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      Dissemination of carbapenemase-producing Enterobacterales in the community of Rawalpindi, Pakistan

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          Abstract

          Carbapenems are considered last-line beta-lactams for the treatment of infections caused by multidrug-resistant Gram-negative bacteria. However, their activity is compromised by the rising prevalence of carbapenemase-producing Enterobacterales (CPE), which are especially marked in the Indian subcontinent. In Pakistan, previous reports have warned about the possible spread of CPE in the community, but data are still partial. This study was carried out to analyse the prevalence of CPE, the genetic characterisation, and phylogenetic links among the spreading CPE in the community. In this cohort study, we collected 306 rectal swabs from patients visiting Benazir Bhutto hospital, Rawalpindi. CPEs were screened by using ertapenem-supplemented MacConkey agar. Identification was performed by using conventional biochemical tests, and genomes were sequenced using Illumina chemistry. Antibiotic resistance genes, plasmid incompatibility groups, and Escherichia coli phylogroups were determined in silico. Sequence types were determined by using MLST tool. The prevalence of CPE carriage observed was 14.4% (44/306 samples). The most common carbapenemase-encoding gene was bla- NDM-5 (n = 58) followed by bla NDM-1 (n = 7), bla NDM (non-assigned variant, n = 4), bla OXA-181 (n = 3), bla OXA-232 (n = 3) and bla NDM-7 (n = 1). Most of the CPE were E. coli (55/64, 86%), and the genomic analysis revealed a pauciclonal diffusion of E. coli with ST167 (n = 14), 405 (n = 10), 940 (n = 8), 648 (n = 6) and 617 (n = 5). We obtained a second sample from 94 patients during their hospital stay in whom carriage was negative at admission and found that 7 (7.4%) acquired a CPE. Our results indicate that the prevalence of CPE carriage in the Pakistani urban community was high and driven by the dissemination of some E. coli clones, with ST167 being the most frequent. The high CPE carriage in the community poses a serious public health threat and calls for implementation of adequate preventive measures.

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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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            SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing.

            The lion's share of bacteria in various environments cannot be cloned in the laboratory and thus cannot be sequenced using existing technologies. A major goal of single-cell genomics is to complement gene-centric metagenomic data with whole-genome assemblies of uncultivated organisms. Assembly of single-cell data is challenging because of highly non-uniform read coverage as well as elevated levels of sequencing errors and chimeric reads. We describe SPAdes, a new assembler for both single-cell and standard (multicell) assembly, and demonstrate that it improves on the recently released E+V-SC assembler (specialized for single-cell data) and on popular assemblers Velvet and SoapDeNovo (for multicell data). SPAdes generates single-cell assemblies, providing information about genomes of uncultivatable bacteria that vastly exceeds what may be obtained via traditional metagenomics studies. SPAdes is available online ( http://bioinf.spbau.ru/spades ). It is distributed as open source software.
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              Fast and sensitive protein alignment using DIAMOND.

              The alignment of sequencing reads against a protein reference database is a major computational bottleneck in metagenomics and data-intensive evolutionary projects. Although recent tools offer improved performance over the gold standard BLASTX, they exhibit only a modest speedup or low sensitivity. We introduce DIAMOND, an open-source algorithm based on double indexing that is 20,000 times faster than BLASTX on short reads and has a similar degree of sensitivity.
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                Author and article information

                Contributors
                Role: Formal analysisRole: InvestigationRole: MethodologyRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: MethodologyRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: Formal analysisRole: SoftwareRole: Writing – review & editing
                Role: Formal analysisRole: MethodologyRole: SoftwareRole: ValidationRole: VisualizationRole: Writing – review & editing
                Role: Data curationRole: InvestigationRole: Resources
                Role: ResourcesRole: SupervisionRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: Project administrationRole: ResourcesRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: InvestigationRole: Project administrationRole: ResourcesRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS One
                plos
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                8 July 2022
                2022
                : 17
                : 7
                : e0270707
                Affiliations
                [1 ] Department of Microbiology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan
                [2 ] Université de Paris, IAME, INSERM, Paris, France
                [3 ] APHP, Laboratoire de Bactériologie, Hôpital Bichat, Paris, France
                [4 ] APHP, Laboratoire de Bactériologie, Hôpital Beaujon, Clichy, France
                [5 ] Accident and Emergency Department, Benazir Bhutto Hospital, Rawalpindi, Pakistan
                UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                ‡ ER and RZ also contributed equally to this work as co-last authors.

                Author information
                https://orcid.org/0000-0001-8784-0114
                Article
                PONE-D-21-36109
                10.1371/journal.pone.0270707
                9269877
                35802735
                0d50f003-84af-4f7f-81ec-e7df52d46110
                © 2022 Habib et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 13 November 2021
                : 15 June 2022
                Page count
                Figures: 13, Tables: 0, Pages: 20
                Funding
                Funded by: Higher Education Commission, Pakistan
                Award ID: Pak-France Joint Grant (PERIDOT)
                Award Recipient :
                Funded by: Higher Education Commission, Pakistan
                Award ID: Pak-France Joint Grant (PERIDOT)
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100010221, Higher Education Commision, Pakistan;
                Award ID: IRSIP PIN: 43 BMS 16
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100003763, Ministère des Affaires Etrangères;
                Award ID: Programme Hubert Curien “Peridot”
                Award Recipient :
                Funded by: Fondation pour la Recherche Médicale
                Award ID: Equipe FRM 2016, grant 325 number DEQ20161136698
                Funded by: Direction Générale des Armées
                Award ID: project FastGeneII
                Funded by: funder-id http://dx.doi.org/10.13039/501100010221, Higher Education Commision, Pakistan;
                Award ID: NRPU-9755
                Award Recipient :
                This work was partially supported by the “Fondation pour la Recherche Médicale” (Equipe FRM 2016, grant 325 number DEQ20161136698); the Direction Générale des Armées (project FastGeneII); the Programme Hubert Curien “Peridot” (Ministère des Affaires Etrangères, France) and by the Higher Education Commission, Pakistan under Pak-France Joint Grant (PERIDOT) to RZ and ER, National Research Program for Universities (NRPU - Project 9755) to RZ, International Research Support Initiative Program (IRSIP) (PIN: 43 BMS 16) to AH.
                Categories
                Research Article
                Biology and Life Sciences
                Microbiology
                Microbial Control
                Antimicrobial Resistance
                Antibiotic Resistance
                Medicine and Health Sciences
                Pharmacology
                Antimicrobial Resistance
                Antibiotic Resistance
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                Custom metadata
                The data relevant to this study are available from the National Centre for Biotechnology (NIH) at accession number PRJNA645311 ( https://www.ncbi.nlm.nih.gov/bioproject/PRJNA645311).

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