Mechanistic research:Selenium regulates virulence factors,reducing adhesion ability and inflammatory damage of Helicobacter pylori

BACKGROUND The pathogenicity of Helicobacter pylori is dependent on factors including the environment and the host.Although selenium is closely related to pathogenicity as an environmental factor,the specific correlation between them remains unclear.AIM To investigate how selenium acts on virulence factors and reduces their toxicity.METHODS H.pylori strains were induced by sodium selenite.The expression of cytotoxin-associated protein A(CagA)and vacuolating cytotoxin gene A(VacA)was determined by quantitative PCR and Western blotting.Transcriptomics was used to analyze CagA,CagM,CagE,Cag1,Cag3,and CagT.C57BL/6A mice were infected with the attenuated strains subjected to sodium selenite induction,and H.pylori colonization,inflammatory reactions,and the cell adhesion ability of H.pylori were assessed.RESULTS CagA and VacA expression was upregulated at first and then downregulated in the H.pylori strains after sodium selenite treatment.Their expression was significantly and steadily downregulated after the 5th cycle(10 d).Transcriptome analysis revealed that sodium selenite altered the levels affect H.pylori virulence factors such as CagA,CagM,CagE,Cag1,Cag3,and CagT.Of these factors,CagM and CagE expression was continuously downregulated and further downregulated after 2 h of induction with sodium selenite.Moreover,CagT expression was upregulated before the 3rd cycle(6 d)and significantly downregulated after the 5th cycle.Cag1 and Cag3 expression was upregulated and downregulated,respectively,but no significant change was observed by the 5th cycle.C57BL/6A mice were infected with the attenuated strains subjected to sodium selenite induction.The extent of H.pylori colonization in the stomach increased;however,sodium selenite also induced a mild inflammatory reaction in the gastric mucosa of H.pylori-infected mice,and the cell adhesion ability of H.pylori was significantly weakened.CONCLUSION These results demonstrate that H.pylori displayed virulence attenuation after the 10th d of sodium selenite treatment.Sodium selenite is a low toxicity compound with strong stability that can reduce the cell adhesion ability of H.pylori,thus mitigating the inflammatory damage to the gastric mucosa.

Molecular Epidemiological Analysis of Group A Streptococci Isolated from Children in Chaoyang District of Beijing, 2011:emm Types, Virulence Factor Genes and Erythromycin Resistant Genes

Group A streptococcus （GAS） causes a wide range of diseases in the human population. GAS diseases are more common in children than in adults, with clinical manifestations ranging from pharyngitis and impetigo to invasive infections and post streptococcal sequelae, such as acute rheumatic fever and acute post-streptococcal glomerulonephritis[1]. GAS harbors a host of virulence factors that contribute to its complex pathogenicity and differences in the disease severity and frequency. M protein, one of the major virulence factors, is encoded by the emm gene induces a type of specific host immune response and confers antiphagocytic properties.

Role of nickel-regulated small RNA in modulation of Helicobacter pylori virulence factors

Helicobacter pylori(H.pylori)is a Gram-negative bacterium that infects about half of the world's population.H.pylori infection prevails by several mechanisms of adaptation of the bacteria and by its virulence factors including the cytotoxin associated antigen A(CagA).CagA is an oncoprotein that is the protagonist of gastric carcinogenesis associated with prolonged H.pylori infection.In this sense,small regulatory RNAs(sRNAs)are important macromolecules capable of inhibiting and activating gene expression.This function allows sRNAs to act in adjusting to unstable environmental conditions and in responding to cellular stresses in bacterial infections.Recent discoveries have shown that nickelregulated small RNA(NikS)is a post-transcriptional regulator of virulence properties of H.pylori,including the oncoprotein CagA.Notably,high concentrations of nickel cause the reduction of NikS expression and consequently this increases the levels of CagA.In addition,NikS expression appears to be lower in clinical isolates from patients with gastric cancer when compared to patients without.With that in mind,this minireview approaches,in an accessible way,the most important and current aspects about the role of NikS in the control of virulence factors of H.pylori and the potential clinical repercussions of this modulation.

Extracellular Polysaccharides Matrix—An Often Forgotten Virulence Factor in Oral Biofilm Research

Oral diseases related to dental biofilms continue to afflict the majority of the world＇s population. Among them, dental caries continues to be the single most prevalent and costly oral infectious disease （Marsh, 2003; Dye et al., 2007）. Dental caries results from the interaction of specific bacteria with constituents of the diet within a dental biofilm known as plaque （Bowen, 2002）. Sucrose is considered to be the ＂arch criminal＂ from the dietary aspect because it serves as a substrate for synthesis of extracellular （EPS） and intracellular （IPS） polysaccharides in dental biofilm and is also fermentable （Bowen, 2002）.

HP0953-hypothetical virulence factor overexpresion and localization during Helicobacter pylori infection of gastric epithelium

BACKGROUND The high prevalence and persistence of Helicobacter pylori(H. pylori) infection, as well as the diversity of pathologies related to it, suggest that the virulence factors used by this microorganism are varied. Moreover, as its proteome contains 340hypothetical proteins, it is important to investigate them to completely understand the mechanisms of its virulence and survival. We have previously reported that the hypothetical protein HP0953 is overexpressed during the first hours of adhesion to inert surfaces, under stress conditions, suggesting its role in the environmental survival of this bacterium and perhaps as a virulence factor.AIM To investigate the expression and localization of HP0953 during adhesion to an inert surface and against gastric(AGS) cells.METHODS Expression analysis was performed for HP0953 during H. pylori adhesion. HP0953 expression at 0,3, 12, 24, and 48 h was evaluated and compared using the Kruskal-Wallis equality-of-populations rank test. Recombinant protein was produced and used to obtain polyclonal antibodies for immunolocalization. Immunogold technique was performed on bacterial sections during adherence to inert surfaces and AGS cells, which was analyzed by transmission electron microscopy. HP0953 protein sequence was analyzed to predict the presence of a signal peptide and transmembrane helices, both provided by the ExPASy platform, and using the GLYCOPP platform for glycosylation sites. Different programs, via, I-TASSER, RaptorX, and HHalign-Kbest, were used to perform three-dimensional modeling.RESULTS HP0953 exhibited its maximum expression at 12 h of infection in gastric epithelium cells.Immunogold technique revealed HP0953 localization in the cytoplasm and accumulation in some peripheral areas of the bacterial body, with greater expression when it is close to AGS cells.Bioinformatics analysis revealed the presence of a signal peptide that interacts with the transmembrane region and then allows the release of the protein to the external environment. The programs also showed a similarity with the Tip-alpha protein of H. pylori. Tip-alpha is an exotoxin that penetrates cells and induces tumor necrosis factor alpha production, and HP0953 could have a similar function as posttranslational modification sites were found;modifications in turn require enzymes located in eukaryotic cells. Thus, to be functional, HP0953 may necessarily need to be translocated inside the cell where it can trigger different mechanisms producing cellular damage.CONCLUSION The location of HP0953 around infected cells, the probable posttranslational modifications, and its similarity to an exotoxin suggest that this protein is a virulence factor.

Corchorus olitorius aqueous extract attenuates quorum sensing-regulated virulence factor production and biofilm formation

Objective:To investigate the effect of Corchorus olitorius aqueous fraction(COAF)on quorum sensing(QS)-regulated virulence factors and biofilm formation in Pseudomonas aeruginosa(PAO1).Methods:The preliminary screening of the anti-QS effect of COAF was performed by evaluating the anti-pathogenic activity against Chromobacterium violaceum CV026 biosensor strain.Next,the inhibitory effects of COAF on QS-regulated pyocyanin production,proteolytic and elastolytic activities,swarming motility,and biofilm formation were evaluated in PAO1.Results:The results showed that the treatment of COAF significantly decreased the biofilm biomass,attenuated virulence factors,and inhibited swarming motility of PAO1 without affecting the growth of the bacteria in a dose-dependent manner.COAF at 2000μg/mL significantly decreased Las B elastase activity in PAO1 culture,exopolysaccharide production,swarming motility,pyocyanin level,and biomass of PAO1 by 55%(P<0.05),60%(P<0.01),61%(P<0.01),65%(P<0.01)and 73%(P<0.01),respectively.In addition,the production of violacein was decreased by 62%(P<0.01)with the treatment of a high dose of COAF.Conclusions:These findings indicate that COAF can be a potential source of anti-QS agents.

Virulence Factor Profile of Staphylococcus aureus Isolated from Bovine Milk from Brazil

This study investigates the biofilm formation, presence and distribution of virulence genes and the capacity to induce an inflammatory response in strains of Staphylococcus aureus isolated from milk samples in Bahia, Brazil. A total of 132 samples of raw milk were collected from four dairy farms (designated A to D) located in southwestern Bahia, in the municipality of Vitória da Conquista, from October/2009 to September/2010. After processing of the samples, 94 (71.2%) S. aureus isolates were obtained. These strains were subjected to the antibiogram method MIC (Minimum Inhibitory Concentration). As for the pathogenicity, tests were performedin vitrobiofilm formation induced by glucose. Moreover, we performed PCR for their virulence genes: sea (enterotoxin A), seb (B), sec (C), pvl (Panton-Valentine Leukocidin), clfA (Clumping Factor A) and spa (protein A) and analysis of cytokine induction in the inflammatory response of J774 macrophages by exocellular lipoteichoic acid. No isolates were resistant to oxacillin and vancomycin. In biofilm production, 5.31% (5/94) isolates did not produce biofilm, 5.31% (5/94) of the samples were poor producers, 15.96% (15/94) strains were moderate producers, 18.09% (17/94) were producers and 55.32% (55/94) of isolates were strong biofilm producers. One (1.06%) isolate expressed the seb gene, one (1.06%) sec, 18 (19.2%) cflA and 44 (46.8%) had spa. There was no expression of sea and pvl between isolates analyzed. The analysis of cytokine induction in the inflammatory response did not show statistical difference in the levels of IL-6, TNF-α and IL-10 induction. However, there was statistical difference in IL-1 induction between isolates from different farms. Thus, it appears that the results obtained in this study show significant effects for the region studied, since it is an important dairy region, hence the need for further studies, with the intent of attracting funding that contributes to improving prevention and control in both dairy farms and dairy industries, since milk contamination poses a serious potential health risk to consumers.

Phylogenetic Analysis of Virulence Factor Genes in Salmonella from Chicken

Salmonella is a common genus of seriously harmful food-borne zoonotic bacteria. Humans and animals may be infected with Salmonella through ingestion of SalmoneUa-contaminated eggs and poultry meat. Therefore, in order to reduce the incidence of Salmonella infections, it is crucial to explore the pathogenic mech- anism of Salmonella. invA and invE are major virulence factor genes that encode invasion proteins of Salmonella. In order to explore the pathogenic mechanism of Salmonella, phylogenetic analysis of major virulence factor genes in 33 Salmonella strains isolated from chicken was analyzed. According to the results, ivnA gene was successfully amplified from 33 Salmonella strains; ivnE gene was successfully amplified from 32 Salmonella strains, ivnA nucleotide sequences shared 72.9% - 97.6% homology among 12 sequenced Salmonella strains and shared 78.9% - 97.2% homology with those in GenBank ; ivnE nucleotide sequences shared over 95.3% homology among 23 sequenced Salmonella strains and shared 89.6% -98.6% homology with those in GenBank, which exhibited no genetic relationship to other organisms. This study provided the basis for rapid molecular detection, epidemiological research and molecular pathogenesis analysis of Salmonella.

Structural Changes of Lignified Tissues from Sugarcane Leaves Induced by Smut （Sporisorium scitamineum） Virulence Factors

Sugarcane leaf shows the classical arrangement of cells, which defines a C4 species. Vascular bundles consist of xylem, phloem and fibres, surrounded by an outer layer ofsclereids and an inner ring of stone cells associated with the phloem. Some sclereids located below and above the vascular bundles act as docking cells and connect the vascular bundle to the internal surfaces of upper and lower layers of the epidermis. A compact mass ofsclereids occupies the total internal volume of the leaf edge. Neither docking cells nor the internal mass of sclereids in the edge were markedly coloured by phloroglucinol, indicating the absence of lignin in their cell walls. However, such staining indicated that fibres of the vascular bundle and the external layer of sclereids were strongly lignified. Incubation of leaf discs with an virulence factors produced by the pathogen Sporisorium scitamineum increased the thickness of the lignified cell walls of sclereids as well as the mid and small xylem vessels, as a possible mechanical defence response to the potential entry of the pathogen. This mechanism was mainly revealed for the resistant cv. Mayari 55-14, whereas lignification decreased for the susceptible cv. B 42231.

Virulence Factors in Methicillin-Resistant Staphylococcus aureus Isolated from ICU Units in Brazil

Species of Staphylococcus are common in hospital infection (HI). Methicillin resistant S. aureus (MRSA) has also become a serious problem in Brazilian HI. The aim of this study was to characterize the pathogenicity of methicillin-resistant S. aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA) isolated in public hospitals. The clinical isolates were obtained from intensive care unit. The MRSA and MSSA strains were genotyped by PCR for detection genes related to virulence factors. Moreover, the strains were tested for biofilm formation and cytokine induction in macrophages. Three strains of MRSA (9.68%) expressed the Sea gene, one (3.23%) Seb, 17 (54.84%) Spa and seven (22.58%) had PVL. Two MSSA strains (2.98%) expressed the Sea gene, three (4.48%) Seb, 18 (26.87%) Spa and 11 (16.42%) showed positive results for the PVL gene. There was no expression of Sec and CflA between MRSA and MSSA strains. Among MRSA and MSSA isolates, none statistical differences were observed in biofilm production. The analysis of cytokine induction in the inflammatory response of J774 macrophages by MRSA and MSSA isolates did not show statistical difference. Understanding the mechanisms of pathogenesis of S. aureus could provide important clues for both preventing and treating infection caused by these organisms.

Characterization of Virulence Factors in Enteroaggregative and Atypical Enteropathogenic Escherichia coli Strains Isolated from Children with Diarrhea

Enteroaggregative (EAEC) and atypical enteropathogenic (EPEC) Escherichia coli are important bacterial etiologic agents causing diarrhea among children. The aim of the present study was to examine the impact of virulence factors predisposes to diarrhea. In this study some virulence properties were examined on 11 EAEC and 8 EPEC strains identified by Polymerase Chain Reaction (PCR), isolated from stool samples of children were analyzed genotypically and phenoltypically for the prevalence of virulence factors. The most frequently detected factor was resistance to serum (94%), followed by curli fimbriae (78%), biofilm production (73%), and gene coding for Extended-Spectrum Beta-Lactamase (ESBL) (68%). EPEC isolates showed at least three of the evaluated properties, while EAEC isolates showed at least two. The prevalence of these virulence factors between the two strains showed no statistical difference. This study showed the heterogeneity of the virulence profile of the isolates of EAEC and atypical EPEC strains and suggests that this diversity may influence in the disease severity.

Two-component signal transduction systems and regulation of virulence factors in Xanthomonas: a perspective

Two-component signal transduction systems(TCSTSs),consisting of a histidine kinase and a response regulator,play a critical role in regulating virulence gene expression in Gram-negative phytopathogenic bacteria Xanthomonas spp..To date,12 TCSTS genes have been identified,accounting for approximately 10%of the TCSTS genes in each genome that have been experimen-tally identified to be related to pathogenesis.These TCSTSs modulate the expression of a number of virulence factors through diverse molecular mechanisms such as interacting with DNA,protein-binding and involvement in second messenger metabolism,which generates a high level of regulatory versatility.Here we summarize the current knowledge in thisfield and discuss the emerging themes and remaining questions that are important in deciphering the signaling network of TCSTSs in Xantho-monas.

Epilithic biofilm as a reservoir for functional virulence factors in wastewater-dominant rivers after WWTP upgrade

Virulence factors(VFs)confer upon pathogens the ability to cause various types of damage or diseases.Wastewater treatment plants(WWTPs)are important point sources for the emission of pathogens and VFs into receiving rivers.Conventional WWTP upgrades are often implemented to improve the water quality of receiving ecosystems.However,knowledge on the pathogens,VFs,and health risks to receiving aquatic ecosystems after upgrade remains limited.In this study,we investigated detailed pathogenic information,including taxa,pathogenicity,and health risk,in two wastewater-dominant rivers after WWTP upgrade.Using 16S rRNA gene sequencing,we screened 14 potential pathogens in water and epilithic biofilm samples,though they were significantly more enriched in the biofilms.Combining 16S rRNA and metagenomic sequencing data,we identified Pseudomonas and Aeromonas as the dominant pathogenic taxa carrying functional VFs(e.g.,mobility and offensive)in the epilithic biofilm.Moreover,strong pathogen-specific VF-host co-occurrence events were observed in the epilithic biofilm samples,indicating the importance of biofilms as reservoirs and vehicles for VFs.Further,we demonstrated that mobility VF is crucial for biofilm formation and pathogens in biofilm carrying offensive VF may be highly invasive.Quantification and health risk assessment suggested that the skin contact risk of P.aeruginosa carrying VFs was higher than the acceptable probability of 10^(-4)in both water and epilithic biofilm samples,which may threaten ecological and human health.

Impact of Helicobacter pylori virulence markers on clinical outcomes in adult populations

BACKGROUND In recent years,associations between specific virulence markers of Helicobacter pylori(H.pylori)and gastrointestinal disorders have been suggested.AIM To investigate the presence of virulence factors including vacuolating cytotoxin A genotypes(s1m1,s1m2,s2m1,and s2m2),cytotoxin-associated gene A(CagA),and urease activity in H.pylori strains isolated from Arab and Jewish populations in northern Israel and to assess associations between these factors and patients’demographics and clinical outcomes.METHODS Patients(n=108)who underwent gastroscopy at the Baruch Padeh Medical Center,Poriya due to symptomatic gastroduodenal pathologies as part of H.pylori diagnosis were enrolled in the study.Gastric biopsy specimens were collected from the antrum of the stomach.Clinical condition was assessed by clinical pathology tests.Bacteria were isolated on modified BD Helicobacter Agar(BD Diagnostics,Sparks,MD,United States).Bacterial DNA was extracted,and PCR was performed to detect CagA and vacuolating cytotoxin A genes.Urease activity was assessed using a rapid urease test.RESULTS A significant correlation was found between disease severity and patient ethnicity(P=0.002).A significant correlation was found between CagA presence and the s1m1 genotype(P=0.02),which is considered the most virulent genotype.Further,a higher level of urease activity was associated with isolates originating from the Jewish population.Moreover,higher urease activity levels were measured among CagA-/s1m1 and CagA-/s2m2 isolates.CONCLUSION Our study highlights the importance of incorporating molecular methods for detection of virulence markers of H.pylori in order to tailor optimal treatments for each patient.Further investigation should be performed regarding associations between H.pylori virulence factors and ethnicity.

Helicobacter pylori's virulence and infection persistence define pre-eclampsia complicated by fetal growth retardation

AIM: To better understand the pathogenic role of Helicobacter pylori (H. pylori) in pre-eclampsia (PE), and whether it is associated or not with fetal growth retardation (FGR). METHODS: Maternal blood samples were collected from 62 consecutive pregnant women with a diagnosis of PE and/or FGR, and from 49 women with uneventful pregnancies (controls). Serum samples were evaluated by immunoblot assay for presence of specific antibodies against H. pylori antigens [virulence: cytotoxin-associated antigen A (CagA); ureases; heat shock protein B; flagellin A; persistence: vacuolating cytotoxin A (VacA)]. Maternal complete blood count and liver enzymes levels were assessed at delivery by an automated analyzer. RESULTS: A significantly higher percentage of H. pyloriseropositive women were found among PE cases (85.7%) compared to controls (42.9%, P < 0.001). There were no differences between pregnancies complicated by FGR without maternal hypertension (46.2%) and controls. Importantly, persistent and virulent infections (VacA/ CagA seropositive patients, intermediate leukocyte blood count and aspartate aminotransferase levels) were exclusively associated with pre-eclampsia complicated by FGR, while virulent but acute infections (CagA positive/ VacA negative patients, highest leukocyte blood count and aspartate aminotransferase levels) specifically correlated with PE without FGR. CONCLUSION: Our data strongly indicate that persistent and virulent H. pylori infections cause or contribute to PE complicated by FGR, but not to PE without feto-placental compromise.

Food Safety Risks and Contributing Factors of Cronobacter spp.

Cronobacter species are a group of Gram-negative opportunistic pathogens,which cause meningitis,sep-ticemia,and necrotizing enterocolitis in neonates and infants,with neurological sequelae in severe cases.Interest in Cronobacter has increased significantly in recent years due to its high virulence in children.In this review,we summarize the current understanding of the prevalence of Cronobacter species in several important food types.We discuss the response mechanisms enabling persistence in adverse growth con-ditions,as well as its pathogenicity.We emphasize the food safety concerns caused by Cronobacter and subsequent control methods and clinical treatments.

Infectious Spondylitis-Associated Staphylococcus aureus with Virulence Gene pvl or tst Causes More Necrosis than Apoptosis in Human Alveolar Basal Epithelial Cell Line A549

Methicillin-sensitive and resistant Staphylococcus aureus (MSSA and MRSA, respectively) can cause non-tuberculosis infectious spondylitis. In 43 cases of bacterial infectious spondylitis, Mycobacterium tuberculosis and S. aureus were the two major causative pathogens. MRSA caused more anterior operations and thoracic infections, while MSSA caused more posterior infections and lumbar infections. Differences between six S. aureus isolates from infectious spondylitis were characterized. MLST and staphylococcal cassette chromosome mec (SCCmec) analysis identified MSSA ST959 and ST30 isolates, MRSA ST239/SCCmec IIIA isolates 2 and 3, ST59/SCCmec IIIA-like isolate 6, and ST30/SCCmec IV isolate 5. While all of the isolates were resistant to penicillin and ampicillin, the MRSA isolates were more resistant than the MSSA isolates. Carbapenem-resistant MRSA ST239/SCCmec IIIA and ST59/SCCmec IIIA-like isolates of the agr1 type were also resistant to clindamycin and erythromycin. Leukocidin genes (pvl or lukED) and hemolysin genes (hla, hld and hlg) were present in all of the isolates. All six isolates caused more necrosis than apoptosis in the human alveolar basal epithelial cell line A549;however, ST59/SCCmec IIIA-like isolate 6, ST30/ SCCmec IV isolate 5 with pvl genes, and MSSA ST30 isolates with tst caused greater than 40% cell death after the 4-h incubation. Regardless of the MRSA isolate and its SCCmec type or the MSSA isolate, the infectious spondylitis-associated S. aureus isolates differed genetically, and the pvl and tst genes may be important genes for cell necrosis.

Construction of Prokaryotic Expressing Vector of Antisense Nucleic Acid of lasR and Its Effect on the Virulence of Pseudomonas Aeruginosus

To construct a pUCP18/lasR^antisense plasmid carrying the reversed gene and analyze its effect on the virulence of Pseudomonas aeruginosus, LasR gene was amplified from the genome of Pseudomonas aeruginosus by PCR and reversely recombined with plasmid pUCP18. The recombinant pUCP18/lasR^antisense was verified by enzyme digestion, PCR and sequencing. The biological effects of pUCP18/lasR^antisense were examined by using RT-PCR, NAD method and the assay of pyocyanin. Our results showed that the expected full length lasR fragment （721 bp） was extended from Pseudomonas aeruginosus gene with PCR. And it is consistent with LasR gene of Pseudomonas aeruginosa in GenBank （No. NC_002516）. The recombinant plasmid was successfully constructed and transferred into Pseudomonas aeruginosus. The antisense nucleic acid of LasR gene could reduce the virulence of Pseudomonas aeruginosus and might serve as a new target site for treatment purpose.

Pathogenesis and drug resistance mechanism of Burkholderia pseudomallei

Burkholderia pseudomallei is the pathogen that causes melioidosis.Melioidosis has a long duration of chronic infection,atypical clinical manifestations at acute onset,and is prone to life-threatening complications and poor prognosis.Understanding the pathogenesis and drug resistance mechanism of Burkholderia pseudomallei will effectively help the diagnosis and treatment of the disease and improve the prognosis.This review focuses on the extracellular movement of Burkholderia pseudomallei in host cells,the way of infecting host cells,virulence factors,and drug resistance mechanisms(efflux pumps,changes in target sites,etc.).This study provides a possible direction for the early diagnosis,treatment and control of melioidosis caused by this bacterium.

A global perspective on microbial risk factors in effluents of wastewater treatment plants

Effective monitoring and management of microbial risk factors in wastewater treatment plants(WWTPs)effluents require a comprehensive investigation of these risks.A global survey on microbial risk factors in WWTP effluents could reveal important insights into their risk features.This study aims to explore the abundance and types of antibiotic resistance genes(ARGs),virulence factor genes(VFGs),the vector of ARG/VFG,and dominant pathogens in global WWTP effluents.We collected 113 metagenomes of WWTP effluents from the Sequence Read Archive of the National Center for Biotechnology Information and characterized the microbial risk factors.Our results showed that multidrug resistance was the dominant ARG type,while offensive virulence factors were the most abundant type of VFGs.The most dominant types of ARGs in the vector of plasmid and phage were both aminoglycoside resistance,which is concerning as aminoglycosides are often a last resort for treating multi-resistant infections.Acinetobacter baumannii was the most dominant pathogen,rather than Escherichia coli,and a weak negative correlation between Escherichia coli and two other dominant pathogens(Acinetobacter baumannii and Bacteroides uniformis)suggests that using Escherichia coli as a biological indicator for all pathogens in WWTP effluents may not be appropriate.The Getah virus was the most dominant virus found in global WWTP effluents.Our study presents a comprehensive global-scale investigation of microbial risk factors in WWTP effluents,providing valuable insights into the potential risks associated with WWTP effluents and contributing to the monitoring and control of these risks.