Staphylococcus Aureus Membrane Vesicles Kill Tumor Cells Through a Caspase-1-Dependent Pyroptosis Pathway.

Introduction: Nanosized outer membrane vesicles (OMVs) from Gram-negative bacteria have attracted increasing interest because of their antitumor activity. However, the antitumor effects of MVs isolated from Gram-positive bacteria have rarely been investigated. Methods: MVs of Staphylococcus aureus USA300 were prepared and their antitumor efficacy was evaluated using tumor-bearing mouse models. A gene knock-in assay was performed to generate luciferase Antares2-MVs for bioluminescent detection. Cell counting kit-8 and lactic dehydrogenase release assays were used to detect the toxicity of the MVs against tumor cells in vitro. Active caspase-1 and gasdermin D (GSDMD) levels were determined using Western blot, and the tumor inhibition ability of MVs was determined in B16F10 cells treated with a caspase-1 inhibitor. Results: The vesicular particles of S. aureus USA300 MVs were 55.23 ± 8.17 nm in diameter, and 5 µg of MVs remarkably inhibited the growth of B16F10 melanoma in C57BL/6 mice and CT26 colon adenocarcinoma in BALB/c mice. The bioluminescent signals correlated well with the concentrations of the engineered Antares2-MVs (R2 = 0.999), and the sensitivity for bioluminescence imaging was 4 × 10-3 µg. Antares2-MVs can directly target tumor tissues in vivo, and 20 µg/mL Antares2-MVs considerably reduced the growth of B16F10 and CT26 tumor cells, but not non-carcinomatous bEnd.3 cells. MV treatment substantially increased the level of active caspase-1, which processes GSDMD to trigger pyroptosis in tumor cells. Blocking caspase-1 activation with VX-765 significantly protected tumor cells from MV killing in vitro and in vivo. Conclusion: S. aureus MVs can kill tumor cells by activating the pyroptosis pathway, and the induction of pyroptosis in tumor cells is a promising strategy for cancer treatment.

LysSYL: a broad-spectrum phage endolysin targeting Staphylococcus species and eradicating S. aureus biofilms.

BACKGROUND: Staphylococcus aureus and its single or mixed biofilm infections seriously threaten global public health. Phage therapy, which uses active phage particles or phage-derived endolysins, has emerged as a promising alternative strategy to antibiotic treatment. However, high-efficient phage therapeutic regimens have yet to be established. RESULTS: In this study, we used an enrichment procedure to isolate phages against methicillin-resistant S. aureus (MRSA) XN108. We characterized phage SYL, a new member of the Kayvirus genus, Herelleviridae family. The phage endolysin LysSYL was expressed. LysSYL demonstrated stability under various conditions and exhibited a broader range of efficacy against staphylococcal strains than its parent phage (100% vs. 41.7%). Moreover, dynamic live/dead bacterial observation demonstrated that LysSYL could completely lyse MRSA USA300 within 10 min. Scan and transmission electron microscopy revealed evident bacterial cell perforation and deformation. In addition, LysSYL displayed strong eradication activity against single- and mixed-species biofilms associated with S. aureus. It also had the ability to kill bacterial persisters, and proved highly effective in eliminating persistent S. aureus when combined with vancomycin. Furthermore, LysSYL protected BALB/c mice from lethal S. aureus infections. A single-dose treatment with 50 mg/kg of LysSYL resulted in a dramatic reduction in bacterial loads in the blood, liver, spleen, lungs, and kidneys of a peritonitis mouse model, which resulted in rescuing 100% of mice challenged with 108 colony forming units of S. aureus USA300. CONCLUSIONS: Overall, the data provided in this study highlight the strong therapeutic potential of endolysin LysSYL in combating staphylococcal infections, including mono- and mixed-species biofilms related to S. aureus.

Sub-MIC vancomycin enhances the antibiotic tolerance of vancomycin-intermediate Staphylococcus aureus through downregulation of protein succinylation.

Bacteria develop tolerance after transient exposure to antibiotics, and tolerance is a significant driver of resistance. The purpose of this study is to evaluate the mechanisms underlying tolerance formation in vancomycin-intermediate Staphylococcus aureus (VISA) strains. VISA strains were cultured with sub-minimum inhibitory concentrations (sub-MICs) of vancomycin. Enhanced vancomycin tolerance was observed in VISA strains with distinct genetic lineages. Western blot revealed that the VISA protein succinylation (Ksucc) levels decreased with the increase in vancomycin exposure. Importantly, Ksucc modification, vancomycin tolerance, and cell wall synthesis were simultaneously affected after deletion of SacobB, which encodes a desuccinylase in S. aureus. Several Ksucc sites were identified in MurA, and vancomycin MIC levels of murA mutant and Ksucc-simulated (MurA(K69E) and MurA(K191E)) mutants were reduced. The vancomycin MIC levels of K65-MurA(K191E) in particular decreased to 1 mg/L, converting VISA strain K65 to a vancomycin-susceptible S. aureus strain. We further demonstrated that the enzymatic activity of MurA was dependent on Ksucc modification. Our data suggested the influence of vancomycin exposure on bacterial tolerance, and protein Ksucc modification is a novel mechanism in regulating vancomycin tolerance.

GehB Inactivates Lipoproteins to Delay the Healing of Acute Wounds Infected with Staphylococcus aureus.

Staphylococcus aureus is one of the most prevalent bacteria found in acute wounds. S. aureus produces many virulence factors and extracellular enzymes that contribute to bacterial survival, dissemination, and pathogenicity. Lipase GehB is a glycerol ester hydrolase that hydrolyzes triglycerides to facilitate the evasion of S. aureus from host immune recognition. However, the role and mechanism of lipase GehB in skin acute wound healing after S. aureus infection remain unclear. In this study, we found that the gehB gene deletion mutant (USA300ΔgehB) stimulated significantly higher levels of pro-inflammatory cytokines in RAW264.7 and Toll-like receptor 2 (TLR2)-transfected HEK293 cells than the wild-type USA300 strain did. Recombinant GehB-His treated lipoprotein (Lpp) reduced stimulation of TLR2-dependent TNF-α production by RAW264.7 macrophages. GehB delayed the skin acute wound healing in BALB/c mice infected with S. aureus, while wound healing was similar in C57BL/6 TLR2-/- mice infected with either wild-type USA300 or USA300ΔgehB. In BALB/c mice, we also observed more bacterial survival, less leukocyte recruitment, lower IL-8 production, and adipocyte differentiation in USA300-infected skin acute wound tissues than those in USA300ΔgehB-challenged ones. Our data indicated that GehB inactivates lipoproteins to shield S. aureus from innate immune killing, resulting in delayed the healing of skin acute wounds infected with S. aureus.

Loaded delta-hemolysin shapes the properties of Staphylococcus aureus membrane vesicles.

Background: Membrane vesicles (MVs) are nanoscale vesicular structures produced by bacteria during their growth in vitro and in vivo. Some bacterial components can be loaded in bacterial MVs, but the roles of the loaded MV molecules are unclear. Methods: MVs of Staphylococcus aureus RN4220 and its derivatives were prepared. Dynamic light scattering analysis was used to evaluate the size distribution, and 4D-label-free liquid chromatography-tandem mass spectrometry analysis was performed to detect protein composition in the MVs. The site-mutation S. aureus RN4220-Δhld and agrA deletion mutant RN4220-ΔagrA were generated via allelic replacement strategies. A hemolysis assay was performed with rabbit red blood cells. CCK-8 and lactate dehydrogenase release assays were used to determine the cytotoxicity of S. aureus MVs against RAW264.7 macrophages. The serum levels of inflammatory factors such as IL-6, IL-1ß, and TNFα in mice treated with S. aureus MVs were detected with an enzyme-linked immunosorbent assay kit. Results: Delta-hemolysin (Hld) was identified as a major loaded factor in S. aureus MVs. Further study showed that Hld could promote the production of staphylococcal MVs with smaller sizes. Loaded Hld affected the diversity of loaded proteins in MVs of S. aureus RN4220. Hld resulted in decreased protein diversity in MVs of S. aureus. Site-mutation (RN4220-Δhld) and agrA deletion (RN4220-ΔagrA) mutants produced MVs (ΔhldMVs and ΔagrAMVs) with a greater number of bacterial proteins than those derived from wild-type RN4220 (wtMVs). Moreover, Hld contributed to the hemolytic activity of wtMVs. Hld-loaded wtMVs were cytotoxic to macrophage RAW264.7 cells and could stimulate the production of inflammatory factor IL-6 in vivo. Conclusion: This study presented that Hld was a major loaded factor in S. aureus MVs, and the loaded Hld played vital roles in the MV-property modification.

Genomic Epidemiology and Phenotypic Characterization of Staphylococcus aureus from a Tertiary Hospital in Tianjin Municipality, Northern China.

Staphylococcus aureus remains a dangerous pathogen and poses a great threat to public health worldwide. The prevalence of the S. aureus clonotype is temporally and geographically variable. The genomic and phenotypic characteristics of S. aureus isolates in Tianjin, which is among the four big municipalities in China, are unclear. In the present study, 201 nonduplicate S. aureus isolates, including 70 methicillin-resistant S. aureus (MRSA) and 131 methicillin-susceptible S. aureus (MSSA), were collected from 2015 to 2021 in a tertiary hospital in Tianjin. Whole-genome sequencing of S. aureus isolates was carried out to investigate bacterial molecular characteristics, genomic phylogeny, antimicrobial resistance (AMR) gene carriage, and virulence factor gene distribution. The antibiotic resistance profiles, hemolytic activities, and biofilm formation abilities of the S. aureus isolates were also determined. In total, 31 distinct sequence types (STs) and 68 spa types were identified. ST59 (15.9%, 32/201) was the predominant clonotype, followed by ST398 (14.9%, 30/201) and several other major STs (ST1, ST5, ST6, ST22, ST25, ST188, and the newly emerging ST5527). ST59 and ST5527 mainly included MRSA isolates, while ST398 and the other major STs mainly included MSSA isolates. The unique characteristics of the S. aureus isolates belonging to the major STs were determined. ST59 isolates exhibited strong hemolytic activity, and ST398 strains had high biofilm formation capacity, while ST5527 isolates presented the greatest AMR. The genomic epidemiology and phenotypic characteristics of S. aureus isolates determined in this study will help in disease control in nosocomial environments. IMPORTANCE Staphylococcus aureus is an important bacterium pathogen in tertiary hospitals, which provide rich medical resources. Tianjin is one of the four municipalities in China with a population of more than 13 million. However, the epidemiology and molecular characteristics of S. aureus isolates in Tianjin are unknown. In this study, the genomic and phenotypic analyses were performed to investigate 201 S. aureus isolates collected from a tertiary hospital in Tianjin over a time span of 6 years. The refined analysis of predominant clones ST59, ST398, the newly emerging clone ST5527, as well as other major clones, will undoubtedly aid in the control and prevention of infections caused by S. aureus in tertiary hospitals.

Optimizing a high-sensitivity NanoLuc-based bioluminescence system for in vivo evaluation of antimicrobial treatment.

Focal and systemic infections are serious threats to human health. Preclinical models enable the development of new drugs and therapeutic regimens. In vivo, animal bioluminescence (BL) imaging has been used with bacterial reporter strains to evaluate antimicrobial treatment effects. However, high-sensitivity bioluminescent systems are required because of the limited tissue penetration and low brightness of the BL signals of existing approaches. Here, we report that NanoLuc (Nluc) showed better performance than LuxCDABE in bacteria. However, the retention rate of plasmid constructs in bacteria was low. To construct stable Staphylococcus aureus reporter strains, a partner protein enolase (Eno) was identified by screening of S. aureus strain USA300 for fusion expression of Nluc-based luciferases, including Nluc, Teluc, and Antares2. Different substrates, such as hydrofurimazine (HFZ), furimazine (FUR), and diphenylterazine (DTZ), were used to optimize a stable reporter strain/substrate pair for BL imaging. S. aureus USA300/Eno-Antares2/HFZ produced the highest number of photons of orange-red light in vitro and enabled sensitive BL tracking of S. aureus in vivo, with sensitivities of approximately 10 CFU from mouse skin and 750 CFU from mouse kidneys. USA300/Eno-Antares2/HFZ was a powerful combination based on the longitudinal evaluation of the therapeutic efficacy of antibiotics. The optimized S. aureus Eno-Antares2/HFZ pair provides a technological advancement for the in vivo evaluation of antimicrobial treatment.

Identification of Lysine Succinylome and Acetylome in the Vancomycin-Intermediate Staphylococcus aureus XN108.

Protein posttranslational modifications (PTMs) play important roles in regulating numerous biological functions of prokaryotic and eukaryotic organisms. Lysine succinylation (Ksucc) and acetylation (Kac) are two important PTMs that have been identified in various bacterial species. However, the biological functions of Ksucc and Kac in vancomycin-intermediate S. aureus (VISA) remain unclear. In this study, we systematically identified 3,260 Ksucc sites in 799 proteins and 7,935 Kac sites across 1,710 proteins in the VISA strain XN108. Functional analyses revealed that both Ksucc and Kac sites were highly enriched in several critical metabolic pathways, including ribosomal metabolism, tricarboxylic acid cycle, and glycolysis. Furthermore, a remarkable cross talk between Ksucc and Kac modifications was observed that almost 75% of the succinylated sites were also frequently acetylated. In addition, we identified SaCobB, a Sirtuin 2-like lysine deacetylase, as a bifunctional enzyme with both deacetylation and desuccinylation activities in S. aureus. We demonstrated the first lysine succinylome and acetylome in a VISA and identified SaCobB, a functional enzyme taking part in the regulation of Ksucc and Kac in S. aureus. Our findings provide valuable information for further study on the regulatory mechanisms of PTMs in S. aureus. IMPORTANCE Lysine succinylation (Ksucc) and acetylation (Kac) are two important protein posttranslational modifications (PTMs) that regulate numerous biological functions in prokaryotes and eukaryotes. However, the functions of Ksucc and Kac in Staphylococcus aureus are seldom described. Understanding of Ksucc and Kac modifications in S. aureus will facilitate the development of new strategies to control infections. Herein, we quantified both Ksucc and Kac in a vancomycin-intermediate S. aureus (VISA) strain XN108, analyzed the interaction between these two PTMs, and identified SaCobB as a bifunctional enzyme with both deacetylation and desuccinylation activities. This study is the first description of dual PTMs, Ksucc and Kac profiles, in the VISA. The findings could provide valuable information for the following researches on the regulatory roles of PTMs in S. aureus.

CcpA Regulates Staphylococcus aureus Biofilm Formation through Direct Repression of Staphylokinase Expression.

Staphylococcus aureus represents a notorious opportunistic pathogen causing various infections in biofilm nature, imposing remarkable therapeutic challenges worldwide. The catabolite control protein A (CcpA), a major regulator of carbon catabolite repression (CCR), has been recognized to modulate S. aureus biofilm formation, while the underlying mechanism remains to be fully elucidated. In this study, the reduced biofilm was firstly determined in the ccpA deletion mutant of S. aureus clinical isolate XN108 using both crystal violet staining and confocal laser scanning microscopy. RNA-seq analysis suggested that sak-encoding staphylokinase (Sak) was significantly upregulated in the mutant ∆ccpA, which was further confirmed by RT-qPCR. Consistently, the induced Sak production correlated the elevated promoter activity of sak and increased secretion in the supernatants, as demonstrated by Psak-lacZ reporter fusion expression and chromogenic detection, respectively. Notably, electrophoretic mobility shift assays showed that purified recombinant protein CcpA binds directly to the promoter region of sak, suggesting the direct negative control of sak expression by CcpA. Double isogenic deletion of ccpA and sak restored biofilm formation for mutant ∆ccpA, which could be diminished by trans-complemented sak. Furthermore, the exogenous addition of recombinant Sak inhibited biofilm formation for XN108 in a dose-dependent manner. Together, this study delineates a novel model of CcpA-controlled S. aureus biofilm through direct inhibition of sak expression, highlighting the multifaceted roles and multiple networks regulated by CcpA.

A vancomycin resistance-associated WalK(S221P) mutation attenuates the virulence of vancomycin-intermediate Staphylococcus aureus.

INTRODUCTION: Vancomycin-intermediate Staphylococcus aureus (VISA) is typically associated with a decline in virulence. We previously reported a WalK(S221P) mutation that plays an important role in mediating vancomycin resistance in VISA XN108. Whether this mutation is implicated in bacterial virulence remains unknown. OBJECTIVES: This study aimed to investigate the effect of WalK(S221P) mutation on the virulence of VISA and the underlying mechanism of this effect. METHODS: The influence of WalK(S221P) mutation on VISA virulence and its underlying mechanism were explored using animal models, RNA-seq analysis, RT-qPCR, hemolytic assay, slide coagulase test, Western blot, ß-galactosidase assay, and electrophoresis mobility shift assay (EMSA). RESULTS: Compared with XN108, WalK(S221P)-reverted strain XN108-R exacerbated cutaneous infections with increased lesion size and extensive inflammatory infiltration in mouse models. The bacterial loads of S. aureus XN108-R in murine kidney increased compared with those of XN108. RNA-seq analysis showed upregulation of a set of virulence genes in XN108-R, which exhibited greater hemolytic and stronger coagulase activities compared with XN108. Introduction of WalK(S221P) to methicillin-resistant S. aureus USA300 and methicillin-susceptible strain Newman increased the vancomycin resistance of the mutants, which exhibited reduced hemolytic activities and decreased expression levels of many virulence factors compared with their progenitors. WalK(S221P) mutation weakened agr promoter-controlled ß-galactosidase activity. EMSA results showed that WalK-phosphorylated WalR could directly bind to the agr promoter region, whereas WalK(S221P)-activated WalR reduced binding to the target promoter. Inactivation of agr in S. aureus did not affect their vancomycin susceptibility but mitigated the virulence alterations caused by WalK(S221P) mutation. CONCLUSION: The results of our study indicate that WalK(S221P) mutation can enhance vancomycin resistance in S. aureus of diverse genetic backgrounds. WalK(S221P)- bearing S. aureus strains exhibit reduced virulence. WalK(S221P) mutation may directly impair the activation of the agr system by WalR, thereby decreasing the expression of virulence factors in VISA.

Microglia activation in the mPFC mediates anxiety-like behaviors caused by Staphylococcus aureus strain USA300.

INTRODUCTION: Staphylococcus aureus (S. aureus) is considered as one of the major causative agents of serious hospital- and community-acquired infections. Recent studies have reported that S. aureus infection induced neuroinflammation and was linked with some mental disorders. To evaluate the effects of S. aureus infection on abnormal behaviors, we conducted the present study. METHODS: A S. aureus USA300-infected mouse model was established using bacterial suspension injection into tail vein. A series of behavioral tests were performed after USA300 infection. The expression of cytokines was detected in serum and mPFC. The number and some morphological parameters of microglia were also evaluated by immunofluorescence staining. RESULTS: Anxiety-like behaviors, instead of locomotor activity impairment or depression-like behaviors, were observed in mice infected with S. aureus USA300 compared with control. S. aureus USA300 infection caused overexpression of IL-6, TNF-α, and IL-1ß in serum, resulted in microglial over-activation and excessive release of proinflammatory cytokines in the mPFC. In addition, overexpression of TLR2 accompanied by increased GLS1 and p-STAT3 was observed in the mPFC of mice infected with S. aureus USA300. CONCLUSION: This study provides evidence that S. aureus USA300 infection can lead to neuroinflammation in the mPFC of mice, which may contribute to the development of anxiety.

Essential Fitness Repertoire of Staphylococcus aureus during Co-infection with Acinetobacter baumannii In Vivo.

Staphylococcus aureus represents a major human pathogen that is frequently involved in polymicrobial infections. However, the prevalence and role of co-infectious microbes on the pathogenesis and fitness essentiality of S. aureus in vivo remain largely unknown. In this study, we firstly performed a retrospective surveillance of 760 clinical samples and revealed a notable predominance of co-infection with S. aureus and Acinetobacter baumannii. The high-density S. aureus transposon mutant library coupled to transposon insertion sequencing (Tn-Seq) further identified a core set of genes enriched in metabolism of inorganic ions, amino acids, and carbohydrates, which are essential for infection and tissue colonization of S. aureus in the murine systemic infection model. Notably, we revealed a differential requirement of fitness factors for S. aureus in tissue-specific (liver and kidney) and infection-type-specific manner (mono- and co-infection). Co-infection with A. baumannii dramatically altered the fitness requirements of S. aureus in vivo; 49% of the mono-infection fitness genes in S. aureus strain Newman were converted to non-essential, and the functionality of ATP-binding cassette (ABC) transporters was significantly elicited during co-infection. Furthermore, the number of genes essential during co-infection (503) outnumbers the genes essential during mono-infection (362). In addition, the roles of 3 infection-type-specific genes in S. aureus during mono-infection or co-infection with A. baumannii were validated with competitive experiments in vivo. Our data indicated a high incidence and clinical relevance of S. aureus and A. baumannii co-infection, and provided novel insights into establishing antimicrobial regimens to control co-infections. IMPORTANCE Polymicrobial infections are widespread in clinical settings, which potentially correlate with increased infection severity and poor clinical outcomes. Staphylococcus aureus is a formidable human pathogen that causes a variety of diseases in polymicrobial nature. Co-infection and interaction of S. aureus have been described with limited pathogens, mainly including Pseudomonas aeruginosa, Candida albicans, and influenza A virus. Thus far, the prevalence and role of co-infectious microbes on the pathogenesis and fitness essentiality of S. aureus in vivo remain largely unknown. Understanding the polymicrobial composition and interaction, from a community and genome-wide perspective, is thus crucial to shed light on S. aureus pathogenesis strategy. Here, our findings demonstrated, for the first time, that a high incidence rate and clinical relevance of co-infection was caused by S. aureus and Acinetobacter baumannii, illustrating the importance of polymicrobial nature in investigating S. aureus pathogenesis. The infection-type-specific genes likely serve as potential therapeutic targets to control S. aureus infections, either in mono- or co-infection situation, providing novel insights into the development of antimicrobial regimens to control co-infections.

Accessory Gene Regulator (agr) Allelic Variants in Cognate Staphylococcus aureus Strain Display Similar Phenotypes.

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.

Pyocyanin biosynthesis protects Pseudomonas aeruginosa from nonthermal plasma inactivation.

Pseudomonas aeruginosa is an important opportunistic human pathogen, which raises a worldwide concern for its increasing resistance. Nonthermal plasma, which is also called cold atmospheric plasma (CAP), is an alternative therapeutic approach for clinical infectious diseases. However, the bacterial factors that affect CAP treatment remain unclear. The sterilization effect of a portable CAP device on different P. aeruginosa strains was investigated in this study. Results revealed that CAP can directly or indirectly kill P. aeruginosa in a time-dependent manner. Scanning electron microscopy and transmission electron microscope showed negligible surface changes between CAP-treated and untreated P. aeruginosa cells. However, cell leakage occurred during the CAP process with increased bacterial lactate dehydrogenase release. More importantly, pigmentation of the P. aeruginosa culture was remarkably reduced after CAP treatment. Further mechanical exploration was performed by utilizing mutants with loss of functional genes involved in pyocyanin biosynthesis, including P. aeruginosa PAO1 strain-derived phzA1::Tn, phzA2::Tn, ΔphzA1/ΔphzA2, phzM::Tn and phzS::Tn, as well as corresponding gene deletion mutants based on clinical PA1 isolate. The results indicated that pyocyanin and its intermediate 5-methyl phenazine-1-carboxylic acid (5-Me-PCA) play important roles in P. aeruginosa resistance to CAP treatment. The unique enzymes, such as PhzM in the pyocyanin biosynthetic pathway, could be novel targets for the therapeutic strategy design to control the growing P. aeruginosa infections.

Fighting Mixed-Species Microbial Biofilms With Cold Atmospheric Plasma.

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.

The outbreak of SARS-CoV-2 pneumonia calls for viral vaccines.

The outbreak of 2019-novel coronavirus disease (COVID-19) that is caused by SARS-CoV-2 has spread rapidly in China, and has developed to be a Public Health Emergency of International Concern. However, no specific antiviral treatments or vaccines are available yet. This work aims to share strategies and candidate antigens to develop safe and effective vaccines against SARS-CoV-2.

Protective Effect of the Golden Staphyloxanthin Biosynthesis Pathway on Staphylococcus aureus under Cold Atmospheric Plasma Treatment.

Staphylococcus aureus infection poses a serious threat to public health, and antibiotic resistance has complicated the clinical treatment and limited the solutions available to solve this problem. Cold atmospheric plasma (CAP) is a promising strategy for microorganism inactivation. However, the mechanisms of microbial inactivation or resistance remain unclear. In this study, we treated S. aureus strains with a self-assembled CAP device and found that CAP can kill S. aureus in an exposure time-dependent manner. In addition, the liquid environment can influence the survival rate of S. aureus post-CAP treatment. The S. aureus cells can be completely inactivated in normal saline and phosphate-buffered saline but not in tryptic soy broth culture medium. Scanning and transmission electron microscopy revealed that the CAP-treated S. aureus cells maintained integrated morphological structures, similar to the wild-type strain. Importantly, the CAP-treated S. aureus cells exhibited a reduced pigment phenotype. Deletion of the staphyloxanthin biosynthetic genes crtM and crtN deprived the pigmentation ability of S. aureus Newman. Both the Newman-ΔcrtM and Newman-ΔcrtN mutants presented high sensitivity to CAP treatment, whereas Newman-ΔcrtO exhibited a survival rate comparable to wild-type Newman after CAP treatment. Our data demonstrated that the yellow pigment intermediates of the staphyloxanthin biosynthetic pathway are responsible for the protection of S. aureus from CAP inactivation. The key enzymes, such as CrtM and CrtN, of the golden staphyloxanthin biosynthetic pathway could be important targets for the design of novel sterilization strategies against S. aureus infections.IMPORTANCEStaphylococcus aureus is an important pathogen that can be widely distributed in the community and clinical settings. The emergence of S. aureus with multiple-antibiotic resistance has complicated staphylococcal infection control. The development of alternative strategies with powerful bactericidal effects is urgently needed. Cold atmospheric plasma (CAP) is a promising strategy for microorganism inactivation. Nevertheless, the underlying mechanisms of microbial inactivation or resistance are not completely illustrated. In this study, we validated the bactericidal effects of CAP on S. aureus, including antibiotic-resistant strains. We also found that the golden staphyloxanthin, as well as its yellow pigment intermediates, protected S. aureus against CAP, and blocking the staphyloxanthin synthesis pathway at the early steps could strengthen the sensitivity of S. aureus to CAP treatment. These data provide insights into the germicidal mechanism of CAP from the aspect of bacteria and suggest new targets against S. aureus infections.

ß-Lactam Antibiotics Enhance the Pathogenicity of Methicillin-Resistant Staphylococcus aureus via SarA-Controlled Lipoprotein-Like Cluster Expression.

Methicillin-resistant Staphylococcus aureus (MRSA) resists nearly all ß-lactam antibiotics that have a bactericidal activity. However, whether the empirically used ß-lactams enhance MRSA pathogenicity in vivo remains unclear. In this study, we showed that a cluster of lipoprotein-like genes (lpl, sa2275 to sa2273 [sa2275-sa2273]) was upregulated in MRSA in response to subinhibitory concentrations of ß-lactam induction. The increasing expression of lpl by ß-lactams was directly controlled by the global regulator SarA. The ß-lactam-induced Lpls stimulated the production of interleukin-6 and tumor necrosis factor alpha in RAW 264.7 macrophages. The lpl deletion mutants (N315Δlpl and USA300Δlpl) decreased the proinflammatory cytokine levels in vitro and in vivo Purified lipidated SA2275-his proteins could trigger a Toll-like-receptor-2 (TLR2)-dependent immune response in primary mouse bone marrow-derived macrophages and C57BL/6 mice. The bacterial loads of N315Δlpl in the mouse kidney were lower than those of the wild-type N315. The ß-lactam-treated MRSA exacerbated cutaneous infections in both BALB/c and C57BL/6 mice, presenting increased lesion size; destroyed skin structure; and easily promoted abscess formation compared with those of the untreated MRSA. However, the size of abscesses caused by the ß-lactam-treated N315 was negligibly different from those caused by the untreated N315Δlpl in C57BL/6 TLR2-/- mice. Our findings suggest that ß-lactams must be used carefully because they might aggravate the outcome of MRSA infection compared to inaction in treatment.IMPORTANCE ß-Lactam antibiotics are widely applied to treat infectious diseases. However, certain poor disease outcomes caused by ß-lactams remain poorly understood. In this study, we have identified a cluster of lipoprotein-like genes (lpl, sa2275-sa2273) that is upregulated in the major clinically prevalent MRSA clones in response to subinhibitory concentrations of ß-lactam induction. The major highlight of this work is that ß-lactams stimulate the expression of SarA, which directly binds to the lpl cluster promoter region and upregulates lpl expression in MRSA. Deletion of lpl significantly decreases proinflammatory cytokine levels in vitro and in vivo The ß-lactam-induced Lpls enhance host inflammatory responses by triggering the Toll-like-receptor-2-mediated expressions of interleukin-6 and tumor necrosis factor alpha. The ß-lactam-induced Lpls are important virulence factors that enhance MRSA pathogenicity. These data elucidate that subinhibitory concentrations of ß-lactams can exacerbate the outcomes of MRSA infection through induction of lpl controlled by the global regulator SarA.

Virulence Determinants Are Required for Brain Abscess Formation Through Staphylococcus aureus Infection and Are Potential Targets of Antivirulence Factor Therapy.

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.