Detection of airborne Legionella while showering using liquid impingement and fluorescent in situ hybridization (FISH)

Abstract

Aerosols of water contaminated with Legionella bacteria constitute the only mode of exposure for humans. However, the prevention strategy against this pathogenic bacteria risk is managed through the survey of water contamination. No relationship linked the Legionella bacteria water concentration and their airborne abundance. Therefore, new approaches in the field of the metrological aspects of Legionella bioaerosols are required. This study was aimed at testing the main principles for bioaerosol collection (solid impaction, liquid impingement and filtration) and the in situ hybridization (FISH) method, both in laboratory and field assays, with the intention of applying such methodologies for airborne Legionella bacteria detection while showering. An aerosolization chamber was developed to generate controlled and reproducible L. pneumophila aerosols. This tool allowed the identification of the liquid impingement method as the most appropriate one for collecting airborne Legionella bacteria. The culturable fraction of airborne L. pneumophila recovered with the liquid impingement principle was 4 and 700 times higher compared to the impaction and filtration techniques, respectively. Moreover, the concentrations of airborne L. pneumophila in the impinger fluid were on average 7.0 x 10(5) FISH-cells m(-3) air with the fluorescent in situ hybridization (FISH) method versus 9.0 x 10(4) CFU m(-3) air with the culture method. These results, recorded under well-controlled conditions, were confirmed during the field experiments performed on aerosols generated by hot water showers in health institutions. This new approach may provide a more accurate characterization of aerobiocontamination by Legionella bacteria.

Evaluation of bioaerosol sampling techniques for Legionella pneumophila coupled with culture assay and quantitative PCR

“…The resulting data may be employed as an estimation of the infectious risk associated with culturable L. pneumophila. Recently, the molecular assays such as in situ hybridization (FISH) and real-time quantitative polymerase chain reaction (qPCR) have also been adopted in airborne Legionella monitoring (Blatny et al, 2008;Bauer et al, 2008;Deloge-Abarkan et al, 2007;Dutil et al, 2007;Mathieu et al, 2006;Medema, Wullings, Roeleveld, & van der Kooij, 2004). It is noted that FISH could fail to detect L. pneumophila due to high detection limit of fluorescence microscope (Buchbinder, Trebesius, & Heesemann, 2002) and background fluorescence noise (Bauer et al, 2008).…”

“…With the popularity and advantage in use, however, the efficiency of the MAS-100 for collecting Legionella has not been quantitatively determined and compared with other bioaerosol samplers, e.g., Andersen one-stage sampler (Andersen 1-STG, Andersen Samplers Inc.), BioSampler (SKC Inc.), and all-glass impinger (AGI-30, Ace Glass Inc.). Moreover, previous studies have illustrated that the AGI-30 and BioSampler recover culturable L. pneumophila greater than the samplers with agar impaction or filtration (Deloge-Abarkan et al, 2007;Li et al, 2003). However, these studies were conducted by testing one of each type of agar-, liquid-, and filter-based samplers.…”

“…Culture-dependent methods are commonly used to assess airborne L. pneumophila in field studies (Blatny et al, 2008;Breiman et al, 1990;Deloge-Abarkan et al, 2007;Nguyen et al, 2006) and laboratory investigations (Deloge-Abarkan et al, 2007;Li, Tseng, Lai, & Chang, 2003). The resulting data may be employed as an estimation of the infectious risk associated with culturable L. pneumophila.…”

“…Among the samplers adopted for Legionella measurement, the microbiological air sampler (MAS-100, Merck), with the benefits of portability and built-in battery and pump, is increasingly used in fields (Bauer et al, 2008;Blatny et al, 2008;Deloge-Abarkan et al, 2007;Medema et al, 2004;Pascual et al, 2001). The MAS-100 is designed to collect bioaerosols on an agar plate.…”

Rapid MPN-Qpcr Screening for Pathogens in Air, Soil, Water, and Agricultural Produce

“…Pathogen detection in aerosols, for example, poses a unique challenge: The sampling is often performed using the settle-plate technique or utilizing a vacuum to suck air into a liquid impinger or onto an agar plate (Napoli et al 2012). However, all air detection methods have their limitations (Chinivasagam and Blackall 2005;Park et al 2014) as there are a variety of different designs each needing calibration for each microorganism (Deloge-Abarkan et al 2007;Landman et al 2013;Ryan et al 2014) and for each nutrient medium used; hence, the results are variable and can be difficult to interpret (Napoli et al 2012).…”