Linda D. Stetzenbach

Enhanced Detection of Surface-Associated Bacteria in Indoor Environments by Quantitative PCR

ABSTRACT Methods for detecting microorganisms on surfaces are needed to locate biocontamination sources and to relate surface and airborne concentrations. Research was conducted in an experimental room to evaluate surface sampling methods and quantitative PCR (QPCR) for enhanced detection of a target biocontaminant present on flooring materials. QPCR and culture analyses were used to quantitateBacillus subtilis (Bacillus globigii) endospores on vinyl tile, commercial carpet, and new and soiled residential carpet with samples obtained by four surface sampling methods: a swab kit, a sponge swipe, a cotton swab, and a bulk method. The initial data showed that greater overall sensitivity was obtained with the QPCR than with culture analysis; however, the QPCR results for bulk samples from residential carpet were negative. The swab kit and the sponge swipe methods were then tested with two levels of background biological contamination consisting of Penicillium chrysogenum spores. The B. subtilis values obtained by the QPCR method were greater than those obtained by culture analysis. The differences between the QPCR and culture data were significant for the samples obtained with the swab kit for all flooring materials except soiled residential carpet and with the sponge swipe for commercial carpet. The QPCR data showed that there were no significant differences between the swab kit and sponge swipe sampling methods for any of the flooring materials. Inhibition of QPCR due solely to biological contamination of flooring materials was not evident. However, some degree of inhibition was observed with the soiled residential carpet, which may have been caused by the presence of abiotic contaminants, alone or in combination with biological contaminants. The results of this research demonstrate the ability of QPCR to enhance detection and enumeration of biocontaminants on surface materials and provide information concerning the comparability of currently available surface sampling methods.

Determination of the efficacy of two building decontamination strategies by surface sampling with culture and quantitative PCR analysis

ABSTRACT The efficacy of currently available decontamination strategies for the treatment of indoor furnishings contaminated with bioterrorism agents is poorly understood. Efficacy testing of decontamination products in a controlled environment is needed to ensure that effective methods are used to decontaminate domestic and workplace settings. An experimental room supplied with materials used in office furnishings (i.e., wood laminate, painted metal, and vinyl tile) was used with controlled dry aerosol releases of endospores of Bacillus atrophaeus (“Bacillus subtilis subsp. niger,” also referred to as BG), a Bacillus anthracis surrogate. Studies were performed using two test products, a foam decontaminant and chlorine dioxide gas. Surface samples were collected pre- and posttreatment with three sampling methods and analyzed by culture and quantitative PCR (QPCR). Additional aerosol releases with environmental background present on the surface materials were also conducted to determine if there was any interference with decontamination or sample analysis. Culture results indicated that 105 to 106 CFU per sample were present on surfaces before decontamination. After decontamination with the foam, no culturable B. atrophaeus spores were detected. After decontamination with chlorine dioxide gas, no culturable B. atrophaeus was detected in 24 of 27 samples (89%). However, QPCR analysis showed that B. atrophaeus DNA was still present after decontamination with both methods. Environmental background material had no apparent effect on decontamination, but inhibition of the QPCR assay was observed. These results demonstrate the effectiveness of two decontamination methods and illustrate the utility of surface sampling and QPCR analysis for the evaluation of decontamination strategies.

Evaluation of the Biological Sampling Kit (BiSKit) for Large-Area Surface Sampling

ABSTRACT Current surface sampling methods for microbial contaminants are designed to sample small areas and utilize culture analysis. The total number of microbes recovered is low because a small area is sampled, making detection of a potential pathogen more difficult. Furthermore, sampling of small areas requires a greater number of samples to be collected, which delays the reporting of results, taxes laboratory resources and staffing, and increases analysis costs. A new biological surface sampling method, the Biological Sampling Kit (BiSKit), designed to sample large areas and to be compatible with testing with a variety of technologies, including PCR and immunoassay, was evaluated and compared to other surface sampling strategies. In experimental room trials, wood laminate and metal surfaces were contaminated by aerosolization of Bacillus atrophaeus spores, a simulant for Bacillus anthracis, into the room, followed by settling of the spores onto the test surfaces. The surfaces were sampled with the BiSKit, a cotton-based swab, and a foam-based swab. Samples were analyzed by culturing, quantitative PCR, and immunological assays. The results showed that the large surface area (1 m2) sampled with the BiSKit resulted in concentrations of B. atrophaeus in samples that were up to 10-fold higher than the concentrations obtained with the other methods tested. A comparison of wet and dry sampling with the BiSKit indicated that dry sampling was more efficient (efficiency, 18.4%) than wet sampling (efficiency, 11.3%). The sensitivities of detection of B. atrophaeus on metal surfaces were 42 ± 5.8 CFU/m2 for wet sampling and 100.5 ± 10.2 CFU/m2 for dry sampling. These results demonstrate that the use of a sampling device capable of sampling larger areas results in higher sensitivity than that obtained with currently available methods and has the advantage of sampling larger areas, thus requiring collection of fewer samples per site.

Measurement of Airborne Fungal Spore Dispersal from Three Types of Flooring Materials

Research was conducted in an experimental roomto measure the effect of human activity onairborne dispersal of settled fungal sporesfrom carpet and vinyl tile flooring. A seriesof experiments were conducted in whichcommercial loop pile carpet, residential cutpile carpet, or vinyl tile installed in theexperimental room were contaminated with Penicillium chrysogenum spores. The flooringmaterials were contaminated to two differentlevels (106 and 107 colony formingunits per square meter [c.f.u./m2] offlooring surface). Airborne culturable andtotal P. chrysogenum concentrations weremeasured using Andersen single-stage impactorsamplers and Burkard personal slide impactorsamplers, respectively. Bioaerosolconcentrations were measured at floor level, 1meter, and the adult breathing zone (1.5 meter)heights before and after human activityconsisting of walking in a prescribed patternfor 1 minute in the room. Airborne P.chrysogenum concentrations were greater withthe higher surface loading for all threeflooring materials. For all flooring materialsthere was no significant difference betweensampler locations, although the data from the1-meter location were the highest, followed bythe floor level and the breathing zonelocations, respectively. The data from theseexperiments indicate that while a very smallfraction of culturable P. chrysogenumspores present on flooring materials wereaerosolized by walking, relatively highairborne concentrations of spores maybere-entrained from contaminated materials. Theairborne P. chrysogenum concentrationswere significantly higher after walking on cutpile carpet than with the other two flooringmaterials at both contamination levels, withthe differences in concentration often ≥ 2orders of magnitude. No differences weremeasured in airborne culturable P.chrysogenum between vinyl flooring and looppile carpet at both contamination levels. Total spore data from the experiments with the107 c.f.u./m2 contamination levelindicated that walking on loop pile carpetproduced higher airborne spore concentrationsthan similarly contaminated vinyl tile althoughno significant difference was observed at the106 c.f.u./m2 level.

Evaluation of two surface sampling methods for detection of Erwinia herbicola on a variety of materials by culture and quantitative PCR

ABSTRACT This research was designed to evaluate surface sampling protocols for use with culture and quantitative PCR (QPCR) amplification assay for detection of the gram-negative bacterial biothreat simulant Erwinia herbicola on a variety of surface materials. Surfaces selected for evaluation were wood laminate, glass and computer monitor screens, metal file cabinets, plastic arena seats, nylon seat cushions, finished concrete flooring, and vinyl tile flooring. Laboratory and test chamber studies were performed to evaluate two sampling methods, a sponge and a macrofoam swab, for detection of E. herbicola on surface materials. In laboratory trials, seven materials were inoculated with a known concentration of E. herbicola cells and samples were collected from the surfaces of the materials to determine sampling efficiencies. Culture analysis was ineffective for assessing E. herbicola collection efficiency because very few culturable cells were obtained from surface samples. QPCR demonstrated that E. herbicola DNA was present in high concentrations on all of the surface samples, and sampling efficiencies ranged from 0.7 to 52.2%, depending on the sampling method and the surface material. The swab was generally more efficient than the sponge for collection of E. herbicola from surfaces. Test chamber trials were also performed in which E. herbicola was aerosolized into the chamber and allowed to settle onto test materials. Surface sampling results supported those obtained in laboratory trials. The results of this study demonstrate the capabilities of QPCR to enhance the detection and enumeration of biocontaminants on surface materials and provide information on the comparability of sampling methods.

The effect of indigenous bacteria on virus survival in ground water

Abstract Over one‐half of the waterborne disease outbreaks in the United States are due to the consumption of contaminated ground water. Although viruses are a major cause of illness in these outbreaks, very little is known about the factors which influence how long viruses can remain infective in ground water. Experiments were conducted using several ground water samples obtained from drinking water wells to determine the effects of the naturally‐occurring bacteria on the survival of coliphage MS‐2 and poliovirus type 1 inoculated into the samples. The numbers of bacteria and viruses were monitored over a 30‐day period. Parallel experiments were conducted using water which had been filtered to remove the bacteria. The increase in bacterial numbers in the first 24 hours of incubation was significantly correlated with the decay rate of coliphage MS‐2. However, consistent trends were found in the ability of the viruses to persist in the presence or absence of bacteria.

An Evaluation of Methicillin-Resistant Staphylococcus aureus Survival on Five Environmental Surfaces

This study evaluated methicillin-resistant Staphylococcus aureus (MRSA) survival on environmental surfaces: glass, wood, vinyl, plastic, and cloth. Effects of relative humidity (RH) and bovine serum albumin (BSA) were examined. Surfaces were inoculated with 10(7)-10(8) colony forming units per milliliter (CFU/ml)of MRSA with and without 1% BSA and incubated at 35°C at 45%-55% and 16% RH. Surfaces were sampled, and each collected sample was re-suspended in phosphate buffer, spread plated, and incubated at 35°C for 24 hrs; resulting colonies were enumerated. Samples were collected immediately on drying, and at 3 hrs, 24 hrs, 2 days, 3 days, 4 days, and 5 days. Results demonstrated that MRSA survived the longest on plastic and vinyl and for the least amount of time on wood (p < 0.001). BSA enabled MRSA to survive for significantly longer duration (p < 0.001). The number of CFU/ml was significantly lesser on surfaces stored in 45%-55% RH versus 16% RH. This study demonstrates that viable MRSA bacteria can remain on surfaces for days, which may impact the public health of occupants in workplace and residential settings.

Evaluation of portable air samplers for monitoring airborne culturable bacteria

Airborne culturable bacteria were monitored at five locations (three in an office/laboratory building and two in a private residence) in a series of experiments designed to compare the efficiency of four air samplers: the Andersen two-stage, Burkard portable, RCS Plus, and SAS Super 90 samplers. A total of 280 samples was collected. The four samplers were operated simultaneously, each sampling 100 L of air with collection on trypticase soy agar. The data were corrected by applying positive hole conversion factors for the Burkard portable, Andersen two-stage, and SAS Super 90 air samplers, and were expressed as log10 values prior to statistical analysis by analysis of variance. The Burkard portable air sampler retrieved the highest number of airborne culturable bacteria at four of the five sampling sites, followed by the SAS Super 90 and the Andersen two-stage impactor. The number of bacteria retrieved by the RCS Plus was significantly less than those retrieved by the other samplers. Among the predominant bacterial genera retrieved by all samplers were Staphylococcus, Bacillus, Corynebacterium, Micrococcus, and Streptococcus.

Dispersal of Fungal Spores from Three Types of Air Handling System Duct Material

Exposure to airborne microorganisms in indoor environments may result in infectious disease or elicit an allergic or irritant response. Air handling system components contaminated by fungi have been implicated in the dispersal of spores into the indoor environment, thereby serving as a route of exposure to occupants. This study was conducted to provide quantitative data on the dispersal of spores from fungal colonies growing on three types of duct material. Galvanized metal, rigid fibrous glass ductboard, and fiberglass duct liner were soiled and contaminated with a known concentration of Penicillium chrysogenum spores. The duct materials were incubated in humidity chambers to provide a matrix of growing, sporulating fungal colonies at a contamination level of 109 colony forming units (CFU) per duct section, consistent for all materials. For each experiment a contaminated duct section was inserted into the air handling system of an experimental room, and the air handling system was operated for three 5-minute cycles with an air flow of 4.2 m3 min−1. The duct air velocity was approximately 2.8 m sec−1. The airborne concentration of culturable P. chrysogenum spores (CFU m−3), total P. chrysogenum spores (spores m−3), and total P. chrysogenum-sized particles (particles m−3) were measured in the room using Andersen single-stage impactor samplers, Burkard slide impactor samplers, and an aerodynamic particle sizer, respectively. The highest airborne concentrations (104 CFU m−3; 105 spores m−3; 104 particles m−3) were measured during the first operating cycle of the air handling system for all duct materials with decreasing airborne concentrations measured during the second and third cycles. There was no significant difference in spore dispersal from the three contaminated duct materials. These data demonstrate the potential exposure for building occupants to high concentrations of spores dispersed from fungal colonies on air handling system duct materials during normal operation of the system.

Specific Detection of Fungi Associated With SBS When Using Quantitative Polymerase Chain Reaction

Publisher Summary Rapid identification and quantitation of fungi in indoor environments are necessary for an assessment of contamination levels and the estimation of the exposure of occupants. Traditional monitoring for fungi and other biocontaminants relies on sampling the air and surfaces and then analyzing the samples either microscopically or by culturing on artificial growth media. However, the inaccuracy of traditional methods and the long analytical time required to characterize bioaerosol and surface contaminant concentrations by these methods underscore the need to develop new analytical techniques that can provide fast, reliable data for fungal exposure monitoring. Methods for enhanced detection of fungi and other micro-organisms have been developed that increase the sensitivity and specificity of detection while reducing the analysis time. Polymerase chain reaction (PCR) is an enzymatic process capable of rapidly amplifying specific DNA sequences. This technique can be used to detect specific micro-organisms by amplifying the DNA sequences unique to the organism of interest regardless of the physiological state of the organism. This is especially useful in the detection of slow-growing organisms, non-culturable organisms, or those that are difficult to grow in the laboratory.