Intra- and Interspecies Effects of Outer Membrane Vesicles from Stenotrophomonas maltophilia on β-Lactam Resistance

Abstract

The treatment of Stenotrophomonas maltophilia infection with β-lactam antibiotics leads to increased release of outer membrane vesicles (OMVs), which are packed with two chromosomally encoded β-lactamases. Here, we show that these β-lactamase–packed OMVs are capable of establishing extracellular β-lactam degradation. We also show that they dramatically increase the apparent MICs of imipenem and ticarcillin for the cohabituating species Pseudomonas aeruginosa and Burkholderia cenocepacia.

TEXT

The multidrug-resistant bacterium Stenotrophomonas maltophilia exploits a variety of mechanisms to resist antibiotic threats, such as the active extrusion of antibiotics by efflux pumps, alteration of cell membrane permeability, shielding by growing as a biofilm, and direct enzymatic inactivation of the antibiotic compounds (1). β-Lactam antibiotics, such as imipenem (IPM), amoxicillin (AMX), and ticarcillin (TIC), are used frequently to treat Gram-negative bacterial infections. Unfortunately, these compounds are often ineffective for S. maltophilia infections due to the presence of two chromosomal β-lactamase genes encoding the L1 metallo-β-lactamase and the L2 serine-β-lactamase, the expression of which immediately increases after exposure (2). In a previous study, we revealed a significant increase in the release of outer membrane vesicles (OMVs) by the clinical S. maltophilia strain 44/98 (LMG 26824) after exposure to the broad-spectrum carbapenem IPM, and we showed that these vesicles are packed with L1 and L2 β-lactamases (3).

In this study, OMVs from penicillin G (PEN)-stimulated cultures (1 mg/ml, sublethal concentration) were used. The culture supernatant was filtered through a syringe-driven 0.22-μm-pore-size polyethersulfone membrane filter unit, and the OMVs were pelleted by ultracentrifugation at 100,000 × g for 1 h. The OMVs were quantified with fluorescent single-particle tracking with the membrane-specific fluorescent PKH67 label (4, 5). The measured increase in levels of secreted membranous particles after exposure to PEN was comparable to that measured after exposure to IPM (17.3-fold increase). PEN-induced OMVs were also subjected to a two-dimensional liquid chromatography–mass spectrometry proteomics study to identify the protein cargo, as previously described (3). The two β-lactamases were again found to be included in the OMVs.

The β-lactamase activity on intact OMVs was examined using a nitrocefin β-lactamase assay. OMVs (isolated from 25 ml PEN-stimulated culture, dissolved in 1 ml phosphate-buffered saline [PBS]) were mixed with 50 μl of a 0.5-mg/ml nitrocefin solution, and the optical density at 490 nm (OD₄₉₀) was measured at different time points. The results show rapid nitrocefin hydrolysis by the β-lactamase–packed OMVs (Fig. 1A). The rate of hydrolysis, derived from the linear part of the curve (5 to 15 min), was calculated as 0.571 μg/min. To assess the contribution of the L1 metallo-β-lactamase, the same assay was performed after incubating the OMVs with the zinc-chelating agent EDTA (6). The initial rate of hydrolysis was then 0.262 μg/min, approximately half of the rate observed without EDTA. This demonstrates the OMV-associated activity of both β-lactamases. The nitrocefin assay was also performed on crude culture lysate. Cells (isolated from 500 μl PEN-stimulated culture, dissolved in 500 μl PBS) were lysed by bead milling, and the clear lysate was transferred to a new Eppendorf tube. The volume was adjusted to 1 ml with PBS for the nitrocefin assay. It was remarkable that the crude culture lysate showed almost complete activity loss after the addition of EDTA (Fig. 1B), which points to a dominant role for L1 in cellular β-lactam resistance, as previously reported (7). However, the OMVs seemed to have had an equal activity distribution between L1 and L2.

FIG 1.

UV visible spectroscopy analysis of nitrocefin hydrolysis (OD₄₉₀) at different time points during incubation with intact PEN-induced OMVs (A) and crude PEN-stimulated culture lysate (B), with and without the addition of EDTA. Error bars show the standard deviation (SD).

As S. maltophilia is often part of polymicrobial communities, we investigated whether OMVs from S. maltophilia influence the tolerance of Pseudomonas aeruginosa, Burkholderia cenocepacia, and itself to the β-lactam antibiotics. Cultures from S. maltophilia strain 44/98, P. aeruginosa strain PAO1 (LMG 24986), and a B. cenocepacia type strain (LMG 16656; 100 μl) were grown in 96-well plates with different concentrations of the antibiotics IPM, AMX, and TIC (0, 0.5, 2.5, 5, 25, 50, 250, 500, and 2,500 μg/ml), with or without OMVs derived from a PEN-stimulated S. maltophilia culture (isolated from a 2.5-ml culture, dissolved in 100 μl fresh Luria Bertani medium). S. maltophilia showed high resistance toward the three β-lactam antibiotics, with growth inhibition at 500, 2,500, and 250 μg/ml of IPM, AMX, and TIC, respectively (Fig. 2, left column). When isolated OMVs (containing β-lactamases) were added, the MICs increased to 2,500 μg/ml for IPM and TIC and were even higher for AMX (>2,500 μg/ml). The effects of the antibiotics and the OMVs on P. aeruginosa and B. cenocepacia were very much alike (Fig. 2, middle and right columns). Both species were as resistant to AMX as S. maltophilia, and the addition of the OMVs also led to an increased MIC (>2,500 μg/ml). P. aeruginosa and B. cenocepacia are naturally less resistant to IPM and TIC, but the presence of S. maltophilia OMVs drastically increased the antibiotic tolerance of these species. A 100-fold increase in MICs was observed, from 2.5 to 250 μg/ml and 25 to 2,500 μg/ml for IPM and TIC, respectively.

FIG 2.

OD₆₀₀ of S. maltophilia, P. aeruginosa, and B. cenocepacia cultures exposed to different concentrations of IPM, AMX, and TIC in the absence and in the presence of PEN-induced OMVs. Error bars show the SD.

We have shown here that S. maltophilia β-lactamase–packed OMVs indeed exhibit β-lactamase activity. Moreover, the OMVs provide the enzymes shelter against proteases, keeping them stable and active for longer periods (8). The extracellular β-lactamase activity associated with the OMVs can thereby also affect other species, possible cohabitants in polymicrobial communities. It was shown previously that S. maltophilia often lives together with the species P. aeruginosa and B. cenocepacia, especially in the lungs of cystic fibrosis patients, where these species are found in polymicrobial biofilm communities (9). In conclusion, the exposure of S. maltophilia to β-lactam antibiotics leads to the secretion of β-lactamase–packed OMVs, which in turn can protect not only other S. maltophilia cells but also P. aeruginosa and B. cenocepacia against β-lactam antibiotics. Although it is not clear whether S. maltophilia can be considered a true cystic fibrosis pathogen (10), its ability to secrete OMVs after antibiotic stress can influence the susceptibility of the pathogens to antibiotic treatment.