Dataset Information

Contribution of a plasmid-borne blaOXA-58 gene with its hybrid promoter provided by IS1006 and an ISAba3-like element to beta-lactam resistance in acinetobacter genomic species 13TU.

ABSTRACT: The contribution of the blaOXA-58 gene and its promoter to beta-lactam resistance has not been validated in Acinetobacter spp. other than Acinetobacter baumannii. We identified a multidrug-resistant (including carbapenem resistance) Acinetobacter genomic species 13TU in which blaOXA-58 was the only detected carbapenemase gene. The blaOXA-58 gene was plasmid located, flanked by ISAba3 (downstream) and an ISAba3-like element (upstream). An IS1006 element was inserted into ISAba3-like (IS1006-DeltaISAba3-like) to generate a hybrid promoter for blaOXA-58, with a -35 promoter located in IS1006 and a -10 promoter in ISAba3-like. The reference strain of Acinetobacter genomic species 13TU, ATCC 17903, revealed higher MICs of amoxicillin, ticarcillin, and piperacillin and heteroresistance to imipenem and meropenem when it was transformed with a shuttle vector containing a fragment encompassing DeltaISAba3-like-blaOXA-58, compared to the same host containing only blaOXA-58. When the fragment was changed from DeltaISAba3-like-blaOXA-58 to IS1006-DeltaISAba3-like-blaOXA-58, the ATCC 17903 transformant revealed a markedly higher level of blaOXA-58 transcription (12-fold), increased cefuroxime and piperacillin-tazobactam MICs, and homoresistance to imipenem and meropenem. Different roles of the insertion elements preceding the blaOXA-58 gene in Acinetobacter genomic species 13TU are demonstrated. The ISAba3-like--blaOXA-58 construct can mediate resistance to penicillin derivatives but only heteroresistance to carbapenems. The insertion of IS1006 into ISAba3-like, generating a hybrid promoter, could further enhance the transcription of blaOXA-58 and mediate homoresistance to carbapenems and also enhanced resistance to piperacillin-tazobactam.

SUBMITTER: Chen TL

PROVIDER: S-EPMC2916316 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Contribution of a plasmid-borne blaOXA-58 gene with its hybrid promoter provided by IS1006 and an ISAba3-like element to beta-lactam resistance in acinetobacter genomic species 13TU.

Chen Te-Li TL Chang Wei-Che WC Kuo Shu-Chen SC Lee Yi-Tzu YT Chen Chien-Pei CP Siu Leung-Kei LK Cho Wen-Long WL Fung Chang-Phone CP

Antimicrobial agents and chemotherapy 20100601 8

The contribution of the blaOXA-58 gene and its promoter to beta-lactam resistance has not been validated in Acinetobacter spp. other than Acinetobacter baumannii. We identified a multidrug-resistant (including carbapenem resistance) Acinetobacter genomic species 13TU in which blaOXA-58 was the only detected carbapenemase gene. The blaOXA-58 gene was plasmid located, flanked by ISAba3 (downstream) and an ISAba3-like element (upstream). An IS1006 element was inserted into ISAba3-like (IS1006-Delta ...[more]

PMID: 20516281

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Project description:We characterize by whole-plasmid-sequence (WPS) two-plasmid-borne blaOXA-58 obtained from Acinetobacter seifertii (Asp-1069) and A. baumannii (Acb-45063) clinical strains recovered 17 years apart from distinct Brazilian regions. Multilocus sequence type (MLST) analysis showed that the Asp-1069 and Acb-45063 strains belong to ST551 and ST15/CC15, respectively. WPS analysis demonstrated that blaOXA-58 was located in two distinct plasmids named pAs1069_a (24,672 bp/44 open reading frames [ORFs]) and pAb45063_b (19,808 bp/24 ORFs), which belong to the GR8/GR23 (repAci23) and GR4 (repAci4) incompatibility groups, respectively. The genetic environments surrounding blaOXA-58 revealed that it was flanked by two intact ISAba3 copies on pAb45063_b, which differed from pAs1069_a. In the latter, the upstream ISAba3 copy was truncated by insertion of ISAba825 element. Although Re27-specific recombination sites were found adjacent to ISAba3-blaOXA-58-ISAba3 arrangement on pAb45063_b, such structures were absent on pAs1069_a. The conserved ISAba125-araC1-lysE arrangement was disrupted by TnaphA6 harboring the aminoglycosides resistance gene aphA6 on pAs1069_a, while an IS26-blaTEM-1-aac(3)-IIa-IS26 genetic structure was found upstream from ISAba3-blaOXA-58-ISAba3 on pAb45063_b. Other two plasmids, pAb45063_a (183,767 bp/209 ORFs) and pAs1069_b (13,129 bp/14 ORFs), were also found in the OXA-58-producing Acinetobacter species strains, harboring the strA and strB genes and the sul2 gene, which confer resistance to streptomycin and sulfonamides, respectively. The plasmid-mediated virulence factors corresponding to genes tonB, spl, glmM, ppa, sulP, and map were found in both strains, as well distinct toxin-antitoxin system-encoding genes stbD and relE (pAs1069_a), brnT and brnA (pAb45063_b), and xreE (pAb45063_a). Although infrequently reported in Brazil, plasmid-borne blaOXA-58 showed a complex and diverse genetic backbone that confers stability in different Acinetobacter species that have been isolated from nosocomial settings over time.IMPORTANCE Although the blaOXA-58 gene has been infrequently described in Brazil, contrasting with other bordering South American countries, we verified the maintenance of this resistance determinant over time among carbapenem-resistant Acinetobacter species isolates, not only in nosocomial settings but also in the environment. In addition, to the best of our knowledge, this is the first study to have used WPS analysis to evaluate the genetic surroundings of blaOXA-58 in Brazil. Moreover, the A. seifertii and A. baumannii clinical strains evaluated in this study were recovered 17 years apart in hospitals located in distinct Brazilian geographic regions.

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:BackgroundThe 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.MethodsCarbapenemase 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.ResultsA 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-ΔISAba3, 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 μg/ml to 16 μg/ml), compared with the majority of blaOXA-23-like-carrying isolates (MICs, 16 μg/ml to 64 μg/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.ConclusionThis 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.

Dataset Information

Contribution of a plasmid-borne blaOXA-58 gene with its hybrid promoter provided by IS1006 and an ISAba3-like element to beta-lactam resistance in acinetobacter genomic species 13TU.

Publications

Contribution of a plasmid-borne blaOXA-58 gene with its hybrid promoter provided by IS1006 and an ISAba3-like element to beta-lactam resistance in acinetobacter genomic species 13TU.

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