"One for All": Functional Transfer of OMV-Mediated Polymyxin B Resistance From Salmonella enterica sv. Typhi ΔtolR and ΔdegS to Susceptible Bacteria.

The appearance of multi-resistant strains has contributed to reintroducing polymyxin as the last-line therapy. Although polymyxin resistance is based on bacterial envelope changes, other resistance mechanisms are being reported. Outer membrane vesicles (OMVs) are nanosized proteoliposomes secreted from the outer membrane of Gram-negative bacteria. In some bacteria, OMVs have shown to provide resistance to diverse antimicrobial agents either by sequestering and/or expelling the harmful agent from the bacterial envelope. Nevertheless, the participation of OMVs in polymyxin resistance has not yet been explored in S. Typhi, and neither OMVs derived from hypervesiculating mutants. In this work, we explored whether OMVs produced by the hypervesiculating strains Salmonella Typhi ΔrfaE (LPS synthesis), ΔtolR (bacterial envelope) and ΔdegS (misfolded proteins and σ E activation) exhibit protective properties against polymyxin B. We found that the OMVs extracted from S. Typhi ΔtolR and ΔdegS protect S. Typhi WT from polymyxin B in a concentration-depending manner. By contrast, the protective effect exerted by OMVs from S. Typhi WT and S. Typhi ΔrfaE is much lower. This effect is achieved by the sequestration of polymyxin B, as assessed by the more positive Zeta potential of OMVs with polymyxin B and the diminished antibiotic's availability when coincubated with OMVs. We also found that S. Typhi ΔtolR exhibited an increased MIC of polymyxin B. Finally, we determined that S. Typhi ΔtolR and S. Typhi ΔdegS, at a lesser level, can functionally and transiently transfer the OMV-mediated polymyxin B resistance to susceptible bacteria in cocultures. This work shows that mutants in genes related to OMVs biogenesis can release vesicles with improved abilities to protect bacteria against membrane-active agents. Since mutations affecting OMV biogenesis can involve the bacterial envelope, mutants with increased resistance to membrane-acting agents that, in turn, produce protective OMVs with a high vesiculation rate (e.g., S. Typhi ΔtolR) can arise. Such mutants can functionally transfer the resistance to surrounding bacteria via OMVs, diminishing the effective concentration of the antimicrobial agent and potentially favoring the selection of spontaneous resistant strains in the environment. This phenomenon might be considered the source for the emergence of polymyxin resistance in an entire bacterial community.

Identification of Genes Involved in Biogenesis of Outer Membrane Vesicles (OMVs) in Salmonella enterica Serovar Typhi.

Outer membrane vesicles (OMVs) are nano-sized proteoliposomes discharged from the cell envelope of Gram-negative bacteria. OMVs normally contain toxins, enzymes and other factors, and are used as vehicles in a process that has been considered a generalized, evolutionarily conserved delivery system among bacteria. Furthermore, OMVs can be used in biotechnological applications that require delivery of biomolecules, such as vaccines, remarking the importance of their study. Although it is known that Salmonella enterica serovar Typhi (S. Typhi), the etiological agent of typhoid fever in humans, delivers toxins (e.g., HlyE) via OMVs, there are no reports identifying genetic determinants of the OMV biogenesis in this serovar. In the present work, and with the aim to identify genes participating in OMV biogenesis in S. Typhi, we screened 15,000 random insertion mutants for increased HlyE secretion. We found 9 S. Typhi genes (generically called zzz genes) determining an increased HlyE secretion that were also involved in OMV biogenesis. The genes corresponded to ompA, nlpI, and tolR (envelope stability), rfaE and waaC (LPS synthesis), yipP (envC), mrcB (synthesis and remodeling of peptidoglycan), degS (stress sensor serine endopeptidase) and hns (global transcriptional regulator). We found that S. Typhi Δzzz mutants were prone to secrete periplasmic, functional proteins with a relatively good envelope integrity. In addition, we showed that zzz genes participate in OMV biogenesis, modulating different properties such as OMV size distribution, OMV yield and OMV protein cargo.

Deletion of the rfaE gene in Haemophilus parasuis SC096 strain attenuates serum resistance, adhesion and invasion.

In Haemophilus parasuis, the lipooligosaccharide (LOS) has been identified as an important virulence factor. The rfa gene cluster encodes enzymes for LOS core biosynthesis. In order to investigate the role of the rfaE gene, we generated an rfaE deficient mutant (ΔrfaE) of a H. parasuis SC096 by a natural transformation method. The purified preparation of LOS from the ΔrfaE mutant strain showed truncated LOS structure on silver-stained SDS-PAGE. Compared to the wild-type SC096 strain, the generation time of ΔrfaE mutant strain was significantly extended from 59 min to 69 min. The ΔrfaE mutant strain caused an approximately 30-fold reductions in survival rate in 50% sera and 36-fold reductions in survival rate in 90% sera, respectively (p < 0.001). In adhesion and invasion assays, the ΔrfaE mutant strain had 10-fold less efficient adherence and 12-fold reductions in invasion of the porcine umbilicus vein endothelial cells (PUVEC) and porcine kidney epithelial cells (PK-15), respectively (p < 0.001). However, the complemented strain could restore the above phenotypes. Hence, the above results suggested that the rfaE gene participated in the pathogenicity of H. parasuis SC096 strain.