Detection of plasmid-borne extended-spectrum β-lactamase (ESBL) genes in Escherichia coli isolates from bovine mastitis

Extended-spectrum β-lactamase (ESBL)-producing isolates have been increasingly reported during recent years. The aims of this study were to characterize ESBL-producing Escherichia coli from bovine mastitis as well as their ESBL gene-carrying plasmids. A culture collection of E. coli isolated from bovine quarter milk samples (2009-2013), was screened for ESBL production using ESBL selective agar plates. Putative ESBL producers (n = 16) were investigated by phenotypic confirmatory tests and were characterized by the detection/sequencing of ESBL genes, XbaI macrorestriction analysis, multilocus sequence typing (MLST), phylotyping and antimicrobial susceptibility testing. ESBL gene-carrying plasmids were investigated by transfer experiments, PCRs for the detection of co-located antimicrobial resistance genes, PCR-based replicon typing and S1-nuclease pulsed-field gel electrophoresis. Twelve ESBL-producing isolates were found. They showed eleven different XbaI patterns and were distributed among eight MLST types [ST10 (n = 3), ST117 (n = 2), ST361 (n = 1), ST362 (n = 1), ST540 (n = 1), ST1431 (n = 2), ST1508 (n = 1), and the novel ST5447 (n = 1)] and the phylogenetic groups A (n = 6), B1 (n = 2), B2 (n = 1) and D (n = 3). ESBL genes blaCTX-M-1 (n = 5), blaCTX-M-2 (n = 2), blaCTX-M-14 and blaCTX-M-15 (n = 4) were found on conjugative plasmids (35–225 kb) of diverse incompatibility groups (e.g. IncF, IncI1 or HI2 + P). Co-located resistance to sulfonamides, tetracycline, trimethoprim, and chloramphenicol/florfenicol was detected on five ESBL gene-carrying plasmids, but seven plasmids conferred solely resistance to β-lactam antibiotics. The presence of additional resistance genes on the ESBL gene-carrying plasmids suggests that co-selection of ESBL genes may occur even in the absence of β-lactam antibiotics.