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referenced object 'lBB56' cannot be found Offset: 4502853 ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - referenced object 'lBB57' cannot be found Offset: 4502853 MIMEtype: application/pdf PDFMetadata: Objects: 549 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: false CenterWindow: false DisplayDocTitle: true NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: RESULTS Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Children: Item: Title: Identification of respiratory bacterial isolates in COPD patients. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Item: Title: Characterization of persistent colonization of the respiratory tract by H. influenzae and H. parainfluenzae. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4eb7f003 Item: Title: Macrolide resistance development during long-term azithromycin therapy. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@eafc191 Item: Title: Phylogeny, population structure, and horizontal gene transfer. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612fc6eb Item: Title: Genetic changes observed during persistent colonization. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1060b431 Item: Title: Gain, loss, and evolution of genome-integrated prophages during persistent infection. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612679d6 Item: Title: DISCUSSION Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@11758f2a Item: Title: MATERIALS AND METHODS Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@e720b71 Children: Item: Title: Study design and bacterial strains. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1b26f7b2 Item: Title: Antimicrobial susceptibility testing. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@491cc5c9 Item: Title: Whole-genome sequencing and assembly. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@74ad1f1f Item: Title: Phylogenetic analysis and antimicrobial resistance determinants. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6a1aab78 Item: Title: Bacterial persistence and genetic adaptation. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@462d5aee Item: Title: Statistical analysis. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@69b0fd6f Item: Title: Ethical statement. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@757942a1 Item: Title: Data availability. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4a87761d Item: Title: SUPPLEMENTAL MATERIAL Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@66d1af89 Item: Title: ACKNOWLEDGMENTS Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@8646db9 Item: Title: REFERENCES Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@37374a5e Info: Title: Genetic Adaptation and Acquisition of Macrolide Resistance in Haemophilus spp. during Persistent Respiratory Tract Colonization in Chronic Obstructive Pulmonary Disease (COPD) Patients Receiving Long-Term Azithromycin Treatment Author: Anna Carrera-Salinas;Aida González-Díaz;Rachel L. Ehrlich;Dàmaris Berbel;Fe Tubau;Xavier Pomares;Junkal Garmendia;M. Ángeles Domínguez;Carmen Ardanuy;Daniel Huertas;Alicia Marín;Conchita Montón;Joshua Chang Mell;Salud Santos;Sara Marti Subject: Research Article Keywords: Haemophilus influenzae; Haemophilus parainfluenzae; persistence; macrolide resistance; azithromycin; adaptation Creator: PTC Arbortext Advanced Print Publisher 11.1.4012/W Producer: Adobe LiveCycle PDF Generator; modified using iText® 5.5.13.2 ©2000-2020 iText Group NV (AGPL-version) CreationDate: Sat Jan 28 06:48:00 CET 2023 ModDate: Wed Feb 01 16:24:47 CET 2023 ID: 0xc7af9fd10c409aaf78dbd29a6ae77856, 0x93ca3308ada79635b1f7e0f92e82f36d Filters: FilterPipeline: FlateDecode Images: Image: NisoImageMetadata: FormatName: image/png ImageWidth: 67 ImageHeight: 67 BitsPerSample: 8 BitsPerSampleUnit: integer Filter: FlateDecode Fonts: Type1: Font: BaseFont: MBPLBC+AdvOT65f8a23b.I FontSubset: true FirstChar: 45 LastChar: 122 FontDescriptor: FontName: MBPLBC+AdvOT65f8a23b.I Flags: Nonsymbolic FontBBox: -112, -250, 894, 873 FontFile3: true 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FirstChar: 44 LastChar: 122 FontDescriptor: FontName: MBPLAP+AdvOT3b30f6db.B Flags: Nonsymbolic FontBBox: -39, -250, 875, 897 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: MBPLBA+AdvOT46dcae81 FontSubset: true FirstChar: 35 LastChar: 252 FontDescriptor: FontName: MBPLBA+AdvOT46dcae81 Flags: Nonsymbolic FontBBox: -46, -250, 838, 879 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: MBPLBB+AdvOT46dcae81+fb FontSubset: true FirstChar: 1 LastChar: 3 FontDescriptor: FontName: MBPLBB+AdvOT46dcae81+fb Flags: Symbolic FontBBox: 0, 0, 742, 721 FontFile3: true EncodingDictionary: Differences: true ToUnicode: true XMP: Genetic Adaptation and Acquisition of Macrolide Resistance in Haemophilus spp. during Persistent Respiratory Tract Colonization in Chronic Obstructive Pulmonary Disease (COPD) Patients Receiving Long-Term Azithromycin Treatment 10.1128/spectrum.03860-22 Anna Carrera-Salinas Aida González-Díaz Rachel L. Ehrlich Dàmaris Berbel Fe Tubau Xavier Pomares Junkal Garmendia M. Ángeles Domínguez Carmen Ardanuy Daniel Huertas Alicia Marín Conchita Montón Joshua Chang Mell Salud Santos Sara Marti Research Article Research Article Haemophilus influenzae Haemophilus parainfluenzae persistence macrolide resistance azithromycin adaptation American Society for Microbiology Carrera-Salinas et al. Copyright © 2022 Carrera-Salinas et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. 20221208 2022 ABSTRACT Patients with chronic obstructive pulmonary disease (COPD) benefit from the immunomodulatory effect of azithromycin, but long-term administration may alter colonizing bacteria. Our goal was to identify changes in Haemophilus influenzae and Haemophilus parainfluenzae during azithromycin treatment. Fifteen patients were followed while receiving prolonged azithromycin treatment (Hospital Universitari de Bellvitge, Spain). Four patients (P02, P08, P11, and P13) were persistently colonized by H. influenzae for at least 3 months and two (P04 and P11) by H. parainfluenzae. Isolates from these patients (53 H. influenzae and 18 H. parainfluenzae) were included to identify, by whole-genome sequencing, antimicrobial resistance changes and genetic variation accumulated during persistent colonization. All persistent lineages isolated before treatment were azithromycin-susceptible but developed resistance within the first months, apart from those belonging to P02, who discontinued the treatment. H. influenzae isolates from P08-ST107 acquired mutations in 23S rRNA, and those from P11-ST2480 and P13-ST165 had changes in L4 and L22. In H. parainfluenzae, P04 persistent isolates acquired changes in rlmC, and P11 carried genes encoding MefE/MsrD efflux pumps in an integrative conjugative element, which was also identified in H. influenzae P11-ST147. Other genetic variation occurred in genes associated with cell wall and inorganic ion metabolism. Persistent H. influenzae strains all showed changes in licA and hgpB genes. Other genes (lex1, lic3A, hgpC, and fadL) had variation in multiple lineages. Furthermore, persistent strains showed loss, acquisition, or genetic changes in prophage-associated regions. Long-term azithromycin therapy results in macrolide resistance, as well as genetic changes that likely favor bacterial adaptation during persistent respiratory colonization. IMPORTANCE The immunomodulatory properties of azithromycin reduce the frequency of exacerbations and improve the quality of life of COPD patients. However, long-term administration may alter the respiratory microbiota, such as Haemophilus influenzae, an opportunistic respiratory colonizing bacteria that play an important role in exacerbations. This study contributes to a better understanding of COPD progression by characterizing the clinical evolution of H. influenzae in a cohort of patients with prolonged azithromycin treatment. The emergence of macrolide resistance during the first months, combined with the role of Haemophilus parainfluenzae as a reservoir and source of resistance dissemination, is a cause for concern that may lead to therapeutic failure. Furthermore, genetic variations in cell wall and inorganic ion metabolism coding genes likely favor bacterial adaptation to host selective pressures. Therefore, the bacterial pathoadaptive evolution in these severe COPD patients raise our awareness of the possible spread of macrolide resistance and selection of host-adapted clones. 20220921 20221121 2165-0497 Pages: Page: Label: 1 Annotations: Annotation: Subtype: Link Rect: 196, 523, 235, 532 Annotation: Subtype: Link Rect: 435, 133, 530, 140 Annotation: Subtype: Link Rect: 400, 124, 456, 131 Annotation: Subtype: Link Rect: 444, 24, 526, 31 Annotation: Subtype: Link Rect: 508, 683, 544, 719 Page: Label: 2 Annotations: Annotation: Subtype: Link Rect: 273, 510, 278, 519 Destination: 245 Annotation: Subtype: Link Rect: 283, 510, 288, 519 Destination: 245 Annotation: Subtype: Link Rect: 81, 474, 86, 483 Destination: 245 Annotation: Subtype: Link Rect: 89, 474, 94, 483 Destination: null Annotation: Subtype: Link Rect: 196, 438, 200, 447 Destination: 245 Annotation: Subtype: Link Rect: 200, 438, 204, 447 Destination: null Annotation: Subtype: Link Rect: 204, 438, 208, 447 Destination: 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