Dr. Tim Sandle - Pharmig

1 Dr. Tim Sandle Pharmaceutical Microbiology: Introduction What is environmental monitoring trying to do? Methods and limitations Problems and myths of incubation Which agar should you select? Should one or two agars be used? Which is the optimal temperature? For how long should you incubate for? What types of microorganisms can you expect to detect? Cleanroom microbiota Pharmaceutical Microbiology: Pharmaceutical Microbiology: Environmental monitoring To assess cleanrooms and controlled environments using viable and particulate counting methods. To verify environmental control (how well is the cleanroom working?). To assess impact of staff behaviours To help evaluate risk in relation to excursions (location and event dependent). To assess event specific incidents HVAC losing power or maintenance works Pharmaceutical Microbiology: Limitations #1 EM methods The classic techniques: Active air-sampling: volumetric air-sampler Passive air-sampling: settle plates Surface samples: contact (RODAC) plates and swabs Personnel samples: Finger plates and gown plates New generation of real time' viable air samplers.

2 Pharmaceutical Microbiology: EM method limitations #2. Settle plates Position in relation to air currents Desiccation Meaningfulness of one CFU. Attempt to quantify by Whyte's deposition rate Active air samplers Particle size cut-off (D50. value). Recovery ~50%. Different instruments Pharmaceutical Microbiology: EM method limitations #3. Contact plates Tidswell's work on organism recovery (~50%). Disinfectant residues on surfaces and neutraliser selection Disinfectant rotation and neutralisers Swabs Plain swabs ~10-20% recovery Flocked swabs 40-60% recovery Pharmaceutical Microbiology: Limitation #2 - The CFU. Often a CFU is not a single bacterium A colony could arise from one cell or several. Issue can occur through: Poor sample mixing bacteria clumping together, Poor plate mixing, Settle plate picking up skin detritus.

3 Pharmaceutical Microbiology: Pharmaceutical Microbiology: 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. Pharmaceutical Microbiology: 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? Pharmaceutical Microbiology: Incubation time Studying incubation time: Studying the effect of desiccation of media Sandle , T.

4 , Skinner, K. and following UDAF exposure Yeandle, E. (2013). Optimal and at the end of conditions for the recovery of incubation: bioburden from pharmaceutical processes: a Sandle , T. (2011): 'Microbial case study, European Journal recovery on settle plates in of Parenteral and unidirectional airflow Pharmaceutical Sciences, 18 cabinets', Clean Air and (3): 84-91 Containment Review, Issue 6, pp8-10. Pharmaceutical Microbiology: 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? Pharmaceutical Microbiology: 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?

5 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. Pharmaceutical Microbiology: Fungal media Pharmaceutical Microbiology: Fungal media study Aims: Outcomes: To assess if there is any Recovery of fungi was significant variation in relatively low. the number of fungal Mean counts varying isolates recovered by between and five selective agars. colonies per sample type. Higher results from active To assess any variation air samplers. in recovery of different Lowest results from species or genera by the surface contact plates. selective agars. More filamentous fungi in the environment than yeasts. Pharmaceutical Microbiology: Outcome of fungal media study Agars Examined for significance using Student's t-test Yeats: MEA agar , followed by SDA, recovered the greatest variety.

6 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. Pharmaceutical Microbiology: Pharmaceutical Microbiology: 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. Pharmaceutical Microbiology: One medium Many sites use one general purpose culture medium incubated at two temperatures: 30-35oC, to encourage the recovery of skin commensurable bacteria.

7 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. Pharmaceutical Microbiology: What is the appropriate order of incubation? Higher temperature: Lower temperature: Majority of Higher temperature may inhibit microorganisms recovered fungal growth. are mesophilic bacteria. Higher temperature may damage fungal enzymes, leading to no Few fungi are recovered. recovery. Filamentous fungi could Avoids overgrowth of mycelia grow and obscure bacteria. obscuring bacterial colonies if Destruction of lytic low temperature first. cellular enzymes in fungi Some bacteria may not grow at if high temperature first. this temperature.

8 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. Pharmaceutical Microbiology: Incubation order 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. Pharmaceutical Microbiology: Incubation order study #2. Two 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 days Incubate at a lower temperature first, followed by the higher temperature: Regime B: 20-25oC for 5 days, followed by 30-35oC for 2 days One of two possible outcomes: That there is a significant difference between the two incubation regimes.

9 That there is not a significant difference between the two incubation regimes. Pharmaceutical Microbiology: Incubation order 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). Pharmaceutical Microbiology: Incubation order 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 2-8 C). Surface samples taken (contact plates). Two enable close to possible' duplicate sampling.

10 136 samples. Pharmaceutical Microbiology: Incubation order study #5. Pharmaceutical Microbiology: Incubation order 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. Pharmaceutical Microbiology: Incubation order 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.