In Vivo Secretion of β-Lactamase-Carrying Outer Membrane Vesicles as a Mechanism of β-Lactam Therapy Failure

Abstract

Outer membrane vesicles carrying β-lactamase (βLOMVs) protect bacteria against β-lactam antibiotics under experimental conditions, but their protective role during a patient’s treatment leading to the therapy failure is unknown. We investigated the role of βLOMVs in amoxicillin therapy failure in a patient with group A Streptococcus pyogenes (GAS) pharyngotonsillitis. The patient’s throat culture was examined by standard microbiological procedures. Bacterial vesicles were analyzed for β-lactamase by immunoblot and the nitrocefin assay, and in vivo secretion of βLOMVs was detected by electron microscopy. These analyses demonstrated that the patient’s throat culture grew, besides amoxicillin-susceptible GAS, an amoxicillin-resistant nontypeable Haemophilus influenzae (NTHi), which secreted βLOMVs. Secretion and β-lactamase activity of NTHi βLOMVs were induced by amoxicillin concentrations reached in the tonsils during therapy. The presence of NTHi βLOMVs significantly increased the minimal inhibitory concentration of amoxicillin for GAS and thereby protected GAS against bactericidal concentrations of amoxicillin. NTHi βLOMVs were identified in the patient’s pharyngotonsillar swabs and saliva, demonstrating their secretion in vivo at the site of infection. We conclude that the pathogen protection via βLOMVs secreted by the flora colonizing the infection site represents a yet underestimated mechanism of β-lactam therapy failure that warrants attention in clinical studies.

Deciphering the evolution of metallo-β-lactamases: A journey from the test tube to the bacterial periplasm

“…OMVs transport SBLs and MBLs in their active form, as well as plasmids coding for these enzymes (39,44,100,(105)(106)(107)(108)(109). OMVs incorporating β-lactamases can protect populations of susceptible bacteria against antibiotics (44,106,110), as recently shown in vivo (111,112), and have been postulated as a novel mechanism of plasmid transfer (113,114). While the secretion of toxic species is nonspecific, the packaging of folded and active MBLs is selective.…”

The biogenesis of β-lactamase enzymes

“…Although serine-β-lactamases have evolved from the membrane-anchored PBPs, they are mostly soluble enzymes localised in the periplasm of Gram-negative bacteria [254][255][256]. Experiments using native and engineered inner-membrane-anchored TEM-1 suggest that the solubilisation of these hydrolases offers long-term expression and survival advantages [13].…”

The role of bacterial membrane vesicles in antibiotic resistance

“…However, coinfection of G. mellonella with carbapenem‐susceptible P. aeruginosa and E. coli expressing NDM‐1 was sufficient to protect both strains during meropenem treatment through a mechanism that correlated with the amount of NDM‐1 secreted within MVs 25 . MVs have also been implicated as a potential contributor to amoxicillin treatment failure in a patient suffering from group A Streptococcus pyogenes pharyngotonsillitis 26 . Despite in vitro sensitivity to amoxicillin, no improvement in symptoms was observed in the patient after 10 days of antibiotic treatment.…”