Occurrence and characterisation of ESBL-encoding plasmids among Escherichia coli isolates from fresh vegetables

Abstract Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates have been increasingly reported in different reservoirs. The aims of this study were to investigate the presence of ESBL-producing E . coli in fresh vegetables and to characterise their ESBL gene-carrying plasmids. Among the 245 samples from vegetables investigated during 2011–2013, seven putative ESBL-producing E . coli (salad n = 2, sprouts n = 5) were found. They were subjected to ESBL phenotypic confirmatory tests, detection/sequencing of ESBL genes, antimicrobial susceptibility testing (AST), phylotyping, XbaI-macrorestriction analysis, multilocus sequence typing and transformation. Transformants were characterised by AST, S1-nuclease PFGE, replicon typing, conjugation and investigated for co-located antimicrobial resistance genes. Two ESBL gene-carrying plasmids were sequenced using a HiSeq 2500 system. The seven isolates were confirmed as ESBL producers, displayed unrelated XbaI-patterns and unique sequence types (STs) and belonged to the phylogroups A, B1 or D. The ESBL genes were located on plasmids. Two plasmids carrying bla CTX-M-14 genes (incompatibility group IncK or IncHI2) were seen in isolates from salad (ST973) and sprout (ST527). Two bla CTX-M-15 - (IncFIB; non-typeable) and the IncN bla CTX-M-65 - and IncHI2 bla CTX-M-125 -carrying plasmids were found in isolates from sprouts (ST410, ST847, ST10, ST542). All plasmids were conjugative, except for the IncFIA-FIB bla CTX-M-2 -carrying plasmid. Sequence analysis of two plasmids identified the ESBL genes in close location to other resistance genes: sulfonamide resistance gene sul2 , streptomycin resistance genes strA and strB , the plasmid-mediated quinolone resistance gene qnrS1 and bla TEM-1 ( sul2 - strA - strB -IS 66 - bla TEM-1 - tnpR -Δ tnpA -IS Ecp1 - bla CTX-M-15 -Δ orf477 -Δ tnpA - qnrS1 ) or the fosfomycin resistance gene fosA3 (ΔIS Ecp1 - bla CTX-M-125 -ΔIS 903B - fosA3 ). These observations underline the importance of vegetables as reservoirs for multidrug resistant ESBL-producing E . coli .