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      Whole-genome sequencing resolves a polyclonal outbreak by extended-spectrum beta-lactam and carbapenem-resistant Klebsiella pneumoniae in a Portuguese tertiary-care hospital

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          Abstract

          Klebsiella pneumoniae has emerged as an important nosocomial pathogen, with whole-genome sequencing (WGS) significantly improving our ability to characterize associated outbreaks. Our study sought to perform a genome-wide analysis of multiclonal K. pneumoniae isolates ( n=39; 23 patients) producing extended spectrum beta-lactamases and/or carbapenemases sourced between 2011 and 2016 in a Portuguese tertiary-care hospital. All isolates showed resistance to third-generation cephalosporins and six isolates (five patients) were also carbapenem resistant. Genome-wide-based phylogenetic analysis revealed a topology representing ongoing dissemination of three main sequence-type (ST) clades (ST15, ST147 and ST307) and transmission across different wards, compatible with missing links that can take the form of undetected colonized patients. Two carbapenemase-coding genes were detected: bla KPC-3 , located on a Tn4401d transposon, and bla GES-5 on a novel class 3 integron. Additionally, four genes coding for ESBLs ( bla BEL-1 , bla CTX-M-8 , bla CTX-M-15 and bla CTX-M-32 ) were also detected. ESBL horizontal dissemination across five clades is highlighted by the similar genetic environments of bla CTX-M-15 gene upstream of IS Ecp1 on a Tn3-like transposon. Overall, this study provides a high-resolution genome-wide perspective on the epidemiology of ESBL and carbapenemase-producing K. pneumoniae in a healthcare setting while contributing for the adoption of appropriate intervention and prevention strategies.

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          Most cited references56

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          Trimmomatic: a flexible trimmer for Illumina sequence data

          Motivation: Although many next-generation sequencing (NGS) read preprocessing tools already existed, we could not find any tool or combination of tools that met our requirements in terms of flexibility, correct handling of paired-end data and high performance. We have developed Trimmomatic as a more flexible and efficient preprocessing tool, which could correctly handle paired-end data. Results: The value of NGS read preprocessing is demonstrated for both reference-based and reference-free tasks. Trimmomatic is shown to produce output that is at least competitive with, and in many cases superior to, that produced by other tools, in all scenarios tested. Availability and implementation: Trimmomatic is licensed under GPL V3. It is cross-platform (Java 1.5+ required) and available at http://www.usadellab.org/cms/index.php?page=trimmomatic Contact: usadel@bio1.rwth-aachen.de Supplementary information: Supplementary data are available at Bioinformatics online.
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            The Sequence Alignment/Map format and SAMtools

            Summary: The Sequence Alignment/Map (SAM) format is a generic alignment format for storing read alignments against reference sequences, supporting short and long reads (up to 128 Mbp) produced by different sequencing platforms. It is flexible in style, compact in size, efficient in random access and is the format in which alignments from the 1000 Genomes Project are released. SAMtools implements various utilities for post-processing alignments in the SAM format, such as indexing, variant caller and alignment viewer, and thus provides universal tools for processing read alignments. Availability: http://samtools.sourceforge.net Contact: rd@sanger.ac.uk
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              Fast and accurate short read alignment with Burrows–Wheeler transform

              Motivation: The enormous amount of short reads generated by the new DNA sequencing technologies call for the development of fast and accurate read alignment programs. A first generation of hash table-based methods has been developed, including MAQ, which is accurate, feature rich and fast enough to align short reads from a single individual. However, MAQ does not support gapped alignment for single-end reads, which makes it unsuitable for alignment of longer reads where indels may occur frequently. The speed of MAQ is also a concern when the alignment is scaled up to the resequencing of hundreds of individuals. Results: We implemented Burrows-Wheeler Alignment tool (BWA), a new read alignment package that is based on backward search with Burrows–Wheeler Transform (BWT), to efficiently align short sequencing reads against a large reference sequence such as the human genome, allowing mismatches and gaps. BWA supports both base space reads, e.g. from Illumina sequencing machines, and color space reads from AB SOLiD machines. Evaluations on both simulated and real data suggest that BWA is ∼10–20× faster than MAQ, while achieving similar accuracy. In addition, BWA outputs alignment in the new standard SAM (Sequence Alignment/Map) format. Variant calling and other downstream analyses after the alignment can be achieved with the open source SAMtools software package. Availability: http://maq.sourceforge.net Contact: rd@sanger.ac.uk
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                Author and article information

                Journal
                Microb Genom
                Microb Genom
                mgen
                mgen
                Microbial Genomics
                Microbiology Society
                2057-5858
                2021
                1 April 2020
                1 April 2020
                : 7
                : 6
                : 000349
                Affiliations
                [ 1] Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa , Portugal
                [ 2] Clinical Pathology Unit. Hospital SAMS , Lisboa, Portugal
                [ 3] Infection Control Commission, Hospital SAMS , Lisboa, Portugal
                [ 4] departmentFaculty of Infectious and Tropical Diseases , London School of Hygiene and Tropical Medicine , London, UK
                [ 5] Genome Institute Singapore , Singapore
                [ 6] Intensive Care Medicine Unit, Hospital SAMS , Lisboa, Portugal
                [ 7] departmentFaculty of Epidemiology and Population Health , London School of Hygiene and Tropical Medicine , London, UK
                [ 8] Faculty of Pharmacy, Universidade de Lisboa , Portugal
                [ 9] Centro de Investigação Interdisciplinar Egas Moniz, Instituto Universitário Egas Moniz , Portugal
                Author notes

                NCBI Bacterial Antimicrobial Resistance Reference Gene Database (GenBank accession PRJNA313047) April 2019.

                [†]

                These authors share senior authorship.

                *Correspondence: Aida Duarte, aduarte@ 123456ff.ulisboa.pt
                *Correspondence: João Perdigão, jperdigao@ 123456ff.ulisboa.pt
                Author information
                https://orcid.org/0000-0002-2579-1799
                https://orcid.org/0000-0003-1368-4651
                https://orcid.org/0000-0001-8323-7019
                https://orcid.org/0000-0002-1548-1121
                https://orcid.org/0000-0003-0258-9616
                https://orcid.org/0000-0001-8985-9265
                https://orcid.org/0000-0002-0616-4650
                Article
                000349
                10.1099/mgen.0.000349
                8627661
                32234124
                7e736d3c-3c60-4fd7-b19e-7c97eb885397
                © 2020 The Authors

                This is an open-access article distributed under the terms of the Creative Commons Attribution License.

                History
                : 02 December 2019
                : 14 February 2020
                Funding
                Funded by: Fundação para a Ciência e a Tecnologia
                Award ID: CEECIND/00394/2017
                Award Recipient : João Perdigão
                Funded by: Medical Research Council
                Award ID: MR/K000551/1, MR/M01360X/1, MR/N010469/1, MR/R020973/1, MR/R025576/1, MR/S01988X/1, MR/S03563X/1
                Award Recipient : Taane G. Clark
                Funded by: Biotechnology and Biological Sciences Research Council
                Award ID: BB/R013063/1
                Award Recipient : Taane G. Clark
                Categories
                Outbreak Reports
                Pathogens and Epidemiology
                Custom metadata
                0

                esbl,kpc,ctx-m,gram-negative,portugal,lisbon
                esbl, kpc, ctx-m, gram-negative, portugal, lisbon

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