b3e8b36ee36235f4cf5a15056e29972c toxins_a2021v13n2p122.pdf 8c9432625192c01a5b808c5248cc873ac93aa734 toxins_a2021v13n2p122.pdf 21a9571b9135fc42f3d827191a288a1e094ba013f0a639c816094b0552b269a1 toxins_a2021v13n2p122.pdf Title: Genotypic and Phenotypic Characterization of Staphylococcus aureus Isolates from the Respiratory Tract in Mechanically-Ventilated Patients Subject: 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. Keywords: Staphylococcus aureus; mechanical ventilation; pneumonia; toxicity; adhesion; persistence Author: Alicia Lacoma, Maisem Laabei, Jose Francisco Sánchez-Herrero, Bernadette Young, Gerard Godoy-Tena, Meissiner Gomes-Fernandes, Lauro Sumoy, Oriol Plans, Fernando Arméstar and Cristina Prat Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Mon Feb 8 08:49:27 2021 CET ModDate: Mon Feb 8 08:53:19 2021 CET Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 23 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 3415807 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- OAGXRN+VnURWPalladioL Type 1 Custom yes yes no 10 0 BNGQBT+URWPalladioL-Ital Type 1 Custom yes yes no 15 0 JFAXRO+URWPalladioL-Bold Type 1 Custom yes yes no 20 0 SJLJLN+URWPalladioL-BoldItal Type 1 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Identity-H yes yes yes 318 0 BBKMJC+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 324 0 BBKMKD+PalatinoLinotype TrueType WinAnsi yes yes no 327 0 BBKMLF+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 330 0 BBLHCI+Arial,Bold CID TrueType Identity-H yes yes yes 333 0 HHBSSZ+CMSY10 Type 1 Builtin yes yes no 342 0 OVNEXW+EURM10 Type 1 Builtin yes yes no 352 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-10-02 03:55:47 CEST RepresentationInformation: toxins_a2021v13n2p122.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2023-03-31 11:50:23 CEST Size: 3415807 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 634 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Results Destination: section.2 Children: Item: Title: Patients and Strains Destination: subsection.2.1 Item: Title: Genomic Characterization Destination: subsection.2.2 Item: Title: Within-Host Variation Destination: subsection.2.3 Item: Title: THP-1 Cytotoxicity Destination: subsection.2.4 Item: Title: Fn Binding Capacity Destination: subsection.2.5 Item: Title: Alpha Toxin Detection by Western Blot Destination: subsection.2.6 Item: Title: Invasion and Persistence Assay Destination: subsection.2.7 Item: Title: Discussion Destination: section.3 Item: Title: Material and Methods Destination: section.4 Children: Item: Title: Patients and Strains Destination: subsection.4.1 Item: Title: Whole Genome Sequencing Destination: subsection.4.2 Item: Title: Genomic Characterization Destination: subsection.4.3 Item: Title: Within-Host Variation Analysis Destination: subsection.4.4 Item: Title: Phenotypic Characterization Destination: subsection.4.5 Item: Title: THP-1 Cytotoxicity Assay Destination: subsection.4.6 Item: Title: Fibronectin Binding Assay Destination: subsection.4.7 Item: Title: Detection of Secreted Hla Destination: subsection.4.8 Item: Title: Invasion Assay Destination: subsection.4.9 Item: Title: Statistical Analysis Destination: subsection.4.10 Item: Title: References Destination: subsection.4.10 Info: Title: Genotypic and Phenotypic Characterization of Staphylococcus aureus Isolates from the Respiratory Tract in Mechanically-Ventilated Patients Author: Alicia Lacoma, Maisem Laabei, Jose Francisco Sánchez-Herrero, Bernadette Young, Gerard Godoy-Tena, Meissiner Gomes-Fernandes, Lauro Sumoy, Oriol Plans, Fernando Arméstar and Cristina Prat Subject: 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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