Antibiotic Resistance of E. coli Isolated From a Constructed Wetland Dominated by a Crow Roost, With Emphasis on ESBL and AmpC Containing E. coli

Abstract

Information on the dissemination of antibiotic resistance mechanisms in the environment as well as wild life is needed in North America. A constructed wetland (where ∼15,000 American crows roost) was sampled on the University of Washington Bothell Campus for the presence of antibiotic resistant E. coli (ARE). Crow droppings from individual birds and grab samples of water were collected in 2014-2015. E. coli were isolated by selective agar plating. The most frequent antibiotic resistance (AR) of the fecal isolates was to ampicillin (AMP) (53%), followed by amoxicillin-clavulanic acid (AMC) (45%), streptomycin (S) (40%), and nalidixic acid (NA) (33%). Water isolates had similar AR pattern and ∼40% were multidrug resistant. Isolates from water samples collected during storm events showed higher resistance than isolates from no rain days to tetracycline, AMP, AMC, NA, and gentamycin. Extended spectrum beta lactamase (ESBL) containing E. coli with the bla _ctx-M was found in three water and nine fecal isolates while bla _cmy-2 in 19 water and 16 fecal isolates. Multilocus Sequence Typing analysis (MLST) yielded 13 and 12 different sequence types (STs) amongst fecal and water isolates, many of which could be correlated to livestock, bird, and humans. MLST identified ESBL E. coli belonging to the clinically relevant ST131 clone in six fecal and one water isolate. Three STs found in feces could be found in water on the same dates of collection but not subsequently. Thus, the strains do not appear to survive for long in the wetland. Phylogenetic analysis revealed similar distribution of the water and fecal isolates among the different phylo-groups, with the majority belonging to the commensal B1 phylo-group, followed by the pathogenic B2 phylo-group. This study demonstrates that corvids can be reservoirs and vectors of ARE and pathogenic E. coli, posing a significant environmental threat.

Keywords: ESBL; ST131; antibiotic resistant genes; blacmy-2; blactx-M; crows; multi-drug resistant E. coli; wetland.

FIGURE 1

Sampling site map showing North Creek, the UW Bothell/Cascadia College campus, and the 58 acre restored floodplain wetland. Red dots indicate locations of surface water sampling sites. Blue arrows indicate direction of water flow. Water sampled at RS1, RS7, and RP3 flows to these locations in a series of catch basins and pipes from the upland (western) portion of the campus. The crow roost boundary fluctuates year to year, though the southern part by the sampling sites is relatively stable. Aerial photograph from Google.

FIGURE 2

Comparison of mean E. coli counts in runoff as it enters (RS2) and leaves the wetland roost zone (SW8). The mean of counts in CFUs, determined 25 times between 2014–2017 at RS2 and 17 times at SW8, is shown. Triplicate samples were collected at each site each time. The error bars represent one standard deviation around the mean for the respective data sets.

FIGURE 3

Percentage of E. coli in water (n = 49) and fecal (n = 98) isolates showing non-susceptibility to 13 selected antimicrobials. AMC, amoxicillin/clavulanic acid; AMP, ampicillin; XNL, ceftiofur; C, chloramphenicol; CAZ, Ceftazidime; CIP, ciprofloxacin; ENO, Enrofloxacin; NA, Nalidixic acid; N, Neomycin; STR, streptomycin; SPT, spectinomycin; TE, tetracycline; SXT, trimethoprim/sulfamethoxazole. Table indicates significant difference in antibiotic resistance between water and fecal isolates for 10 antibiotics according to Z-test.

FIGURE 4

Percentage of E. coli isolates in water on no rain days (n = 62) and post-rain days (n = 63) showing non-susceptibility to 11 selected antimicrobials. AMC, amoxicillin/clavulanic acid; AMP, ampicillin; C, chloramphenicol; CAZ, Ceftazidime; CIP, ciprofloxacin; GM, Gentamycin; K, Kanamycin; NA, Nalidixic acid; STR, streptomycin; TE, tetracycline; SXT, trimethoprim/sulfamethoxazole. Table indicates significant difference in antibiotic resistance between no rain and rain days by Z test of proportionality.

FIGURE 5

Phylo-grouping of the Isolates. Percentage of E. coli isolates from water (n = 46) and feces (n = 92) belonging to each phylo-group. Different groups are represented by different colors. Among the water isolates there were no unknowns. Table indicates significant difference in presence of each phylo-group, between water and fecal samples.