One of the currently most relevant resistance mechanisms in Enterobacteriaceae is the production of enzymes that lead to modern expanded-spectrum cephalosporin and even carbapenem resistance, mainly extended-spectrum β-lactamases (ESBLs) and carbapenemases. A worrisome aspect is the spread of ESBL and carbapenemase producers into the environment. The aim of the present study was to assess the occurrence of ESBL- and carbapenemase-producing Enterobacteriaceae and to further characterize ESBL- and carbapenemase-producing Enterobacteriaceae in rivers and lakes in Switzerland. ESBL-producing Enterobacteriaceae were detected in 21 (36.2%) of the 58 bodies of water sampled. One river sample tested positive for a carbapenemase-producing Klebsiella pneumoniae subsp. pneumoniae strain. Seventy-four individual strains expressing an ESBL phenotype were isolated. Species identification revealed 60 Escherichia coli strains, seven Klebsiella pneumoniae subsp. pneumoniae strains, five Raoultella planticola strains, one Enterobacter cloacae strain, and one Enterobacter amnigenus strain. Three strains were identified as SHV-12 ESBL producers, and 71 strains carried genes encoding CTX-M ESBLs. Of the 71 strains with CTX-M ESBL genes, 8 isolates expressed CTX-M-1, three produced CTX-M-3, 46 produced CTX-M-15, three produced CTX-M-55, one produced CTX-M-79, six produced CTX-M-14, and four produced CTX-M-27. Three of the four CTX-M-27 producers belonged to the multiresistant pandemic sequence type E. coli B2:ST131 that is strongly associated with potentially severe infections in humans and animals.

T he recent identification of members of the family Enterobacte-riaceae harboring the plasmid-mediated transferable colistin resistance mcr-1 gene is of great concern to public health (1-4). Here, we report on the occurrence of mcr-1-harboring extendedspectrum ␤-lactamase (ESBL)-producing members of Enterobacteriaceae from river water in Switzerland and ready-to-eat imported vegetables. For this study, 74 ESBL-producing members of Enterobacteriaceae isolated from 21 rivers and lakes sampled in 2012 in Switzerland (5) and 60 ESBL-producing members of Enterobacteriaceae isolated from 42 imported vegetable samples (11 from the Dominican Republic, 13 from India, 11 from Thailand, and 8 from Vietnam) (6) were screened by PCR for the presence of the mcr-1 gene. The mcr-1 gene was detected in 1 out of 74 water strains (an isolate from the river Birs) and 2 out of 60 vegetable strains (products from Thailand and Vietnam), and sequencing of the amplicons showed a 100% identity with the published mcr-1 sequence (1). The colistin resistance was transferable by transformation experiments into Escherichia coli DH5-alpha. All strains were Escherichia coli and belonged to different multilocus sequence types (MLSTs), harbored different bla ESBL genes, and showed a multiresistance phenotype (Table 1). The diversity of ESBL genes and MLSTs identified among mcr-1-positive isolates suggests that the mcr-1 gene might be carried on different plasmids. The spread of mcr-1-harboring, ESBL-producing members of the Enterobacteriaceae in surface water suggest environmental contamination. Appropriate measures urgently need to be enforced in order to reduce the anthropogenic burden of antibiotic resistance in the environment, such as the judicious use of antibiotics in human and veterinary medicine as well as in agriculture. In addition, improvement of water status is of major concern. New strategies for the treatment of wastewaters, e.g., the use of sand filters or more-stringent chlorine disinfection, need to be taken into consideration to prevent resistant bacteria from being released into the aquatic environment. Moreover, these data show that the international production and trade of fresh vegetables constitute a possible route for the spread of antibiotic-resistant, and particularly colistin-resistant, Enterobacteriaceae.

To examine to what extent fresh vegetables imported into Switzerland represent carriers of extended-spectrum-β-lactamase (ESBL)-producing Enterobacteriaceae , 169 samples of different types of fresh vegetables imported into Switzerland from the Dominican Republic, India, Thailand, and Vietnam were analyzed. Overall, 25.4% of the vegetable samples yielded one or more ESBL-producing Enterobacteriaceae , 78.3% of which were multidrug resistant. Sixty isolates were obtained: Escherichia coli , 26; Klebsiella pneumoniae , 26; Enterobacter cloacae , 6; Enterobacter aerogenes , 1; and Cronobacter sakazakii , 1. We found 29 isolates producing CTX-M-15, 8 producing CTX-M-14, 7 producing CTX-M-55, 3 producing CTX-M-65, 1 each producing CTX-M-1, CTX-M-3, CTX-M-27, and CTX-M-63, 5 producing SHV-2, 3 producing SHV-12, and 1 producing SHV-2a. Four of the E. coli isolates belonged to epidemiologically important clones: CTX-M-15-producing B2:ST131 (1 isolate), D:ST405 (1 isolate), and D:ST38 (2 isolates). One of the D:ST38 isolates belonged to the extraintestinal enteroaggregative E. coli (EAEC) D:ST38 lineage. Two of the K. pneumoniae isolates belonged to the epidemic clones sequence type 15 (ST15) and ST147. The occurrence of antibiotic-resistant pathogenic and commensal Enterobacteriaceae in imported agricultural foodstuffs constitutes a source of ESBL genes and a concern for food safety.

In this study, extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli isolates recovered from the following sources were characterized with regard to the occurrence and distribution of uropathogenic and enteric pathogenic virulence factors: surface waters (rivers and lakes, n=60), the intestines of freshwater fish (n=33), fresh vegetables (n=26), retail poultry meat (n=13) and the fecal samples of livestock (n=28), healthy humans (n=34) and primary care patients (n=13). Among the 207 isolates, 82% tested positive by PCR for one or more of the virulence factors (VF) that predict uropathogenicity, TraT, fyuA, chuA, PAI, yfcv or vat. Uropathogenic E. coli (UPEC) were detected in each of the analyzed sources. Regarding virulence factors for intestinal pathogenic E. coli, these were found more rarely and predominantly associated with the aquatic environment, with aagR (EAEC) found in isolates from surface waters and STp (porcine heat stable enterotoxin) and LT (heat-labile enterotoxin) associated with isolates from fish. Aggregate VF scores (the number of unique virulence factors detected for each isolate) were lowest among isolates belonging to phylogenetic group B1 and highest among group B2. Clustering of the isolates by phylogenetic group, multilocus sequence type (MLST) and ESBL-types revealed clonal overlaps of A:ST10(CTX-M-1) and D:ST350(CTX-M-1) between the sources of livestock, poultry meat and healthy humans, suggesting livestock, in particular poultry, represents a potential reservoir for these particular UPEC clones. The clones A:ST10(CTX-M-55) and B2:ST131(CTX-M-27), harboring uropathogenic virulence factors were significantly associated with fresh vegetables and with fish, respectively. Further clonal complexes with source overlaps included D:ST38(CTX-M-14), D:ST69(CTX-M-15), D:ST405(CTX-M-15) and D:ST648(CTX-M-15), which were found in surface water and healthy humans. Identifying potential reservoirs of UPEC in the environment, animals, food and humans is important in order to assess routes of transmission and risk factors for acquiring UPEC.

Sixty isolates of Enterobacteriaceae resistant to beta-lactam antibiotics were collected over a period of 2 years in Switzerland and screened by hybridization for the carriage of SHV genes. Thirty-four positive strains were found, and their SHV genes were amplified and sequenced. SHV extended-spectrum beta-lactamases (ESBLs) were found: 13 strains contained SHV-2a, 12 harbored SHV-2, and SHV-5 was found twice. Four strains were shown to contain SHV-1. In addition, we report two new SHV variants, termed SHV-11 (non-ESBL) and SHV-12 (ESBL). In spite of the carriage of SHV ESBLs, many strains showed only low resistance to one or more third-generation cephalosporins. In addition, 26 did not transfer the blaSHV gene in mating experiments.