dr. tim sandle - pharmig€¦ · limitations #1 – em methods the classic techniques: active...

Dr. Tim Sandle

Pharmaceutical Microbiology: http://www.pharmamicroresources.com/

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


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 e.g. HVAC losing power or maintenance works


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.


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


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


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 e.g.

bacteria clumping together,

Poor plate mixing,

Settle plate picking up skin detritus.


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


Fungal media study 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.

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.


Outcome of fungal media study Agars Examined for significance

using Student’s 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.


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.


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.

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:

http://www.pharmamicroresources.com/

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.


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.

That there is not a significant difference between the two incubation regimes.


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).


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.

136 samples.


Incubation order study #5


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.


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.

To determine whether this was significant or nor required a second Student's t-test to be constructed.


Incubation order study #8 This time, there the difference was shown to be

statistically significant .

Therefore incubation regime B produces a higher fungal count.

Regime B: 20-25oC for 5 days, followed by 30-35oC 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.


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.


Contamination sources

Product

Adjacent areas

Supply air

Cleanroom air

Machine and

ancillaries

Room surfaces

People

Materials

Water sources


The people factor Different sites on the body have different microbial

communities

Based on nutritional and physicochemical requirements:

Liebig’s law of the minimum

Microbial numbers are determined by the nutrients present

Shelford’s law of tolerance

Non-nutritional factors governing growth, such as pH and temperature

Also antimicrobial factors and mechanical removal factors, like skin shedding


Human skin #1 Average adult has 1.8m2 of skin

Regional variation :

Temperature range 25-37oC

Warmest area under the arms (36.6oC) and the coolest area the fingers (29.5oC).

pH of the skin varies between neutral and alkali,

Forehead: pH 4.8 and under the arm: pH 6.9.

Moisture

High moisture: axilla, groin, and areas between the toes = high population density

Other areas are relatively dry


Human skin #2 Skin #2

Cleanroom risk

With the skin epithelial cells are continually being shed so many microbial communities on the external surface are unstable.


Human skin #3 Why so many Gram-positive bacteria?

Dry environment with high osmotic pressure.

Gram-positive bacteria better adapted (resistant to desiccation).

However, certain occluded regions (such as toe webs), have more variation, including Gram-negative bacteria.


Human skin #4


Why do microorganisms survive in cleanrooms? Varies depending upon the species and different factors:

Temperature

Available water

Concentration of organic compounds

Concentration of hydrogen ions

Concentration of inorganic compounds

Concentration of particles in the air

Redox potential

Pressure

Light intensity

Geographical location and habitat


Types of contamination Variables affect the types of microorganisms

recovered:

Monitoring methods,

Location of the monitoring sample,

Types of agars,

Disinfectant neutralisers,

Incubation temperature,

Incubation time,

Identification method.


What is found in cleanrooms? #1 Description of the human microbiota does not

necessarily predict the types of microorganisms found in cleanrooms.

Variations relating to: Cleanroom types and uses

Grade, temperature and humidity

Geographical location

Gowning requirements

Certain locations of the body are more likely to release organisms than others.


What is found in cleanrooms? #2 Variations also relate to:

Microbial identification method Phenotypic or genotypic

Databases

Size, orientation (clinical or industrial)

Periodic reclassification of microorganisms

Limitations of EM methods

EM agar and incubation regime

Culturability

Some research suggest that less than 10% of bacteria found in cleanrooms are culturable

Seasonality and time of sampling


Study Very few studies of pharmaceutical cleanroom

microflora published.

Mystudy is:

Sandle, T. (2011): ‘A Review of Cleanroom Microflora: Types, Trends, and Patterns’, PDA Journal of Pharmaceutical Science and Technology, Vol. 65, No. 4, July–August 2011, pp392-403


Study #2 Pharmaceutical facility in south-east England

The cleanrooms examined represented:

40 Grade B rooms (of which five had Grade A cleanzones)

35 Grade C cleanrooms

20 Grade D cleanrooms

EM regime used TSA and incubation regime:

20-25oC ≤5 days / 30-35oC ≤2 days

Time period: 2001 - 2010


Study #3 EU GMP Grade A and B clean areas = 6,729 isolates


Study #4 Grade A and B major

species Skin related microflora

represent the most common genera isolated, with the family Micrococcaceae (the genera Micrococci and Staphylococci) representing >90% of the isolates

Order of detection:

Micrococcus luteus

Micrococcus lylae

Staphylococcus spp

Micrococcus spp

Staphylococcus epidermidis

Staphylococcus capitis

Staphylococcus hominis

Bacillus spp

Staphylococcus haemolyticus


Study #5 EU GMP Grade C and D

clean areas = 2, 500 isolates

More diversity due to nature of operations and presence of water

Order of detection:

Micrococcus luteus

Bacillus spp.

Micrococcus lylae

Micrococcus spp.

Staphylococcus spp.

Bacillus spp

Bacillus cereus

Pseudomonads

Corynebacterium spp


Outcomes Association between the microorganisms commonly

found in cleanrooms and those which are transient to (short-term or long term-residents on) human skin .

Low incidents of Bacillus spp.,

Possible transfer into the cleanrooms via personnel, dust, and material transfer .

Occasional, low-level incidences of microorganisms resident within the human body.

Where there is a water source, some microorganisms associated with water detected.


Outcomes Little variation over time

Variation signalled that something had gone wrong e.g. cleaning techniques, changes to personnel, HVAC failure

Sample types: Personnel samples: Gram-positive cocci occur most

frequently

Air samples: Gram-positive cocci occur very frequently (people shedding)

Surfaces: Gram-positive cocci occur quite frequently. Higher levels of Gram-positive rods (possible equipment transfer link) Where Gram-negative rods occur, this is from water on surfaces


Outcomes Majority mesophilic

aerobic or facultatively aerobic bacteria.

Where specialist gases are used some anaerobic found.

Thermophiles and extremotolerant bacteria very rare.

Common fungi in cleanrooms are: Aspergillus, Penicillium and Trychophyton.


Summary Environmental monitoring limitations.

Fungal agars

Whether one or two culture media should be used for environmental monitoring.

The optimal incubation regime, where one medium is used.

Cleanroom microbiota.


dr. tim sandle - pharmig€¦ · limitations #1 – em methods the classic techniques: active...

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