Identification and virulence test of a new pathogen that causes verticillium striping on rapeseed in northwestern China

Five stems of rapeseed with abundant black microsclerotia were collected from Huangyuan County of Qinghai Province,China,and fungal isolates were obtained from the stems.They were identified based on morphology,molecular features and specific PCR detection.The results showed that the 10 fungal isolates belonged to Verticillium longisporum lineage A1/D3.One of the 10 isolates(HW7-1)was tested for virulence on three species of rapeseed,including B.napus Zhongshuang 9,B.rapa Qingyou 9 and B.juncea Tayou 2 by conidia inoculation of HW7-1 on roots of young seedlings.Control seedlings were inoculated with V.dahliae conidia or water alone.The seedlings of these treatments were transplanted in culture mix and incubated in a growth chamber(20℃).Results suggested that the control seedlings of three cultivars appeared quite healthy,while the seedlings inoculated with HW7-1 turned yellowing leaves,seedling stunting or even death after 22 days post-inoculation.V.longisporum was re-isolated from he yellow leaves,thus fulfilling Koch's postulates.Moreover,compared to the control treatments,inoculation with HW7-1 caused flowering delay and seed yield reduction on Tayou 2 with production of microsclerotia on the stems.To our knowledge,this is the first report of V.longisporum lineage A1/D3 on rapeseed in northwestern China.

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.

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 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.

Molecular Detection of Resistance and Virulence Genes in Coagulase Negative Staphylococci Isolated from Blood Cultures at the University Teaching Hospital of Bouake

Introduction: Coagulase-negative staphylococci (CoNS) are currently recognized as genuine pathogens. However, little is known about the resistance and virulence genes that explain their pathogenicity in hospitals in Cte d'Ivoire. The aim of this study was to contribute to the genotypic identification of resistance and virulence genes in CoNS isolated from blood cultures at the University Teaching Hospital (CHU) of Bouak, in order to improve patient management. Material and Methods: This was a descriptive study conducted from September to December 2023. The CoNS isolates studied came from the collection of strains isolated from blood cultures of febrile patients hospitalized or attending consultations at the CHU of Bouak. The strains were analyzed using conventional simplex PCR. Results: Of the 45 isolates analyzed, 46.7% carried both the aacA-aphD and tetK genes and 40% carried the mecA gene. With regard to virulence genes, only the LukS-PV gene was observed in S. epidermidis and S. haemolyticus isolates. Conclusion: The high prevalence of CoNS isolates carrying the mecA gene and the presence of virulence genes observed in this study give cause for concern in hospitals. It is important to develop comprehensive surveillance strategies against nosocomial and multi-resistant infections at the CHU of Bouak.

Susceptibility patterns and virulence genotypes of Helicobacter pylori affecting eradication therapy outcomes among Egyptian patients with gastroduodenal diseases

BACKGROUND Helicobacter pylori(H.pylori)is a significant human pathogen that is responsible for a variety of illnesses,including mucosa-associated lymphoid tissue lymphoma,gastric cancer,peptic ulcers,and gastritis.AIM To investigate the frequency of H.pylori infection and its resistance patterns among Egyptian patients and to determine the influence of H.pylori virulence genetic determinants on the eradication success of 14-d triple therapy regimen.METHODS H.pylori infections were investigated in 72 patients with gastroduodenal complications suggestive of H.pylori infection.The cagA and vacA genotypes of cultured strains were studied using polymerase chain reaction.The patients underwent 14 d of triple-therapy treatment.The treatment response was examined using histology and a rapid urease test 6 wk after therapy discontinuation.RESULTS The intention-to-treat eradication rate was 59.2%(95%CI:48.2%-70.3%).Rates of H.pylori resistance to clarithromycin,amoxicillin,and metronidazole were 52.8%,81.9%,and 100%,respectively.Successful eradication of H.pylori was more significantly associated with vacA s1-positive strains[adjusted odds ratio(aOR)=0.507,95%CI:0.175-0.822].A significant association was found between failed eradication rate and H.pylori strains resistant to clarithromycin(aOR=0.204,95%CI:-0.005 to 0.412)and amoxicillin(aOR=0.223,95%CI:0.026-0.537).CONCLUSION This study’s low H.pylori eradication rate following 14-d triple therapy is concerning and worrying.H.pylori pan-resistance to metronidazole followed by the high resistance to ciprofloxacin,amoxicillin,and clarithromycin in this research is challenging and of great concern.

Molecular epidemiology, characterization of virulence factors and antibiotic resistance profile of Streptococcus agalactiae isolated from dairy farms in China and Pakistan

Streptococcus agalactiae is one of the most common pathogens that cause bovine mastitis worldwide. Identifying pathogen prevalence and virulence factors is critical for developing prevention and control approaches. Herein, 1 161 milk samples from various dairy farms in China(n=558) and Pakistan(n=603) were collected between 2019–2021 and were subjected to S. agalactiae isolation. Prevalence, serotyping, virulence genes, and antibiotic-resistant genes of S. agalactiae were evaluated by PCR assay. All isolates were characterized for haemolysis, biofilm production, cytotoxicity, adhesion, and invasion on bovine mammary epithelial cells. The prevalence of S. agalactiae-induced mastitis in cattle was found to be considerably higher in Pakistan than in China. Jiangsu and Sindh provinces had the highest area-wise prevalence in China and Pakistan, respectively. Serotypes Ia and II were prevalent in both countries, whereas serotype III was found only in Pakistan. Moreover, all isolates tested positive for PI-2b gene but negative for PI-1 and PI-2a genes. All isolates harboured cfb, cylE, hylB, and fbsB virulent genes, whereas many of them lacked bibA, rib and bca. However, the absence of bac and scp genes in Chinese isolates and cspA in Pakistani isolates was noted, while spb1 and lmb were not detected in isolates of both countries. Pakistani isolates, particularly serotype Iapositive, had a considerably higher ability to produce biofilm, haemolysis, cytotoxicity, adhesion, and invasion than Chinese isolates. Most of the isolates were phenotypically resistant to tetracycline, erythromycin, and clindamycin and genotypic resistance was confirmed by the presence of ermA, ermB, tetM and tetO genes. Our study highlights the antimicrobial resistance profile and virulence-related factors contributing to the epidemiological spread of mastitis-causing S. agalactiae in China and Pakistan. The findings may facilitate future studies designed to develop improved treatment and control strategies against this pathogen.

The vital role of CovS in the establishment of Streptococcus equi subsp.zooepidemicus virulence

Streptococcus equi subsp.zooepidemicus(SEZ)is an important zoonotic agent.Here,a virulence-attenuated strain M35246 derived from natural variation of wild-type SEZ ATCC35246 was found.M35246 showed a deletion of 25contiguous genes as well as a loss-of-function mutation in covS.Subsequently,a 25-gene-deleted strain(ΔPI),a covS-mutant strain(Mcov S),and relevant complementary strains were constructed and investigated.M35246 and Mcov S were significantly less encapsulated and exhibited poorer anti-phagocytic capacity compared to wild-type SEZ.McovS was significantly more sensitive toβ-lactams,aminoglycosides,macrolides,and lincosamides than wild-type SEZ.M35246,McovS,andΔPI exhibited an increase in median lethal dose(LD_(50))in mice by 10~5,10~5,and 5 times when compared to wild-type SEZ,respectively.Neither M35246 nor McovS were isolated from mice 48 h after being challenged with approximately 2000 times the LD_(50)of wild-type SEZ.Transcriptome analysis showed that 668 significantly differentially expressed genes existed between McovS and wild-type SEZ.Numerous virulence factor-encoding genes and anabolicrelated genes in McovS that were involved in anti-phagocytosis,capsule formation,pathogenicity,and antibiotic resistance were downregulated significantly relative to the wild-type strain.This study revealed that the CovS plays a vital role in the establishment of SEZ virulence.

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.

The quantity of OA and activity of cellulase and polygalacturonase are involved in variation of virulence in Sclerotium rolfsii

In order to understand the pathogenic mechanisms of Sclerotium rolfsii on peanut and to analyze the variation of virulence in S.rolfsii strains,the highly virulent strain(ZY2)and weakly virulent strain(GP3-1)were investigated under both in vivo and in vitro conditions.The results indicated that S.rolfsii directly infected peanut by producing infection cushions.ZY2 formed infection cushions earlier than GP3-1,and ZY2 produced a greater number of infection cushions compare to GP3-1.Both strains could utilize cellulose,xylose,or polygalacturonic acid in the Czapek medium.The activities of cellulase(CL)and polygalacturonase(PG)in the inoculated peanut stems increased significantly at 9 h after inoculation.The activities of CL and PG produced by ZY2 in the inoculated stems were significantly higher than that produced by GP3-1.Both strains could produce oxalic acid(OA),and the content of OA produced by ZY2 in the inoculated stems was higher than that produced by GP3-1.In summary,it suggested that S.rolfsii destroyed peanut cells through physical and biochemical factors by secreting a large amount of OA,CL and PG during the formation of infection cushions.The difference in OA content,activity of CL and PG produced by highly and weakly virulent strains played important roles in variation of virulence.

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.

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.

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.

Virulence Types of Magnaporthe oryzae to Hybrid Rice in Sichuan,China

A total of 638 isolates of rice blast （Magnaporthe oryzae） were isolated in 2002-2009 from different rice varieties in different regions of Sichuan, China and inoculated onto seven rice varieties （Lijiangxintuanheigu, IR24, Minghui 63, Duohui 1, Chenghui 448, Neihui 99-14 and RHR-1） to differentiate the virulence types of the fungus and trace the changes. The virulence to the seven varieties was respectively scored at 1, 2, 4, 8, 16, 32 and 64. The total scores of individual M. grisea isolates which were the sum of scores infecting differential varieties could, in turn, be used for the nomenclature of the virulence types due to their accordance to the special virulence patterns. The 638 tested isolates were then differentiated into 56 different virulence types. Type 15 virulent to Lijiangxintuanheigu, IR24 and Minghui 63, and Type 127 virulent to all of the seven varieties were the most dominant virulence types respectively with the occurrence frequencies of 15.99% and 15.83%. Type 19 and other seven virulence types were not monitored during 2002-2009. Type 15 was the predominant virulence type in 2002, 2003, 2004 and 2007, whereas Type 127 had been the most dominant virulence type after 2005 except for the year 2007 when the province underwent severe drought. Five hundred and seven out of the 638 tested isolates were virulent to Minghui 63, and 89.58% of the 384 isolates virulent to either Duohui 1, Chenghui 448 or Neihui 99-14 were virulent to Minghui 63, which indicated the impact of the extensive plantation of hybrid rice Minghui 63 as the restorer line on the virulence evolution of M. oryzae in Sichuan. The virulence pattern of the dominant virulence types suggested that the acquiring of virulence to all the major resistant restorer lines was the main routes of the evolution in virulence of M. oryzae to hybrid rice in Sichuan. The virulence frequencies of the 638 tested isolates to IR24, Minghui 63, Duohui 1, Chenghui 448, Neihui 99-14 and RHR-1 were respectively 74.6%, 79.5%, 73.8%, 37.0%, 39.0% and 40.4%. The analysis for the sources of the different virulence type isolates indicated the pathogen on the newly released resistant varieties were stronger than conventional rice varieties which had become susceptible in the field since 1980s.

Stress Physiology and Virulence Characterization of Phomopsis asparagi (Sacc.) Bubák isolated from Asparagus in Jiangxi Province of China

Fungal pathogen of asparagus stem blight was isolated. No significant genetic difference was detected among the three strains with 492 bp long ITS1-5.8S-ITS2 sequence. It was then identified through colony growth, conidia morphology, and molecular characterization. The physiological response to oxidation and osmosis stress, and virulence to Asparagus officinalis L. were analyzed. The results showed that the pathogen causing asparagus stem blight for A. officinalis L. in Jiangxi Province is Phomopsis asparagri (Sacc.) Bubák. Under pure culture conditions, the conidia were oval-shaped (α-type), with colorless single spore and single nucleus, containing 0-2 oil balls. Its vegetative growth rate was higher when cultured on 0.2 × potato dextrose agar (0.2 × PDA) medium than that on oatmeal agar (OA) medium. However, the pycnidia appeared earlier on OA medium than on 0.2 earlier PDA medium. The vegetative growth rate was depressed under oxidation (H2O2) or osmosis (NaCl) stress conditions, and totally inhibited under 7 mmol/L H2O2 or 2.4 mol/L NaCl. All the strains caused typical pathogenic symptoms to Asparagus officinalis L. at 7 days-post-inoculation (dpi) with conidia.

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.

New insights of Helicobacter pylori host-pathogen interactions: The triangle of virulence factors, epigenetic modifications and non-coding RNAs

Helicobacter pylori(H. pylori) is a model organism for understanding host-pathogen interactions and infection-mediated carcinogenesis. Gastric cancer and H. pylori colonization indicates the strong correlation. The progression and exacerbation of H. pylori infection are influenced by some factors of pathogen and host. Several virulence factors involved in the proper adherence and attenuation of immune defense to contribute the risk of emerging gastric cancer, therefore analysis of them is very important. H. pylori also modulates inflammatory and autophagy process to intensify its pathogenicity. From the host regard, different genetic factors particularly affect the development of gastric cancer. Indeed, epigenetic modifications, Micro RNA and long non-coding RNA received more attention. Generally, various factors related to pathogen and host that modulate gastric cancer development in response to H. pylori need more attention due to develop an efficacious therapeutic intervention. Therefore, this paper will present a brief overview of host-pathogen interaction especially emphases on bacterial virulence factors, interruption of host cellular signaling, the role of epigenetic modifications and non-coding RNAs.

Virulence factors of Enterococcus strains isolated from patients with inflammatory bowel disease

AIM: To determine the features of Enterococcus that contribute to the development and maintenance of the inflammatory process in patients with inflammatory bowel disease (IBD). METHODS: Multiplex polymerase chain reaction (PCR) was applied to assess the presence of genes that encode virulence factors [surface aggregating protein (asa1), gelatinase (gelE), cytolysin (cylA), extracellular surface protein (esp) and hyaluronidase (hyl)] in the genomic DNA of 28 strains of Enterococcus isolated from the intestinal tissues of children with IBD (n =16) and of children without IBD (controls; n = 12). Additionally, strains with confirmed presence of the gelE gene were tested by PCR for the presence of quorum sensing genes (fsrA, fsrB, fsrC) that control the gelatinase production. Gelatinase activity was tested on agar plates containing 1.6% gelatin. We also analysed the ability of Enterococcus strains to release and decompose hydrogen peroxide (using Analytical Merckoquant peroxide test strips) and tested their ability to adhere to Caco-2 human gut epithelium cells and form biofilms in vitro. RESULTS: A comparison of the genomes of Enterococcus strains isolated from the inflamed mucosa of patients with IBD with those of the control group showed statistically significant differences in the frequency of theasa1 gene and thegelE gene. Furthermore, the cumulative occurrence of different virulence genes in the genome of a single strain ofEnterococcus isolated from the IBD patient group is greater than in a strain from the control group, although no significant difference was found. Statistically significant differences in the decomposition of hydrogen peroxide and adherence to the Caco-2 epithelial cell line between the strains from the patient group and control group were demonstrated. The results also showed that profuse biofilm production was more frequent amongEnterococcus strains isolated from children with IBD than in control strains. CONCLUSION: Enterococcus strains that adhere strongly to the intestinal epithelium, form biofilms and possess antioxidant defence mechanisms seem to have the greatest influence on the inflammatory process.

Virulence and potential pathogenicity of coccoid Helicobacter pylori induced by antibiotics

AIM: To explore the virulence and the potential pathogenicity of coccoid Helicobacter pylori (H. pylori) transformed from spiral form by exposure to antibiotic. METHODS: Three strains of H. pylori, isolated from gastric biopsy specimens of confirmed peptic ulcer, were converted from spiral into coccoid from by exposure to metronidazole. Both spiral and coccoid form of H. pylori were tested for the urease activity, the adherence to Hep-2 cells and the vacuolating cytotoxicity to Hela cells, and the differences of the protein were analysed by SDS-PAGE and Western blot. The mutation of the genes including ureA, ureB,hpaA, vacA and cagA, related with virulence, was detected by means of PCR and PCR-SSCP. RESULTS: In the coccoid H. pylori,the urease activity, the adherence to Hep-2 cells and the vacuolating cytotoxicity to Hela cells all decreased. In strain F44, the rate and index of adherence reduced from 70.0% +/- 5.3% to 33% +/- 5.1% and from 2.6 +/- 0.4 to 0.96 +/- 0.3 (P 【 0.01), respectively. The invasion of coccoid H. pylori into Hep-2 cell could be seen under electronmicroscope. SDS-PAGE showed that the content of the protein with the molecular weight over Mr 74000 decreased, and the hybriditional signal in band M(r) 125000 weakened, while the band M(r)110000 and M(r)63000 strengthened in coccoid H.pylori as shown in Western blot. The results of PCR were all positive, and PCR-SSCP indicated that there may exist the point mutation in gene hpaA or vacA. CONCLUSION: The virulence and the proteins with molecular weight over M(r)74000 in coccoid H.pylori decrease, but no deletion exists in amplification fragments from ureA, ureB, hpaA, vacA and cagA genes, suggesting that coccoid H.pylori may have potential pathogenicity.

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.