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Multicopy blaOXA-58 gene as a source of high-level resistance to carbapenems in Acinetobacter baumannii.

ABSTRACT: The mechanisms at the origin of heterogeneous carbapenem resistance levels observed among Acinetobacter baumannii isolates collected in 2005 in a large University Hospital of Rome, Italy, were investigated. These isolates were related and possessed similar plasmids carrying the carbapenem-hydrolyzing oxacillinase gene bla(OXA-58) but showed variable levels of resistance to carbapenems. Analysis of sequences surrounding the bla(OXA-58) gene showed genetic variability, with the presence in several isolates of multiple copies of the bla(OXA-58) gene; this extra copy number was likely related to an IS26-mediated transposition or recombination process.

SUBMITTER: Bertini A

PROVIDER: S-EPMC1913244 | biostudies-literature | 2007 Jul

REPOSITORIES: biostudies-literature

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Multicopy blaOXA-58 gene as a source of high-level resistance to carbapenems in Acinetobacter baumannii.

Bertini Alessia A Poirel Laurent L Bernabeu Sandrine S Fortini Daniela D Villa Laura L Nordmann Patrice P Carattoli Alessandra A

Antimicrobial agents and chemotherapy 20070416 7

The mechanisms at the origin of heterogeneous carbapenem resistance levels observed among Acinetobacter baumannii isolates collected in 2005 in a large University Hospital of Rome, Italy, were investigated. These isolates were related and possessed similar plasmids carrying the carbapenem-hydrolyzing oxacillinase gene bla(OXA-58) but showed variable levels of resistance to carbapenems. Analysis of sequences surrounding the bla(OXA-58) gene showed genetic variability, with the presence in several ...[more]

PMID: 17438042

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Project description:Nosocomial infections caused by Acinetobacter spp. resistant to carbapenems are increasingly reported worldwide. Carbapenem-resistant Acinetobacter (CRA) is becoming a serious concern with increasing patient morbidity, mortality, and lengths of hospital stay. Therefore, the rapid detection of CRA is essential for epidemiological surveillance. Polymerase chain reaction (PCR) has been extensively used for the rapid identification of most pathogens. In this study, we have developed two multiplex real-time PCR assays to detect and differentiate A. baumannii and non-A. baumannii Acinetobacter spp, and common carbapenemase genes, including blaNDM, blaOXA-23-like, blaOXA-40-like, blaOXA-51-like, and blaOXA-58-like. We demonstrate the potential utility of these assays for the direct detection of blaNDM-, blaOXA-23-like-, blaOXA-40-like-, blaOXA-51-like-, and blaOXA-58-like-positive CRA in clinical specimens. Primers were specifically designed, and two multiplex real-time PCR assays were developed: multiplex real-time PCR assay1 for the detection of Acinetobacter baumannii 16S-23S rRNA internal transcribed spacer sequence, the Acinetobacter recA gene, and class-B-metalloenzyme-encoding gene blaNDM; and multiplex real-time PCR assay2 to detect class-D-oxacillinase-encoding genes (blaOXA-23-like, blaOXA-40-like, blaOXA-51-like,and blaOXA-58-like). The assays were performed on an ABI Prism 7500 FAST Real-Time PCR System. CRA isolates were used to compare the assays with conventional PCR and sequencing. Known amounts of CRA cells were added to sputum and fecal specimens and used to test the multiplex real-time PCR assays. The results for target and nontarget amplification showed that the multiplex real-time PCR assays were specific, the limit of detection for each target was 10 copies per 20 μL reaction volume, the assays were linear over six log dilutions of the target genes (r2 > 0.99), and the Ct values of the coefficients of variation for intra- and interassay reproducibility were less than 5%. The multiplex real-time PCR assays showed 100% concordance with conventional PCR when tested against 400 CRA isolates and their sensitivity for the target DNA in sputum and fecal specimens was 102 CFU/mL. Therefore, these novel multiplex real-time PCR assays allow the sensitive and specific characterization and differentiation of blaNDM-, blaOXA-23-like-, blaOXA-40-like-, blaOXA-51-like-, and blaOXA-58-like-positive CRA, making them potential tools for the direct detection of CRA in clinical specimens and the surveillance of nosocomial infections.

Project description:The prevalence of carbapenem-resistant Acinetobacter baumannii in hospitals has been increasing worldwide. This study aims to investigate the carbapenemase genes and the clonal relatedness among A. baumannii clinical isolates in a Chinese hospital.Carbapenemase genes and the upstream locations of insertion sequences were detected by polymerase chain reaction (PCR), and the clonal relatedness of isolates was determined by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing.A total of 231 nonduplicate carbapenemase gene-harboring A. baumannii clinical isolates recovered from Shenzhen People's Hospital, were investigated between 2002 and 2009. blaOXA-23-like, blaOXA-58-like, blaOXA-40-like, and ISAba1-blaOXA-51-like were identified in 119, 107, 1, and 4 isolates, respectively. IS1008-?ISAba3, ISAba3, and ISAba1 were detected upstream of the blaOXA-58-like gene in 69, 35, and 3 isolates, respectively. All blaOXA-23-like genes but one had an upstream insertion of ISAba1. blaOXA-58-like was the most common carbapenemase gene in A.baumannii before 2008, thereafter blaOXA-23-like became rapidly prevalent and replaced blaOXA-58-like in 2009. The majority of blaOXA-58-like-carrying isolates showed lower level of resistance to imipenem and meropenem (minimum inhibitory concentrations (MICs), 1 ?g/ml to 16 ?g/ml), compared with the majority of blaOXA-23-like-carrying isolates (MICs, 16 ?g/ml to 64 ?g/ml for both imipenem and meropenem). All 231 blaOXA carbapenemase gene-harboring isolates belonged to 14 PFGE types (A-N), and three dominant clones A, J, and H accounted for 43.3%, 42.0%, and 8.2% of the tested isolates, respectively. Clone A (sequence type ST92/ST208) with blaOXA-58-like was the most prevalent before 2008. Clone H (ST229) with blaOXA-23-like became striking between 2007 and 2008. Clone J (ST381) with blaOXA-23-like rapidly spread and replaced clones A and H in 2009.This study is the first to reveal that the distinct blaOXA-23-like-carrying A. baumannii ST381 displaced the previously prevalent blaOXA-58-like-carrying A. baumannii ST92/ST208, resulting in the rapidly increasing resistance to carbapenems in A. baumannii in Shenzhen People's Hospital in 2009.

Project description:The acquisition of blaOXA genes encoding different carbapenem-hydrolyzing class-D β-lactamases (CHDL) represents a main determinant of carbapenem resistance in the nosocomial pathogen Acinetobacter baumannii. The blaOXA-58 gene, in particular, is generally embedded in similar resistance modules (RM) carried by plasmids unique to the Acinetobacter genus lacking self-transferability. The ample variations in the immediate genomic contexts in which blaOXA-58-containing RMs are inserted among these plasmids, and the almost invariable presence at their borders of non-identical 28-bp sequences potentially recognized by the host XerC and XerD tyrosine recombinases (pXerC/D-like sites), suggested an involvement of these sites in the lateral mobilization of the gene structures they encircle. However, whether and how these pXerC/D sites participate in this process is only beginning to be understood. Here, we used a series of experimental approaches to analyze the contribution of pXerC/D-mediated site-specific recombination to the generation of structural diversity between resistance plasmids carrying pXerC/D-bounded blaOXA-58- and TnaphA6-containing RM harbored by two phylogenetically- and epidemiologically-closely related A. baumannii strains of our collection, Ab242 and Ab825, during adaptation to the hospital environment. Our analysis disclosed the existence of different bona fide pairs of recombinationally-active pXerC/D sites in these plasmids, some mediating reversible intramolecular inversions and others reversible plasmid fusions/resolutions. All of the identified recombinationally-active pairs shared identical GGTGTA sequences at the cr spacer separating the XerC- and XerD-binding regions. The fusion of two Ab825 plasmids mediated by a pair of recombinationally-active pXerC/D sites displaying sequence differences at the cr spacer could be inferred on the basis of sequence comparison analysis, but no evidence of reversibility could be obtained in this case. The reversible plasmid genome rearrangements mediated by recombinationally-active pairs of pXerC/D sites reported here probably represents an ancient mechanism of generating structural diversity in the Acinetobacter plasmid pool. This recursive process could facilitate a rapid adaptation of an eventual bacterial host to changing environments, and has certainly contributed to the evolution of Acinetobacter plasmids and the capture and dissemination of blaOXA-58 genes among Acinetobacter and non-Acinetobacter populations co-residing in the hospital niche.

Dataset Information

Multicopy blaOXA-58 gene as a source of high-level resistance to carbapenems in Acinetobacter baumannii.

Publications

Multicopy blaOXA-58 gene as a source of high-level resistance to carbapenems in Acinetobacter baumannii.

Similar Datasets

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