The roles of cell wall inhibition responsive protein CwrA in the pathogenicity of Staphylococcus aureus.

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 ß-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.

The role of male hormones in bacterial infections: enhancing Staphylococcus aureus virulence through testosterone-induced Agr activation.

Staphylococcus aureus is a notorious pathogen predominantly involved in skin and soft tissue infections, exhibiting a distinct innate sex bias. This study explores the influence of testosterone on the virulence of S. aureus and elucidates its underlying mechanisms. Utilizing a skin abscess model in intact and castrated male mice, we assessed the effects of testosterone on S. aureus pathogenicity. Compared to controls, castrated mice showed significantly reduced abscess sizes and decreased bacterial loads, highlighting the role of testosterone in modulating the severity of S. aureus infections. In vitro experiments revealed that testosterone enhances the hemolytic activity, cytotoxicity, and oxidative stress resistance of S. aureus. Real-time quantitative PCR analysis showed a significant upregulation of the genes encoding α-hemolysin (hla) and phenol-soluble modulin (psmα). Importantly, testosterone treatment significantly enhanced the expression of the accessory gene regulator (Agr) quorum-sensing system components (agrC, agrA, agrB, agrD), while the SaeRS system (saeR, saeS, and sbi) exhibited only slight changes. Gene knockout experiments revealed that deletion of agrC, rather than saeRS and agrBD, abolishes the testosterone-induced enhancement of hemolysis and gene expression, underscoring the key role of AgrC. Molecular docking simulations indicated a direct interaction between testosterone and AgrC protein, with a strong binding affinity at the active site residue SER201. This study provides new insights into the mechanistic basis of how testosterone enhances the pathogenicity of S. aureus, potentially contributing to increased male susceptibility to S. aureus infections and offering a targeted approach for therapeutic interventions.

Insights into small-molecule compound CY-158-11 antibacterial activity against Staphylococcus aureus.

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. IMPORTANCE: The 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.

Phenotypic and genomic analysis of the hypervirulent methicillin-resistant Staphylococcus aureus ST630 clone in China.

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.

Biofilm-producing ability of methicillin-resistant Staphylococcus aureus clinically isolated in China.

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.

Characteristic of KPC-12, a KPC Variant Conferring Resistance to Ceftazidime-Avibactam in the Carbapenem-Resistant Klebsiella pneumoniae ST11-KL47 Clone Background.

Background: Carbapenem-resistant Klebsiella pneumoniae (CRKP) infections are a great threat to public health worldwide. Ceftazidime-avibactam (CZA) is an effective ß-lactam/ß-lactamase inhibitors against CRKP. However, reports of resistance to CZA, mainly caused by Klebsiella pneumoniae carbapenemase (KPC) variants, have increased in recent years. In this study, we aimed to describe the resistance characteristics of KPC-12, a novel KPC variant identified from a CZA resistant K. pneumoniae. Methods: The K. pneumoniae YFKP-97 collected from a patient with respiratory tract infection was performed whole-genome sequencing (WGS) on the Illumina NovaSeq 6000 platform. Genomic characteristics were analyzed using bioinformatics methods. Antimicrobial susceptibility testing was conducted by the broth microdilution method. Induction of resistant strain was carried out in vitro as previously described. The G. mellonella killing assay was used to evaluate the pathogenicity of strains, and the conjugation experiment was performed to evaluate plasmid transfer ability. Results: Strain YFKP-97 was a multidrug-resistant clinical ST11-KL47 K. pneumoniae confers high-level resistance to CZA (16/4 µg/mL). WGS revealed that a KPC variant, KPC-12, was carried by the IncFII (pHN7A8) plasmids (pYFKP-97_a and pYFKP-97_b) and showed significantly decreased activity against carbapenems. In addition, there was a dose-dependent effect of bla KPC-12 on its activity against ceftazidime. In vitro inducible resistance assay results demonstrated that the KPC-12 variant was more likely to confer resistance to CZA than the KPC-2 and KPC-3 variants. Discussion: Our study revealed that patients who was not treated with CZA are also possible to be infected with CZA-resistant strains harbored a novel KPC variant. Given that the transformant carrying bla KPC-12 was more likely to exhibit a CZA-resistance phenotype. Therefore, it is important to accurately identify the KPC variants as early as possible.

TDASD: Generating medically significant fine-grained lung adenocarcinoma nodule CT images based on stable diffusion models with limited sample size.

BACKGROUND AND OBJECTIVES: Spread through air spaces (STAS) is an emerging lung cancer infiltration pattern. Predicting its spread through CT scans is crucial. However, limited STAS data makes this prediction task highly challenging. Stable diffusion is capable of generating more diverse and higher-quality images compared to traditional GAN models, surpassing the dominating GAN family models in image synthesis over the past few years. To alleviate the issue of limited STAS data, we propose a method TDASD based on stable diffusion, which is able to generate high-resolution CT images of pulmonary nodules corresponding to specific nodular signs according to the medical professionals. METHODS: First, we apply the stable diffusion method for fine-tuning training on publicly available lung datasets. Subsequently, we extract nodules from our hospital's lung adenocarcinoma data and apply slight rotations to the original nodule CT slices within a reasonable range before undergoing another round of fine-tuning through stable diffusion. Finally, employing DDIM and Ksample sampling methods, we generate lung adenocarcinoma nodule CT images with signs based on prompts provided by doctors. The method we propose not only safeguards patient privacy but also enhances the diversity of medical images under limited data conditions. Furthermore, our approach to generating medical images incorporates medical knowledge, resulting in images that exhibit pertinent medical features, thus holding significant value in tumor discrimination diagnostics. RESULTS: Our TDASD method has the capability to generate medically meaningful images by optimizing input prompts based on medical descriptions provided by experts. The images generated by our method can improve the model's classification accuracy. Furthermore, Utilizing solely the data generated by our method for model training, the test results on the original real dataset reveal an accuracy rate that closely aligns with the testing accuracy achieved through training on real data. CONCLUSIONS: The method we propose not only safeguards patient privacy but also enhances the diversity of medical images under limited data conditions. Furthermore, our approach to generating medical images incorporates medical knowledge, resulting in images that exhibit pertinent medical features, thus holding significant value in tumor discrimination diagnostics.

Characterisation of highly virulent and colistin-resistant ST367-KL1 Klebsiella quasipneumoniae subsp. similipneumoniae Strain.

OBJECTIVES: To elucidate the characteristics of a colistin-resistant and hypervirulent Klebsiella quasipneumoniae subsp. similipneumoniae strain (KP8) using whole genome sequencing and various phenotypic assays. METHODS: Antimicrobial susceptibility testing was performed using broth microdilution. Whole genome sequencing and comparative genomics were utilised to elucidate genomic characteristics. Phenotypic assays to evaluate virulence factors included measurements of mucosal viscosity, biofilm production, siderophore production, infection of A549 cells, serum-killing assays, and Galleria mellonella infection models. RESULTS: Whole-genome sequencing revealed that the strain (KP8) belongs to sequence type 367 (ST367) and capsular type 1 (KL1), and it harbours several virulence genes, including regulator of mucoid phenotype (rmpA/A2), salmochelin (iroBCDN) and aerobactin (iucABCDiutA). Antibiotic susceptibility tests showed that KP8 was resistant to colistin. Genome analysis showed that the colistin resistance of KP8 might be related to amino acid insertions in pmrB (L215_D217, insL) and pagP (M1_S3, insV). Importantly, KP8 demonstrated comparable mucosal viscosity, biofilm production capacity, siderophore production levels to hvKP. Serum-killing experiments, A549 cell infection models, and G. mellonella infection models further indicated that KP8 displayed high virulence, akin to the hypervirulent strain NUTH-K2044. Notably, global genome analysis of the K. quasipneumoniae subsp. similipneumoniae strains highlighted that the ST367 lineage has a higher tendency to carry virulence-associated genes compared to other sequence types. The prevalence of virulence-associated factors concentrated within Chinese ST367 isolates reinforces this observation. CONCLUSION: These findings further enhance our understanding of the resistance and pathogenicity of ST367 K. quasipneumoniae subsp. similipneumoniae strain and also providing a broader perspective on the global epidemiological landscape.

Novel small-molecule compound YH7 inhibits the biofilm formation of Staphylococcus aureus in a sarX-dependent manner.

The emergence of antibiotic-resistant and biofilm-producing Staphylococcus aureus isolates presents major challenges for treating staphylococcal infections. Biofilm inhibition is an important anti-virulence strategy. In this study, a novel maleimide-diselenide hybrid compound (YH7) was synthesized and demonstrated remarkable antimicrobial activity against methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) in both planktonic cultures and biofilms. The minimum inhibitory concentration (MIC) of YH7 for S. aureus isolates was 16 µg/mL. Quantification of biofilms demonstrated that the sub-MIC (4 µg/mL) of YH7 significantly inhibits biofilm formation in both MSSA and MRSA. Confocal laser scanning microscopy analysis further confirmed the biofilm inhibitory potential of YH7. YH7 also significantly suppressed bacterial adherence to A549 cells. Moreover, YH7 treatment significantly inhibited S. aureus colonization in nasal tissue of mice. Preliminary mechanistic studies revealed that YH7 exerted potent biofilm-suppressing effects by inhibiting polysaccharide intercellular adhesin (PIA) synthesis, rather than suppressing bacterial autolysis. Real-time quantitative PCR data indicated that YH7 downregulated biofilm formation-related genes (clfA, fnbA, icaA, and icaD) and the global regulatory gene sarX, which promotes PIA synthesis. The sarX-dependent antibiofilm potential of YH7 was validated by constructing S. aureus NCTC8325 sarX knockout and complementation strains. Importantly, YH7 demonstrated a low potential to induce drug resistance in S. aureus and exhibited non-toxic to rabbit erythrocytes, A549, and BEAS-2B cells at antibacterial concentrations. In vivo toxicity assays conducted on Galleria mellonella further confirmed that YH7 is biocompatible. Overall, YH7 demonstrated potent antibiofilm activity supports its potential as an antimicrobial agent against S. aureus biofilm-related infections. IMPORTANCE Biofilm-associated infections, characterized by antibiotic resistance and persistence, present a formidable challenge in healthcare. Traditional antibacterial agents prove inadequate against biofilms. In this study, the novel compound YH7 demonstrates potent antibiofilm properties by impeding the adhesion and the polysaccharide intercellular adhesin production of Staphylococcus aureus. Notably, its exceptional efficacy against both methicillin-resistant and methicillin-susceptible strains highlights its broad applicability. This study highlights the potential of YH7 as a novel therapeutic agent to address the pressing issue of biofilm-driven infections.

Metagenomic next-generation sequencing for the identification of infections caused by Gram-negative pathogens and the prediction of antimicrobial resistance.

OBJECTIVE: The aim of this study was to evaluate the efficacy of metagenomic next-generation sequencing (mNGS) for the identification of Gram-negative bacteria (GNB) infections and the prediction of antimicrobial resistance. METHODS: A retrospective analysis was conducted on 182 patients with diagnosis of GNB infections who underwent mNGS and conventional microbiological tests (CMTs). RESULTS: The detection rate of mNGS was 96.15%, higher than CMTs (45.05%) with a significant difference (χ 2 = 114.46, P < .01). The pathogen spectrum identified by mNGS was significantly wider than CMTs. Interestingly, the detection rate of mNGS was substantially higher than that of CMTs (70.33% vs 23.08%, P < .01) in patients with but not without antibiotic exposure. There was a significant positive correlation between mapped reads and pro-inflammatory cytokines (interleukin-6 and interleukin-8). However, mNGS failed to predict antimicrobial resistance in 5 of 12 patients compared to phenotype antimicrobial susceptibility testing results. CONCLUSIONS: Metagenomic next-generation sequencing has a higher detection rate, a wider pathogen spectrum, and is less affected by prior antibiotic exposure than CMTs in identifying Gram-negative pathogens. The mapped reads may reflect a pro-inflammatory state in GNB-infected patients. Inferring actual resistance phenotypes from metagenomic data remains a great challenge.

A novel small-molecule compound S-342-3 effectively inhibits the biofilm formation of Staphylococcus aureus.

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.

Mupirocin enhances the biofilm formation of Staphylococcus epidermidis in an atlE-dependent manner.

The pathogenicity of Staphylococcus epidermidis is largely attributed to its exceptional ability to form biofilms. Here, we report that mupirocin, an antimicrobial agent widely used for staphylococcal decolonization and anti-infection, strongly stimulates the biofilm formation of S. epidermidis. Although the polysaccharide intercellular adhesin (PIA) production was unaffected, mupirocin significantly facilitated extracellular DNA (eDNA) release by accelerating autolysis, thereby positively triggering cell surface attachment and intercellular agglomeration during biofilm development. Mechanistically, mupirocin regulated the expression of genes encoding for the autolysin AtlE as well as the programmed cell death system CidA-LrgAB. Critically, through gene knockout, we found out that deletion of atlE, but not cidA or lrgA, abolished the enhancement of biofilm formation and eDNA release in response to mupirocin treatment, indicating that atlE is required for this effect. In Triton X-100 induced autolysis assay, mupirocin treated atlE mutant displayed a slower autolysis rate compared with the wild-type strain and complementary strain. Therefore, we concluded that subinhibitory concentrations of mupirocin enhance the biofilm formation of S. epidermidis in an atlE dependent manner. This induction effect could conceivably be responsible for some of the more unfavourable outcomes of infectious diseases.

Phenotypic and genomic analysis of the hypervirulent ST22 methicillin-resistant Staphylococcus aureus in China.

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.

Small-Molecule Compound CY-158-11 Inhibits Staphylococcus aureus Biofilm Formation.

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.

Clinical Efficacy and Diagnostic Value of Metagenomic Next-Generation Sequencing for Pathogen Detection in Patients with Suspected Infectious Diseases: A Retrospective Study from a Large Tertiary Hospital.

Purpose: Metagenomic next-generation sequencing (mNGS) is a powerful yet unbiased method to identify pathogens in suspected infections. However, little is known about its clinical effectiveness. The present study aimed to assess the efficacy of mNGS in routine clinical practice. Patients and Methods: In this single-center retrospective cohort study, 518 patients with suspected infectious diseases were assessed for inclusion. Among them, each patient had undergone mNGS testing; 407 patients had undergone both microbial culture and mNGS testing. The result of mNGS testing was compared to microbial culture performed concurrently. The diagnostic performance of mNGS was evaluated using the comprehensive clinical diagnosis as the reference standard. Results: There was a significant difference in the positive detection rates of pathogens between mNGS and culture (331/407, 81.3% vs 79/407, 19.4%, P < 0.001). The sensitivity of mNGS was much higher than the culture method (79.5% vs 21.3%, P < 0.001), especially in sample types of sputum and bronchoalveolar lavage fluid (BALF). Notably, the sensitivity of blood mNGS was relatively lower than other sample types (67.4% vs 88.9-93.8%). Pathogen cfDNA load based on standardized stringently mapped read number at the species level of microorganisms (SDSMRN) was significantly lower in blood than in other sample types from the same patient (P = 0.0003). Importantly, mNGS directly led to a change of treatment regimen in 142 (27.4%) cases, including antibiotic escalation (15.3%), antibiotic de-escalation (9.1%), and early definitive diagnosis to initiate appropriate treatment (3.1%). Conclusion: Our in-house mNGS platform significantly improved the sensitivity for the diagnosis of infectious diseases. mNGS has the potential to improve clinical outcomes by optimizing antimicrobial therapy.

Phylogenetic Analysis and Virulence Characteristics of Methicillin-Resistant Staphylococcus aureus ST45 in China: a Hyper-Virulent Clone Associated with Bloodstream Infections.

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.

Phylogenetic analysis and virulence characteristics of methicillin-resistant Staphylococcus aureus ST764-SCCmec II: an emerging hypervirulent clone ST764-t1084 in China.

Previous 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.

Small-molecule compound SYG-180-2-2 attenuates Staphylococcus aureus virulence by inhibiting hemolysin and staphyloxanthin production.

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.

CGAP-align: a high performance DNA short read alignment tool.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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).