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Molecular Bacteriology for Helicobacter Pylori P724 P725

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Molecular Bacteriology for Helicobacter Pylori P724 P725 Clinical Microbiology and Infection, Volume 10, Supplement 3, 2004 179 Molecular bacteriology for Helicobacter pylori P724 cagA, vacA and babA2 genotypes of Helicobacter different types of environments. The predominant cells’ morphol- pylori strains from patients with gastritis or peptic ulcer in Spain ogy in both clinical and wastewater samples was of helicoidal form. J.A. Garcı´a-Campos, D. Domingo, T. Alarco´n, E. Aznar, Conclusions: The FISH is a specific and rapid culture-independent J. Dı´az-Regan˜o´n, M. Lo´pez-Brea method to determine directly the presence of clarithromycin- Madrid, E resistant H. pylori cells in clinical and environmental samples. Results showed the presence of macrolide resistant cells in water Objectives: In this study we wanted to examine the prevalence of and, therefore, water must be considered a potential route of cagA, vacA and babA2 status in Helicobacter pylori (Hp) isolates H. pylori transmission. from patients with gastritis or peptic ulcer; to compare them and Acknowledgement: This work was supported by Ministerio Espan˜ol to know if there were any relationships between those virulence de Ciencia y Tecnologı´a, Project AGL2002-04480-C03-03. factors in each group. Methods: Gastric biopsy specimens from 44 Hp positive patients with peptic ulcer (25 cases) and gastritis (19 cases) were studied. DNA was extracted and PCR performed to detect cagA, vacA s1/ P726 Effect of the therapy of patients with Helicobacter s2 alleles and babA2 gene. pylori related gastritis is correlated with organisation of CAG Results: Gastritis: In 74% of strains, the expected cagA fragment pathogenicity island was amplified by PCR; 68% carried the s1-allele and 32% the s2- allele; babA2 gene was detected in 37% of strains. Peptic ulcer: V. Simanenkov, D. Bovaeva, O. Solovjova, A. Suvorov 84% of strains were cagA+; 72% were vacA s1-allele and 28% St Petersburg, RUS were vacA s2-allele; babA2 gene was detected in 36% of strains. No significant differences in the prevalence of cagA, vacA or Objectives: Helicobacter pylori is a leading cause of various gastro- babA2 were found in both groups. Neither of them showed rela- intestinal diseases such as atrophic gastritis and gastroduodenal tionship between the presence of babA2 gene and cagA gene or ulcer. The cagA gene product CagA is directly injected into the vacA s1/s2-alleles. bacteria-attached host cells and deregulates intracellular signalling Conclusions: Although the risk of developing more serious gastric pathways and thereby initiates pathogenesis. cagA gene is located lesions increased as the number of virulence factor genes are on pathoginicty island but the function of other genes on the accumulated in a given Hp strain, we did not find any significant island is unknown. The goal of the work was to evaluate the differences or relationship in the cagA, vacA or babA2 status impact of cag island genotype on the outcome of the therapy. between the Hp isolates from patients with gastritis or peptic Materials and methods: Three groups of 25 patients each with total ulcer in this study. A low percentage of babA2 gene was found in number of 75 patients were investigated. First group was taking a both groups. typical antibiotic therapy (amoxicillin, claritromycin, rabeprasol), patients in the second group were treated with the same antibiot- ics together with probiotic Laminolact (E. faecium strain L-3 in the form of bon-bons together with pectin, soy bean amino acids and sea weed), and the third group was taking only Laminolact with- P725 FISH detection of clarithromycin-resistant out any antibiotic. The genotype was determined by PCR with Helicobacter pylori cells in clinical and wastewater samples DNA primers against three H. pylori genes ureB, cagA and cagH. cagH was used as a marker cag island integrity and ureB was a P. Piqueres, Y. Moreno, A. Jimenez, J. Herna´ndez, M. Ferru´ s marker of H. pylori presence. Valencia, E Results: Five different genotypes were determined: ureB+,cagAÀcagHÀ, ure+,cagAÀcagH+, ureB+,cagA+cagH+, Introduction: The presence of viable but non-cultivable Helicobacter ureB+,cagAÀcagH+ and ureB+,cagA+cagHÀ. Treatment with pylori cells in environmental samples may underestimate the antibiotics alone was leading to 68% of eradication. The best importance of this way for its transmission. The determination of eradication percentage (84%) took place in the second group resistance to antibiotics in these strains is important to a better where classical antibiotic treatment was taken together with understanding of the epidemiology of the infection. probiotics. Interestingly, probiotic treatment alone was giving Objectives: We have evaluated the use of a fluorescent in situ 48% of eradication. Results of the therapy were highly consis- hybridisation (FISH) assay directly from biopsies and wastewater tent with cag genotype. Patients were found to be statistically to detect H. pylori and simultaneously its macrolide resistance less susceptible (P < 0.05) to the therapy in case when the genotype. entire cag regulon was present regardless of the therapy used. Methods: A total of 26 gastric biopsies samples from ulcer- This fact suggests immunosuppressant function of CagA or patients were homogenised in 2 mL of selective broth, and a other proteins encoded by the genes on cag pathogenicity 500 lL aliquot was used for FISH detection. Twenty-nine waste- island. water samples collected from different treatment plants were cen- Conclusion: The effect of H. pylori eradication depends on cag trifuged and subsequently fixed with 4% paraformaldehyde pathogenicity island genotype. Probiotics including E. faecium L-3 solution for 4 h at 4C and then washed with 1% PBS buffer. might significantly improve the anti-H. pylori treatment. HPY probe, a 16S rRNA targeted FITC-labelled oligonucleotide sequence was used for the detection of all H. pylori strains. In addition to CLA1–3, a set of three CY3-labelled probes was used for the detection of 23S rRNA mutations associated with resist- ance to clarithromycin. Hybridisation was performed with 35% P727 AP-PCR genotyping of clinical isolates of formamide at 46 C for 2 h. Pseudomonas aeruginosa Results: FISH allowed the detection of H. pylori in 20 out of 26 clinical samples and 12 samples were positive in wastewater. The D.H. Binnet, G. Uraz, E. Senol, N. Akar 35% of the positive biopsies showed the presence of clarithromy- Ankara, TR cin resistant strains and 16.6% of the positive wastewater samples yielded resistance genotype to this macrolide. By using a double Objectives: Arbitrarily Primed Polymerase Chain Reaction (AP- filter set we could observe directly the clarithromicyn resistant PCR) has been used for the epidemiological evolution clinical iso- H. pylori organisms in the samples and its morphology in the lates of P. aeruginosa. This method is recommended for first-pass 180 Abstracts screening of P. aeruginosa isolates. This study was done to deter- P729 Phenotypic and genotypic analysis of Pseudomonas mine if the P. aeruginosa strains isolated from the cultures of aeruginosa strains isolated from cystic fibrosis patients patients hospitalised in the Infectious Diseases Unit were from an individual strain. This technique was preferred because it is cheap S. Scutera, T. Allice, S. Bianco, M.G. Chirillo, M. Zucca, D. Savoia and provides a rapid detection opportunity. Orbassano, I Methods: 45 samples obtained from the clinical specimens of the patients and from the hands of the staff of the Infectious Dis- Objectives: Because of a high prevalence of Pseudomonas aeruginosa eases Unit were cultured. Of the 45 samples, 15 were isolated infections in cystic fibrosis (CF) patients, we conducted a study to from blood, six from sputum, 10 from drainage and 14 from the assess 60 P. aeruginosa isolates collected over 10 years from the hands of the medical staff. P. aeruginosa identification was made sputa of 38 CF adult patients attending an Italian CF centre. Some by API 20NE system. DNA was extracted from the culture phenotypic characters of bacteria (O-serotype, motility, production material by phenol–chloroform extraction method. AP-PCR was of enzymes and resistance to antibiotics) and their PFGE geno- performed by using the primer 5¢-GTT GCG ATCC-3¢, and sub- typic patterns were evaluated to analyse for the presence of epi- jected 8% PAGE. Band patterns were visualised by silver stain- demic strains. Moreover some sequential isolates collected from ing. 15 CF patients were investigated to look for the chronicisation of Results: In none of the isolates of the hospital staff P. aeruginosa the infection. was cultured. Out of the 31 clinical samples of the patients, 15 dif- Methods: The strains were identified biochemically. The ferent genotypes were determined. O-serotype was determined by slide agglutination; the production Conclusions: The P. aeruginosa strains of the patients were individ- of enzymes (protease, elastase, gelatinase, haemolysin, beta- ual strains, neither related to the staff of the department nor to a lactamase) and motility were detected using specific techniques. specific patient. The antibiotic susceptibility was analysed by the Vitek AMS Sys- tem and disc diffusion method. PFGE was used to discriminate the genotypes of P. aeruginosa. Results: In our hands, O serotyping failed to identify 26.3% of isolates, considering the bacteria collected at the onset of colonisa- tion; the most frequent serotypes were O: 10, O: 6 and O: 3. More- over, the percentages of protease, haemolysin, gelatinase and P728 Pseudomonas aeruginosa – microarray for rapid elastase production were respectively 78.9, 52.6, 55.3 and 39.5, determination of antibiotic susceptibility and virulence factors whereas 42.1% of the microorganisms were non-motile. PFGE allowed the typing of all strains except one. The heterogeneity of J. Weile, M. Susa, T. Bachmann, C. Knabbe isolates indicated that cross-infection is unusual; we also observed Stuttgart, D in several strains isolated in the last years a predominant pattern.
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