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

Martina Bielaszewska,Alexander Mellmann,Ondřej Daniel,Otakar Nyč

doi:10.3390/membranes11110806

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.

Highlights

Group A Streptococcus pyogenes (GAS) is the most common cause of acute bacterial pharyngotonsillitis, accounting for 20–30% of cases in children and 5–15% of cases in adults [1]
The throat culture of the patient with pharyngotonsillitis grew GAS, which was susceptible to penicillin, ampicillin, and amoxicillin (Table 2)
Our findings demonstrate that amoxicillin-resistant nontypeable Haemophilus influenzae (NTHi) colonizing the pharyngotonsillar mucosa of the GAS-infected patient secreted in situ β-lactamase-carrying outer membrane vesicles (OMVs), which were inducible by amoxicillin and protected GAS against the antibiotic, thereby accounting for the therapy failure

Summary

Introduction

Group A Streptococcus pyogenes (GAS) is the most common cause of acute bacterial pharyngotonsillitis, accounting for 20–30% of cases in children and 5–15% of cases in adults [1]. GAS is highly susceptible to β-lactam antibiotics, so penicillin and amoxicillin are the treatments of choice [1]. The inabilities of these antibiotics to eradicate GAS from patients with pharyngotonsillitis have been increasingly reported [2]. One cause of the therapy failure is colonization of the pharynx and tonsils by β-lactamase-producing bacteria such as Moraxella catarrhalis, Haemophilus influenzae, and Staphylococcus aureus that protect GAS against β-lactam antibiotics [2]. The mechanisms of this protection are incompletely understood. We investigated the involvement of bacterial outer membrane vesicles (OMVs) in amoxicillin therapy failure

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Results

Discussion

Conclusion