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.

Keywords: GAS protection; Haemophilus influenzae; amoxicillin therapy failure; group A Streptococcus pyogenes (GAS); in vivo secretion; pharyngotonsillitis; β-lactamase-carrying outer membrane vesicles.

Figure 1

Nontypeable Haemophilus influenzae (NTHi) isolated from the patient’s throat culture secretes β-lactamase-carrying OMVs, which are induced by amoxicillin (NTHi OMVs_βL+AMX+): (a) electron microscopy of negatively stained NTHi OMVs_βL+AMX+; scale bar, 200 nm. (b) OMV immunoblot with anti-β-lactamase antibody. Left panel: lane 1, NTHi OMVs_βL+AMX+, lane 2, control β-lactamase-negative OMVs (NTHi OMVs_βL−), lane 3, protein size marker (the marker band sizes are on the right side); β-lactamase is ~32 kDa. Right panel: OMVs_βL+AMX+ subjected to proteinase K (PK) assay, which demonstrates intravesicular localization of β-lactamase. (c,d) β-lactamase activities (c) and the amounts (d) of OMVs produced by NTHi freshly isolated from the patient’s throat culture (fresh isolate), by NTHi passaged twice in BHI broth without amoxicillin (P1 and P2, AMX-), and by NTHi from passage 2 grown in BHI broth with amoxicillin concentrations reported in the tonsils during therapy (0.17 µg/mL, 1.1 µg/mL, or 3.9 µg/mL). Data are presented as means ± standard deviations from three independent experiments; ** p < 0.01 compared to fresh isolate; ^xx p < 0.01 compared to P2 AMX-; ^xxx p < 0.001 compared to P2 AMX- (statistical analysis was performed with one-way ANOVA).

Figure 2

Amoxicillin-induced, β-lactamase-carrying OMVs from amoxicillin-resistant nontypeable Haemophilus influenzae (NTHi OMVs_βL+AMX+) protect GAS against bactericidal concentrations of amoxicillin: (a,b) GAS growth in BHI broth without amoxicillin or with amoxicillin 0.17 µg/mL (a) or 3.9 µg/mL (b) in the absence or presence of the indicated amounts of NTHi OMVs_βL+AMX+ without or with clavulanate, or in the presence of control β-lactamase-negative NTHi OMVs (NTHi OMVs_βL−). Data are means ± standard deviations (SDs) from three independent experiments. (c) Growth of GAS from 24 h cultures shown in (a,b) on BHI agar without or with amoxicillin. Data are means ± SDs from three independent experiments.

Figure 3

Transmission electron microscopy of the patient’s pharyngotonsillar swab, crypt exudate, and saliva demonstrating in vivo secretion of β-lactamase-carrying NTHi OMVs: (a) NTHi secreting β-lactamase-carrying OMVs and released β-lactamase-carrying OMVs (inset) in pharyngotonsillar swab. (b) NTHi bacteria surrounded by large amounts of OMVs (top) and released β-lactamase-carrying OMVs (bottom) in tonsillar crypt exudate. (c) β-lactamase-carrying OMVs in saliva. Pictures are ultrathin cryosections stained (except for (b) top which is unstained) with anti-β-lactamase antibody and 10 nm gold-conjugated secondary antibody. Scale bars: (a,c), 200 nm; (b) top, 1 µm, bottom, 100 nm.