title: Frequent carriage of resistance mechanisms to β-lactams and biofilm formation in Haemophilus influenzae causing treatment failure and recurrent otitis media in young children.
abstract: OBJECTIVE
  Non-typeable Haemophilus influenzae are a major cause of acute otitis media (AOM), including chronic and recurrent otitis in young children. The objective of this study was to determine whether non-typeable H. influenzae isolates causing these infections produce biofilms and carry resistance mechanisms to β-lactams.
  
  
  METHODS
  A collection of 48 H. influenzae isolates was obtained by tympanocentesis or from otorrhoea samples from individual patients <3 years of age and diagnosed with recurrent or treatment failure AOM. Each isolate was surveyed for the presence of blaTEM genes, amino acid substitutions in the transpeptidase domain of penicillin-binding protein 3 (PBP3) and biofilm formation in microtitre plates.
  
  
  RESULTS
  In 43 of the 48 isolates (89.6%), at least one of the three tested conditions was identified: biofilm formation (83.3%) and resistance mechanisms to β-lactams (33.3%), modifications in the transpeptidase domain of PBP3 being the most prevalent (22.9%), followed by β-lactamase production (10.4%). Additionally, 13 (27.1%) isolates had two or more of these three traits. In relation to biofilm formation, those isolates with an amoxicillin MIC ≤ 0.5 mg/L had higher optical density values than isolates with an amoxicillin MIC ≥ 1 mg/L (Mann-Whitney U-test, P=0.048).
  
  
  CONCLUSIONS
  These findings suggest that the successful treatment of non-typeable H. influenzae causing chronic and recurrent AOM in young children may be compromised by the high biofilm-forming capacity of the isolates and the presence of β-lactam resistance mechanisms, particularly PBP3 mutations.
authors.affiliation: Antibiotic and Haemophilus Laboratory, Bacteriology Service, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
authors.forename: Silvia
authors.initials: S
authors.lastname: García-Cobos
authors.affiliation: Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
authors.forename: Miriam
authors.initials: M
authors.lastname: Moscoso
authors.affiliation: Servicio de Otorrinolaringología, Hospital Universitario Vall d'Hebron, Barcelona, Spain.
authors.forename: Félix
authors.initials: F
authors.lastname: Pumarola
authors.affiliation: Antibiotic and Haemophilus Laboratory, Bacteriology Service, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain.
authors.forename: Margarita
authors.initials: M
authors.lastname: Arroyo
authors.affiliation: Antibiotic and Haemophilus Laboratory, Bacteriology Service, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain.
authors.forename: Noelia
authors.initials: N
authors.lastname: Lara
authors.affiliation: Antibiotic and Haemophilus Laboratory, Bacteriology Service, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain.
authors.forename: María
authors.initials: M
authors.lastname: Pérez-Vázquez
authors.affiliation: Antibiotic and Haemophilus Laboratory, Bacteriology Service, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain.
authors.forename: Belén
authors.initials: B
authors.lastname: Aracil
authors.affiliation: Antibiotic and Haemophilus Laboratory, Bacteriology Service, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain.
authors.forename: Jesús
authors.initials: J
authors.lastname: Oteo
authors.affiliation: Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
authors.forename: Ernesto
authors.initials: E
authors.lastname: García
authors.affiliation: Antibiotic and Haemophilus Laboratory, Bacteriology Service, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain Consejo Superior de Investigaciones Científicas, Madrid, Spain jcampos@isciii.es.
authors.forename: José
authors.initials: J
authors.lastname: Campos
mesh_terms: D018441:Biofilms / Q000254:growth & development*; D002675:Child, Preschool; D005798:Genes, Bacterial; D006192:Haemophilus Infections / Q000453:epidemiology / Q000382:microbiology*; D006193:Haemophilus influenzae / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D006801:Humans; D007223:Infant; D010033:Otitis Media / Q000453:epidemiology / Q000382:microbiology*; D012008:Recurrence; D017211:Treatment Failure; D018440:beta-Lactam Resistance
keywords: AOM; H. influenzae; gBLNAR