Identification of bacterial populations in drinking water using 16S rRNA-based sequence analyses.

Intracellular RNA is rapidly degraded in stressed cells and is more unstable outside of the cell than DNA. As a result, RNA-based methods have been suggested to study the active microbial fraction in environmental matrices. The aim of this study was to identify bacterial populations in drinking water by analyzing 16S rRNA-based clone libraries. Hollow-fiber ultrafiltration was used to concentrate bacterial communities from 40l of tap water collected at 12 different times during three different summer months from a single point-of-use. Total RNA was extracted from the microbial concentrates and used to develop 16S rRNA-based clone libraries. Phylogenetic analyses of 1231 partial 16S rRNA gene sequences showed that difficult-to-classify bacterial sequences were the most predominant clones, representing 57.6% of the sequences analyzed. Within these unclassified clades, most sequences were closely related to sequences retrieved from previous DNA- and RNA-based drinking water studies. Other bacterial groups represented in this study included Proteobacteria, cyanobacteria, Actinobacteria, Bacteroidetes, and Planctomycetes. Overall, the results suggest that these bacterial groups are amongst potentially active bacteria in drinking water. Diversity analyses of clones generated show that while overall diversity is similar amongst the different months, membership changes with respect to time. The results from this study further improve our understanding of the molecular diversity and bacterial population dynamics of drinking water microbial communities. Moreover, these results provide the sequence foundation for the development of molecular assays that target active drinking water bacteria.

[1]  D. Lane.  16S/23S rRNA sequencing , 1991 .

[2]  Margaret M. Williams,et al.  Growth of Escherichia coli in Model Distribution System Biofilms Exposed to Hypochlorous Acid or Monochloramine , 2003, Applied and Environmental Microbiology.

[3]  T. Oh,et al.  Porphyrobacter donghaensis sp. nov., isolated from sea water of the East Sea in Korea. , 2004, International journal of systematic and evolutionary microbiology.

[4]  D. Raoult,et al.  Influence of Long Time Storage in Mineral Water on RNA Stability of Pseudomonas aeruginosa and Escherichia coli after Heat Inactivation , 2008, PloS one.

[5]  Harald Meier,et al.  46. ARB: A Software Environment for Sequence Data , 2011 .

[6]  M. Drake,et al.  O 157 : H 7 coli Escherichia and Staphylococcus aureus UV-Irradiated Enterotoxigenic rRNA Stability in Heat-Killed and , 1998 .

[7]  A. Hiraishi,et al.  Proposal of the genus Sphingomonas sensu stricto and three new genera, Sphingobium, Novosphingobium and Sphingopyxis, on the basis of phylogenetic and chemotaxonomic analyses. , 2001, International journal of systematic and evolutionary microbiology.

[8]  S. Molin,et al.  Identification of Bacteria in Biofilm and Bulk Water Samples from a Nonchlorinated Model Drinking Water Distribution System: Detection of a Large Nitrite-Oxidizing Population Associated with Nitrospira spp , 2005, Applied and Environmental Microbiology.

[9]  J. Fry,et al.  Distribution and culturability of the uncultivated 'AGG58 cluster' of the Bacteroidetes phylum in aquatic environments. , 2004, FEMS microbiology ecology.

[10]  J. Tiedje,et al.  Naïve Bayesian Classifier for Rapid Assignment of rRNA Sequences into the New Bacterial Taxonomy , 2007, Applied and Environmental Microbiology.

[11]  L Birch,et al.  Molecular methods for the assessment of bacterial viability. , 2003, Journal of microbiological methods.

[12]  Thomas Huber,et al.  Bellerophon: a program to detect chimeric sequences in multiple sequence alignments , 2004, Bioinform..

[13]  J. Handelsman,et al.  Structures Community Memberships and Comparisons of Microbial Operational Taxonomic Unit-Based Introducing SONS , a Tool for , 2006 .

[14]  M. Höfle,et al.  Composition and Dynamics of Bacterial Communities of a Drinking Water Supply System as Assessed by RNA- and DNA-Based 16S rRNA Gene Fingerprinting , 2006, Applied and Environmental Microbiology.

[15]  E. Geldreich,et al.  A new medium for the enumeration and subculture of bacteria from potable water , 1985, Applied and environmental microbiology.

[16]  L. Richardson,et al.  Enhanced Survival of the Cyanobacterium Oscillatoria terebriformis in Darkness under Anaerobic Conditions , 1987, Applied and environmental microbiology.

[17]  N. Matsumoto,et al.  DNA- versus RNA-based denaturing gradient gel electrophoresis profiles of a bacterial community during replenishment after soil fumigation , 2007 .

[18]  K. Schleifer,et al.  Phylogenetic identification and in situ detection of individual microbial cells without cultivation. , 1995, Microbiological reviews.

[19]  K. Schleifer,et al.  ARB: a software environment for sequence data. , 2004, Nucleic acids research.

[20]  J. Young,et al.  dnaJ is a useful phylogenetic marker for alphaproteobacteria. , 2008, International journal of systematic and evolutionary microbiology.

[21]  T. Curtis,et al.  Presence and activity of ammonia-oxidising bacteria detected amongst the overall bacterial diversity along a physico-chemical gradient of a nitrifying wastewater treatment plant. , 2008, Water research.

[22]  J. S. Santo Domingo,et al.  Identification of active bacterial communities in a model drinking water biofilm system using 16S rRNA-based clone libraries. , 2006, FEMS microbiology letters.

[23]  A. E. Greenberg,et al.  Standard methods for the examination of water and wastewater : supplement to the sixteenth edition , 1988 .

[24]  Eoin L. Brodie,et al.  Greengenes, a Chimera-Checked 16S rRNA Gene Database and Workbench Compatible with ARB , 2006, Applied and Environmental Microbiology.

[25]  J. Regan,et al.  Diversity of nitrifying bacteria in full-scale chloraminated distribution systems. , 2003, Water research.

[26]  D. Lovley,et al.  Quantifying Expression of a Dissimilatory (bi)Sulfite Reductase Gene in Petroleum-Contaminated Marine Harbor Sediments , 2008, Microbial Ecology.

[27]  U. Szewzyk,et al.  Isolation of new bacterial species from drinking water biofilms and proof of their in situ dominance with highly specific 16S rRNA probes , 1997, Applied and environmental microbiology.

[28]  Noha H. Youssef,et al.  Novelty and Uniqueness Patterns of Rare Members of the Soil Biosphere , 2008, Applied and Environmental Microbiology.

[29]  M. Vieira,et al.  Comparison between standard culture and peptide nucleic acid 16S rRNA hybridization quantification to study the influence of physico-chemical parameters on Legionella pneumophila survival in drinking water biofilms , 2009, Biofouling.

[30]  E. Stackebrandt,et al.  Nucleic acid techniques in bacterial systematics , 1991 .

[31]  J. Lalucat,et al.  Identification of culturable bacteria present in haemodialysis water and fluid. , 2005, FEMS microbiology ecology.

[32]  M. Wagner,et al.  Quantification of Target Molecules Needed To Detect Microorganisms by Fluorescence In Situ Hybridization (FISH) and Catalyzed Reporter Deposition-FISH , 2008, Applied and Environmental Microbiology.

[33]  Alla Lapidus,et al.  The enigmatic planctomycetes may hold a key to the origins of methanogenesis and methylotrophy. , 2004, Molecular biology and evolution.

[34]  C. W. Keevil,et al.  Persistence of Helicobacter pylori in Heterotrophic Drinking-Water Biofilms , 2008, Applied and Environmental Microbiology.

[35]  Jothikumar Narayanan,et al.  Development of a Rapid Method for Simultaneous Recovery of Diverse Microbes in Drinking Water by Ultrafiltration with Sodium Polyphosphate and Surfactants , 2005, Applied and Environmental Microbiology.

[36]  I. Head,et al.  Identification of novel bacterial lineages as active members of microbial populations in a freshwater sediment using a rapid RNA extraction procedure and RT-PCR. , 1999, Microbiology.

[37]  H. Ren,et al.  Population dynamics of ammonia-oxidizing bacteria in an aerated submerged biofilm reactor for micropolluted raw water pretreatment , 2008, Applied Microbiology and Biotechnology.

[38]  M. Meckes,et al.  Molecular characterization of bacteria inhabiting a water distribution system simulator. , 2003, Water science and technology : a journal of the International Association on Water Pollution Research.

[39]  M. Meckes,et al.  Phylogenetic diversity of drinking water bacteria in a distribution system simulator , 2004, Journal of applied microbiology.

[40]  U. Szewzyk,et al.  Aquabacterium gen. nov., with description of Aquabacterium citratiphilum sp. nov., Aquabacterium parvum sp. nov. and Aquabacterium commune sp. nov., three in situ dominant bacterial species from the Berlin drinking water system. , 1999, International journal of systematic bacteriology.

[41]  J. Handelsman,et al.  Introducing DOTUR, a Computer Program for Defining Operational Taxonomic Units and Estimating Species Richness , 2005, Applied and Environmental Microbiology.

[42]  P. Gerhardt,et al.  Methods for general and molecular bacteriology , 1994 .