Detection of Shiga toxin- and extended-spectrum -lactamase-producing Escherichia coli O145:NM and Ont:NM from calves with diarrhoea

Abstract

Sir, Shiga toxin-producing Escherichia coli (STEC) are recognized as important human pathogens. Ruminants, in particular bovines, are the primary reservoir of STEC and transmission principally occurs via the food chain. So far, extended-spectrum b-lactamases (ESBLs) have been rarely observed among STEC strains. In humans, such findings are currently limited to five isolates belonging to serogroups O26:NM, O26:H11, O64 and O157:H7, expressing b-lactamases TEM-52, CTX-M-2, CTX-M-3, CTX-M-18 or CTX-M-1, and to the O104:H4-CTX-M-15 strain associated with an outbreak of haemolytic uraemic syndrome (HUS) in Germany. – 7 Even rarer is the finding of ESBL-producing STEC strains from animal sources, where only one O157-CTX-M-2 chicken isolate and one O111:H8-CTX-M-15 bovine isolate have been described so far. In the present study, two E. coli strain collections either known for a positive ESBL phenotype or for possession of Shiga toxin genes were screened for a combination of these two determinants. In detail, 62 ESBL-positive E. coli isolates recovered through routine diagnostics (Vet Med Labor GmbH, Germany) from the faecal contents of cattle/calves with diarrhoea between 2010 and 2012 in Germany, determined as ESBL producers by MIC testing (Vitek 2; AST-GN38 or AST-N062) and the combination disc test (CLSI document M31-A3), were investigated for the possession of Shiga toxin genes in this study. A DNA array (Identibac EC, Alere, Germany) identified the stx2 gene in one strain (VB932491), which was further determined as stx2a by sequence analysis (Table 1). Another 87 E. coli, predominantly isolated from calves with diarrhoea in the past 30 years in the diagnostic laboratory of the Institute of Hygiene and Infectious Diseases of Animals, Germany, and known to harbour either an stx1 or an stx2 gene, as determined by PCR analyses, were screened by overnight incubation on Mueller–Hinton agar containing 4 mg/L cefotaxime. Here, one stx1-positive isolate (IHIT23778), further typed as stx1a by sequence analysis, showed growth, was further examined by MIC testing and could be finally confirmed as an ESBL producer by the combination disc test. The ESBL-producing STEC strains were determined as Ont:NM and O145:NM, respectively. O145 represents a serotype that is counted among the ‘big six’ of non-O157 STEC O antigens, namely O26, O45, O103, O111, O121 and O145. Multilocus sequence typing (MLST) assigned this strain to sequence type (ST) 32, which is currently represented by another seven E. coli isolates from Germany and Norway, six of which were defined as human STEC or enterohaemorrhagic E. coli (EHEC) strains associated with HUS in the years 1996 to 2009, all expressing O145 (http://mlst.ucc.ie/ mlst/dbs/Ecoli). The Ont:NM strain was assigned to ST301. Another two bovine ST301 STEC strains (O80:NM and O4:NM), isolated in 1992 and 1994, respectively, are available in the MLST database. However, none of these strains showed an ESBL phenotype, as confirmed in the present study. Both ESBL-producing STEC strains possessed genes formerly detected in plasmid pO157, i.e. the putative serine protease precursor-encoding gene espP and EHEC haemolysin-encoding gene hlyA. Genes located on the genetic element O island 122 (efa1 and nleABC) initially identified in EHEC strain EDL933 and associated with STEC capable of causing HUS and foodborne outbreaks were further detected. In addition, both strains harboured adherence-conferring protein gene iha, which is located on a chromosomal island recently acquired by O157:H7 STEC strains. Genes aggR, irp2 and pAA, which are related to the 2011 German O104:H4-CTX-M-15 Shiga toxin-producing enteroaggregative haemorrhagic E. coli outbreak strain, were absent. The ESBL-producing STEC strains possessed different types of eae genes (variants j andg). They also differed partly in the genes linked with the type III secretion system and with effector proteins located on the locus of enterocyte effacement (LEE) pathogenicity island (Table 1). Here, O145:NM strain IHIT23778 harboured tccp, which encodes a Tir cytoskeleton coupling protein, and cif, which encodes type III secretion effector cycle inhibiting factor, in addition to esp genes, nleABC and tir. Finally, it differs from the Ont:NM strain in the possession of the catalase-peroxidaseencoding gene katP. These three genes are tightly associated with strains causing complicated EHEC infections underlining the serious threat of the acquisition of an ESBL plasmid by such a strain. PCR and sequence analysis revealed blaCTX-M-1 in our STEC strains. The transferability of ESBL plasmids was explored by filter mating using E. coli K-12 strain J53 as the recipient strain and tryptic soy agar (TSA) plates containing 100 mg/L sodium azide and 4 mg/L cefotaxime. According to PCR-based replicon typing, the donor strains contained four and three plasmids of different sizes and replicon types, respectively. Self-transferable plasmids carrying the blaCTX-M-1 genes were either of replicon type IncFII (VB932491) or Inc1I (IHIT23778).