Helicobacter pylori in the oral cavity is associated with gastroesophageal disease.

BACKGROUND: In Mexico, more than 80% of the population is infected with Helicobacter pylori. The frequency of H. pylori detection in the oral cavity is unknown, as its relationship with gastroesophageal pathology. AIM: To detect the presence of H. pylori in the oral cavity in Mexican population by PCR and to determine its association with gastroesophageal disease. METHODS: Patients were divided into two groups with different clinic conditions from whom gastric biopsy, dental plaque, and saliva samples were taken and analyzed. The first group comprised of hospitalized patients, the majority of whom were diagnosed with gastroesophageal disease, while the second group was selected from a dental clinic (ambulatory population) the majority of whom appeared to be healthy subjects. RESULTS: H. pylori was detected in gastric biopsy, dental plaque and saliva samples by PCR using a set of specific primers for the signal sequence of the vacuolating cytotoxin gene; detection of H. pylori in general was higher in gastric biopsy and dental plaque samples than in saliva samples. Detection of H. pylori in the oral cavity is significantly (P = 0.0001) associated with patients presenting gastroesophageal disease, while healthy subjects and those with other non-gastric disease do not present with H. pylori in their oral cavity. CONCLUSIONS: H. pylori detection in the oral cavity is associated to gastroesophageal disease. In addition, it is suggested that all patients presenting gastric symptoms and H. pylori detection in the oral cavity would begin bacterial treatment immediately.

Identificación de genotipos de Helicobacter pylori, provenientes de muestras de placa dental en la población venezolana

La placa dental ha sido sugerida como un reservorio importante de Helicobacter pylori, pero la hipótesis de que esta bacteria pueda permanecer como parte integrante de la microbiota residente de la cavidad bucal. Estudios previos realizados en nuestro país demostraron la presencia del microorganismo en 12/32 pacientes, lo que representa un 37,6% de positividad. H. pylori se caracteriza por poseer una gran variabilidad genética, y se ha demostrado la presencia de genes bacterianos específicos que están asociados con la virulencia de las especies bacterianas. El objetivo de este estudio fue caracterizar los genotipos vacA y cagA de especies de H. pylori provenientes de placa dental de una muestra de la población venezolana con el fin de determinar los genotipos mas frecuentes de nuestra población a nivel de la cavidad bucal. Fueron evaluadas 69 muestras de placa dental de pacientes con indicación de endoscopia por enfermedad de las vías digestivas superiores, provenientes del Hospital Clínico Universitario de Caracas. Se tomaron muestras de placa dental de cada uno de los pacientes y se realizó la extracción de ADN para el análisis por Reacción en Cadena de la Polimerasa (RCP). Se amplificaron los segmentos de glm M, vac A and cag A. Los resultados demostraron que solo 1/69 muestras (1,4%) fue positiva para la amplificación de glmM. Ninguna de las muestras pudo ser tipificada para las diferentes formas alélicas de vacA , región media de vacA o cagA . Los resultados de la presente investigación demostraron que aún cuando en reportes previos observamos una importante prevalencia de H. pylori en muestras de placa dental, en esta investigación la prevalencia de la bacteria fue muy baja, no pudiéndose identificar los genotipos mas frecuentes a nivel de placa dental.

The aim of this study was to characterize the vacA and cagA genotypes of H. pylori strains from dental plaque of a Venezuelan population. 69 patients attending for routine gastroscopy were evaluated. Dental plaque samples were obtained for DNA extraction and PCR analysis. Amplification of glmM, vacA and cagA segments were performed as previously described. The results demonstrated that only 1/69 (1,4%) was positive for glmM amplification. None of the samples was typeable for vacA signal sequence genotype, vacA mid region or cagA. The results demonstrated that the prevalence of H. pylori in dental plaque of a Venezuelan population was not significant in this study and was not possible to identify the genotypes of H. pylori from dental plaque.

Topographical localisation of cagA positive and cagA negative Helicobacter pylori strains in the gastric mucosa; an in situ hybridisation study.

BACKGROUND: The cagA gene is a marker for the presence of the cag pathogenicity island, and the presence of cagA positive strains of Helicobacter pylori can identify individuals with a higher risk of developing gastrointestinal diseases. AIMS: To study the interaction between H. pylori cagA(+) and cagA(-) strains and the gastric mucosa. METHODS: Patients with H. pylori associated gastritis and peptic ulcers were studied. Biopsies were obtained from the antrum, corpus, fundus, and incisura for H pylori culture, and for in situ hybridisation studies. From each biopsy, multiple single H. pylori colonies were isolated and propagated for DNA isolation, and cagA was detected by the polymerase chain reaction (PCR). For in situ detection of H. pylori an oligonucleotide specific for an H. pylori common antigen and an oligonucleotide specific for cagA were used as probes. Biotinylated probes were incubated with biopsy sections, developed with streptavidin-horseradish peroxidase, and amplified with the tyramide system. RESULTS: PCR results for cagA in isolated colonies confirmed the in situ hydridisation studies. In situ hybridisation identified cagA(+) bacteria in patients with cagA(+) isolates; cagA(-) bacteria in patients with cagA(-) isolates, and cagA(+) and cagA (-) bacteria in patients with both cagA(+) and cagA(-) isolates. CagA(-) bacteria usually colonised the mucous gel or the apical epithelial surface, whereas cagA(+) bacteria colonised the immediate vicinity of epithelial cells or the intercellular spaces. CONCLUSIONS: These results document a different in vivo interaction between H. pylori cagA(+) or cagA(-) strains and the gastric mucosa.

Increasing multidrug resistance in Helicobacter pylori strains isolated from children and adults in Mexico.

The susceptibilities to three antimicrobials of 195 Helicobacter pylori strains isolated from Mexican patients is reported; 80% of the strains were resistant to metronidazole, 24% were resistant to clarithromycin, and 18% presented a transient resistance to amoxicillin. Resistance to two or more antimicrobials increased significantly from 1995 to 1997.

A comparison of Lewis x and Lewis y expression in Helicobacter pylori obtained from children and adults.

There are no reports, to our knowledge, on the expression of Lewis (Le) antigens in Helicobacter pylori isolates from children. The aim of this study was to compare the expression of Le antigens by H. pylori isolates from children and from adults. Totals of 278 clones from 22 children with recurrent abdominal pain and 293 clones from 22 adults with (n=10) or without (n=12) duodenal ulcer were studied. Expression of Le(x) and Le(y) antigens was determined by ELISA, using monoclonal anti-Le antibodies. The Le phenotype of the patients was determined in gastric juice with a hemagglutination assay. Clones expressing Le(x) were more common in children than in adults (55.4% vs. 33.4%, respectively; P<.001), and Le(y) was more common in adults than in children (81.6% vs. 66%, respectively; P<.01). A trend analysis showed a significant decline in frequency of clones expressing Le(x) with age (P=.021). In this community, expression of Le antigens differs in H. pylori isolates obtained from children versus adults.

Helicobacter pylori vacA and cagA genotypes in Mexican adults and children.

Studies examining associations between Helicobacter pylori virulence markers and disease have concentrated on adults in developed countries. This study assessed adults and children in Mexico. Ninety patients were recruited, 56 adults (37 with active peptic ulceration and 19 with no ulcers) and 34 children (all with recurrent abdominal pain and no ulcers). H. pylori was cultured from gastric biopsy specimens, and vacA alleles and cagA were typed by use of polymerase chain reaction from multiple colony sweeps. Multiple vacA types were common in single-biopsy isolates and were more frequent in adults with ulcers (95%) than in adults without ulcers (37%; P<.001) or in children (52%; P<.01). vacA s1b and cagA+ strains were more frequent in adults than in children. vacA s1 and cagA+ strains had similar frequencies in adults with and without ulcers. In conclusion, infection with multiple H. pylori strains, defined by different vacA genotypes, is common in Mexico. Such mixed infection is associated with ulcer disease. Strain populations infecting Mexican adults and children differ.

Vacuolating cytotoxin (vacA) alleles of Helicobacter pylori comprise two geographically widespread types, m1 and m2, and have evolved through limited recombination.

Vacuolating cytotoxin (vacA) alleles of Helicobacter pylori vary, particularly in their mid region (which may be type m1 or m2) and their signal peptide coding region (type s1 or s2). We investigated nucleotide diversity among vacA alleles in strains from several locales in Asia, South America, and the USA. Phylogenetic analysis of vacA mid region sequences from 18 strains validated the division into two main groups (m1 and m2) and showed further significant divisions within these groups. Informative site analysis demonstrated one example of recombination between m1 and m2 alleles, and several examples of recombination among alleles within these groups. Recombination was not sufficiently extensive to destroy phylogenetic structure entirely. Synonymous nucleotide substitution rates were markedly different between regions of vacA, suggesting different evolutionary divergence times and implying horizontal transfer of genetic elements within vacA. Non-synonymous/synonymous rate ratios were greater between m1 and m2 sequences than among m1 sequences, consistent with m1 and m2 alleles encoding functions fitting strains for slightly different ecological niches.

Colonization of Mexican patients by multiple Helicobacter pylori strains with different vacA and cagA genotypes.

Helicobacter pylori virulence determinants have not previously been studied in detail in Latin Americans with H. pylori infections. We characterized the vacA (vacuolating cytotoxin gene A) and cagA (cytotoxin-associated gene A) types of more than 400 single-colony isolates from 20 patients in Mexico City. For 17 patients H. pylori strains of two or more different vacA genotypes were isolated from gastric biopsy specimens, indicating infection with two or more strains of H. pylori. The most frequent vacA genotype was s1b/m1. vacA diversity was more marked than that described previously, in that isolates from seven patients had untypeable vacA midregions and isolates from nine patients had type s1 signal sequence coding regions which could not be further subtyped. Previously undescribed vacA type s2/m1 strains were found in five patients. All patients were infected with cagA-positive strains, but occasionally, these coexisted with small numbers of cagA-negative strains. In conclusion, coinfection with multiple H. pylori strains is common in Mexico, and vacA in these strains is genetically more diverse than has been described in other populations.

Susceptibility of Helicobacter pylori to the bactericidal activity of human serum.

BACKGROUND: Human serum represents an important barrier to the entry of most mucosal organisms into tissues and to the systemic circulation. If at all present, Helicobacter pylori within gastric tissue is rare, and bacteremia for this organism has been described only once. METHODS: To assess the susceptibility of H. pylori to the bactericidal activity present in normal human serum (NHS), we examined 13 H. pylori isolates. To assess the contributions of the classical and alternative complement pathways to killing, we added either C2-deficient or factor B-deficient serum, respectively, to heat-inactivated NHS. Also we assessed the ability of the strains to bind 125I-C3. RESULTS: After incubation for 60 minutes at 37 degrees C, all 13 H. pylori strains were killed by NHS; heating to 56 degrees C for 30 minutes ablated killing, indicating complement dependence for this phenomenon. In the absence of an antibody source, there was no killing when either an alternative or classical complement pathway source was used. Adding B-deficient serum to heat-inactivated normal human serum did not restore killing, but adding C2-deficient serum permitted partial killing. All of the 13 strains bound 125I-C3. Although the kinetics varied from strain to strain, C3 bound was significantly correlated (r = 0.61, p = 0.03) with serum susceptibility. CONCLUSIONS: H. pylori are susceptible to complement, alternative pathway activation appears critical, and C3 binding is a major locus of variability.

Production of cytotoxins and enterotoxins by strains of Shigella and Salmonella isolated from children with bloody diarrhea.

The role of toxins in the pathogenesis of bloody diarrhea caused by Shigella and Salmonella isolated from children with bloody diarrhea was studied for production of toxins active on cells in culture and in rat intestinal loops. Human epithelial cells from colon carcinoma (HT-29), Chinese hamster ovary cells (CHO) and kidney fibroblast from rhesus monkey (Vero) were used to detect cytotoxins. On HT-29 almost 50% of the Shigella and about 20% of the Salmonella strains caused rounding of cells; on CHO over 50% of Salmonella and 20% of Shigella strains caused elongation of cells, some strains caused also rounding of these cells whereas on Vero over 60% of Salmonella and 40% of Shigella strains caused rounding of cells. Cytotoxicity on Vero and CHO cells was strongly inhibited with cholera toxin antiserum, whereas that on HT-29 was inhibited with C. difficile toxin B antiserum. Cytotonic activity on CHO cells and rounding on Vero cells seem to be suitable models to detect toxins cross-reacting with cholera toxin. Both species, Shigella and Salmonella, produce cytotoxins and enterotoxins which could play a role in intestinal disease.

Toxigenicity and adherence in Clostridium difficile strains isolated from patients with and without diarrhoea.

The mechanisms by which Clostridium difficile causes diarrhoea are unknown. The expression of putative virulence factors by 44 Clostridium difficile strains isolated from patients with and without diarrhoea was studied. Toxins A and B were tested in CHO and MRC-5 cells, respectively; adherence was measured in two substrates: HEp-2 cells and polystyrene plates. The in vitro expression of toxins A and B by strains isolated from patients with diarrhoea was not significantly different from that by strains isolated from patients without diarrhoea. The ability of adherence to both HEp-2 cells and polystyrene by strains isolated from patients with diarrhoea was not significantly different when compared with strains isolated from patients without diarrhoea; however, strains isolated from adults with diarrhoea seem to adhere to a greater extent to both substrates than strains isolated from adults without diarrhoea. Twenty three strains which did not produce toxins A and B were tested for enterotoxicity in rat small intestine. Eight such strains induced fluid accumulation and seven of them were isolated from children. Adherence to cells and to polystyrene might be an important virulence factor in strains causing diarrhoea in adults; whereas the production of toxins other than A and B might be an important pathogenic mechanism in strains causing diarrhoea in children.