Horizontal transfer of blaCMY-bearing plasmids among clinical Escherichia coli and Klebsiella pneumoniae isolates and emergence of cefepime-hydrolyzing CMY-19

Abstract

Nine Escherichia coli and 5 Klebsiella pneumoniae clinical isolates resistant to various cephalosporins and cephamycins were identified in a Japanese general hospital between 1995 and 1997. All nine E. coli isolates and one K. pneumoniae isolate carried bla(CMY-9), while the other four K. pneumoniae isolates harbored a variant of bla(CMY-9), namely, bla(CMY-19). The pulsed-field gel electrophoresis patterns of the nine CMY-9-producing E. coli isolates were almost identical, suggesting their clonal relatedness, while those of the five K. pneumoniae isolates were divergent. Plasmid profiles, Southern hybridization, and conjugation assays revealed that the genes for the CMY-9 and the CMY-19 beta-lactamases were located on very similar conjugative plasmids in E. coli and K. pneumoniae. The genetic environment of bla(CMY-19) was identical to that of bla(CMY-9). A single amino acid substitution, I292S, adjacent to the H-10 helix region was observed between CMY-9 and CMY-19. This substitution was suggested to be responsible for the expansion of the hydrolyzing activity against several broad-spectrum cephalosporins, and this finding was consistent with the kinetic parameters determined with purified enzymes. These findings suggest that the bla(CMY-19) genes found in the four K. pneumoniae isolates might have originated from bla(CMY-9) gene following a point mutation and dispersed among genetically different K. pneumoniae isolates via a large transferable plasmid.

FIG. 1.

Alignments of amino acid residues near the H-10 helix. A partial amino acid sequence alignment of CMY-9 (12), CMY-19 (this study), CMY-11 (21), FOX-1 (15), AmpC of E. cloacae Ear1 and Ear2 (5), AmpC of E. cloacae P99 and HD (6), AmpC of E. coli K-12 and HKY28 (13), and AmpC of S. marcescens S3 and HD (22) is shown. Square boxes show the amino acid substitutions or deletions that are predicted to affect the hydrolyzing activity of cefepime. The conserved motif KTG is underlined. Dashes indicate deletions of amino acid residues. CMY-11- and FOX-type enzymes have a serine residue at amino acid position 292, but no observation about their property against cefepime was described in the articles. The numbering of the amino acid residues is in reference to that of the mature CMY-1 reported by Bauernfeind et al. (7).

FIG. 2.

PFGE analysis of K. pneumoniae and E. coli isolates. (A) Lanes: M, PFGE marker; 1, K. pneumoniae HKY209; 2, K. pneumoniae HKY327; 3, K. pneumoniae HKY363; 4, K. pneumoniae HKY466; 5, K. pneumoniae HKY474. (B) Lanes: M, PFGE marker; A, E. coli HKY154; B, E. coli HKY191; C, E. coli HKY200; D, E. coli HKY215; E, E. coli HKY224; F, E. coli HKY297; G, E. coli HKY315; H, E. coli HKY334; and I, E. coli HKYM68.

FIG. 3.

Plasmid profiles and Southern hybridization. (A) Plasmid profiles of clinical isolates and their tranconjugants; (B) hybridization with the probe specific for the CMY-1- and MOX-1-type β-lactamase gene. Lanes: M, HindIII-digested DNA marker; 1, K. pneumoniae HKY209; 2, E. coli CSH-2/pK209; 3, K. pneumoniae HKY327; 4, E. coli CSH-2/pK327; 5, K. pneumoniae HKY363; 6, E. coli CSH-2/pK363; 7, K. pneumoniae HKY466; 8, E. coli CSH-2/pK466; 9, K. pneumoniae HKY474; 10, E. coli CSH-2/pK474; 11, E. coli HKY154; 12, E. coli CSH-2/pE154; 13, E. coli HKYM68; and 14, E. coli CSH-2/pEM68.

FIG. 4.

Plasmid patterns after restriction enzyme digestion and Southern hybridization. (A) SacI-digested plasmid DNAs prepared from the representative transconjugants; (B) hybridization patterns with the probe specific for CMY-1- and MOX-1-type β-lactamase gene. Lanes: M, HindIII-digested DNA marker; 1, E. coli CSH-2/pK209; 2, E. coli CSH-2/pK327; 3, E. coli CSH-2/pK363; 4, E. coli CSH-2/pK466; 5, E. coli CSH-2/pK474; 6, E. coli CSH-2/pE154; and 7, E. coli CSH-2/pEM68.

FIG. 5.

Gene organization around bla_CMY genes. The bla_CMY gene on the conjugative plasmid found in the K. pneumoniae and E. coli clinical isolates located just downstream of orf513 is shown as it was found in our previous study on a CMY-9 producing E. coli HKHM68 (12). Open circle, position of the 59-base element; CS, conserved segment of a class 1 integron. orf513 is speculated to encode a putative transposase, and various antimicrobial resistance genes tend to be integrated just downstream the orf513. The product from the yqgF gene encodes a hypothetical protein very similar to the YqgF identified in Aeromonas hydrophila (EMBL accession no. AJ276030), but the function is unknown.