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      Overproduction of active efflux pump and variations of OprD dominate in imipenem-resistant Pseudomonas aeruginosa isolated from patients with bloodstream infections in Taiwan

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          Abstract

          Background

          The emergence of imipenem-resistant Pseudomonas aeruginosa (IRPA) has become a great concern worldwide. The aim of this study was to investigate resistance mechanisms associated with bloodstream isolated IRPA strains in Taiwan.

          Results

          A total of 78 non-duplicated IRPA isolates were isolated from patients with bloodstream infection. The average prevalence of imipenem-resistance in those isolates was 5.9 % during a 10-year longitudinal surveillance in Taiwan. PFGE results showed high clonal diversity among the 78 isolates. VIM-2, VIM-3, OXA-10, and OXA-17 β-lactamases were identified in 2 (2.6 %), 3 (3.8 %), 2 (2.6 %), and 1 (1.3 %) isolates, respectively. Active efflux pumps, AmpC β-lactamase overproduction, and extended-spectrum AmpC cephalosporinases (ESACs) were found in 58 (74.4 %), 25 (32.1 %) and 15 (19.2 %) of IRPA isolates, respectively. oprD mutations with amino acid substitution, shortened putative loop L7, premature stop codon caused by point mutation, frameshift by nucleotide insertion or deletion, and interruption by insertion sequence were found in 19 (24.4 %), 18 (23.1 %), 15 (19.2 %), 14 (17.9 %), and 10 (12.8 %) of isolates, respectively.

          Conclusions

          This study suggests that alterations in the OprD protein and having an active efflux pump are the main mechanisms associated with bloodstream isolated IRPA. Overproduction of AmpC, ESACs, and the presence of VIM- and OXA-type β-lactamases play additional roles in reduced susceptibility to imipenem in P. aeruginosa isolates in Taiwan.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s12866-016-0719-2) contains supplementary material, which is available to authorized users.

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          Most cited references26

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          ISfinder: the reference centre for bacterial insertion sequences

          ISfinder () is a dedicated database for bacterial insertion sequences (ISs). It has superseded the Stanford reference center. One of its functions is to assign IS names and to provide a focal point for a coherent nomenclature. It is also the repository for ISs. Each new IS is indexed together with information such as its DNA sequence and open reading frames or potential coding sequences, the sequence of the ends of the element and target sites, its origin and distribution together with a bibliography where available. Another objective is to continuously monitor ISs to provide updated comprehensive groupings or families and to provide some insight into their phylogenies. The site also contains extensive background information on ISs and transposons in general. Online tools are gradually being added. At present an online Blast facility against the entire bank is available. But additional features will include alignment capability, PsiBLAST and HMM profiles. ISfinder also includes a section on bacterial genomes and is involved in annotating the IS content of these genomes. Finally, this database is currently recommended by several microbiology journals for registration of new IS elements before their publication.
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            Emergence of oxacillinase-mediated resistance to imipenem in Klebsiella pneumoniae.

            Klebsiella pneumoniae strain 11978 was isolated in Turkey in 2001 and was found to be resistant to all beta-lactams, including carbapenems. Cloning and expression in Escherichia coli identified five beta-lactamases, including two novel oxacillinases. The beta-lactamase OXA-48 hydrolyzed imipenem at a high level and was remotely related (less than 46% amino acid identity) to the other oxacillinases. It hydrolyzed penicillins and imipenem but not expanded-spectrum cephalosporins. The bla(OXA-48) gene was plasmid encoded and not associated with an integron, in contrast to most of the oxacillinase genes. An insertion sequence, IS1999, was found immediately upstream of bla(OXA-48). Another plasmid that encoded a second oxacillinase gene, bla(OXA-47), located inside a class 1 integron was identified in K. pneumoniae 11978. OXA-47 had a narrow spectrum of hydrolysis activity and did not hydrolyze ceftazidime or imipenem, as is found for the beta-lactamase (OXA-1) to which it is related. In addition, beta-lactamases TEM-1 and SHV-2a were expressed from the same K. pneumoniae isolate. Analysis of the outer membrane proteins of this isolate revealed that it lacked a porin of ca. 36 kDa. Thus, the high-level resistance to beta-lactams of this clinical isolate resulted from peculiar beta-lactamases and modification of outer membrane proteins.
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              Interplay of efflux system, ampC, and oprD expression in carbapenem resistance of Pseudomonas aeruginosa clinical isolates.

              Carbapenems are important agents for the therapy of infections due to multidrug-resistant Pseudomonas aeruginosa; the development of carbapenem resistance hampers effective therapeutic options. To assess the mechanisms leading to resistance, 33 clinical isolates with differing degrees of carbapenem susceptibility were analyzed for the expression of the chromosomal beta-lactamase (ampC), the porin that is important for the entry of carbapenems (oprD), and the proteins involved in four efflux systems (mexA, mexC, mexE, and mexX). Real-time reverse transcriptase PCR was performed using primers and fluorescent probes for each of the target genes. The sequencing of regulatory genes (ampR, mexR, nalC, nalD, mexT, and mexZ) was also performed. Diminished expression of oprD was present in all imipenem- and meropenem-resistant isolates but was not required for ertapenem resistance. Increased expression of ampC was not observed in several isolates that were overtly resistant to carbapenems. Increased expression of several efflux systems was observed in many of the carbapenem-resistant isolates. Increased efflux activity correlated with high-level ertapenem resistance and reduced susceptibility to meropenem and aztreonam. Most isolates with increased expression of mexA had mutations affecting nalC and/or nalD. Two isolates with mutations leading to a premature stop codon in mexZ had markedly elevated mexX expressions, although mutations in mexZ were not a prerequisite for overexpression. beta-Lactam resistance in clinical isolates of P. aeruginosa is a result of the interplay between diminished production of oprD, increased activity of ampC, and several efflux systems.
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                Author and article information

                Contributors
                886-6-2353535 , 886-6-2363956 , jjwu@mail.ncku.edu.tw
                Journal
                BMC Microbiol
                BMC Microbiol
                BMC Microbiology
                BioMed Central (London )
                1471-2180
                13 June 2016
                13 June 2016
                2016
                : 16
                : 107
                Affiliations
                [ ]Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan
                [ ]Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan
                [ ]Department of Internal Medicine, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan
                [ ]Department of Pathology, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan
                [ ]Department of Biotechnology and Laboratory Science in Medicine, School of Biomedical Science and Engineering, National Yang-Ming University, Taipei, Taiwan
                Article
                719
                10.1186/s12866-016-0719-2
                4906909
                27296461
                674ae26a-15cb-45e9-9c20-f9fd1f8ea21c
                © The Author(s). 2016

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 13 August 2015
                : 30 May 2016
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100001868, National Science Council Taiwan (TW);
                Award ID: NSC99-3112-B-006-015
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100001868, National Science Council Taiwan (TW);
                Award ID: NSC101-2320-B-006-020-MY3
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100001868, National Science Council Taiwan (TW);
                Award ID: NSC101-2320-B-006-029-MY3
                Award Recipient :
                Categories
                Research Article
                Custom metadata
                © The Author(s) 2016

                Microbiology & Virology
                imipenem,pseudomonas aeruginosa,β-lactamase,efflux pump,oprd
                Microbiology & Virology
                imipenem, pseudomonas aeruginosa, β-lactamase, efflux pump, oprd

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