Marie-France I. Palepou

Outbreak of Klebsiella pneumoniae Producing a New Carbapenem-Hydrolyzing Class A β-Lactamase, KPC-3, in a New York Medical Center

ABSTRACT 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 β-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.

Occurrence of Carbapenem-Resistant Acinetobacter baumannii Clones at Multiple Hospitals in London and Southeast England

ABSTRACT From late 2003 to the end of 2005, the Health Protection Agencys national reference laboratories received approximately 1,600 referrals of Acinetobacter spp., including 419 and 58 examples, respectively, of two carbapenem-resistant Acinetobacter baumannii lineages, designated OXA-23 clones 1 and 2. Representatives of these clones were obtained from 40 and 8 hospitals, respectively, in London or elsewhere in Southeast England. Both clones had blaOXA-23-like genes, as well as the intrinsic (but downregulated) blaOXA-51-like carbapenemase genes typical of A. baumannii. Both were highly multiresistant: only colistin and tigecycline remained active versus OXA-23 clone 1 isolates; OXA-23 clone 2 isolates were also susceptible to amikacin and minocycline. These lineages increase the burden created by the southeast (SE) clone, a previously reported A. baumannii lineage with variable carbapenem resistance contingent on upregulation of the blaOXA-51-like gene. Known since 2000, the SE clone had been referred from over 40 hospitals by the end of 2005, with 627 representatives received by the reference laboratories. The OXA-23 clone 2 is now in decline, but OXA-23 clone 1 continues to be referred from new sites, as does the SE clone. Their spread is forcing the use of unorthodox therapies, principally colistin and tigecycline, although the optimal regimens remain uncertain.

IMP-4, a Novel Metallo-β-Lactamase from Nosocomial Acinetobacter spp. Collected in Hong Kong between 1994 and 1998

ABSTRACT Between 1994 and 1998, 97 imipenem-resistantAcinetobacter isolates were identified at the Prince of Wales Hospital, Hong Kong, China. A blaIMP PCR product was obtained from 23 of 35 viable cultures; 12 isolates belonged to genomic DNA group 3, 8 belonged to group 2 (Acinetobacter baumannii), 2 belonged to group 13TU, and 1 belonged to group 1. The blaIMP homologues were sequenced from two isolates from genomic DNA group 2 and one isolate each from groups 3 and 13TU. The four sequences included an identical 738-bp open reading frame, predicted to encode a polypeptide of 246 amino acids, with 95.6% homology to IMP-1 and 89.3% homology to IMP-2. The new enzyme, designated IMP-4, was partially purified. It had a pI of 8.0 and was strongly active against imipenem and meropenem, with Vmax values 53 and 8% of that for penicillin G, respectively. Strong activity was also seen against oxyimino-aminothiazolyl cephalosporins but not against aztreonam. Hydrolytic activity was inhibited by EDTA but not by clavulanate or tazobactam. Carbapenem MICs for mostblaIMP-positive isolates were 4 to 32 μg/ml, but one isolate with the intact gene was susceptible, with imipenem and meropenem MICs of 0.25 and 0.5 μg/ml, respectively. The latter isolate did not produce the band with a pI of 8.0, and gene expression was inferred to have been lost. None of the isolates studied in detail contained extrachromosomal DNA, and carbapenem resistance was not transmissible to Escherichia coli. Nevertheless, the presence of blaIMP-4 in different genomic DNA groups implies horizontal transfer, and sequences resembling a GTTRRRY integrase-dependent recombination motif were identified in the flanking regions of blaIMP-4.

Nosocomial Outbreak of Carbapenem-Resistant Pseudomonas aeruginosa with a New blaIMP Allele, blaIMP-7

ABSTRACT 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-β-lactamase, designated IMP-7, with 91% identity to IMP-1. blaIMP-7 was not detected with standard blaIMP-specific primers, owing to mismatches in the forward primer.

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.

Phenotypic and Enzymatic Comparative Analysis of the Novel KPC Variant KPC-5 and Its Evolutionary Variants, KPC-2 and KPC-4

ABSTRACT A novel Klebsiella pneumoniae carbapenemase (KPC) variant, designated blaKPC-5, was discovered in a carbapenem-resistant Pseudomonas aeruginosa clinical isolate from Puerto Rico. Characterization of the upstream region of blaKPC-5 showed significant differences from the flanking regions of other blaKPC 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 (Pro103→Arg), while KPC-4 contained Pro103→Arg plus an additional amino acid change (Val239→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.

Carbapenemases of Chryseobacterium (Flavobacterium) meningosepticum: distribution of blaB and characterization of a novel metallo-beta-lactamase gene, blaB3, in the type strain, NCTC 10016.

ABSTRACT Genes encoding carbapenemases in 15 reference strains ofChryseobacterium (Flavobacterium)meningosepticum from the United Kingdom National Collection of Type Cultures and in one recent clinical isolate were investigated. All the strains hydrolyzed imipenem, but their levels of resistance to carbapenems varied, with imipenem and meropenem MICs ranging from 2 to >32 μg/ml. The blaB gene, which encodes a molecular-class B carbapenemase, was detected in only six reference strains and in clinical isolate 97/P/5448. The gene from 97/P/5448 had 98% nucleotide identity with the published sequence ofblaB (from strain NCTC 10585) and was designatedblaB2. A distinct carbapenemase gene, designatedblaB3, was cloned from the type strain of C. meningosepticum, NCTC 10016. blaB3 had an open reading frame of 750 bp with 82% nucleotide identity toblaB and blaB2 and encoded a β-lactamase of 249 amino acids, including the putative signal peptide. This β-lactamase showed 87.6 and 86.7% amino acid homology with BlaB and BlaB2, respectively. blaB3 was detected in one other reference strain besides NCTC 10016, but the genetic basis of the carbapenemase activity detected in the other seven reference strains was not defined. Thus, neither blaB nor blaB3was ubiquitous in the strains of C. meningosepticumstudied, indicating that the reference strains may represent more than one bacterial species, each with its own intrinsic metallo-β-lactamase. Further taxonomic studies of C. meningosepticum are necessary to resolve this topic.Chryseobacterium spp. are environmental organisms and occasional opportunist pathogens. They apparently represent a reservoir of diverse metallo-β-lactamases, which potentially spread to gram-negative bacteria of greater clinical significance.

Characterization of a Divergent vanD-Type Resistance Element from the First Glycopeptide-Resistant Strain of Enterococcus faecium Isolated in Brazil

ABSTRACT Enterococcus faecium 10/96A from Brazil was resistant to vancomycin (MIC, 256 μg/ml) but gave no amplification products with primers specific for known van genotypes. A 2,368-bp fragment of a van cluster contained one open reading frame encoding a peptide with 83% amino acid identity to VanHD, and a second encoding a d-alanine-d-lactate ligase with 83 to 85% identity to VanD. The divergent glycopeptide resistance phenotype was designated VanD4.

Prevalence of the vanB2 Gene Cluster in VanB Glycopeptide-Resistant Enterococci in the United Kingdom and the Republic of Ireland and Its Association with a Tn5382-Like Element

A total of 204 vanB enterococcal isolates, isolated between 1989 and 1999 from patients in 59 different hospitals in England, Wales, and Scotland and from a single hospital in the Republic of Ireland, were examined. Nucleotide sequencing and HhaI digestion of a fragment of the vanB gene was used to distinguish between the vanB1, vanB2, andvanB3 gene clusters (7). Based on theirvanB-HhaI restriction fragment length polymorphism (RFLP) profiles, 202 (99%) isolates contained vanB2. The presence of vanB1 in two isolates was confirmed by sequence analysis.

Effects of the Movement of Insertion Sequences on the Structure of VanA Glycopeptide Resistance Elements in Enterococcus faecium

ABSTRACT A Tn1546-related element with IS1216V at position 8839 underwent a structural change after storage of the host strain of Enterococcus faecium at 4°C. The element acquired IS1542 at position 3932, nucleotides 8732 to 8831 were deleted, and the first 3417 nucleotides were lost and replaced by an inverted copy of the IS1216V–vanY–vanZ-inverted-repeat block from the 3′ end. Insertion sequence movement is likely to play a key role in the evolution of VanA resistance elements.