Prevalence of SHV-12 among clinical isolates of Klebsiella pneumoniae producing extended-spectrum beta-lactamases and identification of a novel AmpC enzyme (CMY-8) in Southern Taiwan

Abstract

Twenty (8.5%) of 234 nonrepetitive clinical isolates of Klebsiella pneumoniae from southern Taiwan were found to produce extended-spectrum beta-lactamases (ESBLs): 10 strains produced SHV-12, 4 produced SHV-5, 2 produced a non-TEM non-SHV ESBL with a pI of 8.3, 3 produced a novel AmpC beta-lactamase designated CMY-8 with a pI of 8.25, and 1 produced SHV-12 and an unidentified AmpC enzyme with a pI of 8.2. The CMY-8 enzyme confers a resistance phenotype similar to CMY-1 and MOX-1, and sequence comparisons showed high homologies (>95%) of nucleotide and amino acid sequences among these three enzymes. Plasmid and pulse-field gel electrophoresis analyses revealed that all isolates harboring an SHV-derived ESBL were genetically unrelated, indicating that dissemination of resistance plasmids is responsible for the spread of SHV ESBLs among K. pneumoniae in this area. All three isolates carrying CMY-8 had identical genotypic patterns, suggesting the presence of an epidemic strain.

FIG. 1

Complete nucleotide sequence and predicted amino acid sequence of the bla_CMY-8 β-lactamase gene. The primers for amplification of the gene were synthesized according to the nucleotide sequence of CMY-1 (4, 14) and are double underlined. DNA sequencing was performed with a sequencing primer (underlined) and the PCR primers as well. Arrows indicate the directions of DNA sequencing. The stop codon is indicated with three asterisks. The β-lactamase active site S-V-S-K, the conserved triad K-T-G, and the class C typical motif Y-X-N are underlined.

FIG. 2

Alignment of deduced amino acid sequences of CMY-8 with those of CMY-1 and MOX-1. Identical amino acids are marked with dots. The stop codon is indicated with an asterisk. The β-lactamase active site S-V-S-K, the conserved triad K-T-G, and the class C typical motif Y-X-N are shown in boldface type.

FIG. 3

PFGE of XbaI-digested genomic DNAs from 19 ESBL-producing K. pneumoniae isolates. Lanes 1, 12, 13, and 23, bacteriophage lambda DNA concatemers (GibcoBRL, Gaithersburg, Md.) which served as molecular size marker; lanes 2 to 11 and 14 to 22, isolates V293, D155, B657, C106, O574, R549, O787, W142, W580, T384, X386, C097, Q381, T923, T386, R436, T986, B262, and T255, respectively. Isolate S276 is not shown here. All ESBL producers except for three isolates carrying CMY-8 (W142, W580, and T384) had different PFGE patterns.