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      Genotypic and Phenotypic Characterization of Staphylococcus aureus Isolates from the Respiratory Tract in Mechanically-Ventilated Patients

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          Abstract

          Staphylococcus aureus is a commensal and frequent colonizer of the upper respiratory tract. When mechanical ventilation disrupts natural defenses, S. aureus is frequently isolated from the lower airways, but distinguishing between colonization and infection is difficult. The objectives of this study were (1) to investigate the bacterial genome sequence in consecutive isolates in order to identify changes related to the pathological adaptation to the lower respiratory tract and (2) to explore the relationship between specific phenotypic and genotypic features with the patient’s study group, persistence of the clinical isolate and clinical outcome. A set of 94 clinical isolates were selected and corresponded to 34 patients that were classified as having pneumonia (10), tracheobronchitis (11) and bronchial colonization (13). Clinical strains were phenotypically characterized by conventional identification and susceptibility testing methods. Isolates underwent whole genome sequencing using Illumina HiSeq4000. Genotypic characterization was performed with an in-house pipeline (BacterialTyper). Genomic variation arising within-host was determined by comparing mapped sequences and de novo assemblies. Virulence factors important in staphylococcal colonization and infection were characterized using previously established functional assays. (1) Toxin production was assessed using a THP-1 cytotoxicity assay, which reports on the gross cytotoxicity of individual isolates. In addition, we investigated the expression of the major virulence factor, alpha-toxin (Hla) by Western blot. (2) Adhesion to the important extracellular matrix molecule, fibronectin, was determined using a standardized microtitre plate assay. Finally, invasion experiments using THP-1 and A539 cell lines and selected clinical strains were also performed. Repeated isolation of S. aureus from endotracheal aspirate usually reflects persistence of the same strain. Within-host variation is detectable in this setting, but it shows no evidence of pathological adaptation related to virulence, resistance or niche adaptations. Cytotoxicity was variable among isolates with 14 strains showing no cytotoxicity, with these latter presenting an unaltered Fn binding capacity. No changes on cytotoxicity were reported when comparing study groups. Fn binding capacity was reported for almost all strains, with the exception of two strains that presented the lowest values. Strains isolated from patients with pneumonia presented a lower capacity of adhesion in comparison to those isolated during tracheobronchitis ( p = 0.002). Hla was detected in 71 strains (75.5%), with most of the producer strains in pneumonia and bronchial colonization group ( p = 0.06). In our cohort, Hla expression (presence or absence) in sequential isolates was usually preserved (70%) although in seven cases the expression varied over time. No relationship was found between low cytotoxicity and intracellular persistence in invasion experiments. In our study population, persistent S. aureus isolation from airways in ventilated patients does not reflect pathological adaptation. There is an important diversity of sequence types. Cytotoxicity is variable among strains, but no association with study groups was found, whereas isolates from patients with pneumonia had lower adhesion capability. Favorable clinical outcome correlated with increased bacterial adhesion in vitro. Most of the strains isolated from the lower airways were Hla producers and no correlation with an adverse outcome was reported. The identification of microbial factors that contribute to virulence is relevant to optimize patient management during lower respiratory tract infections.

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          Basic local alignment search tool.

          A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score. Recent mathematical results on the stochastic properties of MSP scores allow an analysis of the performance of this method as well as the statistical significance of alignments it generates. The basic algorithm is simple and robust; it can be implemented in a number of ways and applied in a variety of contexts including straightforward DNA and protein sequence database searches, motif searches, gene identification searches, and in the analysis of multiple regions of similarity in long DNA sequences. In addition to its flexibility and tractability to mathematical analysis, BLAST is an order of magnitude faster than existing sequence comparison tools of comparable sensitivity.
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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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              Prokka: rapid prokaryotic genome annotation.

              T Seemann (2014)
              The multiplex capability and high yield of current day DNA-sequencing instruments has made bacterial whole genome sequencing a routine affair. The subsequent de novo assembly of reads into contigs has been well addressed. The final step of annotating all relevant genomic features on those contigs can be achieved slowly using existing web- and email-based systems, but these are not applicable for sensitive data or integrating into computational pipelines. Here we introduce Prokka, a command line software tool to fully annotate a draft bacterial genome in about 10 min on a typical desktop computer. It produces standards-compliant output files for further analysis or viewing in genome browsers. Prokka is implemented in Perl and is freely available under an open source GPLv2 license from http://vicbioinformatics.com/. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.
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                Author and article information

                Journal
                Toxins (Basel)
                Toxins (Basel)
                toxins
                Toxins
                MDPI
                2072-6651
                06 February 2021
                February 2021
                : 13
                : 2
                : 122
                Affiliations
                [1 ]Microbiology Department, Hospital Universitari Germans Trias i Pujol, Institut d’Investigació en Ciències de la Salut Germans Trias i Pujol, Universitat Autònoma de Barcelona, 08916 Badalona, Spain; alacoma@ 123456igtp.cat (A.L.); gerardgodoytena@ 123456gmail.com (G.G.-T.); meissinergomes@ 123456gmail.com (M.G.-F.)
                [2 ]CIBER Enfermedades Respiratorias, CIBER, Instituto de Salud Carlos III, 08916 Badalona, Spain
                [3 ]Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK; ml418@ 123456bath.ac.uk
                [4 ]High Content Genomics and Bioinformatics Unit, Institut d’Investigació en Ciències de la Salut Germans Trias i Pujol, 08916 Badalona, Spain; jsanchez@ 123456igtp.cat (J.F.S.-H.); lsumoy@ 123456igtp.cat (L.S.)
                [5 ]Nuffield Department of Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, UK; bernadette.young@ 123456ndm.ox.ac.uk
                [6 ]Intensive Care Unit, Hospital Universitari Germans Trias i Pujol, Institut Germans Trias i Pujol, Universitat Autònoma de Barcelona, 08916 Badalona, Spain; oriolplans@ 123456hotmail.com (O.P.); farmestarrodriguez@ 123456gmail.com (F.A.)
                [7 ]Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, Utrecht University, 3584 CG Utrecht, The Netherlands
                Author notes
                [* ]Correspondence: cprata@ 123456igtp.cat ; Tel.: +34-93-4978894
                Author information
                https://orcid.org/0000-0002-2049-3872
                https://orcid.org/0000-0002-8425-3704
                https://orcid.org/0000-0001-6771-4807
                https://orcid.org/0000-0001-6071-6770
                https://orcid.org/0000-0003-3832-5794
                https://orcid.org/0000-0001-5717-4288
                https://orcid.org/0000-0002-6374-1418
                https://orcid.org/0000-0001-6974-9165
                Article
                toxins-13-00122
                10.3390/toxins13020122
                7915691
                33562023
                92738248-140e-4d98-84c1-ce6f2d30201d
                © 2021 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 26 January 2021
                : 03 February 2021
                Categories
                Article

                Molecular medicine
                staphylococcus aureus,mechanical ventilation,pneumonia,toxicity,adhesion,persistence

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