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Abstract Background Staphylococcus aureus, a commensal bacterium, colonizes the skin and mucous membranes of approximately 30% of the human population. Apart from conventional resistance mechanisms, one of the pathogenic features of S. aureus is its ability to survive in a biofilm state on both biotic and abiotic surfaces. Due to this characteristic, S. aureus is a major cause of human infections, with Methicillin-Resistant Staphylococcus aureus (MRSA) being a significant contributor to both community-acquired and hospital-acquired infections. Results Analyzing non-repetitive clinical isolates of MRSA collected from seven provinces and cities in China between 2014 and 2020, it was observed that 53.2% of the MRSA isolates exhibited varying degrees of ability to produce biofilm. The biofilm positivity rate was notably high in MRSA isolates from Guangdong, Jiangxi, and Hubei. The predominant MRSA strains collected in this study were of sequence types ST59, ST5, and ST239, with the biofilm-producing capability mainly distributed among moderate and weak biofilm producers within these ST types. Notably, certain sequence types, such as ST88, exhibited a high prevalence of strong biofilm-producing strains. The study found that SCCmec IV was the predominant type among biofilm-positive MRSA, followed by SCCmec II. Comparing strains with weak and strong biofilm production capabilities, the positive rates of the sdrD and sdrE were higher in strong biofilm producers. The genetic determinants ebp, icaA, icaB, icaC, icaD, icaR, and sdrE were associated with strong biofilm production in MRSA. Additionally, biofilm-negative MRSA isolates showed higher sensitivity rates to cefalotin (94.8%), daptomycin (94.5%), mupirocin (86.5%), teicoplanin (94.5%), fusidic acid (81.0%), and dalbavancin (94.5%) compared to biofilm-positive MRSA isolates. The biofilm positivity rate was consistently above 50% in all collected specimen types. Conclusions MRSA strains with biofilm production capability warrant increased vigilance.

Staphylococcus aureus (S. aureus) is an opportunistic pathogen that could cause life-threatening bloodstream infections. The objective of this study was to identify potential diagnostic biomarkers of S. aureus bloodstream infection. Gene expression dataset GSE33341 was optimized as the discovery dataset, which contained samples from human and mice. GSE65088 dataset was utilized as a validation dataset. First, after overlapping the differentially expressed genes (DEGs) in S. aureus infection samples from GSE33341-human and GSE33341-mice samples, we detected 63 overlapping genes. Subsequently, the hub genes including DRAM1, PSTPIP2, and UPP1 were identified via three machine-learning algorithms: random forest, support vector machine-recursive feature elimination, and least absolute shrinkage and selection operator. Additionally, the receiver operating characteristic curve was leveraged to verify the efficacy of the hub genes. DRAM1 (AUC=1), PSTPIP2 (AUC=1), and UPP1 (AUC=1) were investigated and demonstrated significant expression differences (all P < 0.05) and diagnostic efficacy in the training and validation datasets. Furthermore, the relationship between the diagnostic markers and the abundance of immune cells was assessed using cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT). These three diagnostic indicators also correlated with multiple immune cells to varying degrees. The expression of DRAM1 was significantly positively correlated with B cell naive and mast cell activation, and negatively correlated with NK cells and CD4/CD8+ T cells. The expression of PSTPIP2 was significantly positively correlated with macrophage M0, macrophage M1, B cell naive, and dendritic cell activation, while the expression of PSTPIP2 was negatively correlated with NK cells and CD4/CD8+ T cells. Significant negative correlations between UPP1 expression and T cell CD4 memory rest and neutrophils were also observed. Finally, we established a mouse model of S. aureus bloodstream infection and collected the blood samples for RNA-Seq analysis and RT-qPCR experiments. The analysis results in RNA-Seq and RT-qPCR experiments further confirmed the significant expression differences (all P < 0.05) of these three genes. Overall, three candidate hub genes (DRAM1, PSTPIP2, and UPP1) were identified initially for S. aureus bloodstream infection diagnosis. Our study could provide potential diagnostic biomarkers for S. aureus bloodstream infection patients.

ABSTRACT The widespread prevalence and dissemination of antibiotic-resistant bacteria, coupled with the diminishing supply of new antibiotics, emphasize the pressing necessity for the exploration of innovative antibacterial agents. Previously, we detailed the impact of the small-molecule compound CY-158-11 on S. aureus biofilm. By hindering adhesion and PIA-mediated biofilm formation, subinhibitory concentrations of CY-158-11 exhibit antibiofilm activity toward S. aureus. Here, we sought to elucidate the antibacterial activity and mode of action of this compound. Upon CY-158-11 treatment in culture, the inhibition of bacterial growth, coupled with MBC to MIC of >4, indicated that CY-158-11 exerted a bacteriostatic effect. Particularly, CY-158-11 showed strong antibacterial activity against a wide variety of S. aureus, including multidrug-resistant bacteria. We found that CY-158-11 promoted the permeability of cell membrane and propidium iodide absorption as well as caused the dissipation of membrane potential. The effect of CY-158-11 on the mammalian cytoplasmic membrane was measured using hemolytic and cytotoxicity assays, and the skin irritation and systemic toxicity of the drug were measured by injecting the compound into the skin and tail vein of mice. Moreover, CY-158-11 exhibited considerable efficacy in a subcutaneous abscess mouse model of S. aureus infection. In conclusion, CY-158-11 possesses antibacterial properties, including inhibition of bacterial growth, damage to cell membranes, and treatment of skin abscesses, which can be a promising therapeutic option for combating S. aureus.IMPORTANCEThe combination of the rising incidence of antibiotic resistance and the shrinking antibiotic pipeline has raised concern about the postantibiotic era. New antibacterial agents and targets are required to combat S. aureus-associated infections. In this study, we identified a maleimide-diselenide hybrid compound CY-158-11 exhibiting antibacterial activity against S. aureus in vitro and in vivo at relatively low concentrations. Furthermore, the investigation of its mode of action revealed that CY-158-11 can selectively perturb the cytoplasmic membrane of bacteria without harming mammalian cells or mouse organs. Thus, CY-158-11 is a compelling novel drug for development as a new therapy for S. aureus infections.

BackgroundDaptomycin non-susceptible Staphylococcus aureus (DNS) strains pose a serious clinical threat, yet their characteristics remain poorly understood.MethodsDNS derivatives were generated by exposing S. aureus strains to subinhibitory concentrations of daptomycin. Competition experiment and growth kinetics experiment were used to observe the growth of bacteria. Galleria mellonella larvae and mouse skin abscess models were used to observe the virulence of bacteria. Transmission electron microscopy (TEM), cytochrome C experiment and biofilm formation experiment were used to observe the drug resistance phenotype. And homologous recombination was used to study the role of mutations.ResultsPhenotypic profiling of DNS strains revealed impaired growth, increased cell wall thickness, enhanced biofilm formation, reduced negative surface charge, and attenuated virulence compared to their wild-type strains. Whole genome sequencing identified mutations in mprF, cls2, and saeR in DNS strains. Allelic replacement experiments validated the roles of MprF L341F and Cls2 F60S substitutions in augmenting daptomycin non-susceptibility in Newman. Deletion of saeR in the NewmanMprFL341F strain and complementation of saeR in the Newman-DNS strain did not directly alter daptomycin susceptibility. However, the deletion of saeR was found to enhance competitive fitness under daptomycin pressure.ConclusionThis work validates adaptive laboratory evolution (ALE) for modeling clinical DNS strains and uncovers contributions of mprF, cls2, and saeR mutations to the adaptation and resistance mechanisms of S. aureus against daptomycin. These findings enrich our understanding of how S. aureus acquired resistance to daptomycin, thus paving the way for the development of more effective treatment strategies and offering potential molecular markers for resistance surveillance.

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) sequence type 630 (ST630) is a rarely reported lineage worldwide. This study aimed to trace the dissemination of the emerging MRSA ST630 clones in China and investigate their virulence potential. We collected 22 ST630-MRSA isolates from across China and performed whole-genome sequencing analysis and virulence characterization on these isolates. Epidemiological results showed that MRSA ST630 isolates were primarily isolated from pus/wound secretions, mainly originating from Jiangxi province, and carried diverse virulence and drug resistance genes. Staphylococcal cassette chromosome mec type V (SCCmec V) predominated (11/22, 50.0%) among the MRSA ST630 isolates. Interestingly, nearly half (45.5%) of the 22 ST630-MRSA isolates tested lacked intact SCCmec elements. Phylogenetic analysis demonstrated that ST630-MRSA could be divided into two distinct clades, with widespread dissemination mainly in Chinese regions. Five representative isolates were selected for phenotypic assays, including hemolysin activity, real-time fluorescence quantitative PCR, western blot analysis, hydrogen peroxide killing assay, blood killing assay, cell adhesion and invasion assay, and mouse skin abscess model. The results showed that, compared to the USA300-LAC strain, ST630 isolates exhibited particularly strong invasiveness and virulence in the aforementioned phenotypic assays. This study described the emergence of a highly virulent ST630-MRSA lineage and improved our insight into the molecular epidemiology of ST630 clones in China.IMPORTANCEMethicillin-resistant Staphylococcus aureus (MRSA) sequence type 630 (ST630) is an emerging clone with an increasing isolation rate in China. This study raises awareness of the hypervirulent MRSA ST630 clones in China and alerts people to their widespread dissemination. ST630-staphylococcal cassette chromosome mec V is a noteworthy clone in China, and we present the first comprehensive genetic and phenotypic analysis of this lineage. Our findings provide valuable insights for the prevention and control of infections caused by this emerging MRSA clone.

The ability to form robust biofilms and secrete a diverse array of virulence factors are key pathogenic determinants of Staphylococcus aureus, causing a wide range of infectious diseases. Here, we characterized cwrA as a VraR-regulated gene encoding a cell wall inhibition-responsive protein (CwrA) using electrophoretic mobility shift assays. We constructed cwrA deletion mutants in the genetic background of methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) strains. Phenotypic analyses indicated that deletion of cwrA led to impaired biofilm formation, which was correlated with polysaccharide intercellular adhesin (PIA). Besides, the results of real-time quantitative PCR (RT-qPCR) and β-galactosidase activity assay revealed that CwrA promoted biofilm formation by influence the ica operon activity in S. aureus. Furthermore, cwrA deletion mutants released less extracellular DNA (eDNA) in the biofilm because of their reduced autolytic activity compared to the wild-type (WT) strains. We also found that cwrA deletion mutant more virulence than the parental strain because of its enhanced hemolytic activity. Mechanistically, this phenotypic alteration is related to activation of the SaeRS two-component system, which positively regulates the transcriptional levels of genes encoding membrane-damaging toxins. Overall, our results suggest that CwrA plays an important role in modulating biofilm formation and hemolytic activity in S. aureus.

ABSTRACT Staphylococcus aureus is an important human pathogen in both community and hospital settings that often causes persistent and recurrent infections. The continuous emergence of multidrug-resistant strains and the lag in antibiotic development make the treatment of S. aureus more challenging. The biofilm formation of S. aureus is an important reason for persistent infection. In this study, we demonstrated that a small-molecule compound S-342-3 can effectively inhibit the biofilm formation of S. aureus. The crystal violet semi-quantitative assays revealed that at a sub-minimum inhibitory concentration of 4 µg/mL, S-342-3 reduced S. aureus biofilm mass by 57.43%, 52.14%, and 25.49%. Using confocal laser scanning microscopy, we observed that the biofilm was more incompact and less uniform upon the treatment of S-342-3. At a sub-inhibitory concentration (4 µg/mL), the S-342-3 can effectively reduce the production of polysaccharide intercellular adhesin (PIA) and suppress the initial adhesion of cells in the biofilm. Consistently, the results of RT-qPCR revealed that the expression of genes associated with biofilm formation was downregulated by S-342-3 (P < 0.05). However, we found that treatment with S-342-3 resulted in a significant decrease in the expression of global regulatory genes agrA and sarA (P < 0.05), which play a key role in promoting cell surface attachment and PIA production in S. aureus biofilms. Also importantly, we experimentally proved that the S-342-3 was not toxic to A549 alveolar epithelial cells and the Galleria mellonella larvae. Collectively, these results suggest that the S-342-3 may be a promising anti-biofilm drug candidate with excellent biosafety, which can be further explored for its practical application in health care. IMPORTANCE Biofilms are an important virulence factor in Staphylococcus aureus and are characterized by a structured microbial community consisting of bacterial cells and a secreted extracellular polymeric matrix. Inhibition of biofilm formation is an effective measure to control S. aureus infection. Here, we have synthesized a small molecule compound S-342-3, which exhibits potent inhibition of biofilm formation in both MRSA and MSSA. Further investigations revealed that S-342-3 exerts inhibitory effects on biofilm formation by reducing the production of polysaccharide intercellular adhesin and preventing bacterial adhesion. Our study has confirmed that the inhibitory effect of S-342-3 on biofilm is achieved by downregulating the expression of genes responsible for biofilm formation. In addition, S-342-3 is non-toxic to Galleria mellonella larvae and A549 cells. Consequently, this study demonstrates the efficacy of a biologically safe compound S-342-3 in inhibiting biofilm formation in S. aureus, thereby providing a promising antibiofilm agent for further research.

ABSTRACTPrevious studies have shown that the increased prevalent ST764 clone in China, Japan, and other Asian areas. However, the knowledge of the genetic features and virulence characteristics of methicillin-resistant Staphylococcus aureus (MRSA) ST764 in China is still limited. In this study, we identified 52 ST764-SCCmec type II isolates collected from five cities in China between 2014 and 2021. Whole genome sequencing showed that the most common staphylococcal protein A (spa) types of ST764 in China were t002 (55.78%) and t1084 (40.38%). Virulence assays showed that ST764-t1084 isolates had high haemolytic activity and α-toxin levels. Of the critical regulatory factors affecting α-toxin production, only the SaeRS was highly expressed in ST764-t1084 isolates. Mouse abscess model indicated that the virulence of ST764-t1084 isolates was comparable to that of S. aureus USA300-LAC famous for its hypervirulence. Interestingly, ST764-t002 isolates exhibited stronger biofilm formation and cell adhesion capacities than ST764-t1084 isolates. This seems to explain why ST764-t002 subclone has become more prevalent in China in recent years. Phylogenetic analysis suggested that all ST764 isolates from China in Clade III were closely related to KUN1163 (an isolate from Japan). Notably, genomic analysis revealed that the 52 ST764 isolates did not carry arginine catabolic mobile element (ACME), which differed from ST764 isolates in Japan. Additionally, most ST764 isolates (69.23%) harboured an obvious deletion of approximately 5 kb in the SCCmec II cassette region compared to KUN1163. Our findings shed light on the potential global transmission and genotypic as well as phenotypic characteristics of ST764 lineage.

ABSTRACT ST22 MRSA (methicillin-resistant Staphylococcus aureus) strains are only sporadically reported in China. Through the phylogenetic reconstruction of 30 ST22 strains from China and 480 ST22 strains from global sources, we found that the global ST22 strains can be divided into three clades (I, II, and III). The China ST22 strains were found primarily in clade II (IIb and IIc) and also in clade III, indicating that the China ST22-MRSA clones have different origins. The China subclade IIb strains (SCCmec Vb-t309) may evolve from the native ST22 MSSA clone, while the China IIc strains may have spread from other countries. Subclade IIc (SCCmecIVa-t309) strains exhibited particularly strong lethality and invasiveness in Galleria mellonella infection and mouse skin abscess models in comparison to USA300 and other dominant China HA-MRSA (ST5 and ST239) or CA-MRSA (ST59) strains. This study described the emergence of a highly virulent ST22 MRSA subclade and improved our insight into the molecular epidemiology of ST22 strains in China. IMPORTANCE ST22 is a successful hospital-associated MRSA lineage which first appeared in the United Kingdom as EMRSA-15. At present, ST22 MRSA clones are spreading rapidly around the world and even replaced other dominant clones in some regions. We placed the Chinese ST22 in the worldwide phylogeny of ST22, demonstrating a distinctive molecular epidemiology and to our knowledge, this is the first time that a novel clade of ST22 has been found in China. Among the 15 ST22 MRSA strains belonging to the novel clade, 14 ST22 SCCmecIVa strains from different regions carried both pvl and tst and displayed significantly higher in vitro and in vivo virulence in comparison to other clade/subclade ST22 strains as well as other common China HA-MRSA or CA-MRSA strains. The further spread of this subclade of strains could pose a serious threat to the health system in China and other regions.

ABSTRACT Staphylococcus aureus is an important human pathogen and brings about many community-acquired, hospital-acquired, and biofilm-associated infections worldwide. It tends to form biofilms, triggering the release of toxins and initiating resistance mechanisms. As a result of the development of S. aureus tolerance to antibiotics, there are few drugs can availably control biofilm-associated infections. In this study, we synthesized a novel small-molecule compound CY-158-11 (C22H14Cl2NO2Se2) and proved its inhibitory effect on the biofilm formation of S. aureus at a subinhibitory concentration (1/8 MIC). The subinhibitory concentration of CY-158-11 not only did not affect the growth of bacteria but also had no toxicity to A549 cells or G. mellonella. Total biofilm biomass was investigated by crystal violet staining, and the results were confirmed by SYTO 9 and PI staining through confocal laser scanning microscopy. Moreover, CY-158-11 effectively prevented initial attachment and repressed the production of PIA instead of autolysis. RT-qPCR analysis also exhibited significant suppression of the genes involved in biofilm formation. Taken together, CY-158-11 exerted its inhibitory effects against the biofilm formation in S. aureus by inhibiting cell adhesion and the expression of icaA related to PIA production. IMPORTANCE Most bacteria exist in the form of biofilms, often strongly adherent to various surfaces, causing bacterial resistance and chronic infections. In general, antibacterial drugs are not effective against biofilms. The small-molecule compound CY-158-11 inhibited the biofilm formation of S. aureus at a subinhibitory concentration. By hindering adhesion and PIA-mediated biofilm formation, CY-158-11 exhibits antibiofilm activity toward S. aureus. These findings point to a novel therapeutic agent for combating intractable S. aureus-biofilm-related infections.

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) sequence type 45 (ST45) was rarely found in China. This study was conducted to trace the transmission and evolution of emerging MRSA ST45 strains in mainland China and explore its virulence. A total of 27 ST45 isolates were included for whole-genome sequencing and genetic characteristic analysis. Epidemiological results showed that MRSA ST45 isolates were often obtained from blood, primarily originated in Guangzhou, and carried diverse virulence and drug resistance genes. Staphylococcal cassette chromosome mec type IV (SCCmec IV) dominated in MRSA ST45 (23/27, 85.2%). ST45-SCCmec V was located on a phylogenetic clade distinct from the SCCmec IV cluster. We selected two representative isolates, MR370 (ST45-SCCmec IV) and MR387 (ST45-SCCmec V), and performed hemolysin activity, a blood killing assay, a Galleria mellonella infection model, and a mouse bacteremia model, as well as real-time fluorescence quantitative PCR. MR370 was proved to have extreme virulence in the phenotypic assays and at the mRNA level compared with ST59, ST5, and USA300 MRSA strains. MR387 was comparable to USA300-LAC on the phenotype and was verified to have higher expression of scn, chp, sak, saeR, agrA, and RNAIII than USA300-LAC. The results emphasized the extraordinary performance of MR370 and the good potential of MR387 in virulence causing bloodstream infection. Meanwhile, we conclude that China MRSA ST45 showed two different clonotypes, which may be widespread in the future. The entire study is valuable as a timely reminder and reports virulence phenotypes of China MRSA ST45 for the first time. IMPORTANCE Methicillin-resistant Staphylococcus aureus ST45 is epidemic worldwide. This study contributed to the awareness of the Chinese hyper-virulent MRSA ST45 strains and served as a timely reminder of its wide dissemination of clonotypes. Further, we provide novel insights for prevention from the perspective of bloodstream infections. ST45-SCCmec V is a clonotype deserving special attention in China, and we performed genetic and phenotypic analyses for the first time on it.

Multi-drug resistant Staphylococcus aureus infection is still a serious threat to global health. Therefore, there is an urgent need to develop new antibacterial agents based on virulence factor therapy to overcome drug resistance. Previously, we synthesized SYG-180-2-2 (C21H16N2OSe), an effective small molecule compound against biofilm. The aim of this study was to investigate the anti-virulence efficacy of SYG-180-2-2 against Staphylococcus aureus. MIC results demonstrated no apparent antibacterial activity of the SYG-180-2-2. The growth curve assay showed that SYG-180-2-2 had nonlethal effect on S. aureus. Besides, SYG-180-2-2 strongly inhibited the hemolytic activity and staphyloxanthin synthesis in S. aureus. Inhibition of staphyloxanthin by SYG-180-2-2 enhanced the sensitivity of S. aureus to oxidants and human whole blood. In addition, SYG-180-2-2 significantly decreased the expression of saeR-mediated hemolytic gene hlb and staphyloxanthin-related crtM, crtN and sigB genes by quantitative polymerase chain reaction (qPCR). Meanwhile, the expression of oxidative stress-related genes sodA, sodM and katA also decreased. Galleria Mellonella assay revealed that SYG-180-2-2 was not toxic to larvae. Further, the larvae infection model showed that the virulence of bacteria was significantly reduced after 4 μg/mL SYG-180-2-2 treatment. SYG-180-2-2 also reduced skin abscess formation in mice by reducing bacterial burden and subcutaneous inflammation. In conclusion, SYG-180-2-2 might be a promising agent to attenuate the virulence of S. aureus by targeting genes associated with hemolytic activity and staphyloxanthin synthesis.

In recent years, Collaborative Filtering (CF) methods have yielded immense success on recommender systems. They mainly use the similarity between users and items, or the interactions between users and items to predict the unknown ratings. However, the social conformity phenomenon received little notice, which means: 1) individuals in a social network can have multiple characteristics and hence tend to belong to multiple overlapping groups or communities and 2) when confronted with conformity pressure, people often adjust their responses to conform to others' opinions to obtain social approval and belonging in the community. In this paper, we propose a new collaborative filtering-based recommendation framework, called ACCF, which explicitly exploits social conformity of users. We incorporate such social conformity phenomenon into the latent factor model, using a weighted average of community preference profiles as the adjusting factor, and learn the weight of each community's influence through an attention network. Compared with the seven state-of-the-art methods on three real-world datasets, our method achieves the best performance.

Next generation sequencing platforms have greatly reduced sequencing costs, leading to the production of unprecedented amounts of sequence data. BWA is one of the most popular alignment tools due to its relatively high accuracy. However, mapping reads using BWA is still the most time consuming step in sequence analysis. Increasing mapping efficiency would allow the community to better cope with ever expanding volumes of sequence data.We designed a new program, CGAP-align, that achieves a performance improvement over BWA without sacrificing recall or precision. This is accomplished through the use of Suffix Tarray, a novel data structure combining elements of Suffix Array and Suffix Tree. We also utilize a tighter lower bound estimation for the number of mismatches in a read, allowing for more effective pruning during inexact mapping. Evaluation of both simulated and real data suggests that CGAP-align consistently outperforms the current version of BWA and can achieve over twice its speed under certain conditions, all while obtaining nearly identical results.CGAP-align is a new time efficient read alignment tool that extends and improves BWA. The increase in alignment speed will be of critical assistance to all sequence-based research and medicine. CGAP-align is freely available to the academic community at http://sourceforge.net/p/cgap-align under the GNU General Public License (GPL).