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Objective:To investigate the relationship between Helicobacter pylori(HP) cytotoxin-associated gene A (HP-CagA), HP isolate vacuole-forming toxin gene A (HP-VacA) and gastric cancer occurrence and clinical pathological factors.Methods:Eighty-eight patients with gastric cancer from January 2018 to January 2020 in Suzhou Hospital Affiliated of Anhui Medical University was selected as the observation group, 80 patients with benign gastric lesions during the same period was selected as the benign control group, and 80 healthy patients was selected as the healthy control group. The clinical data, HP-CagA, HP-VacA positive expression rates of the three groups were compared, the risk factors of gastric cancer were analyzed, and the relationship between HP-CagA, HP-VacA and gastric cancer clinicopathological factors were evaluated.Results:Family history of gastric cancer, high-salt diet, preference for hot food, decreased pepsinogen (PG)Ⅰ/PGⅡ, combined with fatty liver, increased triglyceride, total cholesterol and low density lipoprotein cholesterin, smoking and depression were risk factors of gastric cancer ( P<0.05). The positive rate of HP-CagA, HP-VacA in the observation group were higher than those in the benign control group and the healthy control group: 82.93%(73/88) vs. 62.50%(50/80) and 26.25%(21/80), 30.68%(27/88) vs. 7.50%(6/80) and 0, the differences were statistically significant ( P<0.05). The positive of HP-CagA and HP-VacA had correlation with age, pathological type, and degree of differentiation of gastric cancer ( P<0.05). The 1-year survival rate of HP-CagA and HP-VacA positive patients was lower than that of negative patients by Kaplan-Meier analysis ( P<0.05). Conclusions:The positive of HP-CagA and HP-VacA in HP infections are closely related togastric cancer. Strengthening the treatment of HP infection patients with positive HP-CagA and HP-VacA has important clinical value and social significance for cutting off the early stage of gastric cancer and improving prognosis.
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Objective To detection the carrying situation of Clostridium difficile toxin gene A/B in stool specimens of the pa-tients with ulcerative colitis (UC) and persons undergoing physical examination and to research the relationship between Clostridium difficile toxin gene carrying and UC.Methods The stool specimens were collected from 53 cases of UC(32 cases of active stage and 21 cases of resting stage) and 45 persons undergoing physical examination.Total DNA was extracted from stool specimens.The Clostridium difficile toxin gene cdtA and cdtB were detected by real-time PCR ,then the PCR products were amplified and performed the agarose gel electrophoresis and gene sequencing for conducting the amplification products verification.Results 7 cases of cdtA and 4 cases of cdtB were checked out in the UC group ,in which 5 cases of cdtA and 2 cases of cdtB were in the UC active stage group ,2 cases of cdtA and 2 cases of cdtB were in the UC resting stage group.In the healthy control group ,5 cases of cdtA and 2 cases of cdtB were checked out.The detection rate of cdtA and cdtB had no statistically significant difference between the UC group and healthy control group(P>0.05).There was no statistically significant difference between UC active group and inactive group (P>0.05).Conclusion There is no appearant correlation between the carrying situation of cdtA and cdtB in stool with UC onset and UC stage.
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Clostridium perfringens type A has been incriminated as the etiologic agent in jejunal hemorrhage syndrome (JHS), which is a disease that affects dairy cattle. Although this microorganism is considered an important enteropathogen the pathogenesis of JHS is still not clear, and there have been no reports of its occurrence in Brazil so far. The aim of this study was to describe the occurrence of JHS by infection with a C. perfringens type A strain carrying the beta-2 toxin gene in a zebu cow in Brazil, for the first time.
Clostridium perfringens tipo A tem sido considerado agente etiológico da síndrome do jejuno hemorrágico (SJH), que é uma doença que afeta comumente os rebanhos de gado. Embora este microrganismo seja considerado um importante enteropatógeno, a patogênese da SJH ainda não foi elucidada, e não havia sido reportada no Brasil até então. O alvo deste estudo foi descrever pela primeira vez a ocorrência da SJH causada por C. perfringens tipo A, carreador do gene da toxina beta-2, em um zebuíno no Brasil.
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Objective To develop a real-time PCR assay for the rapid identification of Clostridium(C.)difficile and its toxin. Methods TaqMan real-time PCR was developed for the rapid identification of species specific gene(tpi)of C. difficile strains and the toxins A(TcdA),B(TcdB) and truncated toxin A(TcdAT). Sensitivity,specificity and anti-interference ability of these methods were estimated,as well. Feces sampled from fifty diarrhea patients were tested by real-time PCR and compared to the results from VIDAS assay. Results The detection limits of tpi were 6×10-2 CFU/μl and 6 × 10-1 CFU/μl in the non-oxin producing and toxin producing strains,respectively. The coefficients of variability (CV) of intra-assay and inter-assay for the detection limits of tpi in the non-toxin producing strain were 2.1% and 2.3%. The CVs of intra-assay and inter-assay for the detection limit of tpi,tcdA,tcdB and tcdAT in the toxin producing strain were 3.0%and 3.4%,2.9%and 3.2%,5.3%and 5.7%,2.7%and 2.8%,respectively. No interferance was detected from other genus or species in clostridium. From 50 clinical samples,thirty-nine of them were negative and six of them were positive under the TaqMan-MGB probe technique in accordance with VIDAS. Five samples appeared positive using the TaqMan-MGB probe technique,in which 3 were dubious and 2 were negative under VIDAS. Conclusion The newly developed method was a sensitive and reliable assay for rapid identification of C. difficile and its toxin. This method could be used to screen C. difficile isolates harboring truncated toxin A to avoid misdiagnosis,clinically.
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Objective To develop a real-time PCR assay for the rapid identification of Clostridium(C.)difficile and its toxin. Methods TaqMan real-time PCR was developed for the rapid identification of species specific gene(tpi)of C. difficile strains and the toxins A(TcdA),B(TcdB) and truncated toxin A(TcdAT). Sensitivity,specificity and anti-interference ability of these methods were estimated,as well. Feces sampled from fifty diarrhea patients were tested by real-time PCR and compared to the results from VIDAS assay. Results The detection limits of tpi were 6×10-2 CFU/μl and 6 × 10-1 CFU/μl in the non-oxin producing and toxin producing strains,respectively. The coefficients of variability (CV) of intra-assay and inter-assay for the detection limits of tpi in the non-toxin producing strain were 2.1% and 2.3%. The CVs of intra-assay and inter-assay for the detection limit of tpi,tcdA,tcdB and tcdAT in the toxin producing strain were 3.0%and 3.4%,2.9%and 3.2%,5.3%and 5.7%,2.7%and 2.8%,respectively. No interferance was detected from other genus or species in clostridium. From 50 clinical samples,thirty-nine of them were negative and six of them were positive under the TaqMan-MGB probe technique in accordance with VIDAS. Five samples appeared positive using the TaqMan-MGB probe technique,in which 3 were dubious and 2 were negative under VIDAS. Conclusion The newly developed method was a sensitive and reliable assay for rapid identification of C. difficile and its toxin. This method could be used to screen C. difficile isolates harboring truncated toxin A to avoid misdiagnosis,clinically.
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Clostridium perfringens food poisoning ranks among the most common gastrointestinal diseases in developed countries. In Korea, C. perfringens food poisoning gradually increases. Using PCR, 72 strains of C. perfringens isolated in Seoul, 2013 were tested for the presence of toxin genes. Of the tested strains, 32 isolates carried the cpe gene, 37 isolates carried the cpb2 gene and 3 isolates carried the cpe and cpb2 genes, respectively. 32 cpe-positive strains were isolated from the food poisoning patient, whereas among 37 cpb2-positive strains, 22 strains were isolated from asymptomatic person. To investigate epidemiological relationship between the isolates, Pulsed-filed gel electrophoresis (PFGE) was performed. The genetic relatedness of the isolates ranged from 55.9% to 100% and 47 distinct PFGE profiles were observed. The results show that the cpe-positive outbreak strains showed close genetic relation, whereas the cpb2-positive isolates revealed a wide genetic diversity.
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Humans , Clostridium perfringens , Developed Countries , Electrophoresis , Foodborne Diseases , Gastrointestinal Diseases , Genetic Variation , Korea , Molecular Epidemiology , Polymerase Chain Reaction , SeoulABSTRACT
This study aimed at characterizing the phenotypic and toxigenic status of circulating strains of cholera during outbreaks in Nigeria, employing molecular typing techniques. Two hundred and one samples of rectal swabs, stool, vomitus, water (from the well, borehole, sachet, stream, and tap) and disinfectants (sodium hypochlorite) were collected from three states in the country. The samples were inoculated on thiosulphate-citrate bile salt-sucrose (TCBS), Cary-Blair transport medium and smeared on glass slides for direct examination. The Vibrio cholerae isolates were serotyped, biotyped, and characterized using PCR of the cytotoxin gene A (ctxA), wbeO1, and wbfO139 gene primer. Of the 201 samples screened, 96 were positive for V. cholerae O1 (48%), with 69 (72%) positive for ctxA gene. The results from this study showed that the circulating strains of cholera in Nigeria were of Ogawa serotype, also observed in other outbreaks in Nigeria (1991, 1992, and 1996). However, the strains were of the Classical biotype and were mainly (72%) ctxA gene-positive. This current investigation has confirmed the production of cholera toxin by the circulating strains, and this could be harnessed for possible cholera vaccine production in Nigeria.
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Objective To investigate the pathogenic characteristics of Vibrio cholera strains isola-ted from Hubei province in 2012 , and to identify the source of infection by analyzing their genetic correla-tions.Methods The biochemical identification , toxin gene detection and drug susceptibility test were car-ried out to analyze a total of 35 Vibrio cholera strains isolated from three epidemic areas .Comparison of ge-nomic DNA fingerprints and cluster analysis among isolates of Vibrio cholera was conducted by using pulsed-field gel electrophoresis ( PFGE ) .Results All of the 35 strains were Vibrio cholera O139 , of which 71.42%were toxic strains.The drug resistance rates of Vibrio cholera strains to tetracycline, cotrimoxazole and rifampincin were 57.14%, 88.57%and 80.00%, respectively.Analysis of genomic DNA fingerprints of the isolates showed highly similar with similarity values ranging from 80%-100%.Most of the strains iso-lated from the same epidemic area fell into the same one cluster with 100% homology in genome Only a strain isolated from turtle in Jingzhou area was belong to a different cluster .Conclusion The Vibrio cholera O139 strains were the dominant strains causing the outbreaks of cholera in Hubei province in 2012 .Most of them were toxigenic strains .A large majority of the strains had developed resistance to tetracycline , cotri-moxazole and rifampincin , but all strains showed high susceptibility to ceftriaxone and imipenem .Vibrio cholera strains isolated from the same epidemic area were mainly belonged to the same one cluster , sugges-ting the same source of infection .However, the strains varied among different epidemic area .Follow-up in-vestigations of three outbreaks of cholera in this study were all associated with food infection .Therefore , more attention should be paid to food sanitation and safety measurement .Although a non-toxigenic strain iso-lated from turtle was not associated with the epidemic of cholera , surveillance for seafood and aquatic prod-ucts would still be necessary .
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Aims: Bordetella bronchiseptica is an etiologic agent of bronchopneumonia and progressive atrophic rhinitis (PAR) in swine. Both toxigenic and nontoxigenic B. bronchiseptica strains have been associated with bronchopneumonia. Monitoring and investigation of outbreaks involving these bacteria require sensitive and accurate identification and reliable determination of the isolates. In the present study, we report the development, optimization and performance characteristics of polymerase chain reaction (PCR) for B. bronchiseptica strains. Methodology and Results: A total of 47 isolates of B. bronchiseptica were biochemically identified from 90 pigs suffering from bronchopneumonia maintained in a semi intensive rearing system of organized piggery in Meghalaya. PCR was employed with filamentous hemagglutinin toxin genes (fhaB and fhaC) and fimbrial toxin genes (fim2 and fim3) primers to identify the specific toxin types of B. bronchiseptica. All the 47 isolates were positive for all the toxin genes. The specifity of designed primer pairs was tested by screening some common bacterial species related to the respiratory tract namely, Pasteurella multocida, Staphylococcus aureus and Streptococcus spp. No DNA amplifications of the organisms tested could be seen in the specificity test. Amplicon mobility in agarose gels indicate the amplicons are highly stable. Conclusion, significance and impact of study: The data presented, establish this PCR as a reliable method for identification and study of adhesins of B. bronchiseptica that may greatly simplify investigations of swine bronchopneumonia and PAR for Indian isolates.
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BACKGROUND: The skin of atopic dermatitis (AD) patients has a high susceptibility to Staphylococcus aureus colonization, and the toxins produced by S. aureus may aggravate AD by acting as superantigens. OBJECTIVE: The purpose of this study was to evaluate the relationship of the skin barrier function, colonization of S. aureus, and the clinical severity of AD. We also examined the predominant toxin genes produced in Korean AD patients. METHODS: Thirty-nine patients with AD were evaluated for clinical severity and skin barrier function by using Severity Scoring of Atopic Dermatitis (SCORAD) index and transepidermal water loss (TEWL). S. aureus was isolated from the forearm, popliteal fossa, and anterior nares of AD patients (n=39) and age-matched controls (n=40); the toxin genes were analyzed by performing multiplex polymerase chain reaction. RESULTS: TEWL showed a statistically significant correlation with clinical severity in patients with AD (p<0.05). TEWL was correlated with the number of S. aureus colonization sites and the presence of nasal colonization, but these results were not statistically significant. S. aureus strains were isolated in 64.1% of the 39 AD patients. The SCORAD index and AD severity were strongly correlated with the number of colonization sites. The predominant toxin gene found in AD patients was staphylococcal enterotoxin a (sea) only, which was produced in 52.6% of patients. The toxin genes sea and toxic shock syndrome toxin-1 (tsst-1) were found together in 42.1%, while tsst-1 only was found in 5.3% of the patients. CONCLUSION: S. aureus strains were isolated in 64.1% of the 39 AD patients. Skin barrier function, as measured by TEWL, revealed a statistically significant correlation with clinical severity in AD patients. The SCORAD index and severity of AD was strongly correlated with the number of colonization. The most common toxin gene was sea in the Korean AD patients and this gene might have an important role in the pathogenesis of AD.
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Humans , Bacterial Toxins , Colon , Dermatitis, Atopic , Enterotoxins , Exotoxins , Forearm , Shock, Septic , Skin , Staphylococcus , Staphylococcus aureus , Superantigens , Water Loss, InsensibleABSTRACT
Shiga toxin genes (<I>stx</I>) harbouring <I>Escherichia coli</I> (STEC) strains were isolated and identified from diarrhoeal patients visiting the Dhaka Hospital of ICDDR,B: Centre for Health and Population Research, Dhaka, Bangladesh. Of the 189 <I>E. coli </I>strains isolated from 775 diarrhoeal stool specimens, 19 harboured <I>stx1</I>, and one isolate was revealed to have amplicons for both <I>stx1</I> and <I>stx2</I> by a PCR assay. Sequence analysis of the 349-bp <I>stx1</I> from representative isolates revealed 100% homology with the sequence of <I>stx1</I> available in the GenBank. Among the <I>stx1</I> positive isolates, two harboured the <I>eae</I> but none were positive for <I>hlyA, katP, etpD</I> or <I>saa</I> genes. Fifteen of the 20 <I>stx</I> positive strains could be categorized into 13 non-O157 serogroups while 4 were untypable and one was a rough strain. Most of the STEC strains were resistant to ampicillin, cephalothin, co-trimoxazole, tetracycline, and nalidixic acid. In the Vero cell assay, all the strains were negative for expression of Shiga toxin (Stx). Randomly amplified polymorphic DNA (RAPD) PCR analysis demonstrated genetic diversity. This is one of the first reports to show the presence of STEC in diarrhoeal patients in Bangladesh.
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Alpha-toxin gene was amplified from chromosomal DNA of Clostridium perfringens type C by polymerase chain reaction(PCR). PCR product was inserted into vector pGEM-T directively. The cloned recombinant plasmid pXCPA1 possesses positive nucleotide sequence of alpha-toxin. A 1.2 kb alpha-toxin gene fragment was cleaved with restriction endonucleases NcoI/EcoRI from plasmid pXCPA1, and then inserted into an expression vector pET-28c which cleaved with NcoI/EcoRI by blunt-end ligation. The recombinant expression plasmid pETXA1 was studied in detail by restriction endonucleases analysis and nucleotide sequencing. The results showed that the recombinant expression pETXA1 possessed a positive alpha-toxin gene sequence and reading frame. BL21(DE3) (pETXA1) could produce alpha-toxin and the expressed products were recognized by alpha-toxin monoclonal antibodies, and the expression level of the alpha-toxin proteins were about 16.28% of total cellular protein by SDS-PAGE and thin-layer gel scanning analysis.
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Non-pathogenic, environmental strain of Vibrio cholerae, ELTOR Ogawa EW6 carries a copy of the cholera toxin gene in its chromosome. Restriction enzyme digestion followed by Southern blot analysis revealed that the structure of the cholera toxin gene in this organism is different from that found in the virulent strains. The xbaI site which has been found to be conserved in the cholera toxin of the virulent strains examined so far, is absent here. Results of the RNA dot blot analysis indicated that the cholera toxin gene in EW6 is transcribed much less efficiently compared to the cholera toxin gene present in the virulent strain Vibrio cholerae classical Inaba 569B.