Rapid Detection and Determination of the Aerodynamic Size Range of Airborne Mycobacteria Associated with Whirlpools

Abstract

Novel environmental air and water mycobacteria sampling and analytical methods are needed to circumvent difficulties associated with the use of culture-based methodologies. To implement this objective, a commercial, clinical, genus DNA amplification method utilizing the polymerase chain reaction (PCR) was interfaced with novel air sampling strategies in the laboratory. Two types of air samplers, a three-piece plastic, disposable filter cassette and an eight-stage micro-orifice uniform deposit impactor (MOUDI), were used in these studies. In both samplers, 37-mm polytetrafluoroethylene (PTFE) filters were used. Use of the MOUDI sampler permitted the capture of airborne mycobacteria in discrete size ranges, an important parameter for relating the airborne mycobacteria cells to potential respirable particles (aerodynamic diameter <10 microm) capable of causing health effects. Analysis of the samples was rapid, requiring only 1-1.5 days, as no microbial culturing or DNA purification was required. This approach was then used to detect suspected mycobacteria contamination associated with pools at a large public facility. PCR was also used to analyze various water samples from these pools. Again, no culturing or sample purification was required. Water samples taken from all ultraviolet light/hydrogen peroxide-treated whirlpools tested positive for the presence of mycobacteria. No mycobacteria were detected in the chlorine-treated pools and the water main supply facility. All air samples collected in the proximity of the indoor whirlpools and the associated changing rooms were strongly positive for airborne mycobacteria. The airborne mycobacteria particles were predominantly collected on MOUDI stages 1-6 representing an aerodynamic size range of 0.5 to 9.9 microm. In conclusion, using this approach permits the rapid detection of mycobacteria contamination as well as the routine monitoring of suspected pools. The approach circumvents problems associated with culture-based methods such as fungal overgrowth on agar plates, and the presence of nonculturable or difficult to culture mycobacteria strains.

Bioaerosol Mass Spectrometry for Rapid Detection of Individual Airborne Mycobacterium tuberculosis H37Ra Particles

“…1D. As previously described (35)(36)(37), the NIOSH bioaerosol chamber (Torrington Research Company, Torrington, CT) was constructed of stainless steel and measured 30 in. by 30 in.…”

“…Aerosolized M. bovis BCG and the avirulent M. tuberculosis H37Ra, surrogates for pathogenic M. tuberculosis, have previously been successfully sampled in a laboratory bioaerosol chamber (CDC-NIOSH, Cincinnati, OH) (35)(36)(37). These studies used viable Andersen six-stage samplers and eight-stage micro-orifice uniform deposit impactors containing Teflon filters for particle size fractionation.…”

“…Another study investigated aerosols of artificial saliva containing M. avium, M. intracellulare, and M. tuberculosis that were generated in a class III safety cabinet and sampled and analyzed by using viable Andersen six-stage samplers and viable all-glass impingers (21). Some of these techniques were subsequently applied, in the field, to the detection and characterization of mycobacteria associated with whirlpools at a large public facility (37). Also for the first time, the size range and concentrations of infectious tuberculosis aerosols were directly measured from culturable cough-generated aerosols of M. tuberculosis from infected human test subjects by using Andersen six-stage samplers with culturing (6).…”

Release of Free DNA by Membrane-Impaired Bacterial Aerosols Due to Aerosolization and Air Sampling

“…Very often bioaerosols are collected directly into liquid by using impingers or first collected onto filters and then transferred into liquid. To concentrate such samples, liquid is centrifuged, and only the pelleted cells are used for DNA extraction, while the rest of the liquid is discarded (18,(27)(28)(29)(30). However, if we consider that a large fraction of cells experience severe stress during aerosolization and air sampling, leading to the loss of their structural integrity, it becomes highly likely that genomic DNA from the ruptured cells is released into the liquid.…”

Microbial structure and chemical components of aerosols caused by rotating brushes in a wastewater treatment plant

“…Polymerase chain reaction (PCR)-based methods have been used to detect and quantify aerosolized bacteria and fungus Williams et al 2001;Rintala et al 2004), as well as estimate airborne microorganisms present in WWTPs (Pascual et al 2003) and rapidly detect and determine airborne Mycobacteria associated with whirlpools (Schafer et al 2003). PCRbased methods have also been applied to distinguish seasonal microbial aerosol emissions from the surfaces of aeration tanks in industrial and municipal WWTPs (Ranalli et al 2000), determine the presence of Varicella zoster virus in the atmosphere (Sawyer et al 1994), and analyze environmental sample assays in response to bioterrorism attack (Higgins et al 2003).…”