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Genetic structures at the origin of acquisition of the beta-lactamase bla KPC gene.

ABSTRACT: Genetic structures surrounding the carbapenem-hydrolyzing Ambler class A bla KPC gene were characterized in several KPC-positive Klebsiella pneumoniae and Pseudomonas aeruginosa strains isolated from the United States, Colombia, and Greece. The bla KPC genes were associated in all cases with transposon-related structures. In the K. pneumoniae YC isolate from the United States, the beta-lactamase bla KPC-2 gene was located on a novel Tn3-based transposon, Tn4401. Tn4401 was 10 kb in size, was delimited by two 39-bp imperfect inverted repeat sequences, and harbored, in addition to the beta-lactamase bla KPC-2 gene, a transposase gene, a resolvase gene, and two novel insertion sequences, ISKpn6 and ISKpn7. Tn4401 has been identified in all isolates. However, two isoforms of this transposon were found: Tn4401a was found in K. pneumoniae YC and K. pneumoniae GR from the United States and Greece, respectively, and differed by a 100-bp deletion, located just upstream of the bla KPC-2 gene, compared to the sequence of Tn4401b, which was found in the Colombian isolates. In all isolates tested, Tn4401 was flanked by a 5-bp target site duplication, the signature of a recent transposition event, and was inserted in different open reading frames located on plasmids that varied in size and nature. Tn4401 is likely at the origin of carbapenem-hydrolyzing beta-lactamase KPC mobilization to plasmids and its further insertion into various-sized plasmids identified in nonclonally related K. pneumoniae and P. aeruginosa isolates.

SUBMITTER: Naas T

PROVIDER: S-EPMC2292522 | biostudies-literature | 2008 Apr

REPOSITORIES: biostudies-literature

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Genetic structures at the origin of acquisition of the beta-lactamase bla KPC gene.

Naas Thierry T Cuzon Gaelle G Villegas Maria-Virginia MV Lartigue Marie-Frédérique MF Quinn John P JP Nordmann Patrice P

Antimicrobial agents and chemotherapy 20080128 4

Genetic structures surrounding the carbapenem-hydrolyzing Ambler class A bla KPC gene were characterized in several KPC-positive Klebsiella pneumoniae and Pseudomonas aeruginosa strains isolated from the United States, Colombia, and Greece. The bla KPC genes were associated in all cases with transposon-related structures. In the K. pneumoniae YC isolate from the United States, the beta-lactamase bla KPC-2 gene was located on a novel Tn3-based transposon, Tn4401. Tn4401 was 10 kb in size, was del ...[more]

PMID: 18227185

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Project description:As resistance determinants, KPC beta-lactamases demonstrate a wide substrate spectrum that includes carbapenems, oxyimino-cephalosporins, and cephamycins. In addition, clinical strains harboring KPC-type beta-lactamases are often identified as resistant to standard beta-lactam-beta-lactamase inhibitor combinations in susceptibility testing. The KPC-2 carbapenemase presents a significant clinical challenge, as the mechanistic bases for KPC-2-associated phenotypes remain elusive. Here, we demonstrate resistance by KPC-2 to beta-lactamase inhibitors by determining that clavulanic acid, sulbactam, and tazobactam are hydrolyzed by KPC-2 with partition ratios (kcat/kinact ratios, where kinact is the rate constant of enzyme inactivation) of 2,500, 1,000, and 500, respectively. Methylidene penems that contain an sp2-hybridized C3 carboxylate and a bicyclic R1 side chain (dihydropyrazolo[1,5-c][1,3]thiazole [penem 1] and dihydropyrazolo[5,1-c][1,4]thiazine [penem 2]) are potent inhibitors: Km of penem 1, 0.06+/-0.01 microM, and Km of penem 2, 0.006+/-0.001 microM. We also demonstrate that penems 1 and 2 are mechanism-based inactivators, having partition ratios (kcat/kinact ratios) of 250 and 50, respectively. To understand the mechanism of inhibition by these penems, we generated molecular representations of both inhibitors in the active site of KPC-2. These models (i) suggest that penem 1 and penem 2 interact differently with active site residues, with the carbonyl of penem 2 being positioned outside the oxyanion hole and in a less favorable position for hydrolysis than that of penem 1, and (ii) support the kinetic observations that penem 2 is the better inhibitor (kinact/Km=6.5+/-0.6 microM(-1) s(-1)). We conclude that KPC-2 is unique among class A beta-lactamases in being able to readily hydrolyze clavulanic acid, sulbactam, and tazobactam. In contrast, penem-type beta-lactamase inhibitors, by exhibiting unique active site chemistry, may serve as an important scaffold for future development and offer an attractive alternative to our current beta-lactamase inhibitors.

Project description:BackgroundThis study was designed to characterize the dissemination mechanism and genetic context of Klebsiella pneumoniae carbapenemase (KPC) genes in carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates.MethodsA retrospective analysis was performed on CRKP strains isolated from a teaching hospital of Wenzhou Medical University during 2015-2017. Polymerase chain reaction (PCR)-based amplification and whole-genome sequencing (WGS) were used to analyze the genetic context of the bla KPC-2 gene. Conjugation experiments were performed to evaluate the transferability of bla KPC-2-bearing plasmids. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were performed to investigate the clonal relatedness of bla KPC-2-producing strains.ResultsThe bla KPC-2 gene was identified from 13.61% (40/294) of clinical K. pneumoniae isolates. Three different sequence types (ST11, ST15 and ST656) and 5 PFGE subtypes (A to E) were classified among them. ST11 was the dominant sequence type (92.50%, 37/40). Plasmid-oriented antibiotic resistance genes, such as extended spectrum-?-lactamases (ESBLs) and other antimicrobial resistance genes, were also found in KPC-positive K. pneumoniae (KPC-Kp) isolates. Mapping PCR and genomic sequencing revealed that the bla KPC-2-bearing sequence regions, which are related to different mobile elements, including Tn1721- and IS26-based transposons, were mainly located in but not restricted to IncFII-like plasmids and were structurally divergent.ConclusionThe bla KPC-2 genes related to divergent mobile genetic elements encoded on transferable plasmids may transfer widely, facilitating the spread of carbapenem resistance among bacteria with different genetic backgrounds. The dissemination of bla KPC-bearing plasmids that collectively carry additional multidrug resistance genes has caused widespread public concern, further limiting the antibiotics available to treat infections caused by KPC-producing pathogens.

Dataset Information

Genetic structures at the origin of acquisition of the beta-lactamase bla KPC gene.

Publications

Genetic structures at the origin of acquisition of the beta-lactamase bla KPC gene.

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