Repeated Isolation of Extended-Spectrum-β-Lactamase-Positive Escherichia coli Sequence Types 648 and 131 from Community Wastewater Indicates that Sewage Systems Are Important Sources of Emerging Clones of Antibiotic-Resistant Bacteria

Abstract

Antibiotic resistance in bacteria is an emerging problem globally. Resistant bacteria are found in human and animal microbiota, as well as in the environment. Wastewater receives bacteria from all these sources and thus can provide a measurement of abundance and diversity of antibiotic-resistant bacteria circulating in communities. In this study, water samples were collected from a wastewater pump station in a Norwegian suburban community over a period of 15 months. A total of 45 daily samples were cultured and analyzed for the presence of Escherichia coli Eighty E. coli-like colonies were collected from each daily sample and then phenotyped and analyzed for antibiotic resistance using the PhenePlate-AREB system. During the sampling period, two unique E. coli phenotypes with resistance to cefotaxime and cefpodoxime indicating carriage of extended-spectrum β-lactamases (ESBL) were observed repeatedly. Whole-genome sequencing of 15 representative isolates from the two phenotypes identified these as two distinct clones belonging to the two globally spread E. coli multilocus sequence types (STs) ST131 and ST648 and carrying bla_CTX-M-15 The number of ESBL-positive E. coli strains in the community wastewater pump station was 314 of 3,123 (10%) analyzed E. coli strains. Of the ESBL-positive isolates, 37% belonged to ST648, and 7% belonged to ST131. Repeated findings of CTX-M-15-positive ST648 and ST131 over time indicate that these STs are resident in the analyzed wastewater systems and/or circulate abundantly in the community.

Keywords: ESBL E. coli; Norway; ST131; ST648; antibiotic resistance; antibiotic surveillance; persistence; sewage; wastewater; whole-genome sequencing.

FIG 1

PhP (dendrogram and PhP type) and AREB typing for 15 isolates selected for whole-genome sequencing. Abbreviations: amp, ampicillin; ctx, cefotaxime; chl, chloramphenicol; cip, ciprofloxacin; gen, gentamicin; nal, nalidixic acid; pod, cefpodoxime; tet, tetracycline; tmp, trimethoprim; R, resistant; S, sensitive.

FIG 2

Core genome phylogenetic tree of ST648 and ST131 isolates compared to reference strains. Isolates are labeled by origin and year of isolation. Norwegian ST648 and ST131 isolates are indicated in bold text.

FIG 3

Biofilm formation analysis by E. coli ST131 and ST648. Crystal violet (CV) (A) and FITC-CoA (B) staining of biofilm adherent to polystyrene microplates plates on after incubation for 48 h at 28 or 37°C at the liquid-air interface. For CV staining, retained CV in adherent bacteria is equivalent to the production of biofilm. For FITC-ConA staining, the fluorescence intensity (arbitrary units [a.u.]) represents the EPS production. Horizontal dotted lines indicate low/medium/high biofilm production. Error bars indicate the standard errors of the mean. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.5.

FIG 4

Biofilm phenotypes of Norwegian ST131 and ST648 E. coli isolates. The figure shows rdar morphotype formation of ST131 and ST648 isolates after 48 h of incubation at 28°C on Congo red LB without salt plates.