Molecular characterization of extended-spectrum-β-lactamase-producing and plasmid-mediated AmpC β-lactamase-producing Escherichia coli isolated from stray dogs in South Korea

Abstract

A total of 47 extended-spectrum-cephalosporin-resistant Escherichia coli strains isolated from stray dogs in 2006 and 2007 in the Republic of Korea were investigated using molecular methods. Extended-spectrum β-lactamase (ESBL) and AmpC β-lactamase phenotypes were identified in 12 and 23 E. coli isolates, respectively. All 12 ESBL-producing isolates carried bla(CTX-M) genes. The most common CTX-M types were CTX-M-14 (n = 5) and CTX-M-24 (n = 3). Isolates producing CTX-M-3, CTX-M-55, CTX-M-27, and CTX-M-65 were also identified. Twenty-one of 23 AmpC β-lactamase-producing isolates were found to carry bla(CMY-2) genes. TEM-1 was associated with CTX-M and CMY-2 β-lactamases in 4 and 15 isolates, respectively. In addition to bla(TEM-1), two isolates carried bla(DHA-1), and one of them cocarried bla(CMY-2). Both CTX-M and CMY-2 genes were located on large (40 to 170 kb) conjugative plasmids that contained the insertion sequence ISEcp1 upstream of the bla genes. Only in the case of CTX-M genes was there an IS903 sequence downstream of the gene. The spread of ESBLs and AmpC β-lactamases occurred via both horizontal gene transfer, accounting for much of the CTX-M gene dissemination, and clonal spread, accounting for CMY-2 gene dissemination. The horizontal dissemination of bla(CTX-M) and bla(CMY-2) genes was mediated by IncF and IncI1-Iγ plasmids, respectively. The clonal spread of bla(CMY-2) was driven mainly by E. coli strains of virulent phylogroup D lineage ST648. To our knowledge, this is the first report of bla(DHA-1) in E. coli strains isolated from companion animals. This study also represents the first report of CMY-2 β-lactamase-producing E. coli isolates from dogs in the Republic of Korea.

Fig 1

Gel electrophoresis of plasmid DNAs extracted from bla_CTX-M-positive transconjugants and selected donor E. coli strains (A) and from bla_CMY-2-positive transconjugants (B). Southern hybridization was performed with probes specific for bla_CTX-M-9 cluster genes (C) and the bla_CMY-2 gene (D). Lanes: 1, pE-033J (lacking bla_CTX-M-9 cluster gene); 2, pE-102J; 3, pE-120J; 4, pE-121J; 5, pE-123J; 6, pE-126J; 7, pE-128J; 8, pE-129J; 9, E-006 (lacking bla_CTX-M-9 cluster gene); 10, E-124; 11, E-127; A, pE-008J; B, pE-040J; C, pE-045J; D, pE-099J; E, pE-117J; F, pE-118J; G, pE-119J; H, pE-131J; I, pE-132J; J, pE-133J; K, pE-134J; M, BAC Tracker supercoiled DNA marker.

Fig 2

Dendrograms generated by Bionumerics software showing the cluster analysis of XbaI-PFGE patterns of CTX-M (A)- and CMY-2 (B)-producing E. coli strains isolated from stray dogs. Similarity analysis was performed by using the Dice coefficient, and clustering was done by the unweighted-pair group method using average linkages (UGPMA). For both the CTX-M- and CMY-2-producing strains, details given include the strain, sequence type of each strain, phylogenetic group of each strain, age of host dog, geographical origin of host dog, and bla genes carried by each strain. XbaI-macrorestriction analysis yielded a few or no DNA banding patterns for four E. coli strains due to autodegradation of the genomic DNA during agarose plug preparation, and thus a cluster formed by these strains was ignored throughout this paper. Abbreviations: M, month; Y, year.