tir- and stx-positive Escherichia coli in stream waters in a metropolitan area

Abstract

Diarrheagenic Escherichia coli, which may include the enteropathogenic E. coli and the enterohemorrhagic E. coli, are a significant cause of diarrheal disease among infants and children in both developing and developed areas. Disease outbreaks related to freshwater exposure have been documented, but the presence of these organisms in the urban aquatic environment is not well characterized. From April 2002 through April 2004 we conducted weekly surveys of streams in the metropolitan Baltimore, Md., area for the prevalence of potentially pathogenic E. coli by using PCR assays targeting the tir and stx(1) and stx(2) genes. Coliforms testing positive for the presence of the tir gene were cultured from 653 of 1,218 samples (53%), with a greater prevalence associated with urban, polluted streams than in suburban and forested watershed streams. Polluted urban streams were also more likely to test positive for the presence of one of the stx genes. Sequence analysis of the tir amplicon, as well as the entire tir gene from three isolates, indicated that the pathogenic E. coli present in the stream waters has a high degree of sequence homology with the E. coli O157:H7 serotype. Our data indicate that pathogenic E. coli are continually deposited into a variety of stream habitats and suggest that this organism may be a permanent member of the gastrointestinal microflora of humans and animals in the metropolitan Baltimore area.

FIG. 1.

Map depicting the core stream sampling sites in the Gwynns Falls watershed (∼163 km²) in Western Baltimore County/Baltimore City. Identities of site acronyms are given in the Materials and Methods. (Left) Number of samples assayed over the period from April 2002 to March 2004 and percentage of samples determined to be positive by E. coli tir gene PCR; (right) percentage of samples (n = 31) collected from July 2003 to March 2004 testing positive for either E. coli stx₁ or stx₂ genes.

FIG. 2.

Results of E. coli tir gene PCR conducted on coliforms cultured from stream water samples taken on 27 April 2004. Each lane represents a given stream site: DRKR, GFGB, Gwynns Falls up from Gwynns Run (GFuGR), Jennifer Bridge Harford Hills (JBHH), Jennifer Bridge Northwind Rd. (JBNW), and the Isco sampler from Rognel Heights (ISCO). Lanes: L, DNA ladder; ntc, no template control; lane Odw, E. coli O157:H7 Odwalla strain positive control.

FIG. 3.

Bar graph depicting percentage of weekly stream water-derived coliforms testing positive via tir gene PCR (y axis) during the period from 25 April 2002 to 27 April 2004 (x axis). The total number of samples was 1,218, with 653 (53%) positive. The mean number of samples per week was 12.7 (range, 9 to 21, n = 91 weeks).

FIG. 4.

Citrobacter rodentium-rooted neighbor-joining tree constructed from the alignment of a translated, 140-amino-acid region of the tir gene amplified from 40 stream water-derived coliform cultures. The identities of the stream sampling sites and USDA and GenBank reference sequences (i.e., taxa labeled as E.c. and C. rodentium) are given in Materials and Methods. Numbers associated with branches represent the percentage of bootstrapped replicates supporting that branching pattern. Numbers after stream samples represent the time (MM/DD/YY) the sample was collected; six-digit numbers after select E. coli O111 strains are reference numbers of the E. coli Reference Center.

FIG. 5.

MDS plot constructed from the phylogeny inferred from the alignment of the translated amino acid sequences (∼582 amino acids in length) for the entire tir gene for three stream water samples (GFGL, SB, and BARN), E. coli O157:H7 Odwalla and E. coli O111 cow strains, and various E. coli reference sequences from GenBank (described in the Materials and Methods). Panels A and B represent different three-dimensional perspectives of the same dendrogram. Taxa are represented by colored spheres; the proximity of these spheres to one another reflects degrees of relatedness derived from amino acid sequence similarities. Note that the E. coli O26:H− and O103:H2 amino acid sequences are 100% identical; thus, their spheres are fused into one bicolored sphere.

FIG. 6.

CLUSTAL W-generated alignment of translated amino acid sequences for the tir gene of E. coli showing insertions unique to the BARN sequence at positions 208 to 219 and positions 245 to 248 (equivalent to amino acids 199 to 210 and amino acids 236 to 241 of E. coli O157:H7 strain 86-24, accession no. AF125993). E. coli SB, Spring Branch isolate; E. coli GFGL, Gwynns Falls Glyndon isolate. Descriptions of accompanying reference serotypes and strains of E. coli are provided in Materials and Methods.