全部 标题 作者
关键词 摘要

PLOS ONE  2013 

The Efficient Method for Simultaneous Monitoring of the Culturable as Well as Nonculturable Airborne Microorganisms

DOI: 10.1371/journal.pone.0082186

Full-Text   Cite this paper   Add to My Lib

Abstract:

Cultivation-based microbiological methods are a gold standard for monitoring of airborne micro-organisms to determine the occupational exposure levels or transmission paths of a particular infectious agent. Some highly contagious microorganisms are not easily culturable but it is becoming evident that cultivation and molecular methods are complementary and in these cases highly relevant. We report a simple and efficient method for sampling and analyzing airborne bacteria with an impactor-type high-flow-rate portable air sampler, currently used for monitoring culturable bacteria or fungi. A method is reported for extraction of nucleic acids from impacted cells without prior cultivation and using agarose as a sampling matrix. The DNA extraction efficiency was determined in spiked samples and, samples taken from a wastewater treatment plant and an alpine area. The abundance, diversity and quantity of total bacteria were analysed by a quantitative polymerase chain reaction, and by construction and analysis of clone libraries. The method does not interfere with downstream PCR analysis and can cover the gap between traditional culture and molecular techniques of bioaerosol monitoring.

References

[1]  Alvarez AJ, Buttner MP, Stetzenbach LD (1995) PCR for bioaerosol monitoring: sensitivity and environmental interference. Appl Environ Microbiol 61: 3639–3644.
[2]  D'Arcy N, Canales M, Spratt DA, man Lai K (2012) Healthy schools: standardisation of culturing methods for seeking airborne pathogens in bioaerosols emitted from human sources. Aerobiologia 28: 413–422.
[3]  Angenent LT, Kelley ST, Amand AS, Pace NR, Hernandez MT (2005) Molecular identification of potential pathogens in water and air of a hospital therapy pool. Proc Natl Acad Sci U S A 102: 4860–4865.
[4]  Delgado-Viscogliosi P, Simonart T, Parent V, Marchand G, Dobbelaere M, et al. (2005) Rapid method for enumeration of viable Legionella pneumophila and other Legionella spp. in water. Appl Environ Microbiol 71: 4086–4096.
[5]  Chang C-W, Hung P-Y (2012) Evaluation of sampling techniques for detection and quantification of airborne legionellae at biological aeration basins and shower rooms. J Aerosol Sci 48: 63–74.
[6]  Chang C-W, Hung P-Y (2012) Methods for Detection and Quantification of Airborne Legionellae Around Cooling Towers. Aerosol Sci Technol 46: 369–379.
[7]  Rinsoz T, Duquenne P, Greff-Mirguet G, Oppliger A (2008) Application of real-time PCR for total airborne bacterial assessment: Comparison with epifluorescence microscopy and culture-dependent methods. Atmos Environ 42: 6767–6774.
[8]  Han Y, Li L, Liu J (2013) Characterization of the airborne bacteria community at different distances from the rotating brushes in a wastewater treatment plant by 16S rRNA gene clone libraries. J Environ Sci 25: 5–15.
[9]  Li K (2011) Molecular comparison of the sampling efficiency of four types of airborne bacterial samplers. Sci Total Environ 409: 5493–5498.
[10]  Carducci A, Verani M, Lombardi R, Casini B, Privitera G (2011) Environmental survey to assess viral contamination of air and surfaces in hospital settings. J Hosp Infect 77: 242–247.
[11]  Nehme B, Létourneau V, Forster RJ, Veillette M, Duchaine C (2008) Culture-independent approach of the bacterial bioaerosol diversity in the standard swine confinement buildings, and assessment of the seasonal effect. Environ Microbiol 10: 665–675.
[12]  He Q, Yao M (2011) Integration of high volume portable aerosol-to-hydrosol sampling and qPCR in monitoring bioaerosols. J Environ Monit 13: 706–712.
[13]  Joly-Duhamel C, Hellio D, Djabourov M (2002) All gelatin networks: 1. Biodiversity and physical chemistry. Langmuir 18: 7208–7217.
[14]  Wilson IG (1997) Inhibition and facilitation of nucleic acid amplification. Appl Environ Microbiol 63: 3741.
[15]  Smith HL, Goodner K (1958) Detection of bacterial gelatinases by gelatin-agar plate methods. J Bacteriol 76: 662–665.
[16]  Gibb AP, Wong S (1998) Inhibition of PCR by agar from bacteriological transport media. J Clin Microbiol 36: 275–276.
[17]  Reading F (2002) Inhibitory effects of agarose gel and LB medium on DNA sequencing. BioTechniques 33: 282–284.
[18]  Team RDC (2005) R: A language and environment for statistical computing. ISBN 3-900051-07-0. R Foundation for Statistical Computing. Vienna, Austria, 2013. url: http://www. R-project. org.
[19]  Karra S, Katsivela E (2007) Microorganisms in bioaerosol emissions from wastewater treatment plants during summer at a Mediterranean site. Water Res 41: 1355–1365.
[20]  Bauer H, Fuerhacker M, Zibuschka F, Schmid H, Puxbaum H (2002) Bacteria and fungi in aerosols generated by two different types of wastewater treatment plants. Water Res 36: 3965–3970.
[21]  Bauer H, Kasper-Giebl A, L?flund M, Giebl H, Hitzenberger R, et al. (2002) The contribution of bacteria and fungal spores to the organic carbon content of cloud water, precipitation and aerosols. Atmospheric Res 64: 109–119.
[22]  Nübel U, Engelen B, Felske A, Snaidr J, Wieshuber A, et al. (1996) Sequence heterogeneities of genes encoding 16S rRNAs in Paenibacillus polymyxa detected by temperature gradient gel electrophoresis. J Bacteriol 178: 5636–5643.
[23]  Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, et al. (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75: 7537–7541.
[24]  Cole JR, Wang Q, Cardenas E, Fish J, Chai B, et al. (2009) The Ribosomal Database Project: improved alignments and new tools for rRNA analysis. Nucleic Acids Res 37: D141–D145.
[25]  Willner D, Furlan M, Haynes M, Schmieder R, Angly FE, et al. (2009) Metagenomic analysis of respiratory tract DNA viral communities in cystic fibrosis and non-cystic fibrosis individuals. Plos One 4: e7370.
[26]  An HR, Mainelis G, Yao M (2004) Evaluation of a high-volume portable bioaerosol sampler in laboratory and field environments. Indoor Air 14: 385–393.
[27]  Yao M, Mainelis G (2006) Investigation of cut-off sizes and collection efficiencies of portable microbial samplers. Aerosol Sci Technol 40: 595–606.
[28]  Hospodsky D, Yamamoto N, Peccia J (2010) Accuracy, precision, and method detection limits of quantitative PCR for airborne bacteria and fungi. Appl Environ Microbiol 76: 7004–7012.
[29]  Yamamoto N, Kimura M, Matsuki H, Yanagisawa Y (2010) Optimization of a real-time PCR assay to quantitate airborne fungi collected on a gelatin filter. J Biosci Bioeng 109: 83–88.30.
[30]  Le Goff O, Godon J-J, Milferstedt K, Bacheley H, Steyer J-P, et al. (2012) A new combination of microbial indicators for monitoring composting bioaerosols. Atmos Environ
[31]  Ziros PG, Kokkinos PA, Legaki E, Vantarakis A (2011) Development of an optimized method for the detection of airborne viruses with real-time PCR analysis. Virol J 8: 1–6.
[32]  Shannon KE, Lee D-Y, Trevors JT, Beaudette LA (2007) Application of real-time quantitative PCR for the detection of selected bacterial pathogens during municipal wastewater treatment. Sci Total Environ 382: 121–129.
[33]  Brodie EL, DeSantis TZ, Parker JPM, Zubietta IX, Piceno YM, et al. (2007) Urban aerosols harbor diverse and dynamic bacterial populations. Proc Natl Acad Sci 104: 299–304.
[34]  Lee JA, Thorne PS, Reynolds SJ, O'Shaughnessy PT (2007) Monitoring risks in association with exposure levels among wastewater treatment plant workers. J Occup Environ Med 49: 1235–1248.

Full-Text

comments powered by Disqus