Patterns of Adherence of Helicobacter pylori Clinical Isolates to Epithelial Cells, and its Association with Disease and with Virulence Factors.

BACKGROUND AND AIM: Adherence to the gastric epithelium is one of the most important steps of Helicobacter pylori to remain and cause disease. The aim of this study was to analyze whether H. pylori isolates from patients with different gastroduodenal diseases present differences in the pattern of adherence to gastric epithelial cells (AGS), in the ability to induce IL-8, and in the presence of virulence genes. METHODS: We tested 75 H. pylori strains isolated from nonatrophic gastritis, gastric cancer, and duodenal ulcer patients. The adhesion pattern and IL-8 induction were determined in AGS cells, and invasion of AGS cells was studied using a gentamicin protection assay. The IL-8 levels induced were determined by ELISA. RESULTS: Helicobacter pylori strains presented diffuse adherence (DA) and localized (LA) adherence patterns, similar to those described for enteropathogenic E. coli (EPEC), were observed in AGS cells. A DA pattern was observed in 57% and LA in 43% of the strains, and DA was more frequent in isolates from patients with gastric cancer (p = 0.044). Strains with a LA pattern induced higher levels of IL-8 (p = 0.042) in AGS cells. CONCLUSION: The adherence pattern was not associated with neither invasiveness nor with the presence of virulence genes. Our study shows that H. pylori strains present adherence patterns to AGS cells resembling those observed in EPEC and that these patterns may be associated with disease and with activity on AGS cells.

Detection of the glmM gene in Helicobacter pylori isolates with a novel primer by PCR.

A novel reverse primer (GLM MR1) was designed for detection of the glmM gene in Helicobacter pylori by PCR. The percentage of amplification in clinical isolates using GLM MR1 was 100% for detection of the glmM gene and 86.36% for the ureA gene. The primer designed is useful for the identification of H. pylori.

Evaluation of in vivo and in vitro biological activity of a Vibrio cholerae 01 hemolysin.

PURPOSE: To evaluate the hemolysin effect by ileal loop model produced by Vibrio cholerae O1 strains, compared with the cellular lysis or cytotoxic activity (CA) observed in cell culture. METHOD: We studied nine V. cholerae O1 strains, obtained during the Mexican outbreak of cholera (1990-1993), which had CA in Vero and CHO cells. Hemolysin was monitored with the hemolysis test. Titers of CA were calculated by CD50, and the association between CA and cholera toxin (CT) production was discarded by means of neutralization tests using an anti-CT polyclonal antibody. The CT production was measured with ELISA test. The LAL assay was performed in order to study relationships between the CA and bacterial lipopolysaccharide. Strains with CA were evaluated in rabbit and rat ileal loop models; hemorrhagic fluid was also measured. Tissues from ileal loop were included in paraffin to detect intestinal epithelial damage. RESULTS: The hemolysin CA was not neutralized with the anti-CT polyclonal antibody. However, the associated factor of CA was heat labile. CA in cell cultures was not related to the bacterial lipopolysaccharide. The ileal loop test exhibited the presence of hemorrhagic tissue with inflammation. CONCLUSION: The V. cholerae O1 strains isolated were able to secrete hemolysin which, in turn, caused CA in cell cultures and produced the hemorrhagic and inflammatory effects observed in the ileal loop of rabbit and rat models.

Anticapsular polysaccharide IgG concentrations in Mexican children formerly immunized with Haemophilus influenzae b PRP-T vaccine.

BACKGROUND: In 1999 H. influenzae b (Hib) PRP-T vaccine was introduced into primary immunization schedule in Mexico. There have been no studies evaluating antibody response after widespread immunization in our country. It is now recognized that Hib conjugates induce significant initial antibody levels that in some cases wane over time. This study relies on the measurement of IgG serum antibody concentrations to Hib capsular polysaccharide (PS) and is interpreted in the light of the accepted levels > or =0.15 microg/mL for short-term protection against Hib invasive disease and > or =5.0 microg/mL for protection against Hib oropharyngeal carriage. METHODS: Using a validated ELISA assay, we measured the IgG serum antibody concentrations in 115 children between 7 and 93 months of age who had received three doses of PRP-T. We used the standard reference serum US FDA 1983 for quantification of PS antibody levels. Concentrations were estimated using 3(rd) degree polynomial regression lines. As there was no unvaccinated group available (>95% of Mexican children have received the Hib vaccine), the study was designed as a cross-sectional. RESULTS: All children had serum IgG concentrations > or =0.15 microg/mL [range 0.24-54.64 microg/mL]; 69.6 % (80/115) had > or =1.0 microg/mL and 14 % (16/115) showed concentrations > or =5.0 microg/mL. The vaccine elicited geometric mean concentrations (GMC) of 4.0, 1.6, 1.4 and 2.4 microg/mL in groups of 7-12, 13-24, 25-48 and 49-93 month-old respectively. CONCLUSIONS: PRP-T vaccination in this group of Mexican children has resulted in serum IgG concentrations > or =0.15 microg/mL, suggesting that Hib immunization has conferred protection against invasive disease.

[Microbiologic, serologic diagnosis, and genotypification of Helicobacter pylori isolated from biopsies in children and adult people. Molecular detection of the cag pathogenicity island of Helicobacter pylori]. / Diagnóstico microbiologico, serológico, genotipifi- cación de Helicobacter pylori aislado de biopsias de niños y adultos. Detección molecular de la isla de pato- genicidad cag de Helicobacter pylori.

Hp diagnostic is made by invasive methods using gastric biopsy of antrum, for culture and histological study. Non invasive are serology and urea breath test. 152 Hp from 19 children's with acute gastritis (46. 1%); 9 Hp from an adult. There had ampicillin resistance, 27. 4%, claritromicin 21. 8%, metronidazol 58. 4% and tetracycline 31. 5%, the 21 % susceptible to four anti-microbial. RAPD-PCR with 4 primers gave 44 profiles, related with clinical profile. In 7 children, there were two profiles RAPD. Three were similar but different each other. AFLP 23 adults 151 Hp; cagA and vacA genotypes gave unique patterns for each patient. The genes cagA, babA and oipA, secuencing and compared with reported Hp (Genbank); gave high polymorfism. Everything indicates that there are colonization with multiple Hp strains in Mexicans. In 645 sera from 352 children: 36.9% with Hp, the 46. 9% gave him/her anti-shits and 16. 2% antibodies to urease. Adults 293 (89. 1%) with Hp, 78. 9% gave anti-CagA and 59% antibodies to urease. The expression of IL-8 was bigger in infected children that in not infected and more significant in peptic ulcer. Genomic library Cag-PAI revealed 90% (Hp) positive, they had the complete PAI, 2 were incomplete and three negatives. PCR -LiPA LiPA (LineProbe Assay) is suggestive for genotyping assays.

Serogroups, K1 antigen, and antimicrobial resistance patterns of Aeromonas spp. strains isolated from different sources in Mexico.

A total of 221 strains of Aeromonas species isolated in Mexico from clinical (161), environmental (40), and food (20) samples were identified using the automated system bioMérieux-Vitek. Antisera for serogroups O1 to 044 were tested using the Shimada and Sakazaki scheme. The K1 antigen was examined using as antiserum the O7:K1C of Escherichia coli. Besides, we studied the antimicrobial patterns according to Vitek AutoMicrobic system. Among the 161 clinical strains 60% were identified as A. hydrophila, 20.4% as A. caviae, and 19.25% as A. veronii biovar sobria. Only A. hydrophila and A. veronii biovar sobria were found in food (55 and 90% respectively) and environmental sources (45 and 10% respectively). Using "O" antisera, only 42.5% (94/221) of the strains were serologically identified, 55% (121/221) were non-typable, and 2.5% (6/221) were rough strains. Twenty-two different serogroups were found, O14, O16, O19, O22, and O34 represented 60% of the serotyped strains. More than 50% of Aeromonas strain examined (112/221) expressed K1 encapsulating antigen; this characteristic was predominant among Aeromonas strains of clinical origin. Resistance to ampicillin/sulbactam and cephazolin was detected in 100 and 67% of Aeromonas strain tested for their susceptibility to antibiotics. In conclusion, antibiotic-resistant Aeromonas species that possess the K1 encapsulating antigen and represent serogroups associated with clinical syndrome in man are not uncommon among Aeromonas strains isolated from clinical, food and environmental sources in Mexico.

Caracterización bioquímica y ultraestructural del efecto de la interleucina 1-beta como mediador de degradación de tejido conectivo en membranas corioamnióticas humanas en cultivo / Biochemical and ultrastructural characterization of the effect of interleukin 1-beta as mediator of the degradation of connective tissue in human chorioamniotic membranes in culture

OBJECTIVE: To characterize the effect of IL-1 beta, on connective tissue metabolism in a human chorioamniotic membrane tissue culture (CAM). TYPE OF STUDY: Experimental, in an in vitro model. MATERIAL AND METHODS: CAM explants obtained from cesarean sections were cultured. The presence of local infection was excluded by microbiological methods. An XTT viability essay of the explants was carried out. Explants were stimulated with different doses of IL-1 beta within a 0-10 ng/mL range. After the stimulation, protein content was measured, MMP-9 production was determined by zymography, and each explant was divided in two parts: one was used for collagen measurement and the other analyzed by electronic microscopy. RESULTS: CAMs kept adequate viability and functionality. IL-1 beta stimulation produced an increase in the amount of MMP-9 expressed, as determined by the zymography method with a maximum effect 36 hours after stimulation. Collagen content decreased in a progressive manner after IL-1 beta stimulation and reached its minimum after 36 hours. The characteristic pattern of collagen fibers gradually lost its organization, and could not be observed any more after 36 hours. CONCLUSIONS: The information presented here allows us to conclude that IL-1 beta is capable of inducing an enzymatic expression affecting connective tissue, thus confirming its participation in membrane degradation processes under inflammatory conditions.