Detection and enumeration of airborne biocontaminants

Detection and enumeration of airborne biocontaminants Linda D Stetzenbach, Mark P Buttner and Patricia Cruz The sampling and analysis of airborne microorganisms has received attention in recent years owing to concerns with mold contamination in indoor environments and the threat of bioterrorism. Traditionally, the detection and enumeration of airborne microorganisms has been conducted using light microscopy and/or culture-based methods; however, these analyses are time-consuming, laborious, subjective and lack sensitivity and specificity. The use of molecular methods, such as quantitative polymerase chain reaction amplification, can enhance monitoring strategies by increasing sensitivity and specificity, while decreasing the time required for analysis. (IAQ) concern (Table 1). Exposure of building occupants to certain microorganisms and/or elevated concentrations of environmental organisms could result in allergenic reactions, irritant responses, toxicosis, respiratory illness and other health effects. In this article, an overview of bioaerosol sampling methods and a characterization of common airborne contaminants is presented. In addition, the potential use of enhanced monitoring of bioaerosols with quantitative polymerase chain reaction (QPCR) is discussed. Bioaerosols Addresses Harry Reid Center for Environmental Studies, 4505 South Maryland Parkway, University of Nevada-Las Vegas, Las Vegas, Nevada 89154-4009, USA  e-mail: [email protected] Current Opinion in Biotechnology 2004, 15:170–174 This review comes from a themed issue on Environmental biotechnology Edited by Michael Y Galperin and Alan JM Baker Available online 10th May 2004 0958-1669/$ – see front matter ß 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2004.04.009 Abbreviations IAQ indoor air quality IPC internal positive control PCR polymerase chain reaction QPCR quantitative polymerase chain reaction WMD weapons of mass destruction Introduction Bioaerosols are collections of airborne biological material. Components of the airborne material might result in health effects to exposed individuals in both the outdoor and indoor environment [1]. The threat of the airborne release of pathogenic organisms and microbial toxins as weapons of mass destruction (WMD) has increased awareness of the importance of bioaerosols [2]. Francisella tularensis, Yersinia pestis, Bacillus anthracis and smallpox virus are the causative agents of tularemia, plague, anthrax and smallpox, respectively. These organisms and others are cited as possible WMD, because of their potential for acute illness in the exposed public following an aerosol release (Table 1). Additionally, fungal spores, mycotoxins, endotoxin and other non-infectious agents released into the air in buildings are of indoor air quality Current Opinion in Biotechnology 2004, 15:170–174 Bioaerosols can consist of bacterial cells and cellular fragments, fungal spores and by-products of microbial metabolism, which can be present as particulate, liquid or volatile organic compounds. The particulate in a bioaerosol is generally 0.3–100 mm in diameter; however, the respirable size fraction of 1–10 mm is of primary concern [1]. Single bacterial cells range in size from 0.5–2.0 mm and are commonly spheres (cocci), rods (bacilli) or spirals, but airborne microorganisms are often present as aggregate formations of larger particles [3]. Bioaerosols that range in size from 1.0–5.0 mm generally remain in the air, whereas larger particles are deposited on surfaces [4]. Physical and environmental factors affect the settling of aerosols. Air currents, relative humidity and temperature are the most important environmental parameters affecting bioaerosol settling. The most significant physical parameters are particle size, density and shape [4,5]. Monitoring of bioaerosols Monitoring of airborne microbial populations to assess the IAQ is hampered by the lack of methods that provide precise, accurate and representative exposure estimates for bioaerosols [6]. Air sampling for bioaerosols has been conducted for decades with classical monitoring that relies on collection using forced air samplers and analysis by either culture on artificial growth media or microscopy [7]. Measurement of airborne organisms using forced airflow sampling provides a means to determine the concentration of organisms per unit volume of air, but differences between the various collection and analysis methods used affect the results and make data comparison difficult [7]. Impaction samplers that deposit airborne particulate onto a semi-solid agar surface are used with culture-based analysis. The Andersen single-stage impactor sampler (Andersen Instruments, Smyrna, GA), which is routinely used for the collection of culturable airborne bacteria and fungi, is operated at an air flow rate of 28.3 L/min. Collection of a 2 min sample and enumeration of a single colony in an air sample corresponds to a lower detection limit of 18 colony forming units per cubic www.sciencedirect.com Detecting airborne biocontaminants Stetzenbach, Buttner and Cruz 171 Table 1 Microorganisms associated with an airborne route of exposure that result in adverse human health effects. Organism Health effect Exposure Aspergillus fumigatus Aspergillus versicolor Bacillus anthracis Chaetomium species Francisella tularensis Legionella pneumophila Mycobacterium tuberculosis Penicillium species Stachybotrys chartarum Trichoderma species Variola virus Yersinia pestis Infection, allergy Allergy, toxicosis Anthrax Toxicosis Tularemia Pneumonia Tuberculosis Allergy Toxicosis Allergy, toxicosis Smallpox Plague Mold-contaminated building, compost Mold-contaminated building Bioterrorism, animal handlers, veterinarians Mold-contaminated building Potential WMD, infected rodents Aerosols from water spray Person-to-person Mold-contaminated building Mold-contaminated building Mold-contaminated building Potential WMD Potential WMD, infected fleas meter of air sampled (CFU/m3). The upper quantitation limit of this sampler is 104 CFU/m3, owing to space limitations on the agar collection surface, but considerable enumeration error is introduced at higher concentrations as multiple cells are deposited on the same location of the agar surface. When the sampling time is increased beyond 5 min the agar surface dries, thereby decreasing the physical collection efficiency of airborne particles owing to particle bounce and decreasing the viability of the bacterial cells due to desiccation [7]. Impaction samplers that deposit airborne particulate onto an adhesive-coated microscope slide rather than agar are used with light microscopy analysis to measure both viable and non-viable organisms; however, this methodology is usually limited to the measurement of pollen grains and fungal spores with identification to the genus level. Impingement samplers collect airborne cells and other particulate into a liquid [7]. Commonly used impingement samplers are operated at an air flow rate of 10–12 L/ min, but high velocity samplers have been developed for sampling larger volumes of air over extended sampling times [8]. Impingement collection permits processing of the sample by dilution or concentration to maximize accuracy in quantitation. A liquid sample can also be used with a variety of analytical methods, including culture, microscopy, immunoassay, flow cytometry and molecular methods [7]. Extended collection times with impingement sampling can result in increased sampling stress, thereby decreasing the viability of the collected bacteria [9,10]. Filtration sampling consists of the collection of bioaerosols by passing air through a porous filter material. Filtration sampling results in desiccation of vegetative bacterial cells; therefore, this technique is generally used for sampling of airborne dust, fungal spores and pollen [11]. Settling or gravity plates do not result in a representative sample of airborne cells, because of the differential settling of particles from the air; for this reason the method is not recommended for the determination of airborne microbial populations [12]. www.sciencedirect.com Air sampling is traditionally used to determine the concentrations of airborne contaminants and to evaluate the risk of exposure to individuals [6]. However, air sampling alone does not provide assurance that an area is free of biological contamination [13], because organisms may become re-aerosolized from surfaces during routine activity [14,15]. Therefore, surface sampling in addition to air sampling is used to locate areas of contamination and in identifying the source(s) of biocontamination. Surface sampling is also used in determining the effectiveness of remediation and clean-up of contaminated indoor environments [13,16,17]. The convex sampling surface of agar-filled RODAC (Replicate Organism Detection and Counting) plates is commonly used as a contact sampling method in hospital infection control to validate the efficacy of disinfection of surfaces, but sterile swabs are more often used to sample hard, smooth surfaces in buildings [17,13]. Vacuuming with dust sample collection bags or filter cassettes is used to sample porous (e.g. carpeting, upholstery and clothing) and non-porous (e.g. flooring, horizontal surfaces and furnishings) materials indoors. Vacuuming can sample large surface areas and be used with a variety of analysis methods after processing the collected material with a buffer solution [18]. The culturability of airborne and surface-associated biocontaminants can be affected by environmental factors, sampling practices, sample transport and laboratory culture conditions. The use of culture-based analysis methods underestimates populations of bioaerosols owing to the enumeration and identification of only those organisms that are culturable, while non-culturable organisms go undetected [10]. However, microorganisms such as fungi are capable of causing health effects whether they are in the culturable or non-culturable state [19]. Culture also requires time for the organisms to grow into recognizable colonies, often taking days to weeks depending on the microorganism. Light microscopy is tedious and lacks identification specificity, unless accompanied by specialized staining or immunological assay [7]. Biochemical assays that measure (1–3)b-D-glucan [20,21] Current Opinion in Biotechnology 2004, 15:170–174 172 Environmental biotechnology and ergosterol [22] indicate the presence of fungal biomass, which is useful in assessing the overall levels of fungal contamination, but these indicators do not demonstrate the presence of specific organisms. The inaccuracy of classical methods, lack of specificity, and lengthy analysis time required to characterize airborne biocontaminant concentrations and populations underscore the need to develop new sampling and analysis techniques that can provide rapid, reliable data for bioaerosol exposure. Enhanced analysis using quantitative polymerase chain reaction Polymerase chain reaction (PCR) amplification was developed in 1985 to rapidly amplify specific DNA sequences and to provide a qualitative or semi-quantitative assessment using gel electrophoresis to visualize the resulting PCR products [23]. Genetic material (i.e. DNA or RNA) from a single organism can provide a template for PCR amplification when combined with optimal sampling and analysis conditions. PCR was first demonstrated as a means to detect bacteria [24] and viruses [25] in air samples in 1994, and was later used to detect airborne Pneumocystis carinii [26]. Since that time, QPCR has been used for the enhanced detection of bacterial WMD surrogates [17], an actual bioterrorism agent [13], and fungi commonly associated with respiratory illness and water-damaged indoor environments [27,28]. Currently, there are two main methods capable of providing reproducible, accurate measurements of initial target DNA concentrations in samples: real-time QPCR using fluorogenic probes and competitive PCR [29,30]. The first method, QPCR, is a real-time assay that measures product accumulation utilizing a fluorogenic probe. Realtime QPCR has a high degree of sensitivity and accuracy, is less labor intensive than competitive PCR, and is amenable to high sample throughput. The TaqManTM fluorogenic nuclease QPCR assay is used in conjunction with a sequence detector to amplify and quantitate PCR products; the need for post-PCR gel electrophoresis, which is a requirement of competitive PCR, is eliminated. A fluorescently labeled oligonucleotide probe anneals between the primers of choice as the QPCR amplification reaction occurs, allowing for the determination of starting copy number of target DNA. Because this probe fluoresces only when the target DNA sequence is present, it provides confirmation of the target product. Other real-time QPCR instruments use the fluorescent dye SYBR Green or two fluorescently labeled probes. The second method, competitive PCR, relies on the presence of a known amount of internal control DNA competitor in each PCR reaction. This method requires that a co-amplifying organism (having identical primerbinding sites and amplification efficiency) be added to the sample. Quantitation is accomplished by comparing the quantity of the target product to that of the competitor product. Because the initial concentration of target DNA Current Opinion in Biotechnology 2004, 15:170–174 is unknown, multiple concentrations of the internal standard must be amplified in separate reaction tubes along with constant concentrations of the sample. Therefore, a significant effort must be applied in the development and testing of the competitor DNA [31]. Primers and probes are generally designed for the detection of a given genus (genus-specific primers) or for the detection of a single species (species-specific primers). The 18S or 16S ribosomal RNA genes can be used for the design of genus-specific primers and probes because they contain sequences that are highly conserved between members of the same genus, but the sequences are variable among different genera. Species-specific primers and probes can be designed to target the internal transcribed spacer regions and intergenic spacer of the nuclear rRNA gene, because these are highly variable areas within a genus or among populations [32]. The similarity (homology) in the DNA of closely related species makes the design of species-specific primers and probes difficult. A DNA repository such as GenBank can be used to find known DNA sequences. Using a sequence homology computer program such as the Basic Local Alignment Search Tool (BLAST) algorithm [33] unique DNA regions can be found on the target sequence. A primer and probe design software package such as Primer Express (Applied Biosystems, Foster City, CA) can then be used in which parameters such as melting temperature, amplicon size, base content, and primer/probe length can be selected. Validation studies are needed to ensure that there is no cross-reactivity with other species and to determine the lower detection limit of the assay with the primer and probe set. These studies comprise testing numerous strains of the target organism, as well as related and non-related microorganisms. In addition to specificity testing, the PCR should be optimized for maximum amplification of DNA. Optimization studies involve testing various concentrations of the primers and other PCR reagents with DNA from the target organism. Quantitation is achieved through the amplification of standards containing known concentrations of DNA or DNA extracted from suspensions of known concentrations of the target organism. A range of 100 to 105 templates per reaction provides a standard curve over five orders of magnitude. Using suspensions of the target organism to prepare the standards and extracting the standards in the same manner as samples provides absolute quantitation of target template and corrects for DNA losses during sample extraction. Amplification of standards at the same time and under the same conditions as the replicate unknown samples assists in quality control and quality assurance. Automated QPCR systems provide software that will construct a standard curve of CT value versus concentration; CT is the PCR cycle at which fluorescence (i.e. amplification product) is first detected and is inversely proportional to the concentration of the www.sciencedirect.com Detecting airborne biocontaminants Stetzenbach, Buttner and Cruz 173 initial DNA template. The concentration of the unknown samples can be extrapolated from the standard curve by the software and reported as the mean of two replicates. The presence of environmental background such as dust may inhibit the PCR reaction, resulting in false negatives. An internal positive control (IPC) should be used with all QPCR assays to determine whether PCR inhibitors are present in the sample. An IPC kit consisting of control non-target DNA, primers and a specific fluorescent probe labeled with a dye that is different from the target DNA probe allows for the differentiation of fluorescent signals generated during amplification. When a known amount of IPC DNA is amplified with the sample, inhibition can be detected by a change in amplification of control DNA. Conclusions Recent terrorism events and concern for fungal contaminants in indoor environments have increased the need for enhanced detection of airborne microorganisms, but established protocols that use QPCR in routine monitoring are not yet available. Unresolved issues that require additional research include the development of optimal sampling methods and protocols to effectively minimize interference owing to environmental background. These advances would allow QPCR to be routinely used for the detection of purposefully released biocontaminants and organisms of IAQ concern. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:  of special interest  of outstanding interest 1. Cox CS, Wathes CM: Bioaerosols in the environment. In Bioaerosols Handbook. Boca Raton, FL: Lewis Publishers; Edited by CS Cox and CM Wathes; 1995:11-14. 2. Sheeran TJ: Bioterrorism. In Encyclopedia of Environmental Microbiology, Edited by Bitton G. New York: Wiley; 2002:771-782. 3. Lighthart B: Physics of bioaerosols. In Atmospheric Microbial Aerosols: Theory and Applications. Edited by Lighthart B, Mohr J. New York: Chapman and Hall;1994:5-27. 4.  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