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      Detection of Plasmid-Mediated β-Lactamase Genes and Emergence of a Novel AmpC (CMH-1) in Enterobacter cloacae at a Medical Center in Southern Taiwan

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          Abstract

          The plasmid-mediated extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases in Enterobacter spp. have increasingly been reported. In this study, we investigated the prevalence of the plasmid-mediated β-lactamases in Enterobacter cloacae from bloodstream isolates at a medical center in southern Taiwan. ESBL and ampC genes were detected by PCRs and DNA sequencing. Conjugation experiments were conducted to confirm the transferability of the genetic resistance trait. Among 41 non-repetitive blood isolates of cefuroxime-resistant E. cloacae, eight isolates exhibited ESBL phenotype confirmed by double-disk synergistic tests. Nearly all the strains were susceptible to carbapenems. The prevalence rate of the plasmid-mediated bla ampC genes was 73% (30/41), including one bla DHA-1, one bla MIR-6, two novel bla CMH-1 genes and other bla ACT-like genes. Coexistence of plasmid-mediated bla ACT and ESBL genes (10 with bla SHV-12 and one with bla CTX-M-3) was observed. Successful transmissions of the bla ACT and bla CMH-1 were demonstrated in some transconjugants. The inducible or derepressed CMH-1 had expanded activity of isolates versus ceftazidime. Enterobacterial repetitive intergenic consensus (ERIC)-PCR analysis and pulsotype showed distinct patterns suggesting non-clonal relationship. In conclusion, plasmid-mediated bla ACT-like ampC genes in E. cloacae isolates have been highly prevalent in southern Taiwan and may continue genetic evolution, contributing to the complexities in antibiotic-resistant mechanisms.

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          Most cited references45

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          Extended-Spectrum β-Lactamases: a Clinical Update

          Extended-spectrum β-lactamases (ESBLs) are a rapidly evolving group of β-lactamases which share the ability to hydrolyze third-generation cephalosporins and aztreonam yet are inhibited by clavulanic acid. Typically, they derive from genes for TEM-1, TEM-2, or SHV-1 by mutations that alter the amino acid configuration around the active site of these β-lactamases. This extends the spectrum of β-lactam antibiotics susceptible to hydrolysis by these enzymes. An increasing number of ESBLs not of TEM or SHV lineage have recently been described. The presence of ESBLs carries tremendous clinical significance. The ESBLs are frequently plasmid encoded. Plasmids responsible for ESBL production frequently carry genes encoding resistance to other drug classes (for example, aminoglycosides). Therefore, antibiotic options in the treatment of ESBL-producing organisms are extremely limited. Carbapenems are the treatment of choice for serious infections due to ESBL-producing organisms, yet carbapenem-resistant isolates have recently been reported. ESBL-producing organisms may appear susceptible to some extended-spectrum cephalosporins. However, treatment with such antibiotics has been associated with high failure rates. There is substantial debate as to the optimal method to prevent this occurrence. It has been proposed that cephalosporin breakpoints for the Enterobacteriaceae should be altered so that the need for ESBL detection would be obviated. At present, however, organizations such as the Clinical and Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards) provide guidelines for the detection of ESBLs in klebsiellae and Escherichia coli . In common to all ESBL detection methods is the general principle that the activity of extended-spectrum cephalosporins against ESBL-producing organisms will be enhanced by the presence of clavulanic acid. ESBLs represent an impressive example of the ability of gram-negative bacteria to develop new antibiotic resistance mechanisms in the face of the introduction of new antimicrobial agents.
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            Carbapenemases: the versatile beta-lactamases.

            Carbapenemases are beta-lactamases with versatile hydrolytic capacities. They have the ability to hydrolyze penicillins, cephalosporins, monobactams, and carbapenems. Bacteria producing these beta-lactamases may cause serious infections in which the carbapenemase activity renders many beta-lactams ineffective. Carbapenemases are members of the molecular class A, B, and D beta-lactamases. Class A and D enzymes have a serine-based hydrolytic mechanism, while class B enzymes are metallo-beta-lactamases that contain zinc in the active site. The class A carbapenemase group includes members of the SME, IMI, NMC, GES, and KPC families. Of these, the KPC carbapenemases are the most prevalent, found mostly on plasmids in Klebsiella pneumoniae. The class D carbapenemases consist of OXA-type beta-lactamases frequently detected in Acinetobacter baumannii. The metallo-beta-lactamases belong to the IMP, VIM, SPM, GIM, and SIM families and have been detected primarily in Pseudomonas aeruginosa; however, there are increasing numbers of reports worldwide of this group of beta-lactamases in the Enterobacteriaceae. This review updates the characteristics, epidemiology, and detection of the carbapenemases found in pathogenic bacteria.
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              Development of a set of multiplex PCR assays for the detection of genes encoding important beta-lactamases in Enterobacteriaceae.

              To develop a rapid and reliable tool to detect by multiplex PCR assays the most frequently widespread beta-lactamase genes encoding the OXA-1-like broad-spectrum beta-lactamases, extended-spectrum beta-lactamases (ESBLs), plasmid-mediated AmpC beta-lactamases and class A, B and D carbapenemases. Following the design of a specific group of primers and optimization using control strains, a set of six multiplex PCRs and one simplex PCR was created. An evaluation of the set was performed using a collection of 31 Enterobacteriaceae strains isolated from clinical specimens showing a resistance phenotype towards broad-spectrum cephalosporins and/or cephamycins and/or carbapenems. Direct sequencing from PCR products was subsequently carried out to identify beta-lactamase genes. Under optimized conditions, all positive controls confirmed the specificity of group-specific PCR primers. Except for the detection of carbapenemase genes, multiplex and simplex PCR assays were carried out using the same PCR conditions, allowing assays to be performed in a single run. Out of 31 isolates selected, 22 strains produced an ESBL, mostly CTX-M-15 but also CTX-M-1 and CTX-M-9, SHV-12, SHV-5, SHV-2, TEM-21, TEM-52 and a VEB-type ESBL, 6 strains produced a plasmid-mediated AmpC beta-lactamase (five DHA-1 and one CMY-2) and 3 strains produced both an ESBL (two SHV-12, one CTX-M-15) and a plasmid-mediated AmpC beta-lactamase (DHA-1). We report here the development of a useful method composed of a set of six multiplex PCRs and one simplex PCR for the rapid screening of the most frequently encountered beta-lactamases. This method allowed direct sequencing from the PCR products.
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                Author and article information

                Journal
                J Clin Med
                J Clin Med
                jcm
                Journal of Clinical Medicine
                MDPI
                2077-0383
                20 December 2018
                January 2019
                : 8
                : 1
                : 8
                Affiliations
                [1 ]Division of Infectious Disease, Department of Internal Medicine, Chi Mei Medical Center-Liouying, Tainan 710, Taiwan; althrisas@ 123456gmail.com
                [2 ]Laboratory of Environmental Health, Research Center of Humanities and Technology, National University of Kaohsiung, Kaohsiung 811, Taiwan; mfphd@ 123456yahoo.com.tw
                [3 ]Department of Medical Research, Chi Mei Medical Center, Tainan 710, Taiwan; chuangkenneth@ 123456hotmail.com
                [4 ]Department of Intensive Care Medicine, Chi Mei Medical Center, Tainan 710, Taiwan
                [5 ]Department of Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei 110, Taiwan
                Author notes
                [* ]Correspondence: yu2231@ 123456gmail.com ; Tel.: +886-6-281-2811 (ext. 52605); Fax: +886-6-251-7849
                Author information
                https://orcid.org/0000-0002-5101-2760
                Article
                jcm-08-00008
                10.3390/jcm8010008
                6352282
                30577544
                ec48e3d2-7934-41d9-87ee-fceff07ae6b3
                © 2018 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 20 November 2018
                : 18 December 2018
                Categories
                Article

                act,ampc gene,cmh-1,enterobacter cloacae,plasmid
                act, ampc gene, cmh-1, enterobacter cloacae, plasmid

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