Horizontal Transfer of bla _CMY -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 ␤-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.Resistance to ␤-lactam antibiotics in gram-negative bacilli is mainly mediated by the production of ␤-lactamases, which are divided into four major molecular classes, classes A, B, C, and D (1, 10). Genes for AmpC (class C) ␤-lactamases are generally encoded on the chromosomes in many gram-negative microbes, including Enterobacter spp., Citrobacter freundii, Serratia marcescens, Morganella morganii, and Pseudomonas aeruginosa (27). Chromosomal AmpC enzymes are usually inducible and are often responsible for resistance to cephalosporins (27) as well as to penicillins. Plasmid-mediated class C ␤-lactamases have mainly been described in Klebsiella spp., Escherichia coli, and Salmonella spp. throughout the world (25). A cephamycin-resistant Klebsiella pneumoniae strain producing a plasmid-mediated class C ␤-lactamase, CMY-1, was first reported in 1989 in Korea (7,8). Plasmid-mediated class C enzymes are currently divided into at least five clusters (25) on the basis of amino acid sequence similarities, together with their putative progenitor chromosomal AmpC enzymes. In Japan, MOX-1 (16), CMY-8 (unpublished data), CMY-9 (12), CMY-2 (unpublished data), CFE-1 (23), and DHA-1 (unpublished data) have so far been found as plasmid-mediated AmpC ␤-lactamases, mainly in nosocomial isolates of the family Enterobacteriaceae.Between 1995 and 1997, eight additional E. coli isolates and five K. pneumoniae isolates resistant to both oximino-cephalosporins and cephamycins were isolated in the same hospital where the first CMY-9-producing E. coli strain (strain HKYM68) was isolated in 19...

Extended-spectrumβ-Lactamases: Implications for the Clinical Laboratory and Therapy

“…Although it has been determined that such mutants were mainly located on bacterial chromosome [14][15][16], two plasmid-mediated extended-spectrum cephalosporinases, CMY-19 [17] and CMY-10 [18], have been reported. Some specialists may insist on regarding such enzymes as ESBLs because of their wide spectrum of activity.…”

Scientific Opinion on the public health risks of bacterial strains producing extended-spectrum β-lactamases and/or AmpC β-lactamases in food and food-producing animals

“…In addition, constant pressure by the use of various β-lactams, including penicillins and first-generation cephalosporins, could have contributed to the in vivo evolution of ESBLs (Blazquez et al, 2000). As a result of use, mutations occurring in AmpC-encoding genes can extend their spectrum of activity to fourth generation cephalosporins, giving rise to the Extended Spectrum Cephalosporinases (ESCs) (Ahmed and Shimamoto, 2008;Mammeri et al, 2008;Mammeri et al, 2007;Wachino et al, 2006).…”

Identification of Plasmid-Mediated AmpC β-Lactamases in Escherichia coli , Klebsiella spp., and Proteus Species Can Potentially Improve Reporting of Cephalosporin Susceptibility Testing Results

“…As noted above, in contrast to bacterial isolates containing ESBLs, isolates containing AmpC ␤-lactamases typically remain susceptible to ASCs (13,14,26,27,31). Although the occurrence is rare, AmpC ␤-lactamases, such as CMY-19, show modest cefepime hydrolyzing activity (31) even though the organisms carrying this ␤-lactamase remain fully susceptible to both cefepime and cefpirome in vitro.…”