Phenotypic and enzymatic comparative analysis of the novel KPC variant KPC-5 and its evolutionary variants, KPC-2 and KPC-4.

A novel Klebsiella pneumoniae carbapenemase (KPC) variant, designated bla(KPC-5), was discovered in a carbapenem-resistant Pseudomonas aeruginosa clinical isolate from Puerto Rico. Characterization of the upstream region of bla(KPC-5) showed significant differences from the flanking regions of other bla(KPC) variants. Comparison of amino acid sequences with those of other KPC enzymes revealed that KPC-5 was an intermediate between KPC-2 and KPC-4, differing from KPC-2 by a single amino acid substitution (Pro(103)-->Arg), while KPC-4 contained Pro(103)-->Arg plus an additional amino acid change (Val(239)-->Gly). Transformation studies with an Escherichia coli recipient strain showed differences in the properties of the KPC variants. KPC-4 and KPC-5 both had pIs of 7.65, in contrast with the pI of 6.7 for KPC-2. KPC-2 transformants were less susceptible to the carbapenems than KPC-4 and KPC-5 transformants. These data correlated with higher rates of imipenem hydrolysis for KPC-2 than for KPC-4 and KPC-5. However, KPC-4 and KPC-5 transformants had higher ceftazidime MICs, and the enzymes from these transformants had slightly better hydrolysis of this drug than KPC-2. KPC-4 and KPC-5 were more sensitive than KPC-2 to inhibition by clavulanic acid in both susceptibility testing and hydrolysis assays. Thus, KPC enzymes may be evolving through stepwise mutations to alter their spectra of activity.

Carbapenem-resistant Escherichia coli and Klebsiella pneumoniae isolates from Turkey with OXA-48-like carbapenemases and outer membrane protein loss.

Treatment options are limited in infections caused by extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae, with carbapenems generally preferred. Disturbingly, however, carbapenem-resistant strains are emerging worldwide. Here we report two clinical isolates, one Escherichia coli and one Klebsiella pneumoniae, each with high-level carbapenem resistance (imipenem minimum inhibitory concentration of 32 microg/mL). They were isolated following imipenem therapy from two hospital patients who had received imipenem therapy in different regions of Turkey. Both isolates produced OXA-48-like carbapenemases, enzymes so far reported only from Turkey. Both isolates also had group 1 CTX-M-type ESBLs and had lost major outer membrane proteins. OXA-48-like carbapenemases appear to be scattered in Turkey and surveillance to determine their prevalence is warranted.

Ertapenem resistance among Klebsiella and Enterobacter submitted in the UK to a reference laboratory.

The emergence of carbapenem resistance in Enterobacteriaceae represents a major public health concern. We investigated ertapenem-resistant clinical isolates of Klebsiella spp. and Enterobacter spp. referred to the UK's national reference laboratory for antibiotic resistance. Minimum inhibitory concentrations (MICs) were determined and interpreted according to British Society for Antimicrobial Chemotherapy guidelines. Genes for carbapenemases and CTX-M extended-spectrum beta-lactamases (ESBLs) were sought by polymerase chain reaction, and imipenem hydrolysis was determined by spectrophotometry with crude extracts. From June 2004 to April 2006, 95 Klebsiella spp. and 76 Enterobacter spp. isolates resistant to ertapenem (MICs >2mg/L) were received, 40% of which (38 Klebsiella spp. and 30 Enterobacter spp.) were highly resistant to ertapenem (MICs >16mg/L). Imipenem and meropenem were active (geometric mean MICs <2mg/L) against most isolates with low-level ertapenem resistance but were less active against highly ertapenem-resistant isolates. Only one ertapenem-resistant isolate produced a defined carbapenemase, a Klebsiella pneumoniae with IMP-1 enzyme; one Enterobacter sp. also hydrolysed imipenem, but its carbapenemase remains to be identified. Geometric mean MICs of ertapenem for the collection were reduced five-fold by clavulanic acid for Klebsiella spp. compared with eight-fold by cloxacillin for Enterobacter spp. This study highlights the fact that ertapenem resistance is being detected in Klebsiella spp. and Enterobacter spp. in the UK, but that it is rarely mediated by true carbapenemases. Rather, it probably results from combinations of a beta-lactamase - often a CTX-M ESBL in Klebsiella spp. or an AmpC enzyme in Enterobacter spp. - plus impermeability and/or increased efflux. Imipenem and meropenem often remain moderately active against isolates with low-level ertapenem resistance.

Wide geographic spread of diverse acquired AmpC beta-lactamases among Escherichia coli and Klebsiella spp. in the UK and Ireland.

OBJECTIVES: To determine the distribution of acquired AmpC beta-lactamases in 173 isolates of Escherichia coli and Klebsiella spp. submitted to the UK's national reference laboratory for antibiotic resistance. METHODS: MICs were determined and interpreted according to BSAC guidelines. Candidate isolates were those resistant to cefotaxime and/or ceftazidime, irrespective of addition of clavulanic acid. Genes encoding six phylogenetic groups of acquired AmpC enzymes were sought by PCR. Selected isolates were compared by pulsed-field gel electrophoresis (PFGE), and one bla(AmpC) amplicon was sequenced. RESULTS: Genes encoding acquired AmpC enzymes were detected in 67 (49%) candidate E. coli and 21 (55%) Klebsiella spp. Sixty isolates produced CIT-type enzymes, 14 had ACC types, 11 had FOX types and 3 had DHA enzymes. The low-level cephalosporin resistance of the remaining isolates (n = 85; 49%) was inferred to result from reduced permeability or, in E. coli, from hyperexpression of chromosomal ampC. Twenty-four E. coli isolates from one hospital produced a CIT-type enzyme, with 20 of these additionally producing a group 1 CTX-M extended-spectrum beta-lactamase. PFGE indicated that these isolates belonged to UK epidemic strain A, which normally produces CTX-M-15, but no acquired AmpC. Sequencing a representative bla(AmpC) amplicon indicated that in one centre this strain had acquired a novel CMY-2 variant, designated CMY-23. CONCLUSIONS: Diverse acquired AmpC enzymes occur in E. coli and Klebsiella spp. isolates in the UK and Ireland, with CIT types the most common. Producers are geographically scattered, but with some local outbreaks. Acquisition of a CMY-2-like enzyme by E. coli epidemic strain A suggests that these enzymes may be poised to become an important public health issue.

Outbreak of Klebsiella pneumoniae producing a new carbapenem-hydrolyzing class A beta-lactamase, KPC-3, in a New York Medical Center.

From April 2000 to April 2001, 24 patients in intensive care units at Tisch Hospital, New York, N.Y., were infected or colonized by carbapenem-resistant Klebsiella pneumoniae. Pulsed-field gel electrophoresis identified a predominant outbreak strain, but other resistant strains were also recovered. Three representatives of the outbreak strain from separate patients were studied in detail. All were resistant or had reduced susceptibility to imipenem, meropenem, ceftazidime, piperacillin-tazobactam, and gentamicin but remained fully susceptible to tetracycline. PCR amplified a blaKPC allele encoding a novel variant, KPC-3, with a His(272)-->Tyr substitution not found in KPC-2; other carbapenemase genes were absent. In the outbreak strain, KPC-3 was encoded by a 75-kb plasmid, which was transferred in vitro by electroporation and conjugation. The isolates lacked the OmpK35 porin but expressed OmpK36, implying reduced permeability as a cofactor in resistance. This is the third KPC carbapenem-hydrolyzing beta-lactamase variant to have been reported in members of the Enterobacteriaceae, with others reported from the East Coast of the United States. Although producers of these enzymes remain rare, the progress of this enzyme group merits monitoring.

Nosocomial outbreak of carbapenem-resistant Pseudomonas aeruginosa with a new bla(IMP) allele, bla(IMP-7).

Pseudomonas aeruginosa isolates from an outbreak in Canada were highly resistant to carbapenems and ceftazidime but not piperacillin. They produced a novel integron-associated metallo-beta-lactamase, designated IMP-7, with 91% identity to IMP-1. bla(IMP-7) was not detected with standard bla(IMP)-specific primers, owing to mismatches in the forward primer.