Dissemination of SHV-12 and characterization of new AmpC-type beta-lactamase genes among clinical isolates of enterobacter species in Korea

Abstract

To determine the prevalence and genotype of an extended-spectrum beta-lactamase and new chromosomal AmpC beta-lactamases among clinical isolates of Enterobacter species, we performed antibiotic susceptibility testing, pI determination, induction tests, transconjugation, enterobacterial repetitive consensus (ERIC) PCR, sequencing, and phylogenetic analysis. Among the 51 clinical isolates collected from a university hospital in Korea, 6 isolates have been shown to produce SHV-12 and inducible AmpC beta-lactamases. These also included three isolates producing TEM-1b and one strain carrying TEM-1b and CMY-type beta-lactamases with a pI of 8.0. The results from ERIC PCR revealed that six isolates were genetically unrelated, suggesting that dissemination of SHV-12 was responsible for the spread of resistance to extended-spectrum beta-lactams in Korea. Six genes of inducible AmpC beta-lactamases that are responsible for the resistance to cephamycins (cefoxitin and cefotetan), amoxicillin, cephalothin, and amoxicillin-clavulanic acid were cloned and characterized. A 1,165-bp DNA fragment containing the ampC genes was sequenced and found to have an open reading frame coding for a 381-amino-acid beta-lactamase. The nucleotide sequence of four ampC genes (bla(EcloK992004.1), bla(EcloK995120.1), bla(EcloK99230), and bla(EareK9911729)) shared considerable homology with that of AmpC-type class C beta-lactamase genes of gram-negative bacteria, especially that of the chromosomal ampC gene (bla(EcloMHN1)) of Enterobacter cloacae MHN1 (99.9, 99.7, 99.6, and 99.6% identity, respectively). The sequences of two ampC genes (bla(EcloK9973) and bla(EcloK9914325)) showed close similarity to the chromosomal ampC gene (bla(EcloQ908R)) of E. cloacae Q908R (99.7% identity). The results from phylogenetic analysis suggested that six ampC genes could originate from bla(EcloMHN1) or bla(EcloQ908R).

FIG. 1.

ERIC PCR patterns of genomic DNA from E. cloacae K9973 (lane 2), E. cloacae K9914325 (lane 3), E. cloacae K99230 (lane 4), E. aerogenes K9911729 (lane 5), E. cloacae K992004.1 (lane 6), and E. cloacae K995120.1 (lane 7). Lanes 1 (HindIII/EcoRI-digested phage λ) and 8 (100-bp stepwise ladder), DNA marker fragments (sizes in base pairs are indicated on the edge of the gel). ERIC PCR was performed with primers ERIC2 and ERIC1R.

FIG. 2.

Sequence of the E. cloacae (EcloK992004.1, EcloK995120.1, EcloK99230, EcloK9973, and EcloK9914325) and E. aerogenes (EareK9911729) ampC genes. Different nucleotides are marked by underlining. Boxed nucleotides indicate start and stop codons. Abbreviations of gene names are given in Materials and Methods.

FIG. 3.

Phylogram of 11 ampC beta-lactamase genes. The branch length value represents relative phylogenetic distance. Abbreviations for taxon labels, accession numbers, gene locations (plasmid or chromosome), and organism names are given in Materials and Methods.