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      Multidrug-resistant Citrobacter freundii ST139 co-producing NDM-1 and CMY-152 from China

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          Abstract

          The emergence of carbapenemase-producing Citrobacter freundii poses a significant threat to public health worldwide. Here, we reported a C. freundii strain CWH001 which was resistant to all tested antimicrobials except tetracycline. Whole genome sequencing and analysis were performed. The strain, which belonged to a new sequence type ST139, showed close relationship with other foreign C. freundii strains through phylogenetic analysis. A novel variant of the intrinsic bla CMY gene located on the chromosome was identified and designated as bla CMY-152. Coexistence of bla NDM-1 with qnrS1 was found on a conjugative IncN plasmid, which had a backbone appearing in various plasmids. Other class A ESBL genes ( bla VEB-3 and bla TEM-1) were also detected on two different novel plasmids. The emergence of multidrug-resistant C. freundii is of major concern, causing great challenges to the treatment of clinical infections. Great efforts need to be taken for the specific surveillance of this opportunistic pathogen.

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          Most cited references33

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          Toward almost closed genomes with GapFiller

          De novo assembly is a commonly used application of next-generation sequencing experiments. The ultimate goal is to puzzle millions of reads into one complete genome, although draft assemblies usually result in a number of gapped scaffold sequences. In this paper we propose an automated strategy, called GapFiller, to reliably close gaps within scaffolds using paired reads. The method shows good results on both bacterial and eukaryotic datasets, allowing only few errors. As a consequence, the amount of additional wetlab work needed to close a genome is drastically reduced. The software is available at http://www.baseclear.com/bioinformatics-tools/.
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            Rapid detection and identification of metallo-beta-lactamase-encoding genes by multiplex real-time PCR assay and melt curve analysis.

            Metallo-beta-lactamase enzymes (MbetaL) are encoded by transferable genes, which appear to spread rapidly among gram-negative bacteria. The objective of this study was to develop a multiplex real-time PCR assay followed by a melt curve step for rapid detection and identification of genes encoding MbetaL-type enzymes based on the amplicon melting peak. The reference sequences of all genes encoding IMP and VIM types, SPM-1, GIM-1, and SIM-1 were downloaded from GenBank, and primers were designed to obtain amplicons showing different sizes and melting peak temperatures (Tm). The real-time PCR assay was able to detect all MbetaL-harboring clinical isolates, and the Tm-assigned genotypes were 100% coincident with previous sequencing results. This assay could be suitable for identification of MbetaL-producing gram-negative bacteria by molecular diagnostic laboratories.
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              Complete sequencing of an IncHI1 plasmid encoding the carbapenemase NDM-1, the ArmA 16S RNA methylase and a resistance-nodulation-cell division/multidrug efflux pump.

              To characterize the pNDM-CIT plasmid identified in Citrobacter freundii carrying genes encoding the metallo-β-lactamase NDM-1 and the 16S RNA methylase ArmA. The complete DNA sequence of pNDM-CIT was obtained by using the 454-Genome Sequencer FLX procedure on a library obtained using plasmid DNA purified from the pNDM-CIT Escherichia coli J53 transconjugant. Contig assembly and predicted gaps were confirmed and filled by PCR-based gap closure. Comparative analysis with IncHI1 incompatibility group plasmids was performed using BLASTN and BLASTP algorithms. Plasmid pNDM-CIT was 288::920 bp and revealed an IncHI1 plasmid scaffold, showing novel resistance and potential virulence determinants. The bla(NDM-1) gene was identified within a novel genetic context, flanked by a duplication of the class 1 integron on both sides. The replicase gene repAciN, originating from Acinetobacter spp. plasmids, was identified in a close association with the Tn1548::armA transposon and the macrolide resistance mel-mph2 cluster. The same structure was identified in silico from a series of enterobacterial plasmids carrying the armA gene. The repAciN gene probably represents a remnant sign of the original occurrence of the armA gene in Acinetobacter plasmids. A CP4-like prophage sequence was identified in pNDM-CIT, containing a resistance-nodulation-cell division/multidrug resistance (RND/MDR) efflux pump cluster surrounded by two IS1-like elements. This resistance determinant, associated with such a prophage sequence, has never been reported on plasmids. Plasmid pNDM-CIT differed significantly from all known bla(NDM-1)-carrying plasmids identified in Enterobacteriaceae, since it combines the metallo-β-lactamase NDM-1, the 16S RNA methylase ArmA and a cryptic prophage carrying the RND/MDR efflux pump.
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                Author and article information

                Contributors
                jiekenlee@126.com
                qiushf0613@hotmail.com
                hongbinsong@263.net
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                13 July 2018
                13 July 2018
                2018
                : 8
                : 10653
                Affiliations
                [1 ]ISNI 0000 0004 1803 4911, GRID grid.410740.6, Academy of Military Medical Sciences, ; Beijing, China
                [2 ]ISNI 0000 0001 2267 2324, GRID grid.488137.1, Institute for Disease Control and Prevention of PLA, ; Beijing, China
                Article
                28879
                10.1038/s41598-018-28879-9
                6045649
                30006537
                3f0dc86d-b9e9-4441-a34c-861d7553510f
                © The Author(s) 2018

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as 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 images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 17 January 2018
                : 2 July 2018
                Funding
                Funded by: FundRef https://doi.org/10.13039/501100005090, Beijing Nova Program;
                Award ID: xx2018042
                Award Recipient :
                Funded by: FundRef https://doi.org/10.13039/501100004826, Natural Science Foundation of Beijing Municipality (Beijing Natural Science Foundation);
                Award ID: 5172029
                Award Recipient :
                Funded by: FundRef https://doi.org/10.13039/501100001809, National Natural Science Foundation of China (National Science Foundation of China);
                Award ID: 31200942
                Award Recipient :
                Funded by: Mega-projects of Science and Technology Research 2017ZX10303405-003
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