Co-occurrence of the blaVIM-1 and blaSHV-12 genes on an IncHI2 plasmid of an Escherichia coli isolate recovered from German livestock.

Sir,

The dissemination of carbapenemase-producing Enterobacterales (CPE) is an important public health issue. The number of human CPE isolates has been steadily increasing during recent years, worldwide. Despite the fact that carbapenems are not licensed for use in veterinary medicine, increasing numbers of CPE from the veterinary sector have been reported. The transmission of CPE between pets/livestock and exposed humans as well as via food has been demonstrated. In this study, a detailed characterization of a carbapenem-resistant porcine Escherichia coli co-harbouring blaVIM-1, blaSHV-12 and blaACC-1 genes, along with other resistance genes, is provided.

Within the German annual monitoring of ESBL/AmpC β-lactamase-producing E. coli from animals and food in 2017–18, the isolate 17-AB02384 was recovered from the caecal content of a fattening pig at slaughter. Based on EUCAST epidemiological cut-off values (https://www.eucast.org/mic_distributions_and_ecoffs/), the isolate showed a non-WT phenotype to carbapenems and other antimicrobials [Table S1 (Table S1 is available as Supplementary data at JAC Online)]. Molecular analysis revealed that E. coli 17-AB02384 belonged to the phylogenetic group A and the multilocus sequence type (MLST) 7593. To our knowledge, this ST has not yet been described in E. coli from German livestock or food chain. However, ST7593 was reported for some NDM-5-producing isolates of retail meat samples in China.

Initial S1-PFGE plasmid profiling and subsequent DNA–DNA hybridization indicated that the blaVIM-1 gene was located on an approximately 300 kb IncHI2 plasmid, designated pEC17-AB02384, which was transferable into the E. coli J53 by conjugation at a transfer rate of 2 × 10−4. For a detailed characterization, the whole-genome sequence of E. coli 17-AB02384 was determined by Illumina (CA, USA) short-read and PacBio (Menlo Park, USA) long-read sequencing, according to the manufacturers’ recommendations. Hybrid assembly of the plasmid sequence was carried out using unicycler v.0.44. The resulting sequence of the plasmid pEC17-AB02384 was deposited at GenBank (NCBI) under the accession number MT163739. MLST, resistance and virulence genes were determined using online tools that were provided by the Danish Technical University (http://www.genomicepidemiology.org) . The annotation was carried out by RAST2 provided by PATRIC (www.patricbrc.org). Characteristics of the isolate and its plasmid are summarized in Table S2.

Besides blaVIM-1, this plasmid harboured the ESBL gene blaSHV-12 and the AmpC β-lactamase gene blaACC-1. Overall, pEC17-AB02384 showed similarity to the VIM-1-encoding plasmids pSE15-SA01028 (90% identity, CP026661.1) and pRH-R178 (93% identity, HG530658.1) from Salmonella enterica subsp. enterica and E. coli, respectively, which were both recovered from German pigs (Figure 1a). In contrast to these plasmids, pEC17-AB02384 harboured three additional resistance gene-carrying segments. The 9773 bp segment 1 comprised the resistance genes sul1 and qnrA1 and was flanked by 124 bp inverted repeats. It was inserted into the region upstream of the blaVIM-1-carrying class 1 multiresistance integron in pEC17-AB02384 (Figure 1b). Downstream of the integrase gene intI1 of the blaVIM-1-carrying integron, the segment 2 was integrated. It carried the macrolide resistance operon mph(A)-mrx-mphR, the trimethoprim resistance gene dfrA14 and another intI1 gene. Immediately downstream of both intI1 genes, 202 bp direct repeats were found (Figure 1b). It appears possible that a translocatable unit comprising the entire segment 2 was inserted into plasmid pEC17-AB02384 by recombination with the intI1 gene of the blaVIM-1-carrying integron and its adjacent repeat region. The 4979 bp segment 3 was inserted into segment 2 between the Δrec and the mph(A) gene. It carried the ESBL gene blaSHV-12 and was flanked by IS26 elements in opposite orientation. However, no direct repeats were detected at the immediate boundaries of segment 3, suggesting that the IS26-bounded segment 3 does not function as a transposon.

Figure 1

(a) Schematic illustrations of plasmids pRH-R178 and pSE15-SA01028 in comparison with plasmid pEC17-AB02384 described in this sudy. (b) Schematic illustration of the multidrug resistance region of plasmid pEC17-AB02384. The reading frames are displayed as arrows with the arrowhead showing the dirction of transcription. The numbers refer to the whole plasmid sequence of pEC17-AB02384, which is deposited in the GenBank database under accession no. MT163739. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

To our knowledge, the co-occurrence of blaVIM-1 and blaSHV-12 has not yet been described in plasmids from isolates of animal origin. Co-location of these genes was reported only for a single human clinical Aeromonas caviae isolate (KR869764) in 2014. The corresponding plasmid belonged to the replicon type IncA/C and showed no substantial similarities to pEC17-AB02384. SHV-12 is an extended-spectrum β-lactamase that is commonly detected in isolates from poultry, but rarely from pigs. Alonso et al. provided further data on blaSHV-12-carrying plasmids from human, animal and food sources. Among them, the E. coli plasmid pCAZ590 (LT669764) exhibits a similar SHV-12 region as the one identified in pEC17-AB02384. In general, blaSHV-12 seems to be associated with IS26 elements, which might support its mobility. The ability of IS26 to mobilize neighbouring genes might play an important role in the persistence of antimicrobial resistances.

A comparison of the few known blaVIM-1-carrying plasmids from German livestock revealed close relationships. This might indicate that a prototype-plasmid has adapted to bacteria in different animal populations and persists in an unknown reservoir. The characterization of CPE isolates and their plasmids will contribute to the further understanding of reservoirs, potential transmission pathways and persistence factors for plasmid maintenance in bacteria from animal husbandry.

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