Enterobacteriaceae with extended-spectrum- and pAmpC-type β-lactamase-encoding genes isolated from freshwater fish from two lakes in Switzerland.

There is growing evidence that Enterobacteriaceae harboring clinically relevant bla genes, especially genes encoding the CTX-M family of extended-spectrum -lactamases (ESBLs), have spread into the environment (1). The purposes of this study were (i) to investigate the occurrence of expanded-spectrum-cephalosporinresistant Enterobacteriaceae in intestines of fish captured from Lake Zürich and Lake Thun, Switzerland; (ii) to characterize such isolates by their susceptibility to 13 antimicrobial agents (listed in Table 1) (2); (iii) to identify the involved blaESBL/blapAmpC genes (3–5), (iv) for E. coli multilocus sequence typing (MLST) according to Achtman’s scheme (http://mlst.ucc.ie/mlst/dbs/Ecoli); and (v) to determine phylogenetic groups (6). A total of 139 freshwater fish consisting of 64 samples of Coregonus lavaretus (whitefish), 33 samples of Perca fluviatilis (perch), 29 samples of Rutilus rutilus (roach), six samples of Salmo trutta (brown trout), 4 samples of Esox lucius (pike), and one sample each of Abramis brama (bream), Tinca tinca (tench), and Centrarchidae species (sunfish) were captured by net. The fish were gutted immediately upon arrival at the port, and the intestines were placed in sterile bags. Samples were homogenized and incubated at a ratio of 1:10 in Enterobacteriaceae enrichment (EE) broth (BD, Franklin Lakes, NJ, USA) at 37°C over night. One loopful of each of the enrichment cultures was inoculated onto chromogenic Brilliance ESBL agar (Oxoid, Hampshire, United Kingdom). Colonies with typical Enterobacteriaceae morphology were selected and identified using API ID 32 Etest strips (bioMérieux, Marcy l’Etoile, France). Twenty-six (18.7%) of the fish gut samples rendered 33 ESBLor pAmpC-producing isolates (Table 1). Most of the isolates were E. coli. blaCTX-M genes were detected in 30 isolates. Two isolates contained blaSHV-12, and one isolate carried blaCMY-2. Of the E. coli isolates, 13 belonged to commensal phylogroup A or B1, and 19 belonged to pathogenic phylogroup B2 or D. A total of seven E. coli isolates belonged to the multidrug-resistant pandemic extraintestinal pathogenic group B2:ST131, with a majority (four) containing blaCTX-M-27. These characterization data strongly resemble results from recent studies in the human population and river waters in Switzerland (1, 7, 8). In contrast to what is seen in homeothermic animals, E. coli is not a permanent inhabitant of intestinal tracts of fish (9) but reflects the character of the aquatic habitat and the bacterial load in the water (10–12). On account of their motility, fish must be considered potent disseminators of resistant bacteria. Due to their exposure to a plethora of environmental bacteria on their epidermal mucus layer (13), they also provide excellent conditions for the horizontal transfer of antibiotic resistance genes among the aquatic bacterial community. Recent studies have shown that there is a tendency for Aeromonas spp. from the aquatic environment to acquire ESBLs and other resistance mechanisms (14, 15), thus contributing to the dissemination of resistance determinants.