Molecular investigation of exoU and exoY virulence genes in Pseudomonas aeruginosa collected from hospitalized patients in North of Iran:A descriptive-analytical study

Objective:To investigate the frequency of exoU and exoY genes in patients with Pseudomonas aeruginosa infection.Methods:In this study,100 clinical isolates of Pseudomonas aeruginosa were collected from patients hospitalized in educational-therapeutic hospitals and were identified using standard microbiological tests.Then,the antibiotic resistance pattern of the isolates was determined by the disk agar diffusion method.The bacterial DNAs were extracted by the alkaline lysis method.Finally,the presence of exoU and exoY genes was evaluated by the PCR test.Results:In this study,47%,72%,29%,39%,40%,and 44%of the isolates were non-susceptible to piperacillin,aztreonam,ceftazidime,imipenem,tobramycin,and ciprofloxacin,respectively.In addition,95%and 93%of the clinical isolates carried the exoU and exoY genes.Blood and fecal isolates had both virulence genes,while only one wound isolate had neither genes.Meanwhile,all urinary isolates contained the exoY gene and only one isolate lacked the exoU gene.Also,88 isolates simultaneously had both exoU and exoY genes.Conclusions:High prevalence of exoU and exoY genes in this region indicates a significant role of typeⅢsecretion system in pathogenesis of Pseudomonas aeruginosa.The typeⅢsecretion system may be a suitable target to reduce the pathogenicity of this bacterium.

Effects of Microplastics on Expression of Resistance Genes and Virulence Genes of Vibrio alginolyticus

[Objectives]To study the effects of microplastics on antibiotic resistance genes and virulence genes of Vibrio alginolyticus,so as to provide a certain reference for controlling marine pollution,curbing the spread of environmental antibiotic resistance genes and virulence genes,formulating environmental policies,and maintaining food safety.[Methods]After adding V.alginolyticus into the artificial seawater,they were divided into three groups,namely blank control group(BLK),polyvinyl chloride microplastic group(PVC group)and polyvinyl alcohol microplastic group(PVA group).Aerated culture experiments were carried out,and the effects of microplastics on the expression of resistance genes and virulence genes of V.alginolyticus were studied by PCR and qPCR methods.[Results]The presence of microplastics significantly changed the resistance gene structure of V.alginolyticus.Compared with the control group,the cfxA and cfr resistance genes were detected in the microplastic group.However,only PVC group detected blaZ resistance gene,and only PVA group did not detect aaC resistance gene.In addition,compared with the control group,the expressions of virulence genes in the microplastic group were all down-regulated(P<0.01).[Conclusions]This study provides some reference for curbing the spread of environmental antibiotic resistance genes and virulence genes,formulating environmental policies,and maintaining food safety,but the specific mechanisms of drug resistance and virulence need further research.

PCR Detection of Virulence Genes Colv,Stxs and HlyE of Escherichia coli

[Objective] This study aimed to explore the presence of three causative genes Colv,Stxs and HlyE of the pathogenic E.coli from chickens,pigs and food.[Method] By using 44 E.coli strains from chickens,24 from pigs and 26 from food as the experimental materials,virulence genes Colv,Stxs（stx2,stx2e） and HlyE were detected with polymerase chain reaction（PCR） method.[Result] Among all the E.coli strains,the detection rate of Colv was 25% from chickens,4.2% from pigs,and 0 from food;the detection rate of Stx2（Stx2e） from all E.coli strains was 0;the detection rate of HlyE was 2.27% from chickens,0 from pigs,and 11.5% from food.[Conclusion] Virulence gene Colv shows relatively high carrying rate in E.coli from chickens and pigs;HlyE also shows a certain degree of presence in E.coli from chickens and food.

Helicobacter pylori virulence genes

Helicobacter pylori(H.pylori)is one of the most important human pathogens,infecting approximately half of the global population.Despite its high prevalence,only a subset of H.pylori infected individuals develop serious gastroduodenal pathology.The pathogenesis of H.pylori infection and disease outcome is thus thought to be mediated by an intricate interplay between host,environmental and bacterial virulence factors.H.pylori has adapted to the harsh milieu of the human stomach through possession of various virulence genes that enable survival of the bacteria in the acidic environment,movement towards the gastric epithelium,and attachment to gastric epithelial cells.These virulence factors enable successful colonization of the gastric mucosa and sustain persistent H.pylori infection,causing chronic inflammation and tissue damage,which may eventually lead to the development of peptic ulcers and gastric cancer.Numerous studies have focused on the prevalence and role of putative H.pylori virulence genes in disease pathogenesis.While several virulence factors with various functions have been identified,disease associations appear to be less evident,especially among different study populations.This review presents key findings on the most important H.pylori virulence genes,including several bacterial adhesins and toxins,in children and adults,and focuses on their prevalence,clinical significance and potential relationships.

Factors Influencing Induction of Agrobacterium tumefaciens Virulence Genes

Agrobacterium species are routinely employed for plant genetic modification due to the relatively simple procedures, cost-competitiveness, low copy num- ber, independence to vector DNAs, and targeted integration into transcriptionally active regions of plant chromosomes with defined T-DNA. However, to date, there are still a great number of plant species reluctant to Agrobacterium-mediated transformation. Evidence suggests that the infection capability of Agrobacterium is deter- mined by virulence （vir） genes of Ti plasmid outside ofA. tumefaciens chromosome. Among all v/r genes, virA and virG are constitutively expressed, while the ex- pression of other vir genes is induced by phenolic compounds. In addition, carbohydrates can enhance vir induction mediated by phenolic compounds, while low phosphate and acidic pH conditions may also enhance the induction of vir genes. To improve Agrobacterium-mediated transformation efficiency for potential applica- tions in research and industry, molecular mechanisms of vir induction by factors such as phenolic compounds, carbohydrates, low phosphate, acidic pH and incuba- tion temperature are discussed in this review.

Genetic Diversity, Antimicrobial Resistance, and Virulence Genes of Aeromonas Isolates from Clinical Patients, Tap Water Systems, and Food

Objective This study aimed to evaluate the genetic diversity,virulence,and antimicrobial resistance of Aeromonas isolates from clinical patients,tap water systems,and food.Methods Ninety Aeromonas isolates were obtained from Ma’anshan,Anhui province,China,and subjected to multi-locus sequence typing(MLST)with six housekeeping genes.Their taxonomy was investigated using concatenated gyr B-cpn60 sequences,while their resistance to 12 antibiotics was evaluated.Ten putative virulence factors and several resistance genes were identified by PCR and sequencing.Results The 90 Aeromonas isolates were divided into 84 sequence types,80 of which were novel,indicating high genetic diversity.The Aeromonas isolates were classified into eight different species.PCR assays identified virulence genes in the isolates,with the enterotoxin and hemolysin genes act,aer A,alt,and ast found in 47(52.2%),13(14.4%),22(24.4%),and 12(13.3%)of the isolates,respectively.The majority of the isolates(≥90%)were susceptible to aztreonam,imipenem,cefepime,chloramphenicol,gentamicin,tetracycline,and ciprofloxacin.However,several resistance genes were detected in the isolates,as well as a new mcr-3 variant.Conclusions Sequence type,virulence properties,and antibiotic resistance vary in Aeromonas isolates from clinical patients,tap water systems,and food.

Molecular Characterization and Virulence Genes of Aeromonas hydrophila Isolated from the Chinese Giant Salamander (Andrias davidianus)

The Chinese giant salamander(Andrias davidianus) is the largest living amphibian in the world. Aeromonas hydrophila strain L602 was isolated from A. davidianus. The 16S rDNA gene of this isolate was amplified using PCR,and the phylogenetic tree was constructed by the neighbor-joining method. Four virulence genes(aerA,aha1,hly and alt) of A. hydrophila were amplified by PCR and drug resistances were tested using Kirby-Bauer disk diffusion method. The results showed that the length of this 16S rDNA sequence was 1453 bp,which showed 99% homology with A. hydrophila. The GenBank accession number was JX155398. Phylogenetic analysis indicated it grouped together with A. hydrophila. Four virulence genes were all detected,indicating that strain L602 was highly virulent. This stain was resistant to four antibiotics(vibramycin,furazolidone,ampicillin and erythromycin),while it was insensitive to streptomycin. Furthermore,this strain was susceptible to six antibiotics(sulfafurazole,ciprofloxacin,penbritin,norfloxacin,florfenicol and enrofloxacin). This study will help to validate the classification and virulence of pathogenic bacteria in amphibians.

Isolation,identification,and virulence gene analysis of pathogenic Aeromonas dhakensis in Macrobrachium rosenbergii and histopathological observation

To identify the cause of mass mortality of adult Macrobrachium rosenbergii in a farm in Gaoyou City,Jiangsu Province,China,a dominant strain named DKQ-1 was isolated from the hepatopancreas of dying M.rosenbergii and identified as Aeromonas dhakensis by purification culture,biochemical characterization,and 16S rRNA and gyrB gene sequence analysis.The results of the challenge test revealed that the strain was highly pathogenic and the 50%lethal dose(LD_(50))in 72 h to M.rosenbergii was 1.54×10^(5)CFU/mL.The amplification results of virulence genes show that strain DKQ-1 carried 9 virulence genes,including ascV,aexT,aer,act,lip,ompAI,gcaT,acg,and exu,supporting the strong virulence of strain DKQ-1 to M.rosenbergii.Histopathological observation of the hepatopancreas,gills,and intestines indicated that DKQ-1 injection into M.rosenbergii could cause serious tissue damage,which further supported the strong virulence of this strain.In addition,a drug susceptibility test revealed that strain DKQ-1 was sensitive to 16 kinds of antibiotics,resistant to 9 kinds of antibiotics,and had intermediate resistance to spectinomycin and kanamycin.This study is the first report of A.dhakensis isolated from M.rosenbergii and provided a reference for the pathogen identification of bacterial diseases in M.rosenbergii,and for the prevention and treatment caused by A.dhakensis.

psk1 virulence gene-induced pulmonary and systemic tuberculosis in a young woman with normal immune function:A case report

BACKGROUND Tuberculosis is a chronic infectious disease and an important public health pro-blem.Despite progress in controlling tuberculosis,the incidence of tuberculosis in China is still very high,with 895000 new cases annually.This case report des-cribes the investigation of a case of severe disseminated tuberculosis in a young adult with normal immune function,conducted to ascertain why a Mycobacterium tuberculosis(M.tuberculosis)strain caused such severe disease.CASE SUMMARY A previously healthy 28-year-old woman presented to our hospital with a 1-mo-nth history of fever and fatigue.She was diagnosed with severe disseminated pulmonary tuberculosis,spinal tuberculosis with paravertebral abscesses,and tuberculous meningitis.M.tuberculosis was isolated from bronchoal-veolar lavage fluid.She was treated with standard antituberculous therapy and underwent debridement,bone graft,and internal fixation surgery for spinal tuberculosis.She responded to therapy and regained her ability to walk following the surgery.We analysed the whole-genome sequence of the strain and designated it BLM-A21.Additional M.tuberculosis genomes were selected from the Virulence Factor Database(http://www.mgc.ac.cn/cgi-bin/VFs/genus.cgi?Genus=Mycobacterium)for comparison.An evolutionary tree of the BLM-A21 strain was built using PhyML maximum likelihood software.Further gene analysis revealed that,except for the pks1 gene,BLM-A21 had similar virulence genes to the CDC 1551 and H37Rv strains,which have lower dissemination.CONCLUSION We speculate that the pks1 virulence gene in BLM-A21 may be the key virulence gene responsible for the wide-spread dissemination of M.tuberculosis infection in this previously healthy adult with normal immune function.

Virulence Factors and Biofilm Formation in Vancomycin Resistant Enterococcus faecalis and Enterococcus faecium Isolates in Brazil

In this work, we evaluated biofilm formation of Vancomycin Resistant of E. faecalis and E. faecium (VRE) in different culture media and adhesion substrate, as well as cellular hydrophobicity and presence of virulence genes. For this, 35 isolates were collected from a public hospital in Recife, Pernambuco, Brazil and identified by the Matrix-Assisted Laser Desorption Ionization - Time-of-flight - Mass Spectrometry (MALDI-TOF-MS) technique. Biofilm formation was analyzed by the Crystal Violet (CV) method and fluorescence microscopy, cellular hydrophobicity by hydrocarbon interaction and the presence of gelE, esp and asa1 genes by Polymerase Chain Reaction (PCR). 12 isolates were identified as E. faecalis and 23 as E. faecium. Most were obtained in Coronary Units (40.0%) and Intensive Care Unit (31.4%). E. faecium isolates were more resistant to the antibiotics tested than E. faecalis;however, E. faecalis stood out as a biofilm producer. Regarding the presence and gene frequency, it was observed that gelE (54.3%) and esp (54.3%) were the most prevalent, followed by asa1 (22.9%). When comparing the gene frequency, it was observed that gelE and esp were predominant (48.6% for both species), while asa1 was more frequent in E. faecalis (20.0%). The data presented here are worrying, because they reveal the virulence potential of isolates VRE, which contributes to the dissemination and persistence of these pathogens in the hospital environment.

Detection of antimicrobial resistance and virulence-related genes in Streptococcus uberis and Streptococcus parauberis isolated from clinical bovine mastitis cases in northwestern China

The objectives of this study were to investigate antimicrobial resistance of Streptococcus uberis and Streptococcus parauberis isolated from cows with bovine clinical mastitis in China and to examine the distribution of resistance-and virulence-related gene patterns.Antimicrobial susceptibility was determined by the E-test.Genes encoding antimicrobial resistance and invasiveness factors were examined by PCR.A total of 27 strains were obtained from 326 mastitis milk samples.Streptococcus parauberis isolates(n=11)showed high resistance to erythromycin(90.9%),followed by tetracycline(45.5%),chloramphenicol(36.4%)and clindamycin(27.3%).Streptococcus uberis isolates(n=16)were highly resistant to tetracycline(81.3%)and clindamycin(62.5%).Both species were susceptible to ampicillin.The most prevalent resistance gene in S.uberis was tetM(80.0%),followed by blaZ(62.5%)and ermB(62.5%).However,tetM,blaZ,and ermB genes were only found in 27.3,45.5,and 27.3%,respectively,of S.parauberis.In addition,all of the isolates carried at least one selected virulence-related gene.The most prevalent virulence-associated gene pattern in the current study was sua+pauA/skc+gapC+hasC detected in 22.2%of the strains.One S.uberis strain carried 7 virulence-associated genes and belonged to the sua+pauA/skc+gapC+cfu+hasA+hasB+hasC pattern.More than 59.3%of analysed strains carried 4 to 7 virulence-related genes.Our findings demonstrated that S.parauberis and S.uberis isolated from clinical bovine mastitis cases in China exhibited diverse molecular ecology,and that the strains were highly resistant to antibiotics commonly used in the dairy cow industry.The data obtained in the current study contribute to a better understanding of the pathogenesis of bacteria in mastitis caused by these pathogens,and the findings are relevant to the development of multivalent vaccines and targeted prevention procedures.

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.

Correlation between the distribution pattern of virulence genes and virulence of Aeromonas hydrophila strains

Nine strains of Aeromonas hydrophila isolated from diseased fish or soft-shelled tortoise were tested for the presence of three virulence genes including the genes encoding aerolysin,hemolysin,and extracellular serine protease(i.e.,aerA,hlyA,and ahpA,respectively).These genes were investigated using polymerase chain reaction(PCR)with specific primers for each gene.And the pathogenicities to Carrassius auratus ibebio of these strains were also assayed.PCR results demonstrated that the distribution patterns of aerA,hlyA,and ahpA were different in these strains.6/9 of A.hydrophila strains were aerA positive,8/9 of strains hlyA positive,7/9 of strains ahpA positive,respective-ly.However,the assay for pathogenesis showed that two strains(A.hydrophila XS91-4-1 and C2)were strong virulent,two strains(A.hydrophila ST78-3-3 and 58-20-9)avirulent and the rest middle virulent was to the fish.In conclusion,there are significant correlation between the distribution pattern of the three virulence genes and the patho-genicity to Carrassius auratus ibebio.All strong virulent A.hydrophila strains were aerA+hlyA+ahpA+genotype,and all aerA+hlyA+ahpA+strains were virulent.Strains with the genotype of aerA−hlyA−ahpA+have middle pathogenicity.In the present study,we found for the first time that all A.hydrophila isolated from the ahpA positive were virulent to Carrassius auratus ibebio.Additionally,there was a positive correlation between the virulence of A.hydrophila and the presence of aerA and ahpA.

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.

H pylori iceA alleles are disease-specific virulence factors

AIM： To characterize and compare genotype profiles of H pylori strains isolated from patients with chronic gastritis and duodenal ulcer in western part of Turkey. METHODS： A total of 46 patients [30 chronic gastritis （CG） and 16 duodenal ulcer （DU）] who had undergone endoscopy because of dyspeptic complaints were studied. The antral biopsy specimens were evaluated for the presence of Hpylori by rapid urease test and culture, and the genotype profiles were determined by real-time PCR. RESULTS： The cagA gene was observed in 43 （93.5%） isolates. The vacA s1m2 genotype was the predominant subtype, found in 63.3% and 68.7% of isolates in patients with CG and DU, respectively. Twenty （66.6%） isolates from patients with CG were iceA2 positive while the iceA1 was predominant in those with DU （68.8%）. In terms of the association of the ice,4 alleles to other genes, both alleles were significantly associated with the cagA vacA slm2 genotype. CONCLUSION： The prevalent circulating genotypes in CG and DU were cagA vacA slm2 iceA2 and cagA vacA slm2 iceA1 genotype, respectively. It was found that cagA vacA slm2 genotype seems to be common virulence factors in both CG and DU while iceA alleles show specificity for gastroduodenal pathologies in this study.

Comparison of extended spectrum β-lactamasesproducing Escherichia coli with non-ESBLsproducing E.coli:drug-resistance and virulence

The virulent factors of Escherichia coil （E.cofi） play an important role in the process of pathopoiesis. The study aimed to compare drug-resistant genes and virulence genes between extended spectrum β-1actamases （ESBLs）-producing E.coli and non-ESBLs-producing E.cofi to provide a reference for physicians in management of hospital infection. From October 2010 to August 2011,96 drug-resistant strains of E. coli isolated were collected from the specimens in Qingdao Municipal Hospital, Qingdao, China. These bacteria strains were divided into a ESBLs-producing group and a non-ESBLs-producing group. Drug sensitivity tests were performed using the Kirby-Bauer （K-B） method. Disinfectant gene, qacEAl-sull and 8 virulence genes （CNF2, hlyA, eaeA, VT1, est, bfpA, elt, and CNF1） were tested by polymerase chain reaction （PCR）. Among the 96 E.coli isolates, the ESBLs-producing E.coli comprised 46 （47.9%） strains and the non-ESBLs-producing E.cofi consisted of 50 （52.1%） strains. The detection rates of multiple drug-resistant strain, qacEAl-sull, CNF2, hlyA, eaeA,VT1, est, bfpA, elt, and CNF1 in 46 ESBLs-producing E.coli isolates were 89.1%, 76.1%, 6.5%, 69.6%, 69.6%, 89.1%, 10.9%, 26.1%, 8.7%, and 19.6%, respectively. In the non-ESBLs-producing E.cofi strains, the positive rates of multiple drug-resistant strain, qacEAl-sull, CNF2, hlyA, eaeA, VT1, est, bfpA, elt, and CNF1 were 62.0%, 80.0%, 16.0%, 28.0%, 64.0%, 38.0%, 6.0%, 34.0%, 10.0%, and 24.0%, respectively. The difference in the detection rates of multiple drug-resistant strain, hlyA and VT1 between the ESBLs-producing E.cofi strains and the non-ESBLs-producing E.cofi strains was statistically significant （P〈0.05）. The positive rate of multiple drug-resistant strains is higher in the ESBLs-producing strains than in the non-ESBLs-producing strains. The expression of some virulence genes hlyA and VT1 varies between the ESBLs-producing strains and the non-ESBLs-producing strains. Increased awareness of clinicians and enhanced testing by laboratories are required to reduce treatment failures and prevent the spread of multiple drug-resistant strains.

Screening Helicobacter pylori genes induced during infection of mouse stomachs

AIM:To investigate the effect of in vivo environment on gene expression in Helicobacter pylori(H.pylori) as it relates to its survival in the host.METHODS:In vivo expression technology(IVET) systems are used to identify microbial virulence genes.We modified the IVET-transcriptional fusion vector,pIVET8,which uses antibiotic resistance as the basis for selection of candidate genes in host tissues to develop two unique IVET-promoter-screening vectors,pIVET11 and pIVET12.Our novel IVET systems were developed by the fusion of random Sau3A DNA fragments of H.pylori and a tandem-reporter system of chloramphenicol acetyltransferase and beta-galactosidase.Additionally,each vector contains a kanamycin resistance gene.We used a mouse macrophage cell line,RAW 264.7 and mice,as selective media to identify specific genes that H.pylori expresses in vivo.Gene expression studies were conducted by infecting RAW 264.7 cells with H.pylori.This was followed by real time polymerase chain reaction(PCR) analysis to determine the relative expression levels of in vivo induced genes.RESULTS:In this study,we have identified 31 in vivo induced(ivi) genes in the initial screens.These 31 genes belong to several functional gene families,including several well-known virulence factors that are expressed by the bacterium in infected mouse stomachs.Virulence factors,vacA and cagA,were found in this screen and are known to play important roles in H.pylori infection,colonization and pathogenesis.Their detection validates the efficacy of these screening systems.Some of the identified ivi genes have already been implicated to play an important role in the pathogenesis of H.pylori and other bacterial pathogens such as Escherichia coli and Vibrio cholerae.Transcription profiles of allivi genes were confirmed by real time PCR analysis of H.pylori RNA isolated from H.pylori infected RAW 264.7 macrophages.We compared the expression profile of H.pylori and RAW 264.7 coculture with that of H.pylori only.Some genes such as cag A,vac A,lpx C,mur I,tlp C,trx B,sod B,tnp B,pgi,rbf A and inf B showed a 2-20 fold upregulation.Statistically significant upregulation was obtained for all the above mentioned genes(P < 0.05).tlp C,cag A,vac A,sod B,rbf A,inf B,tnp B,lpx C and mur I were also significantly upregulated(P < 0.01).These data suggest a strong correlation between results obtained in vitro in the macrophage cell line and in the intact animal.CONCLUSION:The positive identification of these genes demonstrates that our IVET systems are powerful tools for studying H.pylori gene expression in the host environment.

Virulence Gene Characterization and Serotyping of Major Bacterial Pathogens Isolated from Bovine Respiratory Disease in Ethiopia

Bovine Respiratory Disease (BRD) causes a severe form of pneumonia in all age of cattle. This study was designed to investigate the distribution of capsular types, serotypes, and virulence-associated genes of the major bacterial pathogens from BRD outbreak samples in Ethiopia. In this study 166 samples were collected from clinically sick (n = 107) and pneumonic lung tissue (n = 59). Laboratory assay confirmed isolation of M. haemolytica 37 (22.29%), P. multocida 25 (15.06%), B. trehalosi 12 (7.23%), and H. somni 15 (9.04%). PCR assay of P. multocida capsular typing revealed 21 (84.0%) cap A (hyaD-hyaC) and 4 (16.0%) cap D (dcbF) strains. M. haemolytica serotypes belonged to A: 1, A: 2, and A: 6 from 26 (70.27%), 4 (10.81%), and 7 (18.92%) isolates, respectively. P. multocida biotyping showed isolation of A: 1, A: 2, and A: 3 from 3 (14.29%), 2 (9.52%), and 16 (76.19%) isolates, respectively. M. haemolytica harbored more than 60% ssa gene, and 90.91% sodA while FbpA, TbpA, and lktC genes were found in all isolates. Likewise, all P. multocida exhibited toxA, FbpA, TbpA, and pmSLP genes. The current finding showed that M. haemolytica serotype A: 1 is frequently associated with BRD followed by P. multocida biotype A: 3. These two isolates harbored diverse virulence-associated genes and presented the pathogenic potential of the current isolates. Thus, investigation of pathogenic strains of BRD, virulence genes distribution, and molecular epidemiology of the disease from wider areas of the country are essential. Hence, continuous outbreak surveillance and molecular approaches are indispensable in designing efficient prevention strategies.

The Detection of Pathogenic Porcine E.coli Virulence Gene (astA,stb)

[ Objective] This paper aimed to find out the relationship between pathogenic procine E. coli virulence gene and pathogenicity, and ex- plore the pathogenic mechanism of E. coil [ Methed] The detection of two E. coil virulence genes was performed. PCR method was taken to test the virulence genes, astA and stb, from 39 strains of typical serotype O porcine E. coli which had been separated and identified. [ Result] It＇s found that of the 39 isolates of porcine E. coli, 22 carried virulence gene astA which represented 56.41%, and 27 carried virulence gene stb which repre- sented 69.23%. [ Conclusion] This study has provided scientific data for future E. coil pathogenicity researches.

Farnesol inhibits development of caries by augmenting oxygen sensitivity and suppressing virulence-associated gene expression in Streptococcus mutans

Streptococcus mutans is a primary etiological agent of dental caries.Farnesol,as a potential antimicrobial agent,inhibits the development of S.mutans biofilm.In this study,we hypothesized that farnesol inhibits caries development in vitro and interferes with biofilm fonnation by regulating virulence-associated gene expression.The inhibitory effects of farnesol to S.mutans biofilms on enamel surfaces were investigated by determining micro-hardness and calcium measurements.Additionally,the morphological changes of S.mutans biofilms were compared using field emission scanning electron microscopy and confocal laser scanning microscopy,and the vitality and oxygen sensitivity of S.mutans biofilms were compared using MTT assays.To investigate the molecular mechanisms of farnesol＇s effects,expressions of possible target genes luxS,brpA,ffh,recA,nth,and smx were analyzed using reverse-transcription polymerase chain reaction（PCR） and quantitative PCR.Farnesol-treated groups exhibited significantly higher micro-hardness on the enamel surface and lower calcium concentration of the supernatants as compared to the-untreated control.Microscopy revealed that a thinner film with less extracellular matrix formed in the farnesol-treated groups.As compared to the-untreated control,farnesol inhibited biofilm formation by 26.4%with500 μmol/L and by 37.1%with 1,000 μmol/L（P〈 0.05）.Last,decreased transcription levels of luxS,brpA,ffh,recA,nth,and smx genes were expressed in farnesol-treated biofilms.In vitro farnesol inhibits caries development and S.mutans biofilm formation.The regulation of luxS,brpA,ffh,recA,nth,and smx genes may contribute to the inhibitory effects of farnesol.