Genetic and Biochemical Characterization of OXA-519, a Novel OXA-48-Like β-Lactamase

Dabos, Laura; Bogaerts, Pierre; Bonnin, Rémy A.; Zavala, Agustín; Sacré, Pierre; Iorga, Bogdan; Huang, Daniel Tsung-Ning; Glupczynski, Youri; Naas, Thierry

doi:10.1128/aac.00469-18

Cited by 23 publications

(35 citation statements)

References 35 publications

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“…the expression vector pET41b (ϩ) (Novagen, VWR International, Fontenay-sous-Bois, France), and recombinant plasmid pET41-OXA-535 was electroporated into the electrocompetent E. coli strain BL21(DE3) (Novagen). OXA-535 was overexpressed and purified, and steady-state kinetic parameters were determined and compared with those of OXA-48 as previously described (6,19).…”

Section: Figmentioning

confidence: 99%

Genetic and Biochemical Characterization of OXA-535, a Distantly Related OXA-48-Like β-Lactamase

Dabos

Jousset

Bonnin

et al. 2018

Antimicrob Agents Chemother

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OXA-535 is a chromosome-encoded carbapenemase of JAB-1 that shares only 91.3% amino acid sequence identity with OXA-48. Catalytic efficiencies are similar to those of OXA-48 for most β-lactams, except for ertapenem, where a 2,000-fold-higher efficiency was observed with OXA-535. OXA-535 and OXA-436, a plasmid-encoded variant of OXA-535 differing by three amino acids, form a novel cluster of distantly related OXA-48-like carbapenemases. Comparison of and genetic environments suggests that an IS may be responsible for gene mobilization from the chromosome of spp. to plasmids.

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Section: Figmentioning

confidence: 99%

Genetic and Biochemical Characterization of OXA-535, a Distantly Related OXA-48-Like β-Lactamase

Dabos

Jousset

Bonnin

et al. 2018

Antimicrob Agents Chemother

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“…Detection of CPE. With Enterobacterales, the Revogene Carba C assay detected all KPC, NDM, VIM, IMP, and OXA-48 producers, including OXA-48 variants with carbapenemase activity (OXA-162, -181, -204, and -232), those difficult to detect by phenotypic means (such as OXA-244 and -519) (36,37), and very recently identified variants (OXA-484, -505, -517, and -793) ( Table 1) (38). The chromosomally encoded OXA-535 identified in a Shewanella species (39), which has only 91.3% amino acid identity with OXA-48, has not been detected (data not shown).…”

Section: Resultsmentioning

confidence: 99%

Evaluation of the Revogene Carba C Assay for Detection and Differentiation of Carbapenemase-Producing Gram-Negative Bacteria

et al. 2020

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The Revogene Carba C assay (formerly GenePOC Carba assay) is a multiplex nucleic acid-based in vitro diagnostic test intended for the detection of carbapenemase-producing Enterobacterales (CPE) from cultured colonies. This assay was evaluated directly on colonies of 118 well-characterized Enterobacterales with reduced susceptibility to carbapenems and on 49 multidrug-resistant (MDR) Pseudomonas aeruginosa and 40 MDR Acinetobacter baumannii isolates. The Revogene Carba C assay’s performance was high, as it was able to detect the five major carbapenemases (NDM, VIM, IMP, KPC, and OXA-48). In Enterobacterales, sensitivity and specificity were 100%. When extrapolating the results to the French CPE epidemiology between 2012 and 2018, this assay would have detected 99.28% of the 9,624 CPE isolates sent to the French NRC, missing 69 CPE isolates (2 GES-5, 10 OXA-23, 2 TMB-1, 1 SME-4, 53 IMI, and 1 FRI). The overall sensitivity and specificity for CP P. aeruginosa were 93.7 and 100%, respectively, as two rare IMP variants (IMP-31 and -46) were not detected. Extrapolating these results to the French epidemiology of CP P. aeruginosa in 2017, 93.3% would have been identified, missing only 1 DIM and 10 GES variants. The Revogene Carba C assay accurately identified the targeted carbapenemase genes in A. baumannii, but when extrapolating these results to the French CP A. baumannii epidemiology of 2017, only 12.50% of them could be detected, as OXA-23 is the most prevalent carbapenemase in CP A. baumannii. The Revogene Carba C assay showed excellent sensitivity and specificity for the five most common carbapenemases regardless of the bacterial host. It is well adapted to the CPE and CP P. aeruginosa epidemiology of many countries worldwide, which makes it suitable for use in the routine microbiology laboratory, with a time to result of ca. 85 min for eight isolates simultaneously.

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“…ACT-1-producing K. pneumoniae was used as source of the bla ACT-1 gene for PCR amplification prior to cloning experiments (19). Electrocompetent E. coli TOP10 and E. coli HB4 (a strain with permeability defect due to OmpC and OmpF major porin deficiency) were used as recipients for electroporation experiments, and E. coli BL21(DE3) (Invitrogen, Eragny, France) was used for overexpressing ACT-28 (29).…”

Section: Methodsmentioning

confidence: 99%

“…Illumina read de novo assembly was performed using CLC genomic workbench 10.0 according to the manufacturer's recommendations (Qiagen). The genome was annotated using the RAST tool (http://rast.nmpdr.org/rast.cgi) (29).…”

Section: Methodsmentioning

confidence: 99%

False-Positive Carbapenem-Hydrolyzing Confirmatory Tests Due to ACT-28, a Chromosomally Encoded AmpC with Weak Carbapenemase Activity from Enterobacter kobei

Jousset

Oueslati

Bernabeu

et al. 2019

Antimicrob Agents Chemother

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In Enterobacter cloacae complex (ECC), the overproduction of the chromosome-encoded cephalosporinase (cAmpC) associated with decreased outer membrane permeability may result in carbapenem resistance. In this study, we have characterized ACT-28, a cAmpC with weak carbapenemase activity, from a single Enterobacter kobei lineage. ECC clinical isolates were characterized by whole-genome sequencing (WGS), susceptibility testing, and MIC, and carbapenemase activity was monitored using diverse carbapenem hydrolysis methods. ACT-28 steady-state kinetic parameters were determined. Among 1,039 non-carbapenemase-producing ECC isolates with decreased susceptibility to carbapenems received in 2016-2017 at the French National Reference Center for antibiotic resistance, only 8 had a positive carbapenemase detection test (Carba NP). These eight ECC isolates were resistant to broad-spectrum cephalosporins due to AmpC derepression, showed decreased susceptibility to carbapenems, and were categorized as carbapenemase-producing Enterobacteriaceae (CPE) according to several carbapenemase detection assays. WGS identified a single clone of E. kobei ST125 expressing only its cAmpC, ACT-28. The bla ACT-28 gene was expressed in a wild-type and in a porin-deficient Escherichia coli background and compared to the bla ACT-1 gene. Detection of carbapenemase activity was positive only for E. coli expressing the bla ACT-28 gene. Kinetic parameters of purified ACT-28 revealed a slightly increased imipenem hydrolysis compared to that of ACT-1. In silico porin analysis revealed the presence of a peculiar OmpC-like protein specific to E. kobei ST125 that could impair carbapenem influx into the periplasm and thus enhance carbapenem-resistance caused by ACT-28. We described a widespread lineage of E. kobei ST125 producing ACT-28, with weak carbapenemase activity that can lead to false-positive detection by several biochemical and phenotypic diagnostic tests. E nterobacter cloacae complex (ECC) organisms are highly adapted to the hospital environment and are mostly responsible for hospital-acquired infections such as intravenous-catheter-related bacteremia, urinary tract infections (UTIs), and pulmonary infections (1). The ECC shows a genomic heterogeneity with 13 clusters based on their hsp60 sequences (2) and currently comprises seven different species (3): Enterobacter asburiae, E. cloacae, Enterobacter hormaechei, Enterobacter kobei, Enterobacter ludwigii, Enterobacter mori, and Enterobacter xiangfangensis.Enterobacter spp. naturally exhibit resistance to aminopenicillins, first-and second-

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Genetic and Biochemical Characterization of OXA-519, a Novel OXA-48-Like β-Lactamase

Cited by 23 publications

References 35 publications

Genetic and Biochemical Characterization of OXA-535, a Distantly Related OXA-48-Like β-Lactamase

Genetic and Biochemical Characterization of OXA-535, a Distantly Related OXA-48-Like β-Lactamase

Evaluation of the Revogene Carba C Assay for Detection and Differentiation of Carbapenemase-Producing Gram-Negative Bacteria

False-Positive Carbapenem-Hydrolyzing Confirmatory Tests Due to ACT-28, a Chromosomally Encoded AmpC with Weak Carbapenemase Activity from Enterobacter kobei

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