 2 daysOne 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.

Incubation times had been set by previous examinations

Regime A was designed to recover bacteria first, and then to recover fungi; and Regime B was designed to recover fungi (and some bacteria first), before recovering any other mesophilic bacteria present.

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.

37Order of incubation study #3Taking 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).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).

38Order of incubation study #4Study 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.

With the duplicate samples required for this study, these were samples taken side-by-side. This provided the closest estimate of 'duplicate' that could be achieved with the surface contact plate method (the alternative approach of sampling a given area within one plate and then immediately sampling the area with a second plate was rejected, because the first plate would take up a disproportionate amount of the surface contamination). It was considered that taking air-samples would have introduced too great a variability, given that two air-samplers would be sampling different volumes of air. Moreover, the sampling stress incurred by airborne microorganisms recovered by active air-samplers is relatively high and this can lead to low and variable recoveries 39Order of incubation study #5RegimeNumber and percentage of samples recording higher counts (n=136)Mean total colony count (CFU)A39 (28.7%)8B54 (39.7%)11Data sets where results were equivalent43 (31.6%)