title: Group A streptococci are protected from amoxicillin-mediated killing by vesicles containing β-lactamase derived from Haemophilus influenzae.
abstract: OBJECTIVE
  Group A streptococci (GAS) cause, among other infections, pharyngotonsillitis in children. The species is frequently localized with the Gram-negative respiratory pathogens non-typeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis, which both produce outer membrane vesicles (OMVs). The aim of this study was to investigate whether OMVs isolated from NTHi contain functional β-lactamase and whether the OMVs hydrolyse amoxicillin and thus protect GAS from killing by the antibiotic.
  
  
  METHODS
  The antibiotic susceptibility of isolates was determined using the Etest. The resistance genes blaTEM-1 (encoding NTHi β-lactamase), bro-1 (encoding M. catarrhalis β-lactamase) and ftsI (encoding NTHi penicillin-binding protein 3) were searched for by PCR, followed by sequencing. OMVs were isolated by ultracentrifugation and the presence of β-lactamase was detected by western blots including specific rabbit polyclonal antibodies. The chromogenic substrate nitrocefin was used to quantify and compare the β-lactamase enzyme activity in the OMVs. The hydrolysis of amoxicillin by β-lactamase was estimated by an agar diffusion method.
  
  
  RESULTS
  We showed that OMVs released from β-lactam-resistant M. catarrhalis and NTHi contain functional β-lactamase that hydrolyses amoxicillin and protects GAS from killing by amoxicillin.
  
  
  CONCLUSIONS
  This is the first report of the presence of β-lactamase in NTHi OMVs. We suggest that OMV-derived β-lactamase from coinfecting pathogens such as NTHi and M. catarrhalis may contribute to the occasional treatment failures seen in GAS tonsillitis.
authors.affiliation: Medical Microbiology, Department of Laboratory Medicine Malmö, Lund University, Malmö, Sweden.
authors.forename: Viveka
authors.initials: V
authors.lastname: Schaar
authors.forename: Ida
authors.initials: I
authors.lastname: Uddbäck
authors.forename: Therese
authors.initials: T
authors.lastname: Nordström
authors.forename: Kristian
authors.initials: K
authors.lastname: Riesbeck
mesh_terms: D000658:Amoxicillin / Q000378:metabolism / Q000494:pharmacology*; D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology*; D055354:Exosomes / Q000201:enzymology* / Q000378:metabolism; D006193:Haemophilus influenzae / Q000201:enzymology* / Q000378:metabolism; D006868:Hydrolysis; D008826:Microbial Sensitivity Tests; D050296:Microbial Viability / Q000187:drug effects*; D013297:Streptococcus pyogenes / Q000187:drug effects* / Q000502:physiology; D001618:beta-Lactamases / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D000658:Amoxicillin; D001618:beta-Lactamases
keywords: Streptococcus pyogenes; non-typeable Haemophilus influenzae; outer membrane vesicles