Characterization of nontypable Haemophilus influenzae isolates recovered from adult patients with underlying chronic lung disease reveals genotypic and phenotypic traits associated with persistent infection.
Nontypable Haemophilus influenzae (NTHi) has emerged as an important opportunistic pathogen causing infection in adults suffering obstructive lung diseases. Existing evidence associates chronic infection by NTHi to the progression of the chronic respiratory disease, but specific features of NTHi associated with persistence have not been comprehensively addressed. To provide clues about adaptive strategies adopted by NTHi during persistent infection, we compared sequential persistent isolates with newly acquired isolates in sputa from six patients with chronic obstructive lung disease. Pulse field gel electrophoresis (PFGE) identified three patients with consecutive persistent strains and three with new strains. Phenotypic characterisation included infection of respiratory epithelial cells, bacterial self-aggregation, biofilm formation and resistance to antimicrobial peptides (AMP). Persistent isolates differed from new strains in showing low epithelial adhesion and inability to form biofilms when grown under continuous-flow culture conditions in microfermenters. Self-aggregation clustered the strains by patient, not by persistence. Increasing resistance to AMPs was observed for each series of persistent isolates; this was not associated with lipooligosaccharide decoration with phosphorylcholine or with lipid A acylation. Variation was further analyzed for the series of three persistent isolates recovered from patient 1. These isolates displayed comparable growth rate, natural transformation frequency and murine pulmonary infection. Genome sequencing of these three isolates revealed sequential acquisition of single-nucleotide variants in the AMP permease sapC, the heme acquisition systems hgpB, hgpC, hup and hxuC, the 3-deoxy-D-manno-octulosonic acid kinase kdkA, the long-chain fatty acid transporter ompP1, and the phosphoribosylamine glycine ligase purD. Collectively, we frame a range of pathogenic traits and a repertoire of genetic variants in the context of persistent infection by NTHi.
Instituto de Agrobiotecnología, CSIC-Universidad Pública Navarra-Gobierno Navarra, Mutilva, Spain; Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), Madrid, Spain; Laboratory Microbial Pathogenesis, Fundación Investigación Sanitaria Illes Balears, Bunyola, Spain.
Junkal
J
Garmendia
Instituto de Agrobiotecnología, CSIC-Universidad Pública Navarra-Gobierno Navarra, Mutilva, Spain.
Cristina
C
Viadas
Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), Madrid, Spain; Microbiology Department, University Hospital Bellvitge, IDIBELL, University of Barcelona, Barcelona, Spain.
Laura
L
Calatayud
Department of Zoology, University British Columbia, Vancouver, British Columbia, Canada; Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania, United States of America.
Joshua Chang
JC
Mell
Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), Madrid, Spain; Laboratory Microbial Pathogenesis, Fundación Investigación Sanitaria Illes Balears, Bunyola, Spain.
Pau
P
Martí-Lliteras
Instituto de Agrobiotecnología, CSIC-Universidad Pública Navarra-Gobierno Navarra, Mutilva, Spain; Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), Madrid, Spain.
Begoña
B
Euba
Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), Madrid, Spain; Laboratory Microbial Pathogenesis, Fundación Investigación Sanitaria Illes Balears, Bunyola, Spain.
Enrique
E
Llobet
Instituto de Agrobiotecnología, CSIC-Universidad Pública Navarra-Gobierno Navarra, Mutilva, Spain.
Carmen
C
Gil
Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), Madrid, Spain; Laboratory Microbial Pathogenesis, Fundación Investigación Sanitaria Illes Balears, Bunyola, Spain; Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain.
José Antonio
JA
Bengoechea
Department of Zoology, University British Columbia, Vancouver, British Columbia, Canada.
Rosemary J
RJ
Redfield
Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), Madrid, Spain; Microbiology Department, University Hospital Bellvitge, IDIBELL, University of Barcelona, Barcelona, Spain.
Josefina
J
Liñares
D000328:Adult; D056809:Alveolar Epithelial Cells / Q000382:microbiology; D000704:Analysis of Variance; D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000494:pharmacology; D001422:Bacterial Adhesion / Q000502:physiology; D001483:Base Sequence; D018441:Biofilms / Q000254:growth & development; D017931:DNA Primers / Q000235:genetics; D016521:Electrophoresis, Gel, Pulsed-Field; D005838:Genotype; D006193:Haemophilus influenzae / Q000166:cytology* / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010641:Phenotype; D029424:Pulmonary Disease, Chronic Obstructive / Q000382:microbiology*; D017422:Sequence Analysis, DNA
D023181:Antimicrobial Cationic Peptides; D017931:DNA Primers