Comparative levels and types of microbial contamination detected in industrial clean rooms

The primary objective of this study was to determine quantitatively and qualitatively the predominant types of microbial contamination occurring in conventional and laminar flow clean rooms. One horizontal laminar flow, three conventional industrial clean rooms, and three open factory areas were selected for microbiological tests. The results showed that as the environment and personnel of a clean room were controlled in a more positive manner with respect to the reduction of particulate contamination, the levels of airborne and surface microbial contaminants were reduced accordingly. The chief sources of microbial contamination were associated with the density and activity of clean room personnel. In addition, the majority of microorganisms isolated from the intramural air by air samplers were those indigenous to humans. Studies on the fallout and accumulation of airborne microorganisms on stainless-steel surfaces showed that, although there were no significant differences in the levels of microbial contamination among the conventional clean rooms, the type of microorganism detected on stainless-steel surfaces was consistently and significantly different. In addition, the "plateau phenomenon" occurred in all environments studied. It was concluded that the stainless-steel strip method for detecting microbial accumulation on surfaces is efficient and sensitive in ultra-clean environments and is the most reliable and practical method for monitoring microbial contamination in future class 100 clean rooms to be used for the assembly of spacecraft which will be sterilized.     

Environmental monitoring in stem cell banks

The processing of stem cell lines for application in human therapy requires a physical environment in which air quality (i.e., the number of airborne particles) is controlled to minimize risk of contamination. The processing facility should be constructed and operated to minimise the introduction, generation and retention of particles and microorganisms. A formal program of environmental monitoring should be maintained in each stem cell bank to specify and assess key factors and their influence on the microbiological quality of the process and product. This program should assure the manipulation of cells involved in the derivation of stem cell lines and their culture under established limits for airborne particles and for microbial contamination of the air and surfaces. Environmental monitoring should also address the regulatory requirements in the countries in which the cells will be used. The monitoring programme will depend on local conditions in each processing centre or cell bank. Each centre will need to evaluate its specific needs and establish appropriate monitoring procedures which should not become intrusive to the extent that they might compromise the quality of the cell banks or products.     

Isolation and characterization of the environmental bacterial and fungi contamination in a pharmaceutical unit of mesenchymal stem cell for clinical use

Design and implementation of an environmental monitoring program is vital to assure the maintenance of acceptable quality conditions in a pharmaceutical manufacturing unit of human mesenchymal stem cells. Since sterility testing methods require 14 days and these cells are only viable for several hours, they are currently administered without the result of this test. Consequently environmental monitoring is a key element in stem cell banks for assuring low levels of potential introduction of contaminants into the cell products. The aim of this study was to qualitatively and quantitatively analyze the environmental microbiological quality in a pharmaceutical manufacturing unit of human mesenchymal stem cells production for use in advanced therapies. Two hundred and sixty one points were tested monthly during one year, 156 from air and 105 from surfaces. Among the 6264 samples tested, 231 showed contamination, 76.6% for bacteria and 23.4% for fungi. Microbial genuses isolated were Staphylococcus (89.7%), Microccocus (4.5%), Kocuria (3.2%) and Bacillus (2.6%). In the identification of fungi, three genuses were detected: Aspergillus (56%), Penicillium (26%) and Cladosporium (18%). The origin of the contamination was found to be due to personnel manipulation and air microbiota. For all sampling methods, alert limits were set and corrective measures suggested.     

Comparison of Microbial Contamination Levels Among Hospital Operating Rooms and Industrial Clean Rooms

Microbial contamination in industrial clean rooms was compared quantitatively and qualitatively with that of hospital operating rooms. The number of aerobic mesophilic microorganisms which accumulated on stainless-steel strips exposed for periods up to 21 weeks to the intramural air of four operating rooms was at least 1 log higher than the accumulation on strips exposed in four clean rooms, and was essentially the same as that found in two factory areas. Volumetric air samplings showed that there were significantly higher numbers of airborne viable particles per cubic foot of air in operating rooms than in industrial clean rooms. In contrast to clean rooms, where most of the airborne contaminants were those associated with human hair, skin, and respiratory tract, the hospital operating rooms showed a very high level of microorganisms associated with dust and soil.     

Microbiological contamination in stem cell cultures

Cell therapy and regenerative medicine are potentially two of the most exciting aspects of the novel therapeutic methods currently under development. However, these treatments present a number of important biosafety issues, like the possible transmission of microorganisms to the recipients. The most common potential form of contamination in these cell products is by bacteria (including Mycoplasma), yeast and fungi. In our study, 32 stem cell lines and feeder cell lines were analysed. There were 19 contaminated cell passages (12%). The main contaminants were gram positive cocci and Mycoplasma species, followed by gram negative rods and gram positive rods. The Mycoplasma contamination rate was 4%. Stem cell banks and other research centres aim to screen all processed stem cell lines for these microorganisms, and to assure that no contaminants are introduced in the banking procedures. It is a standard part of current good practice in stem cell banks to carry out routine microbiological controls of the stem cell lines and to work in a controlled environment to reduce the probability of contamination in the final product.     

A study of antifungal antibiotic production by Streptomyces chattanoogensis MTCC 3423 using full factorial design

AIMS: The understanding of the dynamics of surface microbial colonization with concomitant monitoring of biofilm formation requires the development of biofilm reactors that enable direct and real-time evaluation under different hydrodynamic conditions. METHODS AND RESULTS: This work proposes and discusses a simple flow cell reactor that provides a means to monitoring biofilm growth by periodical removing biofilm-attached slides for off-line, both non-destructive and destructive biofilm analyses. This is managed without the stoppage of the flow, thus reducing the contamination and the disturbance of the biofilm development. With this flow cell, biofilm growth and respiratory activity can be easily followed, either in well-defined laboratory conditions or in an industrial environment. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The reproducible and typical biofilm development curves obtained, validated this flow cell and confirmed its potential for different biofilm-related studies, which can include biocidal treatment.     

The rate of isolation of fungi in the genus Candida from the nasopharyngeal mucosa of the those in contact with the products from microbial protein manufacture]

In the mycological study of the air in working rooms at enterprises for the production of paprin and in the airspace of residential areas, as well as the study of the fungal contamination of the upper respiratory ways in workers and residents of the development zone, the isolation rate of the production fungal strains of the genus Candida from the nasopharynx was shown to depend on their content in the air. In the absence of producer microorganisms in the atmospheric air they were not detected among the population. The detection of yeast-like fungi of the genus Candida on the pharyngeal mucosa is of sanitary demonstrative importance for the evaluation of the specific microbial contamination of the air in working rooms and the air space of the development zone. When the stable work of gas purification systems was ensured at modern enterprises for the production of protein vitamin concentrate no production strains were detected in the atmospheric air of the development zone and on the nasopharyngeal mucosa of the residents, which was indicative of the absence of any influence of gas and air discharges from these enterprises on the microbial contamination of the airspace.     

Microbial Characterization during the Early Habitation of the International Space Station

An evaluation of the microbiota from air, water, and surface samples provided a baseline of microbial characterization onboard the International Space Station (ISS) to gain insight into bacterial and fungal contamination during the initial stages of construction and habitation. Using 16S genetic sequencing and rep-PCR, 63 bacterial strains were isolated for identification and fingerprinted for microbial tracking. Of the bacterial strains that were isolated and fingerprinted, 19 displayed similarity to each other. The use of these molecular tools allowed for the identification of bacteria not previously identified using automated biochemical analysis and provided a clear indication of the source of several ISS contaminants. Strains of Bradyrhizobium and Sphingomonas unable to be identified using sequencing were identified by comparison of rep-PCR DNA fingerprints. Distinct DNA fingerprints for several strains of Methylobacterium provided a clear indication of the source of an ISS water supply contaminant. Fungal and bacterial data acquired during monitoring do not suggest there is a current microbial hazard to the spacecraft, nor does any trend indicate a potential health risk. Previous spacecraft environmental analysis indicated that microbial contamination will increase with time and will require continued surveillance.     

Screening site contamination usina pathway exposure factors

Radian Corporation conducted an investigation of 29 waste sites at an air force base in New Mexico in partial fulfillment of the RCRA operating permit requirements for the facility. The contract required that the investigation be conducted under the Installation Restoration Program (IRP/CERCLA). In an effort to satisfy both RCRA and CERCLA requirements, a hybrid approach was taken for the risk assessment. Site contaminants ranged from petroleum and unconventional fuels to solvents, pesticides, and PCBs. A screening method was developed to classify the level of contamination at each of the 29 sites based on soil and groundwater sampling results. Under this method, sites were classified as “dirty,”; “clean,”; or “borderline.”; Dirty sites did not require a full‐scale risk assessment because some form of remedial action would be necessary. However, clean sites and borderline sites required a full‐scale risk assessment. For clean sites, the risk assessment served as justification for no further action; for borderline sites, the risk assessment determined whether or not remedial action would be required. The screening method used previously developed multipathway and multimedia models for estimating potential human exposure to environmental contaminants in the air, water, and soil through inhalation, ingestion, and dermal contact routes. Pathway exposure factors (PEFs), which combined information on human physiology, behavior patterns, and models of environmental transport, were used to determine the relationship between the concentration of environmental contaminants and human exposure. The PEF converts concentrations in environmental media to lifetime‐equivalent chronic daily intakes (CDI). Three exposure pathways contributing the greatest proportion of the risk were considered for screening these sites: (1) incidental ingestion of soil; (2) dermal contact with soil; and (3) ingestion of water. This project demonstrated that a screening approach could be used effectively to limit the number of full‐scale risk assessments required for a multisite investigation.     

Proposal for an integrated approach to microbial environmental monitoring in cultural heritage: experience at the Correggio exhibition in Parma

Microbial environmental monitoring represents one of the most useful methods to assess potential risks related to the integrity of cultural heritage and people’s health. The monitoring plan described in the present work is based on standardized techniques for measuring microbial air and surface contamination. Air contamination is assessed through both active and passive samplings, measuring the concentration of microbes in air (in colony forming units per cubic metre, CFU/m³) and the rate at which microorganisms settle on surfaces (expressed by the Index of Microbial Air Contamination, IMA, CFU/dm²/h). For surface contamination, two parameters are measured using nitrocellulose membranes: the Microbial Buildup (MB, the total number of microorganisms accumulated on a surface in an unknown period of time prior to the sampling) and the Hourly Microbial Fallout (HMF, the number of microorganisms that settle on a specific surface during 1 h). The monitoring plan was implemented at the Pilotta Palace in Parma, Italy, during the Correggio exhibition in 2009. Samplings were taken before and during opening times. Some microbial contamination was already detected before the arrival of visitors: air contamination mean values of 99.1 CFU/m³ and 5.2 CFU/dm²/h were recorded, while MB and HMF mean values for surfaces were 92 and 7 CFU/dm², respectively. A significant increase was recorded in air contamination during opening times, with mean values of 323.7 CFU/m³ and 19.4 CFU/dm²/h; surface contamination values increased as well. This monitoring plan represents a contribution towards the definition of a much needed standardized methodology.     

Oligophilic Bacteria as Tools To Monitor Aseptic Pharmaceutical Production Units

The bacterial loads of air, surfaces, and personnel in clean rooms are routinely monitored using a set of standard media. Bacteria that can grow on these media are a tiny fraction of the total numbers in any environment. A substantial proportion of bacteria long thought to be unculturable were recently shown to be oligophilic. Oligophile counts in clean rooms in our studies exceeded the standard plate counts by up to 2 orders of magnitude. They responded to disinfection routines in ways similar to the responses of conventional bacteria. We suggest that oligophiles are better tools than conventional bacteria for environmental monitoring in aseptic pharmaceutical production units.     

Oligophilic bacteria as tools to monitor aseptic pharmaceutical production units

The bacterial loads of air, surfaces, and personnel in clean rooms are routinely monitored using a set of standard media. Bacteria that can grow on these media are a tiny fraction of the total numbers in any environment. A substantial proportion of bacteria long thought to be unculturable were recently shown to be oligophilic. Oligophile counts in clean rooms in our studies exceeded the standard plate counts by up to 2 orders of magnitude. They responded to disinfection routines in ways similar to the responses of conventional bacteria. We suggest that oligophiles are better tools than conventional bacteria for environmental monitoring in aseptic pharmaceutical production units.     

Microbiological Evaluation of Protected Environments During Patient Occupancy

Microbiological monitoring has been conducted in two life island (LI) units and two laminar airflow (LAF) rooms while they were occupied by patients undergoing cancer chemotherapy. There were only 5 organisms per 1,000 ft(3) of air sampled in LAF rooms, 31 organisms in LI units, and over 3,000 organisms in regular hospital rooms. None of the floor samples obtained from hospital rooms was sterile, compared to over 70% in LAF rooms. The rate of deposition of organisms onto settling plates was one organism per 4.5 hr in LAF rooms compared to one organism per 0.08 hr in hospital rooms. Potential pathogens were isolated much more frequently from environmental samples obtained from hospital rooms than from LI units or LAF rooms. Two sites of persistent contamination arose in the LAF rooms: the vinyl tile flooring and the water supply system. Over half of the potential pathogens cultured from the protected environment units were cultured initially from the patients who occupied the units.     

Isolation and characterization of bacteria capable of tolerating the extreme conditions of clean room environments

In assessing the bacterial populations present in spacecraft assembly, spacecraft test, and launch preparation facilities, extremophilic bacteria (requiring severe conditions for growth) and extremotolerant bacteria (tolerant to extreme conditions) were isolated. Several cultivation approaches were employed to select for and identify bacteria that not only survive the nutrient-limiting conditions of clean room environments but can also withstand even more inhospitable environmental stresses. Due to their proximity to spacefaring objects, these bacteria pose a considerable risk for forward contamination of extraterrestrial sites. Samples collected from four geographically distinct National Aeronautics and Space Administration clean rooms were challenged with UV-C irradiation, 5% hydrogen peroxide, heat shock, pH extremes (pH 3.0 and 11.0), temperature extremes (4 degrees C to 65 degrees C), and hypersalinity (25% NaCl) prior to and/or during cultivation as a means of selecting for extremotolerant bacteria. Culture-independent approaches were employed to measure viable microbial (ATP-based) and total bacterial (quantitative PCR-based) burdens. Intracellular ATP concentrations suggested a viable microbial presence ranging from below detection limits to 10(6) cells/m(2). However, only 0.1 to 55% of these viable cells were able to grow on defined culture medium. Isolated members of the Bacillaceae family were more physiologically diverse than those reported in previous studies, including thermophiles (Geobacillus), obligate anaerobes (Paenibacillus), and halotolerant, alkalophilic species (Oceanobacillus and Exiguobacterium). Non-spore-forming microbes (alpha- and beta-proteobacteria and actinobacteria) exhibiting tolerance to the selected stresses were also encountered. The multiassay cultivation approach employed herein enhances the current understanding of the physiological diversity of bacteria housed in these clean rooms and leads us to ponder the origin and means of translocation of thermophiles, anaerobes, and halotolerant alkalophiles into these environments.     

Detection of microbial contaminants in mammalian cell cultures using a new fluorescence-based staining method

Aims: Microbial contamination of cell culture production processes is a current concern for biopharmaceutical industries. Traditional testing methods require several days to detect contamination and may advantageously be replaced by a rapid detection method. We developed a new method combining membrane filtration to microcolonies fluorescence staining method (MFSM) and compared it to epifluorescence microscopy. Methods and Results: Both methods were used to detect bacteria in CHO cells cultures. The epifluorescence microscopy showed to be limited by filterability, media interference and nonrobustness issues, whereas MFSM enabled consistent detection of Bacillus cereus, Staphylococcus epidermidis and Propionibacterium acnes after, respectively, 8, 9 and 48 h of incubation. Thanks to the nondestructive feature of the MFSM, stained membranes could be reincubated on culture media to yield visible colonies used for identification. Conclusions: The new method described in this study showed its ability to detect microbial contaminants in cell culture samples with time‐to‐results from 2–5 times shorter than the traditional testing method. Significance and Impact of the Study: The MFSM can be used as monitoring tool for cell cultures to significantly shorten detection times of microbial contamination, while preserving the ability to identify the contaminants and their viability.     

Importance of good manufacturing practices in microbiological monitoring in processing human tissues for transplant

Skin allografts represent an important therapeutic resource in the treatment of severe skin loss. The risk associated with application of processed tissues in humans is very low, however, human material always carries the risk of disease transmission. To minimise the risk of contamination of grafts, processing is carried out in clean rooms where air quality is monitored. Procedures and quality control tests are performed to standardise the production process and to guarantee the final product for human use. Since we only validate and distribute aseptic tissues, we conducted a study to determine what type of quality controls for skin processing are the most suitable for detecting processing errors and intercurrent contamination, and for faithfully mapping the process without unduly increasing production costs. Two different methods for quality control were statistically compared using the Fisher exact test. On the basis of the current study we selected our quality control procedure based on pre- and post-processing tissue controls, operator and environmental controls. Evaluation of the predictability of our control methods showed that tissue control was the most reliable method of revealing microbial contamination of grafts. We obtained 100 % sensitivity by doubling tissue controls, while maintaining high specificity (77 %).     

Prevention of Salmonella cross-contamination in an oilmeal manufacturing plant

AIMS: The mechanisms of Salmonella contamination in an oilmeal plant were investigated and the basic data were collected in order to achieve control of Salmonella in oilmeal. METHODS AND RESULTS: Salmonella was detected in all contamination vectors and environmental factors investigated, namely: operators, processing floor, dust in the air and rodents. In particular, high concentrations of Salmonella were detected on the processing floor of the manufacturing area, which has high oil content. Steam was the most effective disinfection method used for the processing floor, as the effects of heat sterilization and disinfection may work in tandem. In addition, restricting the movement of operators of the production chain remarkably reduced Salmonella contamination, even in areas of otherwise high contamination. CONCLUSIONS: Within the oilmeal plant, high Salmonella contamination rates for the processing floor represent the greatest risk of contamination of oilmeal via operators, dust in the air and rodents. Therefore, control of the processing floor is the most important means for reducing the oilmeal contamination rate. SIGNIFICANCE AND IMPACT OF THE STUDY: Specific Salmonella control methods for oilmeal plants have been established.     

Isolation and Characterization of Bacteria Capable of Tolerating the Extreme Conditions of Clean Room Environments

In assessing the bacterial populations present in spacecraft assembly, spacecraft test, and launch preparation facilities, extremophilic bacteria (requiring severe conditions for growth) and extremotolerant bacteria (tolerant to extreme conditions) were isolated. Several cultivation approaches were employed to select for and identify bacteria that not only survive the nutrient-limiting conditions of clean room environments but can also withstand even more inhospitable environmental stresses. Due to their proximity to spacefaring objects, these bacteria pose a considerable risk for forward contamination of extraterrestrial sites. Samples collected from four geographically distinct National Aeronautics and Space Administration clean rooms were challenged with UV-C irradiation, 5% hydrogen peroxide, heat shock, pH extremes (pH 3.0 and 11.0), temperature extremes (4°C to 65°C), and hypersalinity (25% NaCl) prior to and/or during cultivation as a means of selecting for extremotolerant bacteria. Culture-independent approaches were employed to measure viable microbial (ATP-based) and total bacterial (quantitative PCR-based) burdens. Intracellular ATP concentrations suggested a viable microbial presence ranging from below detection limits to 10⁶ cells/m². However, only 0.1 to 55% of these viable cells were able to grow on defined culture medium. Isolated members of the Bacillaceae family were more physiologically diverse than those reported in previous studies, including thermophiles (Geobacillus), obligate anaerobes (Paenibacillus), and halotolerant, alkalophilic species (Oceanobacillus and Exiguobacterium). Non-spore-forming microbes (α- and β-proteobacteria and actinobacteria) exhibiting tolerance to the selected stresses were also encountered. The multiassay cultivation approach employed herein enhances the current understanding of the physiological diversity of bacteria housed in these clean rooms and leads us to ponder the origin and means of translocation of thermophiles, anaerobes, and halotolerant alkalophiles into these environments.     

Microbial contamination of storerooms at the Auschwitz-Birkenau Museum

Biodeterioration of heritage collections caused by microorganisms is a worldwide problem. To avoid degradation caused by biological contaminants transported into the indoor environment by air, proper bioaerosol protection is required. The aim of this study was to assess the level of microbial contamination of the Auschwitz-Birkenau Museum collection based on qualitative and quantitative analyses of bacteria and fungi isolated from the atmosphere and settled dust of museum storerooms. The obtained results demonstrated that a correctly operated air-conditioning system and limiting the number of visitors in the studied storerooms can significantly inhibit microbial contamination of the air and decrease deposition of bacterial and fungal particulates on exhibit surfaces. The performed analyses confirm that an aerobiological assessment of museum premises is a useful tool in their hygienic evaluation and, if necessary, in decision-making regarding interventions to minimize biological decay of collections.     

Recurrent Aspergillus contamination in a biomedical research facility: a case study

Fungal contamination of biomedical processes and facilities can result in major revenue loss and product delay. A biomedical research facility (BRF) culturing human cell lines experienced recurring fungal contamination of clean room incubators over a 3-year period. In 2010, as part of the plan to mitigate contamination, 20 fungal specimens were isolated by air and swab samples at various locations within the BRF. Aspergillus niger and Aspergillus fumigatus were isolated from several clean-room incubators. A. niger and A. fumigatus were identified using sequence comparison of the 18S rRNA gene. To determine whether the contaminant strains isolated in 2010 were the same as or different from strains isolated between 2007 and 2009, a novel forensic approach to random amplified polymorphic DNA (RAPD) PCR was used. The phylogenetic relationship among isolates showed two main genotypic clusters, and indicated the continual presence of the same A. fumigatus strain in the clean room since 2007. Biofilms can serve as chronic sources of contamination; visual inspection of plugs within the incubators revealed fungal biofilms. Moreover, confocal microscopy imaging of flow cell-grown biofilms demonstrated that the strains isolated from the incubators formed dense biofilms relative to other environmental isolates from the BRF. Lastly, the efficacies of various disinfectants employed at the BRF were examined for their ability to prevent spore germination. Overall, the investigation found that the use of rubber plugs around thermometers in the tissue culture incubators provided a microenvironment where A. fumigatus could survive regular surface disinfection. A general lesson from this case study is that the presence of microenvironments harboring contaminants can undermine decontamination procedures and serve as a source of recurrent contamination.