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      Spread of TEM, VIM, SHV, and CTX-M β-Lactamases in Imipenem-Resistant Gram-Negative Bacilli Isolated from Egyptian Hospitals

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          Abstract

          Carbapenem-resistant Gram-negative bacilli resulting from β-lactamases have been reported to be an important cause of nosocomial infections and are a critical therapeutic problem worldwide. This study aimed to describe the prevalence of imipenem-resistant Gram-negative bacilli isolates and detection of bla VIM, bla TEM, bla SHV, bla CTX-M-1, and bla CTX-M-9 genes in these clinical isolates in Egyptian hospitals. The isolates were collected from various clinical samples, identified by conventional methods and confirmed by API 20E. Antibiotic susceptibility testing was determined by Kirby-Bauer technique and interpreted according to CLSI. Production of bla VIM, bla TEM, bla SHV, and bla CTX-M genes was done by polymerase chain reaction (PCR). Direct sequencing from PCR products was subsequently carried out to identify and confirm these β-lactamases genes. Out of 65 isolates, (46.1%) Escherichia coli, (26.2%) Klebsiella pneumoniae, and (10.7%) Pseudomonas aeruginosa were identified as the commonest Gram-negative bacilli. 33(50.8%) were imipenem-resistant isolates. 22 isolates (66.7%) carried bla VIM, 24(72.7%) had bla TEM, and 5(15%) showed bla SHV, while 12(36%), 6(18.2%), and 0(0.00%) harbored bla CTX-M-1, bla CTX-M-9, and bla CTX-M-8/25, respectively. There is a high occurrence of β-lactamase genes in clinical isolates and sequence analysis of amplified genes showed differences between multiple SNPs (single nucleotide polymorphism) sites in the same gene among local isolates in relation to published sequences.

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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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              Carbapenems: past, present, and future.

              In this review, we summarize the current "state of the art" of carbapenem antibiotics and their role in our antimicrobial armamentarium. Among the β-lactams currently available, carbapenems are unique because they are relatively resistant to hydrolysis by most β-lactamases, in some cases act as "slow substrates" or inhibitors of β-lactamases, and still target penicillin binding proteins. This "value-added feature" of inhibiting β-lactamases serves as a major rationale for expansion of this class of β-lactams. We describe the initial discovery and development of the carbapenem family of β-lactams. Of the early carbapenems evaluated, thienamycin demonstrated the greatest antimicrobial activity and became the parent compound for all subsequent carbapenems. To date, more than 80 compounds with mostly improved antimicrobial properties, compared to those of thienamycin, are described in the literature. We also highlight important features of the carbapenems that are presently in clinical use: imipenem-cilastatin, meropenem, ertapenem, doripenem, panipenem-betamipron, and biapenem. In closing, we emphasize some major challenges and urge the medicinal chemist to continue development of these versatile and potent compounds, as they have served us well for more than 3 decades.
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                Author and article information

                Journal
                Int J Microbiol
                Int J Microbiol
                IJMICRO
                International Journal of Microbiology
                Hindawi Publishing Corporation
                1687-918X
                1687-9198
                2016
                31 March 2016
                : 2016
                : 8382605
                Affiliations
                1Department of Botany, Faculty of Science, Zagazig University, Zagazig 44511, Egypt
                2Department of Medical Microbiology & Immunology, Faculty of Medicine, Zagazig University, Zagazig 44511, Egypt
                Author notes
                *El sayed Hamdy Mohammed: anas.hamdy25@ 123456yahoo.com

                Academic Editor: Giuseppe Comi

                Article
                10.1155/2016/8382605
                4830709
                27123005
                80f61cbc-56fc-4731-8c8f-beb1be4ea096
                Copyright © 2016 El sayed Hamdy Mohammed et al.

                This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 26 August 2015
                : 9 December 2015
                : 14 December 2015
                Categories
                Research Article

                Microbiology & Virology
                Microbiology & Virology

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