Group A streptococci are protected from amoxicillin-mediated killing by vesicles containing β-lactamase derived from Haemophilus influenzae.

Objectives: 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 beta-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 bla(TEM-1) (encoding NTHi b-lactamase), bro-1 (encoding M. catarrhalis beta-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 b-lactamase was detected by western blots including specific rabbit polyclonal antibodies. The chromogenic substrate nitrocefin was used to quantify and compare the beta-lactamase enzyme activity in the OMVs. The hydrolysis of amoxicillin by beta-lactamase was estimated by an agar diffusion method. Results: We showed that OMVs released from beta-lactam-resistant M. catarrhalis and NTHi contain functional b-lactamase that hydrolyses amoxicillin and protects GAS from killing by amoxicillin. Conclusions: This is the first report of the presence of beta-lactamase in NTHi OMVs. We suggest that OMV-derived b-lactamase from coinfecting pathogens such as NTHi and M. catarrhalis may contribute to the occasional treatment failures seen in GAS tonsillitis.