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Applied and Environmental Microbiology, December 2007, p. 7757-7758, Vol. 73, No. 23
0099-2240/07/$08.00+0     doi:10.1128/AEM.01333-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Microarray Analysis of Escherichia coli Strains from Interstitial Beach Waters of Lake Huron (Canada){triangledown}

T. Kon,1 S. C. Weir,2 J. T. Trevors,1 H. Lee,1 J. Champagne,3 L. Meunier,3 R. Brousseau,3,4 and L. Masson3,4*

Department of Environmental Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1,1 Ontario Ministry of the Environment, 125 Resources Rd., Toronto, Ontario, Canada M9P 3V6,2 Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Ave., Montreal, Québec, Canada H4P 2R2,3 Département de Microbiologie et Immunologie, Université de Montréal, 2900 Edouard Montpetit Blvd., Montréal, Québec, Canada H3T 1J424

Received 14 June 2007/ Accepted 13 September 2007


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ABSTRACT
 
DNA microarray analyses revealed that clusters of repetitive extragenic palindromic PCR-related Escherichia coli isolates were isogenic only within interstitial Lake Huron beach water samples and not in surrounding waters. This suggested that adaptation and growth occurred within the interstitial water sites tested. All isolates were nonpathogenic, and three lake isolates possessed tetracycline resistance genes.


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INTRODUCTION
 
Escherichia coli is a commensal bacterium in the intestinal tracts of warm-blooded animals; however, some strains have acquired virulence factors causing intestinal or extraintestinal infections (3). Although intestinal E. coli has limited survival capability once it is in the environment, some isolates can persist (1). High numbers of E. coli cells in interstitial waters in the littoral zone of sandy beaches have been confirmed by several researchers (1, 5); however, their distribution and persistence in secondary environments remain poorly understood.

Previous studies, using repetitive extragenic palindromic PCR (REP-PCR) for typing, suggested that different interstitial water sampling sites on the shore of Lake Huron contain clusters of site-specific clonal populations of E. coli (5). Given the limited discriminatory capability of single REP-PCR primers for similar strains, a subset of isolates from earlier research (5) was selected for further characterization with a focused E. coli virulence-antibiotic resistance (2) and/or whole genomic gene (6) DNA microarray. Our objective was to assess the genetic relatedness of E. coli cells in interstitial waters by comparing representatives of various clonal populations from two different interstitial sites with water isolates from Lake Huron possessing similar REP-PCR patterns. Fifty isolates, including two groups of 10 interstitial beach isolates (from sampling sites separated by 30 m) and two groups of 15 isolates having REP-PCR patterns similar to those of either interstitial site, were selected. Colony lysate and fluorescent genomic DNA labeling and microarray hybridization were performed as described elsewhere (2, 6).


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Genetic relatedness of isolates in interstitial water and lake water.
 
Hybridization signals from the virulence microarray were scored as either present or absent for each of the 264 virulence and 38 antibiotic resistance gene probes. Virulence gene profiling results are shown as a dendrogram in Fig. 1. This distance tree was calculated using the Dice coefficient with Phyltools software (Phylogenetics Computer Tools, version 1.3; J. B. Buntjer, Wageningen University, The Netherlands [http://www.dpw.wau.nl/pv/PUB/pt/]). The resulting distances were used to calculate a neighbor-joining tree using PHYLIP (Phylogeny Inference Package, version 3.6; J. Felsenstein, University of Washington, Seattle [http://evolution.genetics.washington.edu/phylip.html]). The results indicated that within an interstitial site isolates with similar REP-PCR patterns had identical virulence gene contents, yet isolates from the two different sites (g10 or g12) were unrelated. After virulence-related genes common to all isolates were eliminated from the analysis, the dominant clonal population at site g10 possessed eight virulence-related genes (b1121, fliC, tspE4C2, lpfA, iss, traT, malX, and cib) (2) as determined with two extra gene probes specific for variants of the major fimbrial subunit of LPF fimbriae (lpfAEHEC and lpfA0113). In common with isolates from site g10, the clonal population from site g12 contained the b1121, fliC, and tspE4C2 genes. Both populations were positive for two lpfA probes, lpfA0113 and lpfAHEC; however, g12 isolates did not hybridize with the lpfA probe. The g12 isolates did not contain iss, traT, malX, or cib but did possess two other genes, flmA54 and wzy(O104).


Figure 1
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  		FIG. 1. Distance tree for genotypic profiles of E. coli isolates from interstitial sampling sites g10 and g12 and isolates from the surrounding lake as assessed by the virulence and antibiotic resistance microarray. The numbers are the isolate designations. All interstitial isolates clustered into two site-specific groups (brackets). A Dice distance value was used to estimate the degree of similarity, with 0 corresponding to complete similarity and 1 representing complete dissimilarity.

Unlike the identical virulence gene profiles observed within interstitial sites, large variations in virulence gene content among the 30 lake water isolates with REP-PCR patterns similar to those of isolates from either interstitial site (g10 or g12) were observed (Fig. 1). This demonstrated that although these selected isolates were related genetically, as suggested by REP-PCR banding, the lake isolates represent different strains.

E. coli strains can be pathogenic if they contain the appropriate combinations of virulence genes belonging to known pathotypes (3). In this study all isolates possessed incomplete pathotype gene sets, and three isolates (32-2, 538-1, and 538-4) possessed a single tetracycline resistance gene (tetE, tetC, and tetA, respectively). This lack of pathogenicity and low antibiotic resistance differ from the results of an earlier study of Lake Ontario, where 29% of water isolates were considered potentially pathogenic and 14% of the strains carried various antibiotic resistance genes (2). Caution should be used in interpreting the low number of virulence and antibiotic resistance genes because lake water isolates were selected based on the similarity of REP-PCR patterns to those of the two interstitial water populations. This basis for selection could potentially bias the virulence/antibiotic resistance gene distribution in this ecosystem.


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Genetic relatedness of isolates in interstitial sampling sites.
 
To ascertain whether interstitial isolates possessing identical REP-PCR profiles were isogenic, genomic DNA from two site g10 isolates (g10-6 and g10-9) were labeled with different cyanine dyes and hybridized to a genomic chip containing 6,057 oligonucleotide locus tags of open reading frames from three E. coli strains (K-12 strain MG1655, EDL933, and Sakai) (6). This process was repeated for two isolates from site g12 (g12-10 and g12-18). Two independent labelings (four technical replicates) of genomic DNA, including dye swaps, were corrected for background and normalized by LOWESS using GeneSpring 7.0 (Agilent Technologies). All log2 ratios of the replicate means for each gene were near 1.0, suggesting that there were identical distributions of genes or there were only slightly divergent genes in the site g10 or g12 pair of isolates. This result was further confirmed by GACK (genomotyping) analysis (4), which showed that all log2 ratios had high estimated probability of presence values (>90%).

The convergence of the REP-PCR, virulence, and whole genomic comparative hybridization data strongly indicates that the clonal populations previously found in interstitial water sites represent isogenic E. coli strains. Our data support the notion that the isolates have adapted to the interstitial beach water environment, where they can survive and eventually grow; however, microcosm studies are needed to confirm this hypothesis.

The low number of virulence and antibiotic resistance genes in our interstitial populations may reflect the potential loss of genes during adaptation to a suboptimal aquatic environment. The beach sand environment is characterized by a low nutrient content that limits bacterial survival and growth. E. coli strains persisting and dividing in such environments would be under selective pressure to express genes necessary for nutrient uptake enabling basal metabolism and then cell division. The ability of E. coli cells to survive in such a freshwater lake environment may have significant implications when E. coli is used as the indicator bacterium for fecal pollution if a background culturable population is always present or is present on a seasonal basis.


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ACKNOWLEDGMENTS
 
We acknowledge financial support from the Best in Science Program of the Ontario Ministry of the Environment. J.T.T. and H.L. acknowledge support from the Canadian Foundation for Innovation and the Ontario Innovation Trust.

We especially thank Todd Howell for his valuable input.


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FOOTNOTES
 
* Corresponding author. Mailing address: Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Ave., Montreal, Quebec, Canada H4P 2R2. Phone: (514) 496-6150. Fax: (514) 496-6213. E-mail: Luke.Masson{at}nrc-cnrc.gc.ca Back

{triangledown} Published ahead of print on 21 September 2007. Back


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REFERENCES
 
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Applied and Environmental Microbiology, December 2007, p. 7757-7758, Vol. 73, No. 23
0099-2240/07/$08.00+0     doi:10.1128/AEM.01333-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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