Low-Temperature Adaptive Single-Atom Iron Nanozymes against Viruses in the Cold Chain.

Outbreaks of viral infectious diseases, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A virus (IAV), pose a great threat to human health. Viral spread is accelerated worldwide by the development of cold chain logistics; Therefore, an effective antiviral approach is required. In this study, it is aimed to develop a distinct antiviral strategy using nanozymes with low-temperature adaptability, suitable for cold chain logistics. Phosphorus (P) atoms are added to the remote counter position of Fe-N-C center to prepare FeN4P2-single-atom nanozymes (SAzymes), exhibiting lipid oxidase (OXD)-like activity at cold chain temperatures (-20, and 4 °C). This feature enables FeN4P2-SAzymes to disrupt multiple enveloped viruses (human, swine, and avian coronaviruses, and H1-H11 subtypes of IAV) by catalyzing lipid peroxidation of the viral lipid envelope. Under the simulated conditions of cold chain logistics, FeN4P2-SAzymes are successfully applied as antiviral coatings on outer packaging and personal protective equipment; Therefore, FeN4P2-SAzymes with low-temperature adaptability and broad-spectrum antiviral properties may serve as key materials for developing specific antiviral approaches to interrupt viral transmission through the cold chain.

Melatonin attenuates lipopolysaccharide-induced immune dysfunction in dendritic cells.

Melatonin, a ubiquitous hormone, is principally secreted from pineal gland in mammals and possesses strong antioxidant and anti-inflammatory properties. However, its specific roles in the immune functions of dendritic cells (DCs) during acute lung injury (ALI) remain unknown. In this study, we found that melatonin restored the body weight, decreased the lung weight/body weight ratio, alleviated the histopathological lung injury, and decreased the levels of cytokines (tumor necrosis factor-α (TNF-α), interleukin (IL)-12p70, IL-17, and IL-10) in bronchoalveolar lavage fluid of the lipopolysaccharide (LPS)-induced ALI murine model. Moreover, melatonin inhibited the major histocompatibility complex II (MHCII) expression of lung CD11b+ DCs after LPS challenge in vivo. In vitro, melatonin reversed the shape index, promoted the endocytosis, and inhibited phenotypic expression of MHCII, CD40, CD80, and CD86 in LPS-activated DCs. Furthermore, melatonin decreased the expression of an activated marker, CD69, and the secretion of pro-inflammatory cytokines (TNF-α, IL-12p70, and IL-17) after LPS challenge. It hampered the LPS-activated DCs migration by downregulating the C-C chemokine receptor 7 (CCR7) expression, and then weakened the ability of LPS-induced DCs to stimulate allogeneic CD4+ T cell proliferation. Melatonin shaped the immune function of DCs in a nuclear factor erythroid-2-related factor 2 (Nrf-2)/heme oxygenase-1 (HO-1) axis-dependent manner. These findings indicate that melatonin protects DCs from ALI-induced immunological stress and may be used to develop novel DC-targeting strategies for ALI therapy.

Bidirectionally Regulating Viral and Cellular Ferroptosis with Metastable Iron Sulfide Against Influenza Virus.

Influenza virus with numerous subtypes and frequent variation limits the development of high-efficacy and broad-spectrum antiviral strategy. Here, a novel multi-antiviral metastable iron sulfides (mFeS) against various influenza A/B subtype viruses is developed. This work finds that mFeS induces high levels of lipid peroxidation and •OH free radicals in the conservative viral envelope, which depends on Fe2+ . This phenomenon, termed as a viral ferroptosis, results in the loss of viral infectibility and pathogenicity in vitro and in vivo, respectively. Furthermore, the decoction of mFeS (Dc(mFeS)) inhibits cellular ferroptosis-dependent intracellular viral replication by correcting the virus-induced reprogrammed sulfur metabolism, a conserved cellular metabolism. Notably, personal protective equipment (PPE) that is loaded with mFeS provides good antiviral protection. Aerosol administration of mFeS combined with the decoction (mFeS&Dc) has a potential therapeutic effect against H1N1 lethal infection in mice. Collectively, mFeS represents an antiviral alternative with broad-spectrum activity against intracellular and extracellular influenza virus.

PA-X protein of H9N2 subtype avian influenza virus suppresses the innate immunity of chicken bone marrow-derived dendritic cells.

H9N2 subtype avian influenza (AI) is an infectious disease associated with immunosuppression in poultry. Here, the regulation function of PA-X protein was determined on the host innate immune response of H9N2-infected chicken bone marrow-derived DCs (chBM-DCs). Based on 2 mutated viruses expressing PA-X protein (rTX) or deficient PA-X protein (rTX-FS), and the established culture system of chBM-DCs, results showed PA-X protein inhibited viral replication in chBM-DCs but not in non-immune chicken cells (DF-1). Moreover, PA-X protein downregulated the expression of phenotypic markers (CD40, CD86, and MHCII) and proinflammatory cytokine (IL-12 and IL-1ß) of chBM-DCs. The mixed lymphocyte reaction between chBM-DCs and chicken T cells showed PA-X protein significantly decreased H9N2-infected chBM-DCs to induce T cell proliferation, implying a suppression of the DC-induced downstream T cell response. Taken together, these findings indicated that PA-X protein is a key viral protein to help H9N2 subtype AIVs escape the innate immunity of chBM-DCs.

Melatonin Suppresses LPS-Induced Oxidative Stress in Dendritic Cells for Inflammatory Regulation via the Nrf2/HO-1 Axis.

Melatonin, an indoleamine synthesized in the pineal gland of mammals, is a natural bioactive compound with powerful antioxidant and anti-inflammatory properties. Here, we evaluated whether melatonin has the capacity to moderate the oxidative stress of dendritic cells (DCs) for inflammatory control in an acute lung injury (ALI) model. Our findings showed that melatonin remarkably inhibited total nitric oxide synthase (T-NOS) activity, nitric oxide (NO) production, intracellular reactive oxygen species (ROS) levels, and lipid peroxidation (MDA detection) levels in both an LPS-induced murine ALI model and LPS-induced DCs. Meanwhile, the reduced glutathione (GSH) level and the GSH/GSSG ratio were recovered. In addition, antioxidant enzymes, such as glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD), were increased in these processes. Moreover, melatonin also inhibited the LPS-induced secretions of IL-1ß, IL-6, and TGF-ß in vivo and in vitro. Finally, we found that the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) axis was required in the inhibition of LPS-induced oxidative stress in DCs by melatonin. Altogether, these data indicate that melatonin strongly suppresses the LPS-induced oxidative stress in DCs, which is a promising DC-targeted strategy via inflammatory control for ALI treatment.

PA-X protein of H1N1 subtype influenza virus disables the nasal mucosal dendritic cells for strengthening virulence.

PA-X protein arises from a ribosomal frameshift in the PA of influenza A virus (IAV). However, the immune regulatory effect of the PA-X protein of H1N1 viruses on the nasal mucosal system remains unclear. Here, a PA-X deficient H1N1 rPR8 viral strain (rPR8-△PAX) was generated and its pathogenicity was determined. The results showed that PA-X was a pro-virulence factor in mice. Furthermore, it reduced the ability of H1N1 viruses to infect dendritic cells (DCs), the regulator of the mucosal immune system, but not non-immune cells (DF-1 and Calu-3). Following intranasal infection of mice, CCL20, a chemokine that monitors the recruitment of submucosal DCs, was downregulated by PA-X, resulting in an inhibition of the recruitment of CD11b+ DCs to submucosa. It also attenuated the migration of CCR7+ DCs to cervical lymph nodes and inhibited DC maturation with low MHC II and CD40 expression. Moreover, PA-X suppressed the maturation of phenotypic markers (CD80, CD86, CD40, and MHC II) and the levels of secreted pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α) while enhancing endocytosis and levels of anti-inflammatory IL-10 in vitro, suggesting an impaired maturation of DCs that the key step for the activation of downstream immune responses. These findings suggested that the PA-X protein played a critical role in escaping the immune response of nasal mucosal DCs for increasing the virulence of H1N1 viruses.

H5N8 Subtype avian influenza virus isolated from migratory birds emerging in Eastern China possessed a high pathogenicity in mammals.

Novel H5N8 highly pathogenic avian influenza viruses (HPAIVs) bearing the clade 2.3.4.4b HA gene have been widely spread through wild migratory birds since 2020. One H5N8 HPAIV (A/Wild bird/Cixi/Cixi02/2020; here after Cixi02) was isolated from migratory birds in Zhejiang Province, Eastern China in 25 November 2020. However, its pathogenicity in avian and mammal remains unknown. Hemagglutinin gene genetic analysis indicated that Cixi02 virus belonged to the branch II of H5 clade 2.3.4.4b originated from Iraq in May 2020. Cixi02 virus showed a binding affinity to both SA α-2, 3-galactose (Gal) and SA α-2, 6 Gal receptors, good pH stability, thermostability, and replication ability in both avian and mammal cells. The poultry pathogenicity indicated that Cixi02 virus was lethal to chickens. Moreover, the mammalian pathogenicity showed that the 50% mouse lethal dose (MLD50 ) is 2.14 lgEID50 /50 µl, indicating a high pathogenicity in mice. Meanwhile, Cixi02 virus was widely detected in multiple organs, including heart, liver, spleen, lung, kidney, turbinate, and brain after nasal infection. In addition, we found high level gene expressions of TNF-α, IL-12p70, CXCL10, and IFN-α in lungs, IL-8 and IL-1ß in brains, and observed severe histopathological change in lungs and brains. Collectedly, this study provided new insights on the pathogenic and zoonotic features of an H5N8 subtype AIV isolated from migratory birds.

PA-X protein assists H9N2 subtype avian influenza virus in escaping immune response of mucosal dendritic cells.

H9N2 subtype low pathogenicity avian influenza virus (AIV) poses a potential zoonotic risk. PA-X, a novel protein generated by PA gene ribosomal frameshift, is considered to be the virulence factor of H9N2 subtype AIVs. Our study found that rTX possessing PA-X protein enhanced the mammalian pathogenicity of H9N2 subtype AIVs compared with PA-X-deficient virus (rTX-FS). Furthermore, PA-X protein inhibited H9N2 subtype AIVs to infect dendritic cells (DCs), but not nonimmune cells (MDCK cells). Meanwhile, PA-X protein suppressed the phenotypic expression (CD80, CD86, CD40 and MHCII), early activation marker (CD69) and pro-inflammatory cytokines (IL-6 and TNF-α), whereas increased anti-inflammatory cytokine (IL-10) in DCs. After intranasally viral infection in mice, we found that PA-X protein of H9N2 subtype AIVs reduced CD11b+ and CD103+ subtype mucosal DCs recruitment to the nasal submucosa by inhibiting CCL20 expression. Moreover, PA-X protein abolished the migratory ability of CD11b+ and CD103+ DCs into draining cervical lymph nodes by down-regulating CCR7 expression. The rTX-infected DCs significantly impaired the allogeneic CD4+ T cell proliferation, suggesting PA-X protein suppressed the immune functions of DCs for hindering the downstream immune activation. These findings indicated that PA-X protein assisted H9N2 subtype AIVs in escaping immune response of mucosal DCs for enhancing the pathogenicity.

Preparation of eugenol nanoemulsions for antibacterial activities.

Eugenol is a versatile plant essential oil, but its high volatility and low water solubility greatly limit its application. Accordingly, this study prepared eugenol nanoemulsions by a high-speed shearing technique. Through visual inspection and a series of characterizations, including dynamic light scattering, and confocal laser scanning microscopy, the optimized formula was determined to be 5% (w/w) oil phase (eugenol) and 8% (w/w) surfactant (Tween-80), and the optimized shearing time was 5 min. The optimized nanoemulsion had good stability, small droplets (85 nm), and uniform distribution. At a concentration of 0.02 mg µL-1, the nanoemulsion showed strong inhibition against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Scanning electron microscopy (SEM) images showed severe deformation and membrane rupture of both bacteria treated by the nanoemulsion. This result was further confirmed by the leakage of proteins in both bacteria after treatment. The results of reactive oxygen species (ROS) and malondialdehyde (MDA) measurements indicated that the increased levels of ROS in both bacteria treated by the nanoemulsion triggered lipid peroxidation, thus increasing the MDA levels, ultimately causing changes in cell membrane permeability and disruption of the membrane structure. In addition, the nanoemulsion had a small effect on the proliferation and apoptosis of hepatocytes (L02) and lung cells (BEAS-2B), indicating its good biocompatibility. In this study, we developed a novel eugenol nanoemulsion with high stability and good biological activity, which may provide a promising and effective method for wound treatment in the healthcare area.

Generation of an avian influenza DIVA vaccine with a H3-peptide replacement located at HA2 against both highly and low pathogenic H7N9 virus.

A differentiating infected from vaccinated animals (DIVA) vaccine is an ideal strategy for viral eradication in poultry. Here, according to the emerging highly pathogenic H7N9 avian influenza virus (AIV), a DIVA vaccine strain, named rGD4HALo-mH3-TX, was successfully developed, based on a substituted 12 peptide of H3 virus located at HA2. In order to meet with the safety requirement of vaccine production, the multi-basic amino acid located at the HA cleavage site was modified. Meanwhile, six inner viral genes from a H9N2 AIV TX strainwere introduced for increasing viral production. The rGD4HALo-mH3-TX strain displayed a similar reproductive ability with rGD4 and low pathogenicity in chickens, suggesting a good productivity and safety. In immuned chickens, rGD4HALo-mH3-TX induced a similar antibody level with rGD4 and provided 100% clinical protection and 90% shedding protection against highly pathogenic virus challenge. rGD4HALo-mH3-TX strain also produced a good cross-protection against low pathogenic AIV JD/17. Moreover, serological DIVA characteristics were evaluated by a successfully established competitive inhibition ELISA based on a 3G10 monoclonal antibody, and the result showed a strong reactivity with antisera of chickens vaccinated with H7 subtype strains but not rGD4HALo-mH3-TX. Collectedly, rGD4HALo-mH3-TX is a promising DIVA vaccine candidate against both high and low pathogenic H7N9 subtype AIV.

Riboflavin as a Mucosal Adjuvant for Nasal Influenza Vaccine.

Intranasal immunization with whole inactivated virus (WIV) is an important strategy used for influenza prevention and control. However, a powerful mucosal adjuvant is required to improve nasal vaccine efficacy. Riboflavin, as a food additive with the advantages of being safe and low-cost, widely exists in living organisms. In this paper, the mucosal adjuvant function of riboflavin was studied. After intranasal immunization with H1N1 WIV plus riboflavin in mice, we found that the mucosal immunity based on the secretory IgA (sIgA) levels in the nasal cavity, trachea, and lung were strongly enhanced compared with H1N1 WIV alone. Meanwhile, the IgG, IgG1, and IgG2a levels in serum also showed a high upregulation and a decreased ratio of IgG1/IgG2a, which implied a bias in the cellular immune response. Moreover, riboflavin strongly improved the protection level of H1N1 inactivated vaccine from a lethal influenza challenge. Furthermore, riboflavin was found to possess the capacity to induce dendritic cell (DC) phenotypic (MHCII, CD40, CD80, and CD86) and functional maturation, including cytokine secretion (TNF-α, IL-1ß, IL-12p70, and IL-10) and the proliferation of allogeneic T cells. Lastly, we found that the DC maturation induced by riboflavin was dependent on the activation of the mitogen-activated protein kinase (MAPK) signaling pathway, which plays an important role in immune regulation. Therefore, riboflavin is expected to be developed as an alternative mucosal adjuvant for influenza nasal vaccine application.

Astaxanthin Provides Antioxidant Protection in LPS-Induced Dendritic Cells for Inflammatory Control.

Astaxanthin, originating from marine organisms, is a natural bioactive compound with powerful antioxidant activity. Here, we evaluated the antioxidant ability of astaxanthin on dendritic cells (DCs), a key target of immune regulation, for inflammatory control in a sepsis model. Our results showed that astaxanthin suppressed nitric oxide (NO) production, reactive oxygen species (ROS) production, and lipid peroxidation activities in LPS-induced DCs and LPS-challenged mice. Moreover, the reduced glutathione (GSH) levels and the GSH/GSSG ratio were increased, suggesting that astaxanthin elevated the level of cellular reductive status. Meanwhile, the activities of antioxidant enzymes, including glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD), were significantly upregulated. Astaxanthin also inhibited the LPS-induced secretions of IL-1ß, IL-17, and TGF-ß cytokines. Finally, we found that the expressions of heme oxygenase 1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf2) were significantly upregulated by astaxanthin in LPS-induced DCs, suggesting that the HO-1/Nrf2 pathway plays a significant role in the suppression of oxidative stress. These results suggested that astaxanthin possesses strong antioxidant characteristics in DC-related inflammatory responses, which is expected to have potential as a method of sepsis treatment.

Astaxanthin Protects Dendritic Cells from Lipopolysaccharide-Induced Immune Dysfunction.

Astaxanthin, originating from seafood, is a naturally occurring red carotenoid pigment. Previous studies have focused on its antioxidant properties; however, whether astaxanthin possesses a desired anti-inflammatory characteristic to regulate the dendritic cells (DCs) for sepsis therapy remains unknown. Here, we explored the effects of astaxanthin on the immune functions of murine DCs. Our results showed that astaxanthin reduced the expressions of LPS-induced inflammatory cytokines (TNF-α, IL-6, and IL-10) and phenotypic markers (MHCII, CD40, CD80, and CD86) by DCs. Moreover, astaxanthin promoted the endocytosis levels in LPS-treated DCs, and hindered the LPS-induced migration of DCs via downregulating CCR7 expression, and then abrogated allogeneic T cell proliferation. Furthermore, we found that astaxanthin inhibited the immune dysfunction of DCs induced by LPS via the activation of the HO-1/Nrf2 axis. Finally, astaxanthin with oral administration remarkably enhanced the survival rate of LPS-challenged mice. These data showed a new approach of astaxanthin for potential sepsis treatment through avoiding the immune dysfunction of DCs.

Mucosal Vaccination for Influenza Protection Enhanced by Catalytic Immune-Adjuvant.

Influenza poses a severe threat to global health. Despite the whole inactivated virus (WIV)-based nasal vaccine being a promising strategy for influenza protection, the mucosal barrier is still a bottleneck of the nasal vaccine. Here, a catalytic mucosal adjuvant strategy for an influenza WIV nasal vaccine based on chitosan (CS) functionalized iron oxide nanozyme (IONzyme) is developed. The results reveal that CS-IONzyme increases antigen adhesion to nasal mucosa by 30-fold compared to H1N1 WIV alone. Next, CS-IONzyme facilitates H1N1 WIV to enhance CCL20-driven submucosal dendritic cell (DC) recruitment and transepithelial dendrite(TED) formation for viral uptake via the toll-like receptor(TLR) 2/4-dependent pathway. Moreover, IONzyme with enhanced peroxidase (POD)-like activity by CS modification catalyzes a reactive oxygen species (ROS)-dependent DC maturation, which further enhances the migration of H1N1 WIV-loaded DCs into the draining lymph nodes for antigen presentation. Finally, CS-IONzyme-based nasal vaccine triggers an 8.9-fold increase of IgA-mucosal adaptive immunity in mice, which provides a 100% protection against influenza, while only a 30% protection by H1N1 WIV alone. This work provides an antiviral alternative for designing nasal vaccines based on IONzyme to combat influenza infection.

Catalytic inactivation of influenza virus by iron oxide nanozyme.

Influenza poses a severe threat to human health in the world. However, developing a universal anti-viral strategy has remained challenging due to the presence of diverse subtypes as well as its high mutation rate, resulting in antigenic shift and drift. Here we developed an antiviral strategy using iron oxide nanozymes (IONzymes) to target the lipid envelope of the influenza virus. Methods: We evaluated the antiviral activities of our IONzymes using a hemagglutination assay, together with a 50% tissue culture infectious doses (TCID50) method. Lipid peroxidation of the viral envelope was analyzed using a maleic dialdehyde (MDA) assay and transmission electron microscopy (TEM). The neighboring viral proteins were detected by western blotting. Results: We show that IONzymes induce envelope lipid peroxidation and destroy the integrity of neighboring proteins, including hemagglutinin, neuraminidase, and matrix protein 1, causing the inactivation of influenza A viruses (IAVs). Furthermore, we show that our IONzymes possess a broad-spectrum antiviral activity on 12 subtypes of IAVs (H1~H12). Lastly, we demonstrate that applying IONzymes to a facemask improves the ability of virus protection against 3 important subtypes that pose a threat to human, including H1N1, H5N1, and H7N9 subtype. Conclusion: Together, our results clearly demonstrate that IONzymes can catalyze lipid peroxidation of the viral lipid envelope to inactivate enveloped viruses and provide protection from viral transmission and infection.

Membrane Vesicles Are the Dominant Structural Components of Ceftazidime-Induced Biofilm Formation in an Oxacillin-Sensitive MRSA.

Methicillin-resistant Staphylococcus aureus (MRSA) has received increasing attention in recent years. However, the characteristics and relevant mechanisms of biofilm formation in oxacillin-sensitive MRSA (OS-MRSA) are poorly understood. This study was designed to characterize biofilm formation in OS-MRSA BWSA15 in response to ceftazidime (TZ) by comparing the methicillin-sensitive S. aureus (MSSA) strain BWSA23 and the oxacillin-resistant MRSA (OR-MRSA) strain BWSA11. The biofilms and biofilm-forming cells were observed by electron microscopy. Biofilms grown on microtiter plates were chemically decomposed and analyzed by Fourier transform infrared spectroscopy. The transcriptional regulation of genes associated with methicillin resistance, surface adhesion, fatty acid biosynthesis, and global regulation (sigma B) was investigated. A significant increase in biofilm formation ability (10.21-fold) and aggregation ability (2.56-fold) was observed in BWSA15 upon the treatment with TZ (16 µg/ml). The TZ-induced biofilm formation in BWSA15 was characterized by a disappearance of polysaccharide-like extracellular substances and an appearance of a large number of intercellular MVs from extracellular matrix. Few MVs were identified in the biofilms formed by BWSA11 and BWSA23. There was a significant upregulation of mecA, sigB, and fatty acid biosynthesis-associated genes and downregulation of icaA, icaD, clfA, clfB, and fnaA in BWSA15 upon the treatment with TZ. The formation of intracellular junctions of MVs in the biofilms of BWSA15 was mediated by a significant increase in the proportion of proteins as well as by an increase in the proportion of non-ionized carboxyl groups in fatty acids. This study demonstrated that beta-lactam antibiotics can induce biofilm formation in OS-MRSA, and the biofilm induction in OS-MRSA can mainly be attributed to exposed MVs with increased hydrophobicity rather than polysaccharide intercellular adhesins, cell wall-anchored surface proteins, and extracellular DNA.

Design and immune characterization of a novel Neisseria gonorrhoeae DNA vaccine using bacterial ghosts as vector and adjuvant.

Gonorrhea, an important sexually transmitted disease, is becoming a growing public health problem around the globe. Vaccination is considered the best long-term approach for control of infection. In this study, we designed a novel Neisseria gonorrhoeae (N. gonorrhoeae) DNA vaccine delivered by bacterial ghosts and characterized its immune responses in vitro and in vivo. Our results demonstrate that bacterial ghosts greatly promoted BMDCs maturation and activation. Bacterial ghosts loaded with N. gonorrhoeae DNA vaccine were efficiently taken up by mouse macrophage RAW264.7 cells. Furthermore, oral immunization with the ghost vaccine candidate elicited greater CD4+ and CD8+ T cell responses and induced higher IgG responses than N. gonorrhoeae DNA vaccine alone. In addition, mice immunized with the vaccine candidate responded with a significant rise in bactericidal antibody titer. These results suggest that bacterial ghosts may function as a vaccine adjuvant by promoting BMDCs maturation, which in turn enhances the immune responses to the vaccine antigens. This study also highlights the potential of using bacterial ghosts as antigen delivery system in the development of an efficacious gonorrhea vaccine.

Vancomycin-induced biofilm formation by methicillin-resistant Staphylococcus aureus is associated with the secretion of membrane vesicles.

Chronic burn wound infections caused by Stapyhylococcus aureus (S. aureus) are largely associated with biofilm formation. However, the mechanism by which S. aureus form biofilm in clinical environments is far less understood. In the present study we addressed the association between biofilm formation and membrane vesicle (MV) secretion of S. aureus during vancomycin treatment. A representative methicillin-resistant S. aureus (MRSA) strain BWMR22 obtained from a chronic burn wound was used in this study. Transmission electron microscope was used to observe the MV secretion. Fourier transform infrared spectroscopy was used to analyze the chemical component of MV. Biofilm formation was assayed under conditions of sub-inhibitory concentrations of vancomycin. Functional potencies of MV in surface adhesion and auto-aggregation were assayed in the presence of additional purified MVs. Biofilm formation by S. aureus BWMR22 was enhanced in the presence of sub-inhibitory concentration of vancomycin. Vancomycin treatment caused an increase in the chemical composition of protein relative to carbohydrates of secreted MVs, a property which was highly associated with bacterial hydrophobicity, surface adhesion, and intercellular aggregation. These findings suggest that MV secretion is correlated with biofilm formation by MRSA especially under clinical conditions with improper vancomycin chemotherapy. This study first demonstrates a potential role of MVs in the biofilm formation by S. aureus, stresses on the importance of avoiding low dose of antibiotic therapy in controlling of S. aureus infections, and provides further information to reveal the mechanisms behind MRSA infections.