Abstract
The question which bacterial species are present in water and if they are viable is essential for drinking water safety but also of general relevance in aquatic ecology. To approach this question we combined propidium iodide/SYTO9 staining (“live/dead staining” indicating membrane integrity), fluorescence-activated cell sorting (FACS) and community fingerprinting for the analysis of a set of tap water samples. Live/dead staining revealed that about half of the bacteria in the tap water had intact membranes. Molecular analysis using 16S rRNA and 16S rRNA gene-based single-strand conformation polymorphism (SSCP) fingerprints and sequencing of drinking water bacteria before and after FACS sorting revealed: (1) the DNA- and RNA-based overall community structure differed substantially, (2) the community retrieved from RNA and DNA reflected different bacterial species, classified as 53 phylotypes (with only two common phylotypes), (3) the percentage of phylotpes with intact membranes or damaged cells were comparable for RNA- and DNA-based analyses, and (4) the retrieved species were primarily of aquatic origin. The pronounced difference between phylotypes obtained from DNA extracts (dominated by Betaproteobacteria, Bacteroidetes, and Actinobacteria) and from RNA extracts (dominated by Alpha-, Beta-, Gammaproteobacteria, Bacteroidetes, and Cyanobacteria) demonstrate the relevance of concomitant RNA and DNA analyses for drinking water studies. Unexpected was that a comparable fraction (about 21%) of phylotypes with membrane-injured cells was observed for DNA- and RNA-based analyses, contradicting the current understanding that RNA-based analyses represent the actively growing fraction of the bacterial community. Overall, we think that this combined approach provides an interesting tool for a concomitant phylogenetic and viability analysis of bacterial species of drinking water.
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References
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410
Bassam BJ, Caetano-Anoll G, Gresshoff PM (1991) Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal Biochem 196:80–83
Berney M, Weilenmann HU, Egli T (2006) Flow-cytometric study of vital cellular functions in Escherichia coli during solar disinfection (SODIS). Microbiology 152:1719–1729
Berney M, Hammes F, Bosshard F, Weilenmann H, Egli T (2007) Assessment and interpretation of bacterial viability by using the LIVE/DEAD BacLight Kit in combination with flow cytometry. Appl Environ Microbiol 73:3283–3290
Berney M, Vital M, Hülshoff I, Weilenmann H, Egli T, Hammes F (2008) Rapid, cultivation-independent assessment of microbial viability in drinking water. Water Res 42:4010–4018
Birch L, Dawson C, Cornett J, Keer J (2001) A comparison of nucleic acid amplification techniques for the assessment of bacterial viability. Lett Appl Microbiol 33:296–301
Boulos L, Prevost M, Barbeau B, Coallier J, Desjardins R (1999) LIVE/DEAD BacLight, application of a new rapid staining method for direct enumeration of viable and total bacteria in drinking water. J Microbiol Methods 37:77–86
Brettar I, Labrenz M, Flavier S, Botel J, Kuosa H, Christen R, Höfle MG (2006) Identification of a Thiomicrospira denitrificans-like epsilonproteobacterium as a catalyst for autotrophic denitrification in the Central Baltic Sea. Appl Environ Microbiol 72:1364–1372
Brettar I, Höfle MG (2008) Molecular assessment of bacterial pathogens—a contribution to drinking water safety. Curr Opin Biotechnol 19:274–280
Cole JR, Chai B, Farris RJ, Wang Q, Kulam-Syed-Mohideen AS, McGarrell DM, Bandela AM, Cardenas E, Garrity GM, Tiedje JM (2007) The ribosomal database project (RDP-II), introducing myRDP space and quality controlled public data. Nucl Acids Res 35:169–172
Czechowska K, Johnson DR, Meer JRVD (2008) Use of flow cytometric methods for single-cell analysis in environmental microbiology. Cur Opin Microbiol 11:205–212
Dumont M, Harmand J, Rapaport A, Godon J (2009) Towards functional molecular fingerprints. Environ Microbiol 11:1717–1727
Eichler S, Weinbauer MG, Dominik K, Höfle MG (2004) Extraction of total RNA and DNA from bacterioplankton, chapter 108. In: Kowalchuk GA, Bruijn FJD, Head IM, Akkermans ADL, van Elsas JD (eds) Molecular microbial ecology manual. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 103–120
Eichler S, Christen R, Höltje C, Westphal P, Bötel J, Brettar I, Mehling A, Höfle MG (2006) Composition and dynamics of bacterial communities of a drinking water supply system as assessed by RNA- and DNA-based 16S rRNA gene fingerprinting. Appl Environ Microbiol 72:1858–1872
Falcioni T, Papa S, Gasol JM (2008) Evaluating the flow-cytometric nucleic acid double-staining protocol in realistic situations of planktonic bacterial death. Appl Environ Microbiol 74:1767–1779
Farrelly V, Rainey F, Stackebrandt E (1995) Effect of genome size and rrn gene copy number on PCR amplification of 16S rRNA genes from a mixture of bacterial species. Appl Environ Microbiol 61:2798–2801
Hammes F, Berney M, Wang Y, Vital M, Köster O, Egli T (2008) Flow-cytometric total bacterial cell counts as a descriptive microbiological parameter for drinking water treatment processes. Water Res 42:269–277
Haugland R (2005) Assays for cell viability, proliferation, and function. In: The handbook: a guide to fluorescent probes and labeling technologies, 10th edn. Invitrogen Corp., USA. pp. 699–776. (http://www.invitrogen.com)
Henne K, Kahlisch L, Draheim J, Brettar I, Höfle MG (2008) Polyvalent fingerprint based molecular surveillance methods for drinking water supply systems. Water Sci Tech Water Supply 8:527–532
Hoefel D, Monis P, Grooby W, Andrews S, Saint C (2005) Profiling bacterial survival through a water treatment process and subsequent distribution system. J Appl Microbiol 99:175–186
Huq A, Rivera I, Colwell RR (2000) Epidemiological significance of viable but non culturable microorganisms In: Colwell RR, Grimes DJ (eds) Nonculturable Microorganisms in the Environment. American Society for Microbiology, Washington DC. pp. 301–323
Joux F, Lebaron P (2000) Use of fluorescent probes to assess physiological functions of bacteria at single-cell level. Microbes Infect 2:1523–1535
Kahlisch L, Henne K, Draheim J, Brettar I, Höfle MG (2010) High-resolution in situ genotyping of Legionella pneumophila populations in drinking water by Multiple-Locus Variable-Number of Tandem Repeat Analysis (MLVA) using environmental DNA. Appl Environ Microbiol 76:6186–6195
Kahlisch L, Henne K, Groebe L, Draheim J, Höfle MG, Brettar I (2010) Molecular analysis of the bacterial drinking water community with respect to live/dead status. Water Sci Tech 61:9–14
Klappenbach JA, Dunbar JM, Schmidt TM (2000) rRNA operon copy number reflects ecological strategies of bacteria. Appl Environ Microbiol 66:1328–1333
La Scola B, Mallet M, Grimont PAD, Raoult D (2003) Bosea eneae sp nov, Bosea massiliensis sp nov and Bosea vestrisii sp nov, isolated from hospital water supplies, and emendation of the genus Bosea (Das et al.). Int J Syst Evol Microbiol 53:15–20
Leuko S, Legat A, Fendrihan S, Stan-Lotter H (2004) Evaluation of the LIVE/DEAD BacLight Kit for detection of extremophilic Archaea and visualization of microorganisms in environmental hypersaline samples. Appl Environ Microbiol 70:6884–6886
Madigan MT, Martinko JM, Dunlap PV, Clark DP (2008) Brock biology of microorganisms, 12th edn. USA, Pearson Higher Education, New York
Mahmood S, Paton GI, Prosser JI (2005) Cultivation-independent in situ molecular analysis ofbacteria involved in degradation of pentachlorophenol in soil. Environ Microbiol 7:1349–1360
Marzorati M, Wittebolle L, Boon N, Daffonchio D, Verstraete W (2008) How to get more out of molecular fingerprints, practical tools for microbial ecology. Environ Microbiol 10:1571–1581
Moreno Y, Alonso JL, Botella S, Ferrús MA, Hernández J (2004) Survival and injury of Arcobacter after artificial inoculation into drinking water. Res Microbiol 155:726–730
Nocker A, Sossa-Fernandez P, Burr MD, Camper AK (2007) Use of propidium monoazide for live/dead distinction in microbial ecology. Appl Environ Microbiol 73:5111–5117
Schwieger F, Tebbe CC (1998) A new approach to utilize PCR–single-strand-conformation polymorphism for 16s rrna gene-based microbial community analysis. Appl Environ Microbiol 64:4870–4876
Steele HL, Streit WR (2005) Metagenomics, advances in ecology and biotechnology. FEMS Microbiol Lett 247:105–111
Weinbauer MG, Beckmann C, Höfle MG (1998) Utility of green fluorescent nucleic acid dyes and aluminum oxide membrane filters for rapid epifluorescence enumeration of soil and sediment bacteria. Appl Environ Microbiol 64:5000–5003
Weinbauer MG, Fritz I, Wenderoth DF, Höfle MG (2002) Simultaneous extraction from bacterioplankton of total RNA and DNA suitable for quantitative structure and function analyses. Appl Environ Microbiol 68:1082–1087
Wilbanks S, Glazer A (1993) Rod structure of a phycoerythrin II-containing phycobilisome. I. Organization and sequence of the gene cluster encoding the major phycobiliprotein rod components in the genome of marine Synechococcus sp WH8020. J Biol Chem 268:1226–1235
Wintzingerode FV, Göbel UB, Stackebrandt E (1997) Determination of microbial diversity in environmental samples, pitfalls of PCR-based rRNA analysis. FEMS Microbiol Rev 21:213–229
Yentsch CM, Horan PK, Muirhead K, Dortch Q, Haugen E, Legendre L, Murphy LS, Perry MJ, Phinney DA, Pomponi SA (1983) Flow cytometry and cell sorting, a technique for analysis and sorting of aquatic particles. Limnol Oceanogr 28:1275–1280
Zwart G, Crump B, Kamst-van-Agterveld MP, Hagen F, Han S (2002) Typical freshwater bacteria, an analysis of available 16S rRNA gene sequences from plankton of lakes and rivers. Aquat Microb Ecol 28:141–155
Acknowledgments
This work was supported by funds from the European Commission for the HEALTHY WATER project (FOOD-CT-2006-036306). The authors are solely responsible for the content of this publication. It does not represent the opinion of the European Commission. The European Commission is not responsible for any use that might be made of data appearing therein. We thank Josefin Draheim and Julia Strömpl for outstanding technical support.
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Kahlisch, L., Henne, K., Gröbe, L. et al. Assessing the Viability of Bacterial Species in Drinking Water by Combined Cellular and Molecular Analyses. Microb Ecol 63, 383–397 (2012). https://doi.org/10.1007/s00248-011-9918-4
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DOI: https://doi.org/10.1007/s00248-011-9918-4