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      Novel Multidrug-Resistant Enterococcal Mobile Linear Plasmid pELF1 Encoding vanA and vanM Gene Clusters From a Japanese Vancomycin-Resistant Enterococci Isolate

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          Abstract

          Vancomycin-resistant enterococci are troublesome pathogens in clinical settings because of few treatment options. A VanA/VanM-type vancomycin-resistant Enterococcus faecium clinical isolate was identified in Japan. This strain, named AA708, harbored five plasmids, one of which migrated during agarose gel electrophoresis without S1 nuclease treatment, which is indicative of a linear topology. We named this plasmid pELF1. Whole genome sequencing (WGS) analysis of the AA708 strain revealed that the complete sequence of pELF1 was 143,316 bp long and harbored both the vanA and vanM gene clusters. Furthermore, mfold analysis and WGS data show that the left end of pELF1 presumably forms a hairpin structure, unlike its right end. The pELF1 plasmid was not digested by lambda exonuclease, indicating that terminal proteins were bound to the 5′ end of the plasmid, similar to the Streptomyces linear plasmids. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis results were also consistent with the exonuclease assay results. In retardation assays, DNAs containing the right end of proteinase K-untreated pELF1 did not appear to move as well as the proteinase K-treated pELF1, suggesting that terminal proteins might be attached to the right end of pELF1. Palindromic sequences formed hairpin structures at the right terminal sequence of pELF1; however, sequence similarity with the well-known linear plasmids of Streptomyces spp. was not high. pELF1 was unique as it possessed two different terminal structures. Conjugation experiments revealed that pELF1 could be transferred to E. faecalis, E. faecium, E. casseliflavus, and E. hirae. These transconjugants exhibited not only high resistance levels to vancomycin, but also resistance to streptomycin, kanamycin, and erythromycin. These results indicate that pELF1 has the ability to confer multidrug resistance to Enterococcus spp. simultaneously, which might lead to clinical hazards.

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          DFAST: a flexible prokaryotic genome annotation pipeline for faster genome publication

          Abstract Summary We developed a prokaryotic genome annotation pipeline, DFAST, that also supports genome submission to public sequence databases. DFAST was originally started as an on-line annotation server, and to date, over 7000 jobs have been processed since its first launch in 2016. Here, we present a newly implemented background annotation engine for DFAST, which is also available as a standalone command-line program. The new engine can annotate a typical-sized bacterial genome within 10 min, with rich information such as pseudogenes, translation exceptions and orthologous gene assignment between given reference genomes. In addition, the modular framework of DFAST allows users to customize the annotation workflow easily and will also facilitate extensions for new functions and incorporation of new tools in the future. Availability and implementation The software is implemented in Python 3 and runs in both Python 2.7 and 3.4—on Macintosh and Linux systems. It is freely available at https://github.com/nigyta/dfast_core/under the GPLv3 license with external binaries bundled in the software distribution. An on-line version is also available at https://dfast.nig.ac.jp/. Contact yn@nig.ac.jp Supplementary information Supplementary data are available at Bioinformatics online.
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            A general method for detecting and sizing large plasmids.

            We have devised a method for detecting and estimating the sizes of large bacterial plasmids in the presence of genomic DNA by pulsed-field gel electrophoresis (PFGE). Bacteria harboring plasmids were embedded in agarose and lysed using a rapid protocol. Plugs were incubated with S1 nuclease and subjected to PFGE in agarose gels. S1 nuclease converted supercoiled plasmids into full-length linear molecules. Large plasmids migrated as discrete bands that were readily observed after ethidium staining. Their sizes were reliably estimated by comparison with linear DNA markers. Without S1 digestion, supercoiled plasmids migrated at rates that were not a simple function of their molecular weights, making size determinations problematic. S1-PFGE detected megaplasmids up to 609 kilobases (kb) in six genera of bacteria (Agrobacterium, Escherichia, Klebsiella, Pseudomonas, Salmonella, and Staphylococcus). The procedure gave size values consistent with previous estimates for characterized megaplasmids. Eight new plasmids between 102 and 316 kb were discovered in Klebsiella and Staphylococcus. S1-PFGE avoids the difficulties of plasmid isolation, eliminates the preparation of probes, and does not require knowledge of restriction enzyme cleavage sites. It detects multiple large plasmids up to the limits of PFGE and can be used to screen for megaplasmids in many strains simultaneously.
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              Multilocus sequence typing scheme for Enterococcus faecium.

              A multilocus sequence typing (MLST) scheme has been developed for Enterococcus faecium. Internal fragments from seven housekeeping genes of 123 epidemiologically unlinked isolates from humans and livestock and 16 human-derived isolates from several outbreaks in the United States, the United Kingdom, Australia, and The Netherlands were analyzed. A total of 62 sequence types were detected in vancomycin-sensitive E. faecium (VSEF) and vancomycin-resistant E. faecium (VREF) isolates. VSEF isolates were genetically more diverse than VREF isolates. Both VSEF and VREF isolates clustered in host-specific lineages that were similar to the host-specific clustering obtained by amplified fragment length polymorphism analysis. Outbreak isolates from hospitalized humans clustered in a subgroup that was defined by the presence of a unique allele from the housekeeping gene purK and the surface protein gene esp. The MLST results suggest that epidemic lineages of E. faecium emerged recently worldwide, while genetic variation in both VREF and VSEF was created by longer-term recombination. The results show that MLST of E. faecium provides an excellent tool for isolate characterization and long-term epidemiologic analysis.
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                Author and article information

                Contributors
                Journal
                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                1664-302X
                13 November 2019
                2019
                : 10
                : 2568
                Affiliations
                [1] 1Department of Bacteriology, Gunma University Graduate School of Medicine , Maebashi, Japan
                [2] 2Oral Microbiome Center, Taniguchi Dental Clinic , Takamatsu, Japan
                [3] 3Graduate School of Bioagricultural Sciences, Nagoya University , Nagoya, Japan
                [4] 4Laboratory of Bacterial Drug Resistance, Gunma University Graduate School of Medicine , Maebashi, Japan
                [5] 5MIROKU Medical Laboratory , Inc., Saku, Japan
                [6] 6Institute of Clinical Pharmacology, Peking University First Hospital , Beijing, China
                Author notes

                Edited by: Yuji Morita, Meiji Pharmaceutical University, Japan

                Reviewed by: Julia Willett, University of Minnesota Twin Cities, United States; Ana P. Tedim, Institute of Health Sciences Studies of Castilla y León (IECSCYL), Spain; Kristin Hegstad, University Hospital of North Norway, Norway

                *Correspondence: Bo Zheng, doctorzhengbo@ 123456163.com

                This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology

                Article
                10.3389/fmicb.2019.02568
                6863802
                31798546
                5aad8925-5dfa-494d-964f-8005b2805072
                Copyright © 2019 Hashimoto, Taniguchi, Uesaka, Nomura, Hirakawa, Tanimoto, Tamai, Ruan, Zheng and Tomita.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 07 August 2019
                : 23 October 2019
                Page count
                Figures: 8, Tables: 2, Equations: 0, References: 69, Pages: 13, Words: 0
                Categories
                Microbiology
                Original Research

                Microbiology & Virology
                enterococci,linear plasmid,conjugation,vancomycin-resistant enterococci,drug resistance

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