Moraxella catarrhalisOuter Membrane Vesicles Carry β-Lactamase and Promote Survival ofStreptococcus pneumoniaeandHaemophilus influenzaeby Inactivating Amoxicillin

Abstract

ABSTRACTMoraxella catarrhalisis a common pathogen found in children with upper respiratory tract infections and in patients with chronic obstructive pulmonary disease during exacerbations. The bacterial species is often isolated together withStreptococcus pneumoniaeandHaemophilus influenzae. Outer membrane vesicles (OMVs) are released byM. catarrhalisand contain phospholipids, adhesins, and immunomodulatory compounds such as lipooligosaccharide. We have recently shown thatM. catarrhalisOMVs exist in patients upon nasopharyngeal colonization. As virtually allM. catarrhalisisolates are β-lactamase positive, the goal of this study was to investigate whetherM. catarrhalisOMVs carry β-lactamase and to analyze if OMV consequently can prevent amoxicillin-induced killing. Recombinant β-lactamase was produced and antibodies were raised in rabbits. Transmission electron microscopy, flow cytometry, and Western blotting verified that OMVs carried β-lactamase. Moreover, enzyme assays revealed thatM. catarrhalisOMVs contained active β-lactamase. OMVs (25 μg/ml) incubated with amoxicillin for 1 h completely hydrolyzed amoxicillin at concentrations up to 2.5 μg/ml. In functional experiments, preincubation of amoxicillin (10× MIC) withM. catarrhalisOMVs fully rescued amoxicillin-susceptibleM. catarrhalis,S. pneumoniae, and type b or nontypeableH. influenzaefrom β-lactam-induced killing. Our results suggest that the presence of amoxicillin-resistantM. catarrhalisoriginating from β-lactamase-containing OMVs may pave the way for respiratory pathogens that by definition are susceptible to β-lactam antibiotics.