Extraintestinal Pathogenic and Antimicrobial-Resistant Escherichia coli Contamination of 56 Public Restrooms in the Greater Minneapolis-St. Paul Metropolitan Area

Abstract

How extraintestinal pathogenic Escherichia coli (ExPEC) and antimicrobial-resistant E. coli disseminate through the population is undefined. We studied public restrooms for contamination with E. coli and ExPEC in relation to source and extensively characterized the E. coli isolates. For this, we cultured 1,120 environmental samples from 56 public restrooms in 33 establishments (obtained from 10 cities in the greater Minneapolis-St. Paul, MN, metropolitan area in 2003) for E. coli and compared ecological data with culture results. Isolates underwent virulence genotyping, phylotyping, clonal typing, pulsed-field gel electrophoresis (PFGE), and disk diffusion antimicrobial susceptibility testing. Overall, 168 samples (15% from 89% of restrooms) fluoresced, indicating presumptive E. coli: 25 samples (2.2% from 32% of restrooms) yielded E. coli isolates, and 10 samples (0.9% from 16% of restrooms) contained ExPEC. Restroom category and cleanliness level significantly predicted only fluorescence, gender predicted fluorescence and E. coli, and feces-like material and toilet-associated sites predicted all three endpoints. Of the 25 E. coli isolates, 7 (28%) were from phylogenetic group B2(virulence-associated), and 8 (32%) were ExPEC. ExPEC isolates more commonly represented group B2 (50% versus 18%) and had significantly higher virulence gene scores than non-ExPEC isolates. Six isolates (24%) exhibited ≥3-class antibiotic resistance, 10 (40%) represented classic human-associated sequence types, and one closely resembled reference human clinical isolates by pulsed-field gel electrophoresis. Thus, E. coli, ExPEC, and antimicrobial-resistant E. coli sporadically contaminate public restrooms, in ways corresponding with restroom characteristics and within-restroom sites. Such restroom-source E. coli strains likely reflect human fecal contamination, may pose a health threat, and may contribute to population-wide dissemination of such strains.

FIG 1

Overall prevalence of positive culture results among environmental samples from public restrooms. (Top) Prevalence by sample (n = 1,120). (Bottom) Prevalence by restroom (n = 56). Culture endpoints of interest included fluorescence (suggesting the presence of β-glucuronidase, hence Escherichia coli), E. coli (i.e., isolated E. coli colonies), and extraintestinal pathogenic E. coli (ExPEC).

FIG 2

Prevalence of positive culture results in relation to restroom category among 1,120 environmental samples from public restrooms. Culture endpoints of interest included fluorescence (suggesting the presence of Escherichia coli), E. coli (i.e., isolated colonies), and extraintestinal pathogenic E. coli (ExPEC). The number of restrooms screened per restroom category was 10 for all but malls/stores, for which the number was 6. The number of specimens per restroom was 20. P values are shown for pairwise restroom category comparisons when P is <0.05, as determined by chi-square test [with (N − 1)/N correction]. Significant pairwise comparisons included those between samples from public parks versus gas stations, the Veterans Affairs Medical Center, and supermarkets (top arrows) and those between samples from fast food outlets versus the Veterans Affairs Medical Center and supermarkets (bottom arrows).

FIG 3

Prevalence of positive culture results in relation to restroom gender among 1,120 environmental samples from public restrooms. Culture endpoints of interest included fluorescence (suggesting the presence of Escherichia coli), E. coli (i.e., isolated colonies), and extraintestinal pathogenic E. coli (ExPEC). P values are shown for comparisons of the three culture endpoints in relation to restroom gender when P is <0.05, as determined by chi-square test [with (N−1)/N correction].

FIG 4

Prevalence of positive culture results in relation to presence of feces-like material among 1,120 environmental samples from public restrooms. Culture endpoints of interest included fluorescence (suggesting the presence of Escherichia coli), E. coli (i.e., isolated colonies), and extraintestinal pathogenic E. coli (ExPEC). P values are shown for comparisons of the three culture endpoints in relation to presence of feces-like material, as determined by chi-square test [with (N−1)/N correction].

FIG 5

Prevalence of positive culture results in relation to proximity to the toilet among 1,120 environmental samples from public restrooms. Culture endpoints of interest included fluorescence (suggesting the presence of Escherichia coli), E. coli (i.e., isolated colonies), and extraintestinal pathogenic E. coli (ExPEC). P values are shown for comparisons of the three culture endpoints in relation to proximity to the toilet, as determined by chi-square test [with (N−1)/N correction]. Sites in close proximity to the toilet were classified as toilet associated.

FIG 6

Virulence genotypes of 25 Escherichia coli isolates from public restrooms. (Left) Extraintestinal pathogenic E. coli (ExPEC) versus non-ExPEC; right, phylogenetic group B2 versus non-B2. The traits shown are those (among 38 total) that yielded P values of <0.05 for the comparisons of ExPEC isolates (pink bars) versus non-ExPEC isolates (blue bars) and/or for group B2 isolates (pink bars) versus non-B2 isolates (blue bars). Traits are arranged from top to bottom in order of descending prevalence among ExPEC isolates. P value symbols are shown adjacent to the higher-prevalence group when P is < 0.05 as follows: *, P < 0.05, **, P < 0.01, and ***, P < 0.001, as determined by chi-square test [with (N-1)/N correction]. Rectangles enclose traits contributing to molecular definition of ExPEC. Trait definitions: afa/draBC, Dr antigen-specific adhesion operons; cdtB, cytolethal distending toxin; fyuA, Yersinia siderophore receptor; ibeA, invasion of brain endothelium; iha, iron-regulated gene homologue adhesin; ireA, iron-regulated element; iroN, catecholate siderophore receptor; iutA, ferric aerobactin receptor; kpsM II, group 2 capsule polysaccharide synthesis (e.g., K1, K5, and K12); kpsM K1 and K2/K100, group 2 capsule variants; malX, pathogenicity-associated island marker; ompT, outer membrane protein T (protease); papA, P fimbrial structural subunit (with papC, papEF, papG, and papG allele II giving the same result as papA); sat, secreted autotransporter toxin; traT, surface exclusion, serum survival-associated; usp, uropathogenic-specific protein (bacteriocin).

FIG 7

Virulence scores among 25 Escherichia coli isolates from public restrooms. (Left) Extraintestinal pathogenic E. coli (ExPEC) (solid squares) versus non-ExPEC (squares) isolates. (Right) Phylogenetic group B2 (solid circles) versus non-B2 (circles) isolates. Horizontal lines, group medians. P values, as determined by the Mann-Whitney U test (two tailed), are for ExPEC versus non-ExPEC and group B2 versus non-B2.

FIG 8

XbaI pulsed-field gel electrophoresis (PFGE)-based dendrogram for 25 Escherichia coli isolates from public restrooms. The dendrogram was inferred within BioNumerics according to the unweighted-pair group method, based on Dice similarity coefficients. Rectangles enclose two isolate pairs with indistinguishable profiles. ExPEC, extraintestinal pathogenic E. coli; STc, sequence type complex (group of closely related STs).

FIG 9

XbaI pulsed-field gel electrophoresis (PFGE)-based dendrogram for 11 Escherichia coli isolates from pulsotype 857, sequence type (ST) 95. Isolate FF5-2 (rectangle) is a public restroom isolate from ST95. The 10 reference isolates, all of which likewise represent ST95, were selected from a large private PFGE database based on their PFGE profile similarity to isolate FF5-2.