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      Molecular Epidemiology and Mechanism of Sulbactam Resistance in Acinetobacter baumannii Isolates with Diverse Genetic Backgrounds in China

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          ABSTRACT

          Sulbactam is a plausible option for treating Acinetobacter infections because of its intrinsic antibacterial activity against the members of the Acinetobacter genus, but the mechanisms of sulbactam resistance have not been fully studied in Acinetobacter baumannii . In this study, a total of 2,197 clinical A. baumannii isolates were collected from 27 provinces in China. Eighty-eight isolates with various MICs for sulbactam were selected on the basis of their diverse clonality and underwent multilocus sequence typing (MLST), antimicrobial susceptibility testing, and resistance gene screening. The copy number and relative expression of bla TEM-1D and ampC were measured via quantitative PCR and quantitative reverse transcription-PCR, respectively. The genetic structure of multicopy bla TEM-1D was determined using the whole-genome sequencing technology. The cefoperazone-sulbactam resistance rate of the 2,197 isolates was 39.7%. The rate of positivity for bla TEM-1D or IS Aba1-ampC in the sulbactam-nonsusceptible group (64.91% and 78.95%, respectively) was significantly higher than that in the sulbactam-susceptible group (0% and 0%, respectively; P < 0.001). The MIC of sulbactam ( P < 0.001) varied considerably between the groups expressing ampC with or without upstream IS Aba1 . Notably, the genetic structure of the multicopy bla TEM-1D gene in strain ZS3 revealed that bla TEM-1D was embedded within four tandem copies of the cassette IS 26-bla TEM-1D -Tn 3 -IS 26 . Therefore, bla TEM-1D and IS Aba1-ampC represent the prevalent mechanism underlying sulbactam resistance in clinical A. baumannii isolates in China. The structure of the four tandem copies of bla TEM-1D first identified may increase sulbactam resistance.

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          Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli.

          Real-time QPCR based methods for determination of plasmid copy number in recombinant Escherichia coli cultures are presented. Two compatible methods based on absolute and relative analyses were tested with recombinant E. coli DH5alpha harboring pBR322, which is a common bacterial cloning vector. The separate detection of the plasmid and the host chromosomal DNA was achieved using two separate primer sets, specific for the plasmid beta-lactamase gene (bla) and for the chromosomal d-1-deoxyxylulose 5-phosphate synthase gene (dxs), respectively. Since both bla and dxs are single-copy genes of pBR322 and E. coli chromosomal DNA, respectively, the plasmid copy number can be determined as the copy ratio of bla to dxs. These methods were successfully applied to determine the plasmid copy number of pBR322 of E. coli host cells. The results of the absolute and relative analyses were identical and highly reproducible with coefficient of variation (CV) values of 2.8-3.9% and 4.7-5.4%, respectively. The results corresponded to the previously reported values of pBR322 copy number within E. coli host cells, 15-20. The methods introduced in this study are convenient to perform and cost-effective compared to the traditionally used Southern blot method. The primer sets designed in this study can be used to determine plasmid copy number of any recombinant E. coli with a plasmid vector having bla gene.
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            InterPro and InterProScan: tools for protein sequence classification and comparison.

            Protein sequence classification and comparison has become increasingly important in the current "omics" revolution, where scientists are working on functional genomics and proteomics technologies for large-scale protein function prediction. However, functional classification is also important for the bench scientist wanting to analyze single or small sets of proteins, or even a single genome. A number of tools are available for sequence classification, such as sequence similarity searches, motif- or pattern-finding software, and protein signatures for identifying protein families and domains. One such tool, InterPro, is a documentation resource that integrates the major players in the protein signature field to provide a valuable tool for annotation of proteins. Protein sequences are searched using the InterProScan software to identify signatures from the InterPro member databases; Pfam, PROSITE, PRINTS, ProDom, SMART, TIGRFAMs, PIRSF, SUPERFAMILY, Gene3D, and PANTHER. The InterPro database can be searched to retrieve precalculated matches for UniProtKB proteins, or to find additional information on protein families and domains. For completely sequenced genomes, the user can retrieve InterPro-based analyses on all nonredundant proteins in the proteome, and can execute user-selected proteome comparisons. This chapter will describe how to use InterPro and InterProScan for protein sequence classification and comparative proteomics.
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              Resistance trends among clinical isolates in China reported from CHINET surveillance of bacterial resistance, 2005-2014.

              With the aim of gathering temporal trends on bacterial epidemiology and resistance from multiple laboratories in China, the CHINET surveillance system was organized in 2005. Antimicrobial susceptibility testing was carried out according to a unified protocol using the Kirby-Bauer method or automated systems. Results were analyzed according to Clinical and Laboratory Standards Institute (CLSI) 2014 definitions. Between 2005 and 2014, the number of bacterial isolates ranged between 22,774 and 84,572 annually. Rates of extended-spectrum β-lactamase production among Escherichia coli isolates were stable, between 51.7 and 55.8%. Resistance of E. coli and Klebsiella pneumoniae to amikacin, ciprofloxacin, piperacillin/tazobactam and cefoperazone/sulbactam decreased with time. Carbapenem resistance among K. pneumoniae isolates increased from 2.4 to 13.4%. Resistance of Pseudomonas aeruginosa strains against all of antimicrobial agents tested including imipenem and meropenem decreased with time. On the contrary, resistance of Acinetobacter baumannii strains to carbapenems increased from 31 to 66.7%. A marked decrease of methicillin resistance from 69% in 2005 to 44.6% in 2014 was observed for Staphylococcus aureus. Carbapenem resistance rates in K. pneumoniae and A. baumannii in China are high. Our results indicate the importance of bacterial surveillance studies.
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                Author and article information

                Journal
                Antimicrobial Agents and Chemotherapy
                Antimicrob Agents Chemother
                American Society for Microbiology
                0066-4804
                1098-6596
                March 2018
                February 23 2018
                January 08 2018
                : 62
                : 3
                Article
                10.1128/AAC.01947-17
                5826149
                29311074
                f6f83c44-be2d-45fd-8c86-0613c519996a
                © 2018
                History

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