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Comparison of Different Fungal Agar for the Environmental Monitoring of Pharmaceutical-Grade Cleanrooms Barbara Gebala and Tim Sandle
PDA J Pharm Sci and Tech 2013, 67 621-633 Access the most recent version at doi:10.5731/pdajpst.2013.00944
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CASE STUDIES
Comparison of Different Fungal Agar for the Environmental Monitoring of Pharmaceutical-Grade Cleanrooms BARBARA GEBALA and TIM SANDLE* ABSTRACT: In relation to a growth in reported incidents of fungal contamination of pharmaceutical products, there has been a developing interest by U.S. and U.K. regulators concerning the risk of fungi. This paper describes a study undertaken to examine the suitability of different commercially available mycological agars for the environmental monitoring of pharmaceutical-grade cleanrooms. Five agars were evaluated in relation to the detection of both numbers and different species of fungi (yeasts and moulds). The objective was to determine if one mycological medium is more suitable than another. Data was collected using different sampling techniques (settle plates, active air samples, and contact plates) from different locations within representative cleanrooms. Samples were taken over a 3 month time period. The study results indicated that fungi are not distributed evenly across cleanrooms and that that the prevalence of fungi partly relates to the room design and operation. In relation to the different agar types, the study indicated that Sabouraud dextrose agar was the most effective at detecting the widest number of different types of isolates, and that Sabouraud dextrose agar and malt extract agar were the most efficient in terms of the numbers of recovered isolates. Other media, notably potato dextrose agar, was relatively less effective. KEYWORDS: Environmental monitoring, Cleanroom, Fungi, Microorganisms, Quality control, Agar, Mycological LAY ABSTRACT: There has been an increased regulatory concern about the presence of fungi in cleanrooms. Some environmental monitoring regimes are not especially orientated towards the examination of fungi, and it may be that special agars are required. Given the choice of different agars available, this paper outlines a case study where different fungal agars were evaluated. The study showed that Sabouraud dextrose agar was the optimal agar.
Introduction Since the 1990s significant issues with contamination of pharmaceutical products (sterile and nonsterile) due to fungi have been noted. From an analysis of U.S. Food and Drug Administration (FDA)-listed recalls, contamination due to mould and yeast account for the second highest cause of nonsterile product recalls at levels (representing 23% of recalls between 2001 and 2011) (1). In addition, the incidents, including fatalities, surrounding the New England Compounding Center (NECC) in 2012, where the pharmacy produced contaminated injections of preservative-free methylprednisolone acetate (2, 3), show the risk from
* Corresponding Author: Dr. Tim Sandle, Bio Products Laboratory, Microbiology, Dagger Lane, Elstree, Hertfordshire, WD6 3BX, U.K. Telephone: ⫹44-208258-2483. E-mail: [email protected] doi: 10.5731/pdajpst.2013.00944
Vol. 67, No. 6, November–December 2013
fungi when prevalent in uncontrolled environments and to improperly validated processes (4). Pharmaceutical facilities are made up of a series of rooms called cleanrooms. Cleanrooms and zones are typically classified according to their use (the main activity within each room or zone) and are confirmed by the cleanliness of the air by the measurement of particles. This air cleanliness is either based on European Union (EU) good manufacturing practice (GMP) guidance for aseptically filled products, and the EU GMP alphabetic notations are adopted (5); or by using the International Standard ISO14644, where numerical classes are adopted (6). The cleanliness of the air is controlled by a heating, ventilation, and air conditioning (HVAC) system. The cleanliness of cleanrooms is controlled through a range of physical controls relating to air filtration, air changes, and air flow. The cleanliness is assessed through microbiological environmental monitoring regimes that are designed to assess numbers of micro621
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organisms and the types of species present. Environmental monitoring involves the collection of samples and the subsequent examination of data relating to the numbers or incidents of microorganisms present on surfaces, in the air, and from people. While reviews of cleanroom microflora have shown that bacteria resident or transient to human skin accounts for the overwhelming majority of isolates, a range of different types of fungi sometimes may be present within cleanrooms, depending upon the grade, use, and environmental conditions (7). The recent increase in notifications of fungal contamination of pharmaceutical products may be related to a lack of specific fungal monitoring as part of a facility’s environmental monitoring regime, together with limited knowledge of expected levels of fungal contamination in drug manufacturing cleanrooms (8). This may be linked with a general uncertainty as to how processes should be controlled and how cleaning and disinfection regimes for cleanrooms are evaluated to show that the product can be protected from environmental fungi (9). Although there is little regulatory guidance in relation to the construction of an environmental monitoring program, the standard environmental monitoring regime is generally reliant upon the use of a highly nutritious general growth medium, such as soya bean casein digest medium, incubated at a temperature designed to encourage mesophilic microorganisms (primarily bacteria) (10). Mesophiles contain those microorganisms that grow best in temperatures between 20 and 40 °C (11). The reasons why monitoring regimes are conventionally select for mesophilic microorganisms is because these microorganisms are most likely to be present, as most cleanroom contaminants are introduced into the area from people and are deposited into the air as skin is shed (12, 13). The weaknesses of environmental monitoring programs are well recognized. Such weaknesses include the imprecision of the monitoring methods used to recover microorganisms (14) and the ability of monitoring programs to only detect a small number of the microorganisms present in the environment (15). These limitations place a far greater reliance, on the part of the pharmaceutical manufacturer, upon environmental controls, such as air supply systems and cleaning and disinfection regimes (16). These limitations notwithstanding, environmental monitoring regimes are an expected and established feature 622
of pharmaceutical quality control. While environmental monitoring regimes for many years have focused on mesophilic monitoring, regulatory agencies, perhaps due to the various product recall incidents discussed above, are requesting pharmaceutical manufacturers to examine environments for microorganisms that may not grow at mesophilic conditions (such as psychrophilic microorganisms) (17) or screen for selective microorganisms, such as fungi. Although regulators express an interest in more comprehensive environmental monitoring regimes, none of the documents produced by regulatory agencies describe the types of media required for the isolation of microorganisms in the pharmaceutical cleanrooms. However, some, such as the FDA guidance for environments used for aseptic filling, require the microbiological agar used to have been tested to show the growth promotion of both bacteria and fungi (18). It also stands that no regulatory document, aside from the Japanese Pharmacopeia (19), which has more limited reach than the United States or European equivalents, requires the use of separate agar for the detection of yeasts and moulds. As a result most monitoring regimes use only a single, general purpose culture medium incubated at two temperatures: a higher one, often 30 –35 °C, to encourage the recovery of skincommensurable bacteria (20); and a lower one, often 20 –25 °C, designed to recover fungi (21). Whether such samples should be incubated at the higher or lower temperature first is a recurrent subject of debate within the industry. Alternatively, a modified general purpose agar at 30 –35 °C could be as effective for the recovery of fungi (22). It may be, however, that some fungi recover better on a selective mycological agar when that agar is incubated at a lower temperature (⬍25 °C) for a prolonged period of time. The optimal outcome for the time, temperature, and medium conundrum depends upon the cleanroom, environmental conditions, and types of fungi expected. If the concept of a culture media designed of the growth and detection of fungi is accepted, there are a range of different “selective” fungal agars available. An examination of these formed the basis of this case study. The study did not set out to argue that a special mycological medium was required for environmental monitoring, but to evaluate different types of media should the microbiologist elect to use a specially designed mycological media as part of the environmental monitoring program. PDA Journal of Pharmaceutical Science and Technology
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This study was focused upon the recovery of fungi from the environmental monitoring of a pharmaceutical processing facility. The objective was to assess the performance of different mycological agars within the environment rather than to assess the cleanliness of a particular facility. This was undertaken through a comparison of commercially available mycological agars. The reason for restricting the study to commercially produced agars was because environmental monitoring regimes, in large facilities, use thousands of agar plates each year and thus there is a need to connect suitable monitoring agars with what can be manufactured on a large scale by media suppliers and therefore be made available for pharmaceutical microbiologists to purchase. In setting out such a study there were complications. The suitability of any media would depend upon whether higher levels of fungi were recovered through the use of mycological culture media compared (otherwise any meaningful statistical comparison would be difficult and the outcome would err towards a qualitative assessment). This is not a straightforward assessment, for there is a range of different mycological media, none of which has been especially developed for environmental monitoring. Furthermore, with any study of cleanroom micoflora there are limitations. The first limitation relates to benchmarking, for there have been very few studies of pharmaceutical cleanroom microflora (or microbiota) published in recent years, and these studies have been largely based upon the use of soya bean casein digest medium subjected to a dual incubation regime (23– 25). However, some studies have indicated that certain types of fungi are more prevalent within cleanroom than others. The types of fungi described are Aspergillus spp., Penicillium spp., Trychophyton spp., Cladosporium spp., Curvularia spp., Fusarium spp., and Alternaria spp. (1, 7). In addition, the findings of Wilson et al. (26), who conducted studies on fungal contamination of high-efficiency particulate air (HEPA) filter air supply into cleanrooms, concluded that the most prevalent contaminants were species of Cladosporiium and that other species, such as Aspergillus spp., Penicillium spp., and Paecilomyces spp., were also present. A weakness with these studies is that little information is provided regarding the incident rates for these contaminants. A limitation in relation to the case study approach is that all cleanrooms differ in design and function, and Vol. 67, No. 6, November–December 2013
with the types of products processed. Therefore the type of cleanroom environment will have an impact upon the range of microorganisms recovered. A further limitation is with the microbial identification method (27). The key variables here are the size and scope of the databases used to compare cleanroom isolates and the types of methods used for analysis (whether the method is phenotypic or genotypic, and then the various technological variations of these methods) (28). Given the limitations with the automated methods, many fungi, particularly those of a filamentous type, continue to be identified either through macroscopic and microscopic study, or using genotypic methods. While the reader should be aware of these limitations before drawing any generalizations from the work presented in this paper, the authors consider that the approach and general findings will be of interest for facility microbiologists who are considering the application of a mycological agar for an environmental monitoring regime. Materials and Methods To examine for the presence of fungi within cleanrooms using different mycological media, a study was set up to examine pharmaceutical cleanrooms used for different activities. The study related to one pharmaceutical facility. It is acknowledged that each facility will differ and the results from the study are not intended to indicate that the facility studied was necessarily representative of other production plants. However, in order to improve the representativeness of the study, a range of cleanrooms dedicated to different uses were selected for the review (such as a wet area, a cold area, an ambient area, and so forth). The cleanrooms within the facility were used for nonsterile processing and they were classified as EU GMP Grade D. The air within the rooms was volumetrically exchanged at a rate of twenty times per hour. During the period of the study all personnel entering the cleanrooms wore cleanroom-certified garments and the rooms were subject to a weekly cleaning and disinfection regime. The aim of this study was to assess if there is any significant variation in the numbers of fungal isolates recovered and in relation to the variety of species in relation to five different mycological agars. For the study, five different commercially available mycological agars were selected: malt extract agar (MEA); 623
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MEA with penicillin and streptomycin supplement (MEP); potato dextrose agar (PDA) containing no antibiotics; Rose Bengal Agar (RBA) with chloramphenicol; and Sabouraud dextrose agar (SDA) (sometimes described as Sabouraud glucose agar) with chloramphenicol. The agar media used to perform environmental monitoring were manufactured by Cherwell Laboratories Limited’s (Bicester, Oxford, U.K.) RediporTM brand. Agar used was in the form of 9 cm diameter settle plates and 2.5 cm diameter contact plates. The inclusion of the antibiotics was based on standard commercial availability, with MEA available with and without penicillin; and RBA and SDA available with the bacteriostatic antimicrobial chloramphenicol. PDA generally does not contain an antimicrobial agent.
Sabouraud Dextrose Agar with Chloramphenicol (SDA) Formula Mycological peptone Glucose Chloramphenicol Agar pH 5.6 ⫾ 0.2
g/L 10.0 40.0 0.1 15.0
Potato Dextrose Agar (PDA) Formula Potato extract Glucose Agar pH 5.6 ⫾ 0.2
g/L 4.0 20.0 15.0
The formulation for each of the agars was as follows (29):
With the exception of RBA, each agar was formulated with a low pH designed to inhibit bacterial growth.
Malt Extract Agar (MEA)
MEA is arguably the most commonly used agar for environmental sampling of airborne fungi (30) and is recommended by the American Conference of Governmental Industrial Hygienists for fungal isolation from indoor environments (31). Other recommendations exist for typical media used for selective fungal monitoring in a variety of environments (such as for nonsterile pharmaceutical products). Various sources (including the main pharmacopeia) recommend SDA (32, 33). PDA is recommended for recovery of yeast and mould isolates (34). Within the food industry, RBA is a common choice (35, 36). The MEP agar was identical to MEA except for an antibiotic supplement. This medium was included in order to determine whether the addition of an antibiotic to the agar will have a significant impact in specifically inhibiting bacterial growth during selective fungal monitoring.
Formula Malt extract Mycological peptone Agar pH 5.4 ⫾ 0.2
g/L 30.0 5.0 15.0
Malt extract agar has a high content of peptone and is acidic. Malt Extract Agar Penicillin and Streptomycin (MEP) Formula Malt extract Agar Penicillin G Na salt Streptomycin sulphate pH 5.4 ⫾ 0.2
g/L 20.0 12.0 0.012 0.05
Rose Bengal Agar with Chloramphenicol (RBA) Formula Mycological peptone Glucose Dipotassium phosphate Magnesium sulphate Rose Bengal Chloramphenicol Agar pH 7.2 ⫾ 0.2 at 25 °C 624
g/L 5.0 10.0 1.0 0.5 0.05 0.1 15.5
In terms of availability, there is little to distinguish between the selected media. Manufacturers charge similar prices, and apart from RBA, which must be kept in darkness and at 2– 8 °C, they are all easy to store. Based on the lack of consensus on appropriate types of media, there is no reason to expect that any one of the above agars will significantly outperform any other. The two research objectives were ●
To assess if there is any significant variation in the number of fungal isolates recovered by five selective agars.
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Table I Cleanrooms Selected for the Study Room code A101 A102 A103 A104 A105 A106 A107 A108 A109
A110
●
Rationale for room selection These are wash up areas used for cleaning of used equipment. The rooms are characterized by a moist environment, which could potentially support fungal growth. In these areas large equipment is cleaned by a clean in place (CIP) process. These rooms are characterized by moist and warm environment, which could support germination of fungal spores. These are 2–8 °C cold rooms used for product processing. These areas were selected as representative cold areas. This is an airlock room, which interfaces to an unclassified area. The area could be at risk of fungal spores from close proximity of outside environment. This is an autoclave and equipment preparation area. This room is characterized by dry and warm conditions. This is a storage area, characterized by dry ambient conditions. The room has access to lift, which leads to an unclassified corridor within the building. The lift could pose a risk of transferring fungal spores from unclassified areas. This is a room has a large number of fixed processing vessels. It is characterized by warm and moist conditions, which could support fungal growth.
To assess any variation in recovery of different species or genera by the selective agars.
Prior to undertaking the study the media used was assessed for growth promotion in order to demonstrate that it could recover a low-level inocula of a yeast (Candida albicans ATCC 10231) and a filamentous fungus (Aspergillus brasiliensis ATCC 16404). For the study, ten process cleanrooms were selected. The rooms differed by temperature, moisture, intended use, and previous history of fungal recovery. The set of selected rooms is shown in Table I. Different types of environmental monitoring samples were taken in the rooms. These samples were designed to recover any fungi present from the air (in the form of settle plates, 9 cm exposed plate for a period of 1 h; active air-samples, taken using an impaction air sampler where 1 m3 of air was sampled using an SAS Super180 sampler (VWR International PBI S.r.l., Milan, Italy); and contact plates, where plates of 25 cm 2 surface area were pressed onto a surface for 10 s). The room–sample combinations are shown in Table II. Each room monitoring was performed in triplicate (in three separate sessions) for each agar. In total, 210 samples, across each room, were sampled for each Vol. 67, No. 6, November–December 2013
agar (with the total number of samples from the three sessions being 630). Mean microbial counts were calculated from each monitoring session. All plates collected as part of environmental monitoring were incubated at 20 –25 °C for a minimum of 8 days to ensure fungal spore germination. After completing the incubation period, all plates were read against an artificial light source and the number of colony-forming units (CFU) was recorded. All nonfilamentous colonies from any plates that showed growth were subcultured onto the same media from which they were initially recovered. These plates were incubated for 24 –72 h to produce a pure culture. Colonies were then Gram-stained to distinguish between yeast and bacterial isolates. Further identification was performed only for yeast isolates and not for the bacteria. Fungal isolates were identified using the GEN III OmniLog威 ID System automated identification system (Biolog Inc., Hayward, CA, USA). Where the automated identification system could not provide a reliable result for filamentous fungi, a visual identification was made by describing the macroscopic characteristics (morphology, pigment, and so forth) and microscopic characteristics (using a lactophenol cotton blue stain). 625
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Table II Room and Sample Combinations Contact plates Room number
Settle plates (n)
Working height (n)
Floor (n)
Air samples (n)
A101 A102 A103 A104 A105 A106 A107 A108 A109 A110
2 3 2 2 1 2 1 2 3 5
2 3 4 2 2 2 1 1 1 1
1 1 1 1 1 2 1 2 2 3
2 1 1 1 1 1 1 1 2 2
Results The mean counts of all fungal results obtained for each sample type (such as active air samples) for each agar were calculated. The recovery of fungi was, in general, low across the cleanroom surveyed with mean counts varying between 0.1 and 7.8 CFU per sample across each of the sampling methods. The highest fungal counts were obtained from active air sampling and the lowest counts from surface sampling using contact plates. It is noted that this is based upon low level recoveries, although a slight bias towards air recoveries was apparent. However, the range of results across agars for individual sampling methods was less pronounced. While the data, because of the low values, does not lend itself to detailed statistical analysis, the variance between media using air samplers was greater in absolute terms compared with the other sampling methods; the lowest result (MEA) was 73% that of
the highest (SDA), and 2.2 CFU was the difference between the mean results for MEA and SDA. However, a higher variance proportionally was recorded from contact plates and settle plates, where the lowest means obtained showed a recovery, for both sample types, of only 53% compared with the highest recoveries. A summary of the results is shown in Table III. The results were compared for statistical significance using an unpaired, two tailed, Student’s t-test with a 5% confidence interval. All results per sample type and per agar were compared to each other using t-test, and from this P-values were calculated. The lowest P-value was obtained between RBA and SDA settle plate results, ⫺0.16; the highest P-value was obtained between MEP and RBA AS results, ⫺1.00. None of the calculated P-values were lower than the 0.05 (5%) confidence interval or, in other words, none
Table III Summary of Data for Each Agar across the Three Sampling Methods (Mean Counts) Sampling Method
626
Medium
Air sample (CFU)
Contact plate (CFU)
Settle plate (CFU)
MEA MEP PDA RBA SDA
5.80 6.69 6.82 6.72 7.80
0.14 0.12 0.22 0.19 0.23
1.60 1.97 1.16 2.10 1.10
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Table IV Summary of Significance Test Results Unpaired T- Test (5%) Media Variant 1 MEA MEA MEA MEA MEP MEP MEP PDA PDA RBA
Sampling Method Air sample
Variant 2 MEP PDA RBA SDA PDA RBA SDA RBA SDA SDA
Settle plate
P-values 0.84 0.84 0.85 0.68 0.98 1.00 0.81 0.98 0.84 0.83
of the calculated values of t were higher than the values of the t-distribution table. A summary of the significance test results is provided in Table IV. Therefore there was no significant difference between the different agars in terms of the numbers of fungi detected. In comparing the different types of cleanrooms, the overwhelming majority of colonies were recovered from one of the rooms (the room coded A110). This room was often warm (25–30 °C), due to the presence of processing equipment, and of higher humidity (⬃60% relative humidity), in comparison to the other rooms studied. The culture media varied in relation to the different types of fungi detected, with some media detecting a greater variety of fungi than others. This is summarized in terms of both yeasts (Table V) and filamentous fungi (Table VI). From a review of Tables V and VI, the MEA agar, followed by SDA, recovered the greatest variety of yeasts. In contrast, the PDA medium recovered the lowest variety. The highest numbers of yeast isolates for each agar were recovered by contact plates, and the lowest numbers of yeast isolates was obtained from settle plate samples. In each case, visible colonies were seen, although no assessment was made as to the size of the colonies or other growth characteristics (the assessment was for detection or no detection). With filamentous fungi, the largest variety of species was recovered on RBA (15 species), and the smallest Vol. 67, No. 6, November–December 2013
Contact plate
0.81 0.44 0.57 0.35 0.35 0.44 0.27 0.79 0.93 0.70
0.64 0.46 0.54 0.41 0.23 0.89 0.21 0.18 0.91 0.16
variety from MEA (7 species). In contrast to the recovery of yeasts, the highest numbers of filamentous fungi were recovered from active air samples. The largest proportions of characterizable fungal species were Cladosporium spp. and Penicillium spp. The other species recovered were Aspergillus spp. and Bionectria sesquicillii Samuels (anam. Clonostachys spp.). In addition to the fungal counts, a note was made about the incidences of bacteria recovered by the different mycological agars. None of the media were wholly selective for fungi, and each recovered some bacteria. The greatest incidences of nonfungal colonies (represented as mean of all samples taken) were recovered on PDA. The mean counts for bacteria for each agar and across each sample type is summarized in Table VII. In order to further illustrate which sample types and which media recovered the greater proportion of fungi to bacteria, a ratio chart was constructed. This is displayed in Figure 1. The lowest relative proportion observed was on PDA contact plates with 15% fungal to 85% nonfungal mean colonies recovered, and RBA with 20% fungal to 80% nonfungal mean colonies recovered. In contrast, the highest ratio is observed for MEA and SDA (fungal 99% to nonfungal 1%). In terms of different sample types, surface sampling, using contact plates, recovered proportionally more nonfungal than fungal colonies, although the absolute 627
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Table V Recovery of Different Species of Yeast, for Each Agar Media Species Name
MEA
MEP
Arthroascus javanensis Candida edax Candida spp. ⻫ Cryptococcus albidus ⻫ Cryptococcus albidus var aerius Cryptococcus marinus ⻫ Cryptococcus spp. ⻫ Endomyces fibulinger ⻫ ⻫ Filobasidiella neoformans bacillisporus Pitchia alni Pitchia guilliermondii B ⻫ Rhodotorula acheniorum Rhodotorula aurantiaca A ⻫ ⻫ Rhodotorula aurantiaca B ⻫ Rhodotorula minuta ⻫ Schizosaccharomyces japonicas var japoni ⻫ Sporidibolus johnsonii A ⻫ Sporidibolus johnsonii B Trichosporon beigelii B ⻫ Other (including nonviable) ⻫⻫* * more than one uncharacterized or nonviable yeast isolate recovered. numbers in both cases are far lower than with the results from air sampling. Discussion When all results are counted, the greatest numbers of filamentous fungus colonies in general (by each agar) were recovered from active air samples and the lowest numbers from surface contact plates. This was expected and is consistent with the nature of filamentous fungi, where studies have shown that in dynamic environments higher numbers of fungal spores are likely to be dispersed in the air, rather than settling onto surfaces, as this is their principal means of transmission (37, 38). The reason why active air samples gave higher recoveries compared with settle plates can perhaps be explained by the nature of the settle plate technique. Settle plates require microbes to physically drop from the air onto a plate by gravitational forces or by moving through the air and coming into contact with an inanimate object (39). In a correctly working, dynamic cleanroom the constant movement of the air should make this difficult to achieve. 628
PDA
RBA
SDA
⻫ ⻫
⻫
⻫ ⻫ ⻫ ⻫ ⻫
⻫ ⻫
⻫ ⻫ ⻫ ⻫
⻫
⻫ ⻫
⻫⻫⻫*
It was noticeable that the overwhelming majority of samples gave a count of zero. This is not to imply that the media selected was weak at recovering environmental fungi, rather it was a reflection of the areas examined. From the data set, one particular room, with a higher temperature and humidity, recorded the highest microbial counts. Given that the facility within which the study was conducted may or may not be representative of other manufacturing facilities, little notice should be taken of the levels, other than the fact that the low levels made statistical analysis more difficult. Although the levels recovered were low and the final conclusion regarding the different media perhaps better rests on a qualitative assessment, the fungal results were compared statistically using Student’s t-test for significant difference. The result of this evaluation indicated that there was no statistically significant difference between the numbers of fungal colonies recovered by the five agars (which may be a consequence of the relatively low data values). However, it was noted, when qualitatively assessing the results, PDA Journal of Pharmaceutical Science and Technology
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Table VI Recovery of Different Species of Rilamentous Fungi, for Each Agar Media Species Name
MEA
Cladosporium spp. Cladosporium sphaerospermum Cladosporium macrocarpum Cladosporium tenuissimum Cladosporium cladosporoides Cladosporium herbarum Penicillium spp. Penicillium rubrum Penicillium melinii Penicillium corylophilum Penicillium chrysogenum Penicillium variabile Aspergillus spp. Bionectria sesquicillii Nonsporing (uncharacterized)*
⻫
MEP
PDA
⻫
RBA
SDA
⻫
⻫⻫⻫
⻫⻫ ⻫ ⻫⻫⻫⻫
⻫ ⻫ ⻫
⻫ ⻫
⻫⻫ ⻫
⻫ ⻫
⻫ ⻫ ⻫ ⻫⻫⻫
⻫ ⻫
⻫⻫⻫
that slightly higher counts on SDA contact plates were obtained compared with the other media. Thus on a simple, numerical basis it might therefore appear that SDA was slightly superior compared with the other agars at collecting fungi. However, this observed difference could not, based on the low recoveries obtained, be statistically proven. Each of the tested agars was shown to support a variety of yeasts and filamentous fungal colonies, although at slightly different levels. Notably, all media supported the growth of common cleanroom fungal contaminants such as Cladosporium spp. and Penicillium spp., fungi identified earlier from the literature as occurring more often in cleanrooms (1, 7, 16). One genera of filamentous fungus often cited as typical
⻫ ⻫ ⻫⻫⻫
⻫
⻫
⻫⻫⻫⻫
⻫⻫⻫⻫
⻫⻫⻫⻫⻫⻫
contaminant of indoor environments and pharmaceutical facilities is Aspergillus spp. In this case study, a limited number of these genera were recovered and then only on MEP, PDA, and RBA. The fact that Aspergillus was found in such small numbers (typically only one colony per agar across the set of settle plates) is more reflective of low levels of Aspergillus spp. in the particular environment sampled rather than inability of the agars to support the growth of this particular fungal species. In terms of recovering the greatest variety of different species, no medium was universally superior. The MEA medium recovered the greatest range of yeasts, whereas the RBA medium recovered the greatest range of filamentous fungi. With yeasts, the MEA
Table VII Summary of Mean Bacterial Colony Counts Recovered on Each Medium Sampling Method Medium
Air sample (CFU)
Contact plate (CFU)
Settle plate (CFU)
MEA MEP PDA RBA SDA
0.051 0.154 1.103 0.103 0.077
0.196 0.137 1.225 0.745 0.245
0.058 0.058 1.159 0.000 0.116
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established mycological agars. Sabouraud agar medium was developed by the French dermatologist Raymond J. A. Sabouraud in the late 1800s to support the growth of dermatophytes over a prolonged incubation time (40).
Figure 1 Relative proportions of bacteria and fungi recovered across different media and for different sample types. AS ⴝ active air samples, CP ⴝ contact plates, SP ⴝ settle plates. recorded 11 different types of yeast and the RBA only recorded 6 different yeasts. However, with filamentous fungi the RBA recorded 15 different species and the MEA only 7 different species. The differences in variety did not mean that the agars that recorded less diversity were not capable of growing a range of fungi. Some cross-agar tests were undertaken where each of the fungi detected were inoculated onto the other media. Although there were some differences in growth, each of the fungi grew on each of the media. These inoculations used the environmentally isolated strains. However, the strains had been subcultured and grown within the laboratory and were therefore in a more healthy state than the isolates originally detected within the cleanrooms (where such isolates would be, as minimum, stressed and possibly sublethally damaged if they had been exposed to cleanroom disinfectants). Therefore, what can be concluded is that some agars were slightly better at detecting certain species of fungi within the environment; this is a different inference than stating the suitability or unsuitability of the agars for growing or storing cultures within the laboratory. While the agar recording the highest diversity of filamentous fungi and yeasts was different, it is noteworthy that SDA featured second in both categories, recording relatively high levels of different yeast and filamentous fungal species. Given that SDA also recorded marginally higher fungal counts, this medium appears qualitatively to be the optimal one. As an historical note, the medium is one of the longest 630
Each agar presented a similar range of different genera in similar proportions. While these numbers do not represent a sufficiently large enough pool of data to be conclusive, they could be used as a basis for the likely range of fungal isolates one might expect to find in a pharmaceutical cleanroom (the limitations of different types of cleanrooms and the fact that different facilities are located in different geographical regions is acknowledged). In drawing this conclusion, results from literature into studies of fungi from controlled environments are noted. For examples, other studies have shown that Cladosporium spp. and Penicillium spp. are common in high numbers, with Aspergillus spp. also found, although in lower proportions (41). It was further noted that there were a number of uncharacterizable fungal isolates. This was limited by the identification methods available. If the study was to be repeated, more sophisticated methods of identification, such as internal transcribed spacer (ITS) regions of fungal ribosomal DNA, would be recommended (42, 43). As well as filamentous fungal isolates, the agars also recovered numbers of nonfilamentous isolates (bacteria); therefore, no agar was truly selective (including the media that contained antibiotics). The agar that recorded the lowest numbers of bacteria was MEA, whereas the agar recording the highest numbers was PDA (notably, this agar also recovered the lowest numbers of fungi). It could be that the low levels of bacteria recovered, should they have originated from personnel, had a resistance to the antibiotics added to the media formulae. This possibility was not explored further, as it fell outside of the objectives of the case study. The findings suggest that potato dextrose agar is the least selective out of all tested agars. While the selectivity of this agar could be improved by the addition of tartaric acid to lower the pH to 3.5, or by the addition of antibiotics, the data suggests that PDA is not capable of increased recovery of fungi in comparison to other agars, so there is little to recommend it, on the basis of this case study, in preference to the other agars tested. PDA Journal of Pharmaceutical Science and Technology
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In drawing conclusions from the study, some caution is required, as there will be variations relating to the formulation of each agar and the way in which it is manufactured. Furthermore, each pharmaceutical facility is different and within the facility there will be differences between cleanroom types and operations; moreover, the cleanroom studied in this investigated may or may not be representative of the industry in general. It also stands that the numbers of fungi present in the environments studied were very low, which made meaningful statistical analysis difficult. However, we offer some tentative conclusions. The research question, that there would not be significant differences between the abilities of various agars to promote fungal growth, was broadly confirmed. This was in terms of numerical values obtained (CFU counted) and the range of species recovered. A marginal argument, based on a qualitative assessment, could be made in favor of SDA in relation to numerical recovery and in consideration of the variety of species recovery. The least appropriate choice of agar appears, of the basis of less diversity of fungi and lower fungal counts, to be PDA. Moreover, while PDA supports the growth of filamentous fungus at similar levels to other selective fungal agars and it is recommended for use in enumeration of fungi by some standards, it is not selective enough (based on high numbers and large variety of bacteria it recovered). In order to clarify more effectively which of the available media, if any, are most suitable for promoting the growth of these species, and to draw more concrete conclusions regarding the prevalent fungal species present in pharmaceutical cleanrooms, future experiments face a number of obstacles. To obtain sufficient isolates, a longer sampling period or further repeated sampling would be required; it would also be prudent to examine other facilities in different geographical regions. As noted above, identifying the resultant isolates would require more complex and sophisticated identification systems than were available to the authors at the time this study was executed. Nonetheless, the study, given the current concerns about fungi in pharmaceutical facilities, does stand as a case study for microbiologists to compare and contrast their monitoring regimes against.
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35. Beuchat, LR. Media for detecting and enumerating yeasts and moulds. Chapter 14 in: Corry, J.E.L.; Curtis, G.D. W. and Baird, R.M. (Eds.) Culture Media for Food Microbiology. 1995, V34, Elsvier Science BV. Amsterdam, The Netherlands. 36. Montville, T. J.; Matthews, K. R. Food Microbiology: An Introduction, 2nd ed.; ASM Press: Herndon, VA, 2008. 37. Ottria, G.; Dallera, M.; Aresu, O.; Manniello, M. A.; Parodi, B.; Spagnolo, A. M.; Cristina, M. L. Environmental monitoring program in the cell therapy facility of a research centre: preliminary investigation. J. Prev. Med. Hyg. 2010, 51 (4), 133–138. 38. Moissl-Eichinger, C.; Rettberg, P.; Pukall R. The first collection of spacecraft-associated microor-
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40. Sabouraud, R. Recherche des Milieux de Culture Propres a la Diffe´renciation des Espe`ces Trichophytiques a Grosse Spore. Les Trichophyties Humaines; Masson et Cie: Paris, France, 1896; pp 49 –55. 41. Kim, K. Y.; Kim, Y. S.; Kim, D. Distribution characteristics of airborne bacteria and fungi in the general hospitals of Korea. Ind. Health 2010, 48 (2), 236 –243. 42. Hibbet, D. S.; Ohman, A.; Glotzer, D.; Huhn, M., Kirk, P.; Nilsson, R. H. Progress in molecular and morphological taxon discovery in Fungi and options for formal classification of environmental sequences. Fungal Biology Reviews 2011, 25 (1), 38 – 47. 43. Nilsson, R. H.; Abarenkov, K.; Veldre, V.; Nylinder, S.; De Wit, P.; Brosche, S.; Alfredsson, J. F.; Ryberg, M.; Kristiansson, E. An open source chimera checker for the fungal ITS region. Molecular Ecology Resources 2010, 10 (6), 1076 – 1081.
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Comparison of Different Fungal Agar for the Environmental Monitoring of Pharmaceutical-Grade Cleanrooms Barbara Gebala and Tim Sandle
PDA J Pharm Sci and Tech 2013, 67 621-633 Access the most recent version at doi:10.5731/pdajpst.2013.00944
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CASE STUDIES
Comparison of Different Fungal Agar for the Environmental Monitoring of Pharmaceutical-Grade Cleanrooms BARBARA GEBALA and TIM SANDLE* ABSTRACT: In relation to a growth in reported incidents of fungal contamination of pharmaceutical products, there has been a developing interest by U.S. and U.K. regulators concerning the risk of fungi. This paper describes a study undertaken to examine the suitability of different commercially available mycological agars for the environmental monitoring of pharmaceutical-grade cleanrooms. Five agars were evaluated in relation to the detection of both numbers and different species of fungi (yeasts and moulds). The objective was to determine if one mycological medium is more suitable than another. Data was collected using different sampling techniques (settle plates, active air samples, and contact plates) from different locations within representative cleanrooms. Samples were taken over a 3 month time period. The study results indicated that fungi are not distributed evenly across cleanrooms and that that the prevalence of fungi partly relates to the room design and operation. In relation to the different agar types, the study indicated that Sabouraud dextrose agar was the most effective at detecting the widest number of different types of isolates, and that Sabouraud dextrose agar and malt extract agar were the most efficient in terms of the numbers of recovered isolates. Other media, notably potato dextrose agar, was relatively less effective. KEYWORDS: Environmental monitoring, Cleanroom, Fungi, Microorganisms, Quality control, Agar, Mycological LAY ABSTRACT: There has been an increased regulatory concern about the presence of fungi in cleanrooms. Some environmental monitoring regimes are not especially orientated towards the examination of fungi, and it may be that special agars are required. Given the choice of different agars available, this paper outlines a case study where different fungal agars were evaluated. The study showed that Sabouraud dextrose agar was the optimal agar.
Introduction Since the 1990s significant issues with contamination of pharmaceutical products (sterile and nonsterile) due to fungi have been noted. From an analysis of U.S. Food and Drug Administration (FDA)-listed recalls, contamination due to mould and yeast account for the second highest cause of nonsterile product recalls at levels (representing 23% of recalls between 2001 and 2011) (1). In addition, the incidents, including fatalities, surrounding the New England Compounding Center (NECC) in 2012, where the pharmacy produced contaminated injections of preservative-free methylprednisolone acetate (2, 3), show the risk from
* Corresponding Author: Dr. Tim Sandle, Bio Products Laboratory, Microbiology, Dagger Lane, Elstree, Hertfordshire, WD6 3BX, U.K. Telephone: ⫹44-208258-2483. E-mail: [email protected] doi: 10.5731/pdajpst.2013.00944
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fungi when prevalent in uncontrolled environments and to improperly validated processes (4). Pharmaceutical facilities are made up of a series of rooms called cleanrooms. Cleanrooms and zones are typically classified according to their use (the main activity within each room or zone) and are confirmed by the cleanliness of the air by the measurement of particles. This air cleanliness is either based on European Union (EU) good manufacturing practice (GMP) guidance for aseptically filled products, and the EU GMP alphabetic notations are adopted (5); or by using the International Standard ISO14644, where numerical classes are adopted (6). The cleanliness of the air is controlled by a heating, ventilation, and air conditioning (HVAC) system. The cleanliness of cleanrooms is controlled through a range of physical controls relating to air filtration, air changes, and air flow. The cleanliness is assessed through microbiological environmental monitoring regimes that are designed to assess numbers of micro621
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organisms and the types of species present. Environmental monitoring involves the collection of samples and the subsequent examination of data relating to the numbers or incidents of microorganisms present on surfaces, in the air, and from people. While reviews of cleanroom microflora have shown that bacteria resident or transient to human skin accounts for the overwhelming majority of isolates, a range of different types of fungi sometimes may be present within cleanrooms, depending upon the grade, use, and environmental conditions (7). The recent increase in notifications of fungal contamination of pharmaceutical products may be related to a lack of specific fungal monitoring as part of a facility’s environmental monitoring regime, together with limited knowledge of expected levels of fungal contamination in drug manufacturing cleanrooms (8). This may be linked with a general uncertainty as to how processes should be controlled and how cleaning and disinfection regimes for cleanrooms are evaluated to show that the product can be protected from environmental fungi (9). Although there is little regulatory guidance in relation to the construction of an environmental monitoring program, the standard environmental monitoring regime is generally reliant upon the use of a highly nutritious general growth medium, such as soya bean casein digest medium, incubated at a temperature designed to encourage mesophilic microorganisms (primarily bacteria) (10). Mesophiles contain those microorganisms that grow best in temperatures between 20 and 40 °C (11). The reasons why monitoring regimes are conventionally select for mesophilic microorganisms is because these microorganisms are most likely to be present, as most cleanroom contaminants are introduced into the area from people and are deposited into the air as skin is shed (12, 13). The weaknesses of environmental monitoring programs are well recognized. Such weaknesses include the imprecision of the monitoring methods used to recover microorganisms (14) and the ability of monitoring programs to only detect a small number of the microorganisms present in the environment (15). These limitations place a far greater reliance, on the part of the pharmaceutical manufacturer, upon environmental controls, such as air supply systems and cleaning and disinfection regimes (16). These limitations notwithstanding, environmental monitoring regimes are an expected and established feature 622
of pharmaceutical quality control. While environmental monitoring regimes for many years have focused on mesophilic monitoring, regulatory agencies, perhaps due to the various product recall incidents discussed above, are requesting pharmaceutical manufacturers to examine environments for microorganisms that may not grow at mesophilic conditions (such as psychrophilic microorganisms) (17) or screen for selective microorganisms, such as fungi. Although regulators express an interest in more comprehensive environmental monitoring regimes, none of the documents produced by regulatory agencies describe the types of media required for the isolation of microorganisms in the pharmaceutical cleanrooms. However, some, such as the FDA guidance for environments used for aseptic filling, require the microbiological agar used to have been tested to show the growth promotion of both bacteria and fungi (18). It also stands that no regulatory document, aside from the Japanese Pharmacopeia (19), which has more limited reach than the United States or European equivalents, requires the use of separate agar for the detection of yeasts and moulds. As a result most monitoring regimes use only a single, general purpose culture medium incubated at two temperatures: a higher one, often 30 –35 °C, to encourage the recovery of skincommensurable bacteria (20); and a lower one, often 20 –25 °C, designed to recover fungi (21). Whether such samples should be incubated at the higher or lower temperature first is a recurrent subject of debate within the industry. Alternatively, a modified general purpose agar at 30 –35 °C could be as effective for the recovery of fungi (22). It may be, however, that some fungi recover better on a selective mycological agar when that agar is incubated at a lower temperature (⬍25 °C) for a prolonged period of time. The optimal outcome for the time, temperature, and medium conundrum depends upon the cleanroom, environmental conditions, and types of fungi expected. If the concept of a culture media designed of the growth and detection of fungi is accepted, there are a range of different “selective” fungal agars available. An examination of these formed the basis of this case study. The study did not set out to argue that a special mycological medium was required for environmental monitoring, but to evaluate different types of media should the microbiologist elect to use a specially designed mycological media as part of the environmental monitoring program. PDA Journal of Pharmaceutical Science and Technology
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This study was focused upon the recovery of fungi from the environmental monitoring of a pharmaceutical processing facility. The objective was to assess the performance of different mycological agars within the environment rather than to assess the cleanliness of a particular facility. This was undertaken through a comparison of commercially available mycological agars. The reason for restricting the study to commercially produced agars was because environmental monitoring regimes, in large facilities, use thousands of agar plates each year and thus there is a need to connect suitable monitoring agars with what can be manufactured on a large scale by media suppliers and therefore be made available for pharmaceutical microbiologists to purchase. In setting out such a study there were complications. The suitability of any media would depend upon whether higher levels of fungi were recovered through the use of mycological culture media compared (otherwise any meaningful statistical comparison would be difficult and the outcome would err towards a qualitative assessment). This is not a straightforward assessment, for there is a range of different mycological media, none of which has been especially developed for environmental monitoring. Furthermore, with any study of cleanroom micoflora there are limitations. The first limitation relates to benchmarking, for there have been very few studies of pharmaceutical cleanroom microflora (or microbiota) published in recent years, and these studies have been largely based upon the use of soya bean casein digest medium subjected to a dual incubation regime (23– 25). However, some studies have indicated that certain types of fungi are more prevalent within cleanroom than others. The types of fungi described are Aspergillus spp., Penicillium spp., Trychophyton spp., Cladosporium spp., Curvularia spp., Fusarium spp., and Alternaria spp. (1, 7). In addition, the findings of Wilson et al. (26), who conducted studies on fungal contamination of high-efficiency particulate air (HEPA) filter air supply into cleanrooms, concluded that the most prevalent contaminants were species of Cladosporiium and that other species, such as Aspergillus spp., Penicillium spp., and Paecilomyces spp., were also present. A weakness with these studies is that little information is provided regarding the incident rates for these contaminants. A limitation in relation to the case study approach is that all cleanrooms differ in design and function, and Vol. 67, No. 6, November–December 2013
with the types of products processed. Therefore the type of cleanroom environment will have an impact upon the range of microorganisms recovered. A further limitation is with the microbial identification method (27). The key variables here are the size and scope of the databases used to compare cleanroom isolates and the types of methods used for analysis (whether the method is phenotypic or genotypic, and then the various technological variations of these methods) (28). Given the limitations with the automated methods, many fungi, particularly those of a filamentous type, continue to be identified either through macroscopic and microscopic study, or using genotypic methods. While the reader should be aware of these limitations before drawing any generalizations from the work presented in this paper, the authors consider that the approach and general findings will be of interest for facility microbiologists who are considering the application of a mycological agar for an environmental monitoring regime. Materials and Methods To examine for the presence of fungi within cleanrooms using different mycological media, a study was set up to examine pharmaceutical cleanrooms used for different activities. The study related to one pharmaceutical facility. It is acknowledged that each facility will differ and the results from the study are not intended to indicate that the facility studied was necessarily representative of other production plants. However, in order to improve the representativeness of the study, a range of cleanrooms dedicated to different uses were selected for the review (such as a wet area, a cold area, an ambient area, and so forth). The cleanrooms within the facility were used for nonsterile processing and they were classified as EU GMP Grade D. The air within the rooms was volumetrically exchanged at a rate of twenty times per hour. During the period of the study all personnel entering the cleanrooms wore cleanroom-certified garments and the rooms were subject to a weekly cleaning and disinfection regime. The aim of this study was to assess if there is any significant variation in the numbers of fungal isolates recovered and in relation to the variety of species in relation to five different mycological agars. For the study, five different commercially available mycological agars were selected: malt extract agar (MEA); 623
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MEA with penicillin and streptomycin supplement (MEP); potato dextrose agar (PDA) containing no antibiotics; Rose Bengal Agar (RBA) with chloramphenicol; and Sabouraud dextrose agar (SDA) (sometimes described as Sabouraud glucose agar) with chloramphenicol. The agar media used to perform environmental monitoring were manufactured by Cherwell Laboratories Limited’s (Bicester, Oxford, U.K.) RediporTM brand. Agar used was in the form of 9 cm diameter settle plates and 2.5 cm diameter contact plates. The inclusion of the antibiotics was based on standard commercial availability, with MEA available with and without penicillin; and RBA and SDA available with the bacteriostatic antimicrobial chloramphenicol. PDA generally does not contain an antimicrobial agent.
Sabouraud Dextrose Agar with Chloramphenicol (SDA) Formula Mycological peptone Glucose Chloramphenicol Agar pH 5.6 ⫾ 0.2
g/L 10.0 40.0 0.1 15.0
Potato Dextrose Agar (PDA) Formula Potato extract Glucose Agar pH 5.6 ⫾ 0.2
g/L 4.0 20.0 15.0
The formulation for each of the agars was as follows (29):
With the exception of RBA, each agar was formulated with a low pH designed to inhibit bacterial growth.
Malt Extract Agar (MEA)
MEA is arguably the most commonly used agar for environmental sampling of airborne fungi (30) and is recommended by the American Conference of Governmental Industrial Hygienists for fungal isolation from indoor environments (31). Other recommendations exist for typical media used for selective fungal monitoring in a variety of environments (such as for nonsterile pharmaceutical products). Various sources (including the main pharmacopeia) recommend SDA (32, 33). PDA is recommended for recovery of yeast and mould isolates (34). Within the food industry, RBA is a common choice (35, 36). The MEP agar was identical to MEA except for an antibiotic supplement. This medium was included in order to determine whether the addition of an antibiotic to the agar will have a significant impact in specifically inhibiting bacterial growth during selective fungal monitoring.
Formula Malt extract Mycological peptone Agar pH 5.4 ⫾ 0.2
g/L 30.0 5.0 15.0
Malt extract agar has a high content of peptone and is acidic. Malt Extract Agar Penicillin and Streptomycin (MEP) Formula Malt extract Agar Penicillin G Na salt Streptomycin sulphate pH 5.4 ⫾ 0.2
g/L 20.0 12.0 0.012 0.05
Rose Bengal Agar with Chloramphenicol (RBA) Formula Mycological peptone Glucose Dipotassium phosphate Magnesium sulphate Rose Bengal Chloramphenicol Agar pH 7.2 ⫾ 0.2 at 25 °C 624
g/L 5.0 10.0 1.0 0.5 0.05 0.1 15.5
In terms of availability, there is little to distinguish between the selected media. Manufacturers charge similar prices, and apart from RBA, which must be kept in darkness and at 2– 8 °C, they are all easy to store. Based on the lack of consensus on appropriate types of media, there is no reason to expect that any one of the above agars will significantly outperform any other. The two research objectives were ●
To assess if there is any significant variation in the number of fungal isolates recovered by five selective agars.
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Table I Cleanrooms Selected for the Study Room code A101 A102 A103 A104 A105 A106 A107 A108 A109
A110
●
Rationale for room selection These are wash up areas used for cleaning of used equipment. The rooms are characterized by a moist environment, which could potentially support fungal growth. In these areas large equipment is cleaned by a clean in place (CIP) process. These rooms are characterized by moist and warm environment, which could support germination of fungal spores. These are 2–8 °C cold rooms used for product processing. These areas were selected as representative cold areas. This is an airlock room, which interfaces to an unclassified area. The area could be at risk of fungal spores from close proximity of outside environment. This is an autoclave and equipment preparation area. This room is characterized by dry and warm conditions. This is a storage area, characterized by dry ambient conditions. The room has access to lift, which leads to an unclassified corridor within the building. The lift could pose a risk of transferring fungal spores from unclassified areas. This is a room has a large number of fixed processing vessels. It is characterized by warm and moist conditions, which could support fungal growth.
To assess any variation in recovery of different species or genera by the selective agars.
Prior to undertaking the study the media used was assessed for growth promotion in order to demonstrate that it could recover a low-level inocula of a yeast (Candida albicans ATCC 10231) and a filamentous fungus (Aspergillus brasiliensis ATCC 16404). For the study, ten process cleanrooms were selected. The rooms differed by temperature, moisture, intended use, and previous history of fungal recovery. The set of selected rooms is shown in Table I. Different types of environmental monitoring samples were taken in the rooms. These samples were designed to recover any fungi present from the air (in the form of settle plates, 9 cm exposed plate for a period of 1 h; active air-samples, taken using an impaction air sampler where 1 m3 of air was sampled using an SAS Super180 sampler (VWR International PBI S.r.l., Milan, Italy); and contact plates, where plates of 25 cm 2 surface area were pressed onto a surface for 10 s). The room–sample combinations are shown in Table II. Each room monitoring was performed in triplicate (in three separate sessions) for each agar. In total, 210 samples, across each room, were sampled for each Vol. 67, No. 6, November–December 2013
agar (with the total number of samples from the three sessions being 630). Mean microbial counts were calculated from each monitoring session. All plates collected as part of environmental monitoring were incubated at 20 –25 °C for a minimum of 8 days to ensure fungal spore germination. After completing the incubation period, all plates were read against an artificial light source and the number of colony-forming units (CFU) was recorded. All nonfilamentous colonies from any plates that showed growth were subcultured onto the same media from which they were initially recovered. These plates were incubated for 24 –72 h to produce a pure culture. Colonies were then Gram-stained to distinguish between yeast and bacterial isolates. Further identification was performed only for yeast isolates and not for the bacteria. Fungal isolates were identified using the GEN III OmniLog威 ID System automated identification system (Biolog Inc., Hayward, CA, USA). Where the automated identification system could not provide a reliable result for filamentous fungi, a visual identification was made by describing the macroscopic characteristics (morphology, pigment, and so forth) and microscopic characteristics (using a lactophenol cotton blue stain). 625
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Table II Room and Sample Combinations Contact plates Room number
Settle plates (n)
Working height (n)
Floor (n)
Air samples (n)
A101 A102 A103 A104 A105 A106 A107 A108 A109 A110
2 3 2 2 1 2 1 2 3 5
2 3 4 2 2 2 1 1 1 1
1 1 1 1 1 2 1 2 2 3
2 1 1 1 1 1 1 1 2 2
Results The mean counts of all fungal results obtained for each sample type (such as active air samples) for each agar were calculated. The recovery of fungi was, in general, low across the cleanroom surveyed with mean counts varying between 0.1 and 7.8 CFU per sample across each of the sampling methods. The highest fungal counts were obtained from active air sampling and the lowest counts from surface sampling using contact plates. It is noted that this is based upon low level recoveries, although a slight bias towards air recoveries was apparent. However, the range of results across agars for individual sampling methods was less pronounced. While the data, because of the low values, does not lend itself to detailed statistical analysis, the variance between media using air samplers was greater in absolute terms compared with the other sampling methods; the lowest result (MEA) was 73% that of
the highest (SDA), and 2.2 CFU was the difference between the mean results for MEA and SDA. However, a higher variance proportionally was recorded from contact plates and settle plates, where the lowest means obtained showed a recovery, for both sample types, of only 53% compared with the highest recoveries. A summary of the results is shown in Table III. The results were compared for statistical significance using an unpaired, two tailed, Student’s t-test with a 5% confidence interval. All results per sample type and per agar were compared to each other using t-test, and from this P-values were calculated. The lowest P-value was obtained between RBA and SDA settle plate results, ⫺0.16; the highest P-value was obtained between MEP and RBA AS results, ⫺1.00. None of the calculated P-values were lower than the 0.05 (5%) confidence interval or, in other words, none
Table III Summary of Data for Each Agar across the Three Sampling Methods (Mean Counts) Sampling Method
626
Medium
Air sample (CFU)
Contact plate (CFU)
Settle plate (CFU)
MEA MEP PDA RBA SDA
5.80 6.69 6.82 6.72 7.80
0.14 0.12 0.22 0.19 0.23
1.60 1.97 1.16 2.10 1.10
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Table IV Summary of Significance Test Results Unpaired T- Test (5%) Media Variant 1 MEA MEA MEA MEA MEP MEP MEP PDA PDA RBA
Sampling Method Air sample
Variant 2 MEP PDA RBA SDA PDA RBA SDA RBA SDA SDA
Settle plate
P-values 0.84 0.84 0.85 0.68 0.98 1.00 0.81 0.98 0.84 0.83
of the calculated values of t were higher than the values of the t-distribution table. A summary of the significance test results is provided in Table IV. Therefore there was no significant difference between the different agars in terms of the numbers of fungi detected. In comparing the different types of cleanrooms, the overwhelming majority of colonies were recovered from one of the rooms (the room coded A110). This room was often warm (25–30 °C), due to the presence of processing equipment, and of higher humidity (⬃60% relative humidity), in comparison to the other rooms studied. The culture media varied in relation to the different types of fungi detected, with some media detecting a greater variety of fungi than others. This is summarized in terms of both yeasts (Table V) and filamentous fungi (Table VI). From a review of Tables V and VI, the MEA agar, followed by SDA, recovered the greatest variety of yeasts. In contrast, the PDA medium recovered the lowest variety. The highest numbers of yeast isolates for each agar were recovered by contact plates, and the lowest numbers of yeast isolates was obtained from settle plate samples. In each case, visible colonies were seen, although no assessment was made as to the size of the colonies or other growth characteristics (the assessment was for detection or no detection). With filamentous fungi, the largest variety of species was recovered on RBA (15 species), and the smallest Vol. 67, No. 6, November–December 2013
Contact plate
0.81 0.44 0.57 0.35 0.35 0.44 0.27 0.79 0.93 0.70
0.64 0.46 0.54 0.41 0.23 0.89 0.21 0.18 0.91 0.16
variety from MEA (7 species). In contrast to the recovery of yeasts, the highest numbers of filamentous fungi were recovered from active air samples. The largest proportions of characterizable fungal species were Cladosporium spp. and Penicillium spp. The other species recovered were Aspergillus spp. and Bionectria sesquicillii Samuels (anam. Clonostachys spp.). In addition to the fungal counts, a note was made about the incidences of bacteria recovered by the different mycological agars. None of the media were wholly selective for fungi, and each recovered some bacteria. The greatest incidences of nonfungal colonies (represented as mean of all samples taken) were recovered on PDA. The mean counts for bacteria for each agar and across each sample type is summarized in Table VII. In order to further illustrate which sample types and which media recovered the greater proportion of fungi to bacteria, a ratio chart was constructed. This is displayed in Figure 1. The lowest relative proportion observed was on PDA contact plates with 15% fungal to 85% nonfungal mean colonies recovered, and RBA with 20% fungal to 80% nonfungal mean colonies recovered. In contrast, the highest ratio is observed for MEA and SDA (fungal 99% to nonfungal 1%). In terms of different sample types, surface sampling, using contact plates, recovered proportionally more nonfungal than fungal colonies, although the absolute 627
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Table V Recovery of Different Species of Yeast, for Each Agar Media Species Name
MEA
MEP
Arthroascus javanensis Candida edax Candida spp. ⻫ Cryptococcus albidus ⻫ Cryptococcus albidus var aerius Cryptococcus marinus ⻫ Cryptococcus spp. ⻫ Endomyces fibulinger ⻫ ⻫ Filobasidiella neoformans bacillisporus Pitchia alni Pitchia guilliermondii B ⻫ Rhodotorula acheniorum Rhodotorula aurantiaca A ⻫ ⻫ Rhodotorula aurantiaca B ⻫ Rhodotorula minuta ⻫ Schizosaccharomyces japonicas var japoni ⻫ Sporidibolus johnsonii A ⻫ Sporidibolus johnsonii B Trichosporon beigelii B ⻫ Other (including nonviable) ⻫⻫* * more than one uncharacterized or nonviable yeast isolate recovered. numbers in both cases are far lower than with the results from air sampling. Discussion When all results are counted, the greatest numbers of filamentous fungus colonies in general (by each agar) were recovered from active air samples and the lowest numbers from surface contact plates. This was expected and is consistent with the nature of filamentous fungi, where studies have shown that in dynamic environments higher numbers of fungal spores are likely to be dispersed in the air, rather than settling onto surfaces, as this is their principal means of transmission (37, 38). The reason why active air samples gave higher recoveries compared with settle plates can perhaps be explained by the nature of the settle plate technique. Settle plates require microbes to physically drop from the air onto a plate by gravitational forces or by moving through the air and coming into contact with an inanimate object (39). In a correctly working, dynamic cleanroom the constant movement of the air should make this difficult to achieve. 628
PDA
RBA
SDA
⻫ ⻫
⻫
⻫ ⻫ ⻫ ⻫ ⻫
⻫ ⻫
⻫ ⻫ ⻫ ⻫
⻫
⻫ ⻫
⻫⻫⻫*
It was noticeable that the overwhelming majority of samples gave a count of zero. This is not to imply that the media selected was weak at recovering environmental fungi, rather it was a reflection of the areas examined. From the data set, one particular room, with a higher temperature and humidity, recorded the highest microbial counts. Given that the facility within which the study was conducted may or may not be representative of other manufacturing facilities, little notice should be taken of the levels, other than the fact that the low levels made statistical analysis more difficult. Although the levels recovered were low and the final conclusion regarding the different media perhaps better rests on a qualitative assessment, the fungal results were compared statistically using Student’s t-test for significant difference. The result of this evaluation indicated that there was no statistically significant difference between the numbers of fungal colonies recovered by the five agars (which may be a consequence of the relatively low data values). However, it was noted, when qualitatively assessing the results, PDA Journal of Pharmaceutical Science and Technology
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Table VI Recovery of Different Species of Rilamentous Fungi, for Each Agar Media Species Name
MEA
Cladosporium spp. Cladosporium sphaerospermum Cladosporium macrocarpum Cladosporium tenuissimum Cladosporium cladosporoides Cladosporium herbarum Penicillium spp. Penicillium rubrum Penicillium melinii Penicillium corylophilum Penicillium chrysogenum Penicillium variabile Aspergillus spp. Bionectria sesquicillii Nonsporing (uncharacterized)*
⻫
MEP
PDA
⻫
RBA
SDA
⻫
⻫⻫⻫
⻫⻫ ⻫ ⻫⻫⻫⻫
⻫ ⻫ ⻫
⻫ ⻫
⻫⻫ ⻫
⻫ ⻫
⻫ ⻫ ⻫ ⻫⻫⻫
⻫ ⻫
⻫⻫⻫
that slightly higher counts on SDA contact plates were obtained compared with the other media. Thus on a simple, numerical basis it might therefore appear that SDA was slightly superior compared with the other agars at collecting fungi. However, this observed difference could not, based on the low recoveries obtained, be statistically proven. Each of the tested agars was shown to support a variety of yeasts and filamentous fungal colonies, although at slightly different levels. Notably, all media supported the growth of common cleanroom fungal contaminants such as Cladosporium spp. and Penicillium spp., fungi identified earlier from the literature as occurring more often in cleanrooms (1, 7, 16). One genera of filamentous fungus often cited as typical
⻫ ⻫ ⻫⻫⻫
⻫
⻫
⻫⻫⻫⻫
⻫⻫⻫⻫
⻫⻫⻫⻫⻫⻫
contaminant of indoor environments and pharmaceutical facilities is Aspergillus spp. In this case study, a limited number of these genera were recovered and then only on MEP, PDA, and RBA. The fact that Aspergillus was found in such small numbers (typically only one colony per agar across the set of settle plates) is more reflective of low levels of Aspergillus spp. in the particular environment sampled rather than inability of the agars to support the growth of this particular fungal species. In terms of recovering the greatest variety of different species, no medium was universally superior. The MEA medium recovered the greatest range of yeasts, whereas the RBA medium recovered the greatest range of filamentous fungi. With yeasts, the MEA
Table VII Summary of Mean Bacterial Colony Counts Recovered on Each Medium Sampling Method Medium
Air sample (CFU)
Contact plate (CFU)
Settle plate (CFU)
MEA MEP PDA RBA SDA
0.051 0.154 1.103 0.103 0.077
0.196 0.137 1.225 0.745 0.245
0.058 0.058 1.159 0.000 0.116
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established mycological agars. Sabouraud agar medium was developed by the French dermatologist Raymond J. A. Sabouraud in the late 1800s to support the growth of dermatophytes over a prolonged incubation time (40).
Figure 1 Relative proportions of bacteria and fungi recovered across different media and for different sample types. AS ⴝ active air samples, CP ⴝ contact plates, SP ⴝ settle plates. recorded 11 different types of yeast and the RBA only recorded 6 different yeasts. However, with filamentous fungi the RBA recorded 15 different species and the MEA only 7 different species. The differences in variety did not mean that the agars that recorded less diversity were not capable of growing a range of fungi. Some cross-agar tests were undertaken where each of the fungi detected were inoculated onto the other media. Although there were some differences in growth, each of the fungi grew on each of the media. These inoculations used the environmentally isolated strains. However, the strains had been subcultured and grown within the laboratory and were therefore in a more healthy state than the isolates originally detected within the cleanrooms (where such isolates would be, as minimum, stressed and possibly sublethally damaged if they had been exposed to cleanroom disinfectants). Therefore, what can be concluded is that some agars were slightly better at detecting certain species of fungi within the environment; this is a different inference than stating the suitability or unsuitability of the agars for growing or storing cultures within the laboratory. While the agar recording the highest diversity of filamentous fungi and yeasts was different, it is noteworthy that SDA featured second in both categories, recording relatively high levels of different yeast and filamentous fungal species. Given that SDA also recorded marginally higher fungal counts, this medium appears qualitatively to be the optimal one. As an historical note, the medium is one of the longest 630
Each agar presented a similar range of different genera in similar proportions. While these numbers do not represent a sufficiently large enough pool of data to be conclusive, they could be used as a basis for the likely range of fungal isolates one might expect to find in a pharmaceutical cleanroom (the limitations of different types of cleanrooms and the fact that different facilities are located in different geographical regions is acknowledged). In drawing this conclusion, results from literature into studies of fungi from controlled environments are noted. For examples, other studies have shown that Cladosporium spp. and Penicillium spp. are common in high numbers, with Aspergillus spp. also found, although in lower proportions (41). It was further noted that there were a number of uncharacterizable fungal isolates. This was limited by the identification methods available. If the study was to be repeated, more sophisticated methods of identification, such as internal transcribed spacer (ITS) regions of fungal ribosomal DNA, would be recommended (42, 43). As well as filamentous fungal isolates, the agars also recovered numbers of nonfilamentous isolates (bacteria); therefore, no agar was truly selective (including the media that contained antibiotics). The agar that recorded the lowest numbers of bacteria was MEA, whereas the agar recording the highest numbers was PDA (notably, this agar also recovered the lowest numbers of fungi). It could be that the low levels of bacteria recovered, should they have originated from personnel, had a resistance to the antibiotics added to the media formulae. This possibility was not explored further, as it fell outside of the objectives of the case study. The findings suggest that potato dextrose agar is the least selective out of all tested agars. While the selectivity of this agar could be improved by the addition of tartaric acid to lower the pH to 3.5, or by the addition of antibiotics, the data suggests that PDA is not capable of increased recovery of fungi in comparison to other agars, so there is little to recommend it, on the basis of this case study, in preference to the other agars tested. PDA Journal of Pharmaceutical Science and Technology
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In drawing conclusions from the study, some caution is required, as there will be variations relating to the formulation of each agar and the way in which it is manufactured. Furthermore, each pharmaceutical facility is different and within the facility there will be differences between cleanroom types and operations; moreover, the cleanroom studied in this investigated may or may not be representative of the industry in general. It also stands that the numbers of fungi present in the environments studied were very low, which made meaningful statistical analysis difficult. However, we offer some tentative conclusions. The research question, that there would not be significant differences between the abilities of various agars to promote fungal growth, was broadly confirmed. This was in terms of numerical values obtained (CFU counted) and the range of species recovered. A marginal argument, based on a qualitative assessment, could be made in favor of SDA in relation to numerical recovery and in consideration of the variety of species recovery. The least appropriate choice of agar appears, of the basis of less diversity of fungi and lower fungal counts, to be PDA. Moreover, while PDA supports the growth of filamentous fungus at similar levels to other selective fungal agars and it is recommended for use in enumeration of fungi by some standards, it is not selective enough (based on high numbers and large variety of bacteria it recovered). In order to clarify more effectively which of the available media, if any, are most suitable for promoting the growth of these species, and to draw more concrete conclusions regarding the prevalent fungal species present in pharmaceutical cleanrooms, future experiments face a number of obstacles. To obtain sufficient isolates, a longer sampling period or further repeated sampling would be required; it would also be prudent to examine other facilities in different geographical regions. As noted above, identifying the resultant isolates would require more complex and sophisticated identification systems than were available to the authors at the time this study was executed. Nonetheless, the study, given the current concerns about fungi in pharmaceutical facilities, does stand as a case study for microbiologists to compare and contrast their monitoring regimes against.
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