Opinion Article | Open Access | Published 15 June 2021
Study of fungi isolated from pharmaceutical cleanrooms: Types and origins
Understanding cleanroom microbiota is of importance for microbiologists and quality personnel to assess risks to products and to the environment, to propose remediation activities, to review changes in trends and to select representative organisms for growth promotion and disinfectant efficacy studies. Shifts with the types of organisms may indicate resistant strains, problems with cleaning practices and objectionable microorganisms. There are few published studies of the typical cleanroom microbiota and extraordinarily little information pertaining to fungi. This paper reviews fungi collected across a ten year period from EU GMP Grade C and D cleanrooms from a pharmaceutical facility. The paper considers the common types of fungi recovered and discusses their potential origins, in relation to people, indoor environments and outdoor environments (temperate oceanic climate). The most common genera of fungi isolates were: Aspergillus, Candida, Cladosporium, Fusarium and Penicillium.
Keywords: Cleanrooms, microorganisms, fungi, yeast, moulds, mycotoxins, pharmaceuticals, contamination control
Of the different types of microorganisms that cause microbial contamination (including those referenced in regulatory reports relating recalls), fungi can be the most challenging. This challenge relates to the environment niches that they occupy; the relative ease of dispersal; the ability of fungi to grow on almost any substrate (1); with the identification of fungi remaining a relatively specialist area when compared to the characterisation of bacteria (and here visual aids can help, and some are provided in this paper). Fungi (fruiting, filamentous, and yeasts) (2) are an important group of microorganisms which are responsible for various infections. Filamentous (moulds) and yeast-like fungi (and dimorphic variants) have, in the context of this paper, an association with the contamination of cleanroom surfaces and the potential contamination of pharmaceutical and healthcare products. Many of the reasons are for fungal contamination within pharmaceutical facilities relates to poor design, ageing facilities (especially fabric damage), poor controls over incoming materials, weaknesses with air handling systems, and process controls. This paper considers some of these issues looking at fungal types and fungal sources. The data relating common types of fungi is drawn from a long-term study of fungi isolated from EU GMP Grade C and Grade D cleanrooms from a pharmaceutical facility located in south-east England (where the climate is variable, often wet, and classed as a ‘temperate oceanic climate’ under the Köppen System) (3). There have been few published studies considering cleanroom microbiota, and limited studies presenting empirical data in relation to fungi (there is one relating to a pharmaceutical facility in Japan (4) and one undertaken in Spain (5); plus another study relating to clean air areas in a South Korean hospital (6)). The published work suggests that the majority of microorganisms found in cleanrooms are those expected to be transient to or resident on human skin. In addition to the skin flora other types of microorganisms present will be those that are present in soil which may be carried into the cleanrooms on shoes, clothing or materials (7). The most common microorganisms in cleanrooms are Gram-positive bacteria. These microorganisms often have a close phylogenetic affiliation as indicated by comparative analysis of partial 16S rDNA studies (such as between the Micrococci and Staphylococci) (8). Nonetheless, pharmaceutical environments will sometimes contain fungi, as evidenced from research that has looked at the extent of product recalls due to fungal contamination (9) (where there appears to be relationship between fungi typically recovered from cleanrooms, as presented in this current study, and with pharmaceutical product recall reports) (10); and from the few published studies into cleanroom microbiota (11-13). Studying the range, types and patterns of fungi found in cleanrooms can provide essential information for microbiologists and quality personnel in understanding cleanroom environments and for assisting with contamination control. This helps to establish a ‘norm’ and provide a measure for trending purposes. Moreover, such analysis enables potential origins to be considered, it enables fungi to be tracked to assess weaknesses in the contamination control cascade, and it enables representative isolates to be selected for activities like growth promotion testing and to assess disinfectant efficacy. One weakness when undertaking such a study is with the lack of a benchmark; this paper aims to provide a starting point.
Fungal contamination risks
Fungi are more evolutionarily advanced forms of microorganisms, as compared to the prokaryotes (such as bacteria).Fungi are commonly divided into two distinct morphological forms: yeasts and hyphae (or filamentous). Yeasts are unicellular fungi which reproduce asexually by blastoconidia formation (budding) or fission. Fungal contamination in pharmaceutical products represents a potential hazard for two reasons. First, it may cause product spoilage; the metabolic versatility of fungi is such that any formulation ingredient from simple sugars to complex aromatic molecules may undergo chemical modification in the presence of a suitable organism (14). Spoilage will not only affect therapeutic properties of the product but may also discourage the patient from taking the medication. Second, product contamination represents a health hazard to the patient, although the extent of the hazard will vary from product to product and patient to patient, depending on the types and numbers of organisms present, the route of administration, and the resistance of the patient to infection. Fungal contamination in any sterile product will always present a direct risk of causing patient harm. With non-sterile pharmaceuticals, the ability of fungi to produce degradative spoilage in products depends on their ability to synthesize appropriate enzymes. Pharmaceuticals, cosmetics, foods, and other products are so much at risk because fungi are extremely versatile and adaptive in their ability to synthesize degradative enzymes. A consequence of degradation is that low-molecular-weight substrates such as sugars, amino acids, organic acids, and glycerol are broken down by primary catabolic pathways. The enzymes for these pathways are constitutive in a wide range of fungi. Fungal contaminants like Aspergillus and Penicillium spp. are the most common source of proteinase and peptidase enzymes causing breakdown of compounds such as gelatine. It has been estimated that around half of the fungi found in the environment could cause infections in people (mycosis) (15). With pharmaceutical products the two major hazards are air, through the inhalation of spores (such as through inhalers), where the risk is with fungal growth or with mycotoxins; and skin, through the rubbing in of creams and ointments (many fungi live off keratin, a protein that makes up the skin, hair and nails), or through injection of a contaminated product (either through the vein, into the muscle, or into the spinal cord). Risks also arise from mycotoxins, which are metabolic products of filamentous fungi. There have been more than 400 different mycotoxins identified to date (16). In nature, mycotoxins are produced for the purposes of host immobilization. Some of the primary mycotoxin producing fungi being the types of fungi with an association with pharmaceutical ingredients and storage or manufacturing environment: species of Aspergillus, Penicillium, and Fusarium (17, 18). Depending upon the type and the level some mycotoxins are harmful, capable of causing acute toxicity, causing damage to liver, kidneys, nervous system, skin, mucous membranes, immune systems. The symptoms of a mycotoxicosis depend on the type of mycotoxin; the concentration and length of exposure; as well as age, health, and sex of the exposed individual (19). In relation to pharmaceuticals, certain types of raw materials may present a hazard should they be contaminated with mycotoxins. Foremost, this is with raw materials deriving from cellulosic or grain material, earth e.g., diatomaceous earth, or with herbal raw materials (20). In addition, the storage conditions of the material (presence of humidity and higher temperatures e.g., non-chilled) presents an additional risk factor.
Types of fungi in pharmaceutical environments
The most commonly isolated species to cleanrooms, according to literature, are Cladosporium spp., Aspergillus spp., Penicillium spp. and Aurebasidium spp (21). In addition to the cleanroom sources, the extent that personnel carry fungi appears to be greater than previously though. U.S. National Institutes of Health researchers sequenced the DNA of fungi at skin sites of healthy adults and found the heel and toes to carry high levels of fungi, with populous areas also relating to the head, neck and eyebrows. The main species found on the human body were Malassezia spp., Penicillium spp. and Aspergillus spp. Such high numbers mean that cleanroom gowning procedures and cleanroom behaviour disciplines need to be of a high standard (22). Despite the references in literature, which tend to veer towards the theoretical, there are few studies that provide actual data. To address this, a study was undertaken of fungi from a facility located in south-east England. The fungi isolated were from samples assessed over a ten year period (2011 – 2020) and the isolates were from EU GMP Grade C (ISO 14644 class 8 in operation) and Grade D cleanrooms. Isolates were drawn from an environmental monitoring regime designed to recover both bacteria and fungi. This was using a general-purpose agar (Tryptone Soy Agar) incubated at 20oC to 25 degrees C, followed by 30 degrees C to 35oC (23). The fungal densities were generally low, typically 2 CFU/unit of measurement where fungi was identified. Across the review period, some 7, 200 identifications were undertaken. Of these, 180 were fungal (the majority - 7, 020 - were bacterial) and 133 were able to be characterised to either genus or species level.
For the study, identification was principally undertaken by visual identification of morphological and microscopic characteristics (such as colony morphology, colour and sporulation, plus available cellular diagnostic features like conidiophores) and by using phenotypic identification systems which look at the carbohydrate utilization pattern (primary the Biolog Onmilog system and Microstation).This technology examines the phenotypic characteristics (based on the microorganisms’ observable characteristics) and requires a grounding in mycological training (24). It is recognised that more accurate identifications may have been obtained from a genotypic system, either looking at the D1/D2 region of the large ribosome subunit or the internal transcribed spacer regions (ITS1/ITS2). However, it was not economically viable to access such systems (25), or more novel assessments like pyrosequencing (26). The different types of fungi and their relative proportions are displayed in the Figure 1 below.
Figure 1: Major genera of fungi recovered across the course of the study, from Grade C and D cleanrooms Fungi are morphologically divided into three forms: those that develop macroscopic fruiting bodies (like mushrooms); those that are filamentous (‘mould’), where a fungus forms multicellular filamentous structures known as hyphae, that extend in three dimensions; and yeast-like, which are single cells. Some yeasts, like Candida albicans, are dimorphic (that is they have two shapes, according to different environmental conditions). In terms of the division between those fungi that are filamentous and those fungi that are yeast-like (which are dimorphic). In relation to the research, the division is as per Figure 2.
Figure 2: Division of the research findings into filamentous fungi (moulds) and yeasts Figure 2 indicates that the majority of fungi recovered are classed as filamentous fungi. In terms of the likely origins of the fungi, Table 1 (below) provides an indication of the most likely origins according to literature. The fungi recovered have been categorised as originating from people (which accounts for most of the yeasts and the filamentous fungus Trichophyton); indoors or outdoors. Some genus of fungi are found predominantly indoors, such as Cladosporium; others, more exclusively outdoors (such as Fusarium and Penicillium). Where fungi are found in both indoor and outdoor environments (such as Aspergillus), the numbers recovered have been divided equally (this is for illustrative purposes, the specific origins cannot be determined). The outcome of this exercise is shown in Figure 3.
Figure 3: Chart categorising fungi by likely origin Figure 3 illustrates the division between fungi associated with the external environment and fungi associated with the as-built environment are almost evenly divided, with a slight inclination towards fungi originating outside. Given the approximate allocation of the Aspergilli it is perhaps more reliable to regard the division as ‘equal’. A sizable proportion (at 20% of isolates) are attributable to people, in terms of naturally residential members of the skin microbiome (like Candida) (27) or transient pathogens (such as Trichophyton) (28). Table 1 lists the fungi in alphabetical order, detailing their possible origins and whether the fungi are considered pathogenic.
Table 1: Detail of identified fungal genera
From the genera detailed in Table 1, an attempt was made to speciate some of the isolates. Characterising specific species is more problematic. However, from the genera identified, the major species that could be characterised are presented in Table 2.
Table 2: Species of fungi from the study, including an indication of potential origins
The majority of the species that could be identified, as per Table 2, are those associated with the outdoor environment, and hence these organisms would suggest transfer into the facility.
With the major fungi recovered, they belong to six main genera. To a degree this matches the ubiquity of certain genera in the south-east of England For example, the filamentous fungi genera Aspergillus and Penicillium are ubiquitous, with more than 300 accepted species. Further detail of these genera is provided below, together with some microscopic and macroscopic images (presented to aid the reader with any benchmarking or characterisation exercise).
Aspergillus is a common fungus that can be found in indoor and outdoor environments. Aspergillus thrives on a variety of substrates such as corn, decaying vegetation and soil. These fungi are also common contaminants in air. The primary disease of concern is aspergillosis and this usually occurs in people with lung diseases (such as cystic fibrosis) or weakened immune systems (54).
Figure 4: Aspergillus species growing on tryptone soya agar, green-to-black pigmentation (Image: Tim Sandle’s laboratory)
Figure 5: Microscopic image of Aspergillus species, methylene blue stain x100 (Image: Tim Sandle’s laboratory)
Candida are thin-walled, small yeasts (4 to 6 microns) that reproduce by budding. Candida species are the most common cause of invasive fungal infections in humans (55).
Figure 6: Microscopic image of Candida albicans, a dimorphic fungus (with filamentous and yeast-like structures) (Image: Tim Sandle’s laboratory)
Cladosporium is a common mould found outdoors, on soil and plants, and indoors, on wet surfaces, including wallpaper and carpet. Many species are cosmopolitan fungi isolated from soil, plant debris and leaf surfaces. Cladosporium is very frequently isolated from air, especially during seasons in which humidity is elevated (56).
Figure 7: Cladosporium growing on tryptone soya agar, black pigmentation (Image: Tim Sandle’s laboratory)
Figure 8: Microscopic image of Cladosporium, phase contrast x 100 (Image: Tim Sandle’s laboratory)
Most Fusarium species are soil fungi and have a worldwide distribution. Some are plant pathogens, causing root and stem rot, vascular wilt or fruit rot.
Figure 9: Fusarium growing on tryptone soya agar, characteristic fluffy white mould (Image: Tim Sandle’s laboratory)
Figure 10: Microscopic image of Fusarium species, methylene blue stain x100 magnification (Image: Tim Sandle’s laboratory)
Penicillium species: the mould that saved millions of lives. Penicillium is a genus of moulds found everywhere world-wide.
Figure 11: Penicillium growing on tryptone soya agar, green pigmentation (Image: Tim Sandle’s laboratory)
Figure 12: Microscopic image on Penicillium species, methylene blue stain x100 (Image: Tim Sandle’s laboratory).
The above images are intended to illustrate represented fungi isolated.
Sources of fungi
Understanding the origin of fungi is important for contextualising the risk posed from different sources. The analysis above divides the cleanroom isolated fungi into human sources, indoor sources, and outdoor sources. With people, our skin is home to millions of bacteria, fungi and viruses that compose the skin microbiota, as detailed in a previous paper published in EJPPS (57). The extent to which the fungi recovered from the cleanrooms match what is expected from studies of the human skin mycobiome is stronger at the morphological level (a predominance of yeasts) than it is with specific genus or species. Human microbiota studies suggest fungi of the genus Malassezia should predominate at core body and arm sites, whereas foot sites were colonized by a more diverse combination of Malassezia spp., Aspergillus spp., Cryptococcus spp., Rhodotorula spp., Epicoccum spp. and Candida spp. Whereas the current study had a stronger bias towards Candida species, with other types of yeast found in smaller numbers and there was no recovery of Malassezia, which is surprising given that some species of this yeast are the most common cause of dandruff and seborrheic dermatitis (58). The absence will relate to the need to use specialist agar of a type not commonly used in environmental monitoring: modified Dixon’s agar ( malt-rich agar containing bile, upon which the colonies of the yeast appear cream to yellowish and smooth or lightly wrinkled). Growth does not normally occur on tryptone soya agar or Sabouraud dextrose agar (59) The extent to which this poses a problem needs to be addressed through effective gowning training and through a reliance o detecting other skin associated fungi that are recovery on the standard media used for cleanroom monitoring.
With the larger recovery of Candida in the current study, this will vary between individuals, given that Candida is more often an opportunistic pathogen (a similar point can be made in relation to Trichophyton) (60).
Research suggests that fungal communities are less stable than bacterial ones and many regions, such as the foot, are of a more transient nature and with more variety of species between individuals (61). Overall, bacteria are found in far greater numbers than fungi from skin sites (62). Given the close association of some fungi with particular niches of human skin, it can be reasoned that their recovery would relate to the quality of cleanroom gowns and the efficacy of gowning procedures. Hence a shift in the recovery of skin related fungi should alert microbiologists and cleanroom managers that greater attention needs to be paid to gowning practices. Where cleanroom clothing may be re-worn (such as with non-sterile manufacturing) changing practices should be defined so that personnel do not contaminate their factory clothing. One study looking at cleanroom contamination transfer metrics found that 68% of microorganisms within a cleanroom had been transferred in via the changing room (63).
With fungi more commonly associated with the outdoor environment, the route into the facility will be either through personnel (something that can be minimised by ensuring that outdoor clothing is removed on entry to the building and scrubs worn for entry to the changing room leading to the Grade C or D cleanroom) or via consumables trough the delivery process. Controls should be introduced where in-coming materials for pharmaceutical processing are held before transfer to the process areas. One key aspect is with storage, and in ensuring that sufficient air can circulate around the storage containers. Air movement will allow outer packaging to dry out if it has become damp. On movement into core production, a risk of cross-contamination from the outer packaging remains a possibility. This can be minimized through effective air handling systems.
Fungi within the cleanroom will have been transferred in at some point and will have survived by finding an ideal niche, being better suited to the conditions. It is less likely that fungi have entered from plant areas or through outer walls, although this is possible should there be damp or fabric damage (this risk will centre on the level of cleanroom care and maintenance). Once within the facility, there are some niches that are ether more favourable for fungi or which will act as suitable vectors for transfer. Industry surveys have shown a number of potential sources (64, 65):
Boxes (especially cardboard)
Cardboard in cleanrooms should be avoided. In contrast, other materials like laminates have smooth impervious surfaces and lower surface microbial counts – as a rule, remove outer packaging.
Controls are needed for storage, to ensure sufficient air can circulate around the storage containers. Air movement allows outer packaging to dry out if it has become damp.
Take care on movement, a risk of cross-contamination from the outer packaging remains a possibility.
Control of the pharmaceutical environment is important in relation to microbiological control in general. With fungi in particular, control should be centred on good cleanroom design, with a focus on minimizing airborne contamination. Fungi can be in the air, either dispersed from the fungus or carried in air-streams or on particles such as dust or in conjunction with water droplets. Air control is achieved through having air filtration and ensuring good turbulent airflow within cleanrooms (and unidirectional airflow as required in cabinets). Rooms of different cleanliness classes should be at different pressure differentials in order to prevent air from the “dirtier” area trailing into the “cleaner” area. There is an additional concern where their is inadequate facility design can lead to areas where fungi remain undiscovered, presenting a contamination risk in the future. Of particular concern here is damage to surfaces which can create niches in which fungi can settle and remain. Surfaces should be designed to minimize contamination and to enable them to be easily cleaned and disinfected. For example, the use of coving and designing chemically resistant surfaces. Particular care should be undertaken during structural alterations to buildings as this may give rise to these contaminants remaining in the vicinity. Thus strict controls should be in place during facility downtime. With cleaning and disinfection, as part of general rotation and where there is concern with fungal spores, an effective sporicidal agent (such as hydrogen peroxide, peracetic acid, chlorine dioxide, or hypochlorous acid) should be used (66).
In reviewing this author’s experience, the primary sources are from materials transferred into a cleanroom area from a non-cleanroom, especially where materials have bene outside (e.g., moved from a warehouse) or where building damage has occurred (such as damage to an outer facing wall, exposing inner materials, such as a torn vinyl covered wall exposing plasterboard). A species of fungus like Cladosporium (a melanized filamentous fungus) has a greater association with the built environment (and, if not addressed, a long residency). Whereas fungi like Penicillium and Fusarium are more likely to be brought into the cleanroom and, if detected frequently, there will be an equally frequent process of transferring in and out. In terms of survival, cooler and damp environments are mor likely to lead to fungal survival. In terms of the proliferation of fungi within cleanrooms, the main risk factors are poorly ventilated areas or cleanrooms with insufficient air changes; where areas are damp; where there are ridges or cracks in finishes. In addition, plaster and paint can provide a nutritional source for moulds, although the addition of a fungistat to paint (such as 5-pentachlorophenol) can be effective at inhibiting fungal growth.
This paper has presented the findings of industrial research where ten years of mycological data has been assessed, with data relating to fungal identifications performed of isolates from environmental monitoring plates. The plates were collected from Grade C and Grade
There are two significant limitations with any type of cleanroom organism review. The first relates to the type of cleanrooms. All cleanrooms differ in design and function. The type of cleanroom, including whether it operates at EU GMP Grades A, B, C, or D, will have an impact upon the range of microorganisms recovered, as will the location in the world (which will shape external environmental challenges). Here there will be differences with the temperature and humidity and whether there is a water source (a future review could assess temperature and humidity in conjunction with viable monitoring, using the thermo-hygrometer). Whilst these limitations are accepted, this author maintains there is meriting seeking to provide a benchmark.
The second limitation relates to the microbial identification method. 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). The database determines which microorganism will be characterised once all of the required tests have been completed. Many identification systems have databases which are biased towards medical microbiology and are more limited with respect to industrial and pharmaceutical microbiology. Phenotypic systems can be influenced by the culture media used. There are other limitations which could be considered in relation to different sampling techniques and in relation to seasonality.
A further limitation that could be levelled at this paper is with how representative the data is? As noted in the introduction, there have been very few studies of cleanroom organisms and even fewer relating to fungi. The two major studies relate to Japan and Spain (as referenced in the introduction). Here the main types of fungi identified were: Aspergillus spp., Penicillium spp., Scopulariopsis spp., and Chaetomium spp. These are all outdoor associated organisms, and two of the main genera are the same. There is, however, an absence of classic indoor fungi (like Cladosporium). This may relate to a different facility design or reflect that the two other published studies presented a far lower number of fungal isolates for review.
Furthermore, this current study did not assess the fungal load in the outdoor air in order to assess the relative impact to the indoor environment, although literature suggests a considerable reduction occurs moving from outside-in (67). Attempts were made to overcome this by drawing upon a range of different types of cleanrooms from different locales. However, there will, however, always be some differences based on the geographical location and the types of operations undertaken in the cleanrooms. Nonetheless, the data collated bears a strong resemblance to that of earlier studies and published work.
In terms of interpreting the findings, the most useful aspect is arguably by relating the data to contamination control. Repeat occurrences will signify that the cleanroom is not working as expected within the norm, and a connection between fungal occurrences between different grades of cleanroom or between different function areas (such as those separated by airlock or changing rooms) is indicative of a contamination transfer and control breakdown. It is important to emphasise this point, given that Cundell finds that insufficient attention is often afforded to fungal isolation and trending during environmental monitoring (68). Another application of the data relates to the quality control of culture media, such as with selecting representative environmental isolates for testing. A further feature of such analysis is whether the microorganisms are ‘objectionable’. Whether a microorganism is objectionable or not depends upon the manufacturer of the pharmaceutical and judgement of the microbiologist, in relation to the type of product and its application (69). Further work following the findings contained here could include an assessment of resistance to different cleanrooms conditions, especially with those fungi that produce conidia or spores as part of their life cycle that are more resistant to environmental impact than the typical fungal coenocytic cell (70). This could be extended to an assessment of disinfectant efficacy in conjunction with molecular characterization.
In drawing together the data trends and data interpretation, this paper has attempted not only to provide a benchmark for microbiologist to compare the cleanroom fungi in their facilities with, it has also attempted to explain why and when the characterisation of fungi is important and what that data means for pharmaceutical quality personnel.
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