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Burkholderia pseudomallei is the causal agent of melioidosis, a deadly tropical infectious disease that lacks a vaccine. On the basis of the attenuated Staphylococcus aureus RN4220-Δagr (RN), we engineered the RN4220-Δagr/pdhB-hcp1 strain (RN-Hcp1) to generate B. pseudomallei hemolysin-coregulated protein 1 (Hcp1)-loaded membrane vesicles (hcp1MVs). The immunization of BALB/c mice with hcp1MVs mixed with adjuvant by a three-dose regimen increased the serum specific IgG production. The serum levels of inflammatory factors, including TNF-α and IL-6, in hcp1MV-vaccinated mice were comparable with those in PBS-challenged mice. The partial adjuvant effect of staphylococcal MVs was observed with the elevation of specific antibody titer in hcp1MV-vaccinated mice relative to those that received the recombinant Hcp1 protein (rHcp1) or MVs derived from RN strain (ΔagrMVs). The hcp1MVs/adjuvant vaccine protected 70% of mice from lethal B. pseudomallei challenge. Immunization with hcp1MVs only protected 60% of mice, whereas vaccination with rHcp1 or ΔagrMVs conferred no protection. Moreover, mice that received hcp1MVs/adjuvant and hcp1MVs immunization had low serum TNF-α and IL-6 levels and no inflammatory infiltration in comparison with other groups. In addition, all surviving mice in hcp1MVs/adjuvant and hcp1MVs groups exhibited no culturable bacteria in their lungs, livers, and spleens five days postinfection. Overall, our data highlighted a new strategy for developing B. pseudomallei vaccine and showed that Hcp1-incorporated staphylococcal MV is a promising candidate for the prevention of acute melioidosis.

The accessory gene regulator (agr) quorum-sensing system is an important global regulatory system of Staphylococcus aureus and contributes to its pathogenicity. The S. aureus agr system is divided into four agr groups based on the amino acid polymorphisms of AgrB, AgrD, and AgrC. The agr activation is group-specific, resulting in variations in agr activity and pathogenicity among the four agr groups. Strains with divergent agr system always have different phenotypes. In the present report, we, respectively, exchanged the agr system of a certain S. aureus with other three agr alleles and assessed the corresponding phenotypes of these congenic strains. Replacement of the agr system led to significant variations in hemolytic activity, protein expression, and virulence gene expression comparing with that of the parental strain. Interestingly, we found that the biological characteristics of these agr congenic strains in the same strain background were highly similar to each other, and the allele-dependent differences of the agr systems were weakened. These findings indicate that the allele-dependent agr predilections of S. aureus are determined by some factors in addition to the polymorphisms of AgrB, AgrD, and AgrC. Future studies may reveal the novel mechanism to improve our understanding of the agr network.

Bacterial membrane vesicles (MVs) are produced by both Gram-positive and Gram-negative bacteria during growth in vitro and in vivo. MVs are nanoscale vesicular structures with diameters ranging from 20 to 400 nm. MVs incorporate bacterial lipids, proteins, and often nucleic acids, and can effectively stimulate host immune response against bacterial infections. As vaccine candidates and drug delivery systems, MVs possess high biosafety owing to the lack of self-replication ability. However, wild-type bacterial strains have poor MV yield, and MVs from the wild-type strains may be harmful due to the carriage of toxic components, such as lipopolysaccharides, hemolysins, enzymes, etc. In this review, we summarize the genetic modification of vesicle-producing bacteria to reduce MV toxicity, enhance vesicle immunogenicity, and increase vesicle production. The engineered MVs exhibit broad applications in vaccine designs, vaccine delivery vesicles, and drug delivery systems.

Most biofilms in nature are formed by multiple microbial species, and such mixed-species biofilms represent the actual lifestyles of microbes, including bacteria, fungi, viruses (phages), and/or protozoa. Microorganisms cooperate and compete in mixed-species biofilms. Mixed-species biofilm formation and environmental resistance are major threats to water supply, food industry, and human health. The methods commonly used for microbial eradication, such as antibiotic or disinfectant treatments, are often ineffective for mixed-species biofilm consortia due to their physical matrix barrier and physiological interactions. For the last decade, an increasing number of investigations have been devoted to the usage of cold atmospheric plasma (CAP), which is produced by dielectric barrier discharges or plasma jets to prevent or eliminate microbial biofilms. Here, we summarized the production of CAP, the inactivation of microorganisms upon CAP treatment, and the microbial factors affecting the efficacy of CAP procedure. The applications of CAP as antibiotic alternative strategies for fighting mixed-species biofilms were also addressed.

Bacterial brain abscesses (BAs) are difficult to treat with conventional antibiotics. Thus, the development of alternative therapeutic strategies for BAs is of high priority. Identifying the virulence determinants that contribute to BA formation induced by Staphylococcus aureus would improve the effectiveness of interventions for this disease. In this study, RT-qPCR was performed to compare the expression levels of 42 putative virulence determinants of S. aureus strains Newman and XQ during murine BA formation, ear colonization, and bacteremia. The alterations in the expression levels of 23 genes were further confirmed through specific TaqMan RT-qPCR. Eleven S. aureus genes that persistently upregulated expression levels during BA infection were identified, and their functions in BA formation were confirmed through isogenic mutant experiments. Bacterial loads and BA volumes in mice infected with isdA, isdC, lgt, hla, or spa deletion mutants and the hla/spa double mutant strain were lower than those in mice infected with the wild-type Newman strain. The therapeutic application of monoclonal antibodies against Hla and SpA decreased bacterial loads and BA volume in mice infected with Newman. This study provides insights into the virulence determinants that contribute to staphylococcal BA formation and a paradigm for antivirulence factor therapy against S. aureus infections.

The emergence of vancomycin-intermediate Staphylococcus aureus (VISA) has raised healthcare concerns worldwide. VISA is often associated with multiple genetic changes. However, the relative contributions of these changes to VISA phenotypes are incompletely defined. We have characterized VISA XN108 with vancomycin MIC of 12 μg/ml. Genome comparison revealed that WalK(S221P), GraS(T136I), and RpoB(H481N) mutations possibly contributed to the VISA phenotype of XN108. In this study, the above mutations were stepwise cured, and the phenotypes between XN108 and its derivates were compared. We constructed four isogenic mutant strains, XN108-WalK(P221S) (termed as K65), XN108-GraS(I136T) (termed as S65), XN108-RpoB(N481H) (termed as B65), and XN108-WalK(P221S)/GraS(I136T) (termed as KS65), using the allelic replacement experiments with the native alleles derived from a vancomycin-susceptible S. aureus isolate DP65. Antimicrobial susceptibility test revealed K65 and S65 exhibited decreased vancomycin resistance, whereas B65 revealed negligibly differed when compared with the wild-type XN108. Sequentially introducing WalK(P221S) and GraS(I136T) completely converted XN108 into a VSSA phenotype. Transmission electronic microscopy and autolysis determination demonstrated that cell wall thickening and decreasing autolysis were associated with the change of vancomycin resistance levels. Compared with XN108, K65 exhibited 577 differentially expressed genes (DEGs), whereas KS65 presented 555 DEGs. Of those DEGs, 390 were common in K65 and KS65, including those upregulated genes responsible for citrate cycle and bacterial autolysis, and the downregulated genes involved in peptidoglycan biosynthesis and teichoic acid modification. In conclusion, a VSSA phenotype could be completely reconstituted from a VISA strain XN108. WalK(S221P) and GraS(T136I) mutations may work synergistically in conferring vancomycin resistance in XN108.