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 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.