Frequency of virulence genes in mixed infections with Helicobacter pylori strains from a Mexican population.

BACKGROUND: Helicobacter pylori (H. pylori) is associated with gastroduodenal diseases. Virulence of clinical isolates is related to clinical outcome. Moreover, with microdiversity studies in clinical isolates from a single patient, but from a different origin (antrum or corpus), it is possible to demonstrate that there are simultaneous mixed infections. AIMS: To genotype H. pylori strains with multiplex PCR, according to their clinical virulence, and in this manner know the frequency of each genotype and relate it to clinical outcome in order to prevent the development of severe diseases. METHODS: A total of 210 patients with gastric alterations were studied. Virulence classification of H. pylori strains was carried out with multiplex PCR and 127 strains were identified as H. pylori by PCR (glmM and cagE). Genotype and clinical outcome were evaluated with the Fisher's exact test. In addition, RAPD-PCR was performed as a fingerprinting method to analyze mixed infections. RESULTS: The cagA, vacAs1, and vacAm1 genes were detected in all the clinical isolates. Strains were classified as: type i, 40.15% (51/127); type ii, 22.04% (28/127); and type iii, 28.4% (36/127), but two new different genotypes were also detected: (1) babA2+, cagA+, vacAs1+, 6.29% (8/127) and (2) babA2+, cagA-, vacAs2/m2+, 3.14% (4/127). The cagE gene was detected in type i strains. CONCLUSIONS: The Fisher's exact test did not support a significant association between clinical outcome and genotype. The main circulating genotypes in the Mexican population studied were: cagA+, vacAs1, and vacAm1. Multiplex PCR can be used as a screening test for H. pylori strains. Furthermore, the cagE gene is a good marker for identifying cag-PAI positive strains.

A PCR procedure for the detection of Giardia intestinalis cysts and Escherichia coli in lettuce.

Giardia intestinalis is a pathogen associated with foodborne outbreaks and Escherichia coli is commonly used as a marker of faecal contamination. Implementation of routine identification methods of G. intestinalis is difficult for the analysis of vegetables and the microbiological detection of E. coli requires several days. This study proposes a PCR-based assay for the detection of E. coli and G. intestinalis cysts using crude DNA isolated from artificially contaminated lettuce. The G. intestinalis and E. coli PCR assays targeted the ß-giardin and uidA genes, respectively, and were 100% specific. Forty lettuces from local markets were analysed by both PCR and light microscopy and no cysts were detected, the calculated detection limit was 20 cysts per gram of lettuce; however, by PCR, E. coli was detected in eight of ten randomly selected samples of lettuce. These data highlight the need to validate procedures for routine quality assurance. These PCR-based assays can be employed as alternative methods for the detection of G. intestinalis and E. coli and have the potential to allow for the automation and simultaneous detection of protozoa and bacterial pathogens in multiple samples. Significance and impact of the study: There are few studies for Giardia intestinalis detection in food because methods for its identification are difficult for routine implementation. Here, we developed a PCR-based method as an alternative to the direct observation of cysts in lettuce by light microscopy. Additionally, Escherichia coli was detected by PCR and the sanitary quality of lettuce was evaluated using molecular and standard microbiological methods. Using PCR, the detection probability of Giardia cysts inoculated onto samples of lettuce was improved compared to light microscopy, with the advantage of easy automation. These methods may be employed to perform timely and affordable detection of foodborne pathogens.

Analysis of the genotypic diversity of strains of Helicobacter pylori isolated from pediatric patients in Mexico.

Genotypic differences in Helicobacter pylori play an important role in infection. We characterized the diversity of the cagA, cagE, babA2, and vacA genes in H. pylori strains isolated from pediatric patients and the relationship between these genes and clinical disease. Additionally, we employed the Neighbor-net algorithm to predict the behavior of the genotypes of the strains isolated from patients. Of 93 patients analyzed, 32 were positive for infection. A total of 160 H. pylori strains (five isolates per positive patient) were analyzed. A total of 91% and 83% of strains possessed the cagA and cagE genes, respectively. For the vacA gene, 84% of strains possessed the s1 allele, 15% the s2 allele, 81% the m1 allele and 13.8% the m2 allele. The babA2 gene was present in 79% of strains. Infection with H. pylori strains with the vacA (s1m1) genotype was associated with risk of esophagitis and gastritis (p=0.0001). The combination of cagA and vacA (s1m1) was significantly associated with abdominal pain (p=0.002); however, EPIYA type was not significantly associated with abdominal pain. A total of 16 different genotypes were identified; the most common genotype was vacAs1m1cagA+cagE+babA2+ (47.5%). A total of 84% of pediatric patients were infected by at least two and up to five different genotypes. The network recovered two genotype groups (A: strains with vacAs1 and B: strains with vacAs2). The presence of multiple paths in the network suggests that reticulate events, such as recombination or reinfection, have contributed to the observed genotypic diversity.

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.

Cell vacuolation caused by Vibrio cholerae hemolysin.

Non-O1 strains of Vibrio cholerae implicated in gastroenteritis and diarrhea generally lack virulence determinants such as cholera toxin that are characteristic of epidemic strains; the factors that contribute to their virulence are not understood. Here we report that at least one-third of diarrhea-associated nonepidemic V. cholerae strains from Mexico cause vacuolation of cultured Vero cells. Detailed analyses indicated that this vacuolation was related to that caused by aerolysin, a pore-forming toxin of Aeromonas; it involved primarily the endoplasmic reticulum at early times (approximately 1 to 4 h after exposure), and resulted in formation of large, acidic, endosome-like multivesicular vacuoles (probably autophagosomes) only at late times (approximately 16 h). In contrast to vacuolation caused by Helicobacter pylori VacA protein, that induced by V. cholerae was exacerbated by agents that block vacuolar proton pumping but not by endosome-targeted weak bases. It caused centripetal redistribution of endosomes, reflecting cytoplasmic alkalinization. The gene for V. cholerae vacuolating activity was cloned and was found to correspond to hlyA, the structural gene for hemolysin. HlyA protein is a pore-forming toxin that causes ion leakage and, ultimately, eukaryotic cell lysis. Thus, a distinct form of cell vacuolation precedes cytolysis at low doses of hemolysin. We propose that this vacuolation, in itself, contributes to the virulence of V. cholerae strains, perhaps by perturbing intracellular membrane trafficking or ion exchange in target cells and thereby affecting local intestinal inflammatory or other defense responses.

The emerging diversity of the electrophoretic types of Vibrio cholerae in the Western Hemisphere.

Since the Latin American cholera epidemic began in 1991, 447 isolates of Vibrio cholerae O1 from the Western Hemisphere have been assayed by multilocus enzyme electrophoresis (MEE) to determine allelic variation among 16 enzyme-encoding genes. Two electrophoretic types (ETs) were identified among toxigenic isolates from Latin America: 323 were ET 4, the ET associated with the Latin American epidemic, and 29 were ET 3. Twenty-three of these ET 3 isolates had a distinctive antimicrobial resistance pattern also seen in isolates imported into the United States from Latin America and Southeast Asia. These resistant isolates had an identical ribotype and nearly identical pulsed-field gel electrophoresis (PFGE) patterns. Most nontoxigenic isolates analyzed were not precursors or descendants of toxigenic epidemic strains. MEE provided a population genetic frame-work for the interpretation of PFGE and ribotype data from the isolates in this study. All three methods identified 2 distinct strains of toxigenic V. cholerae O1 currently epidemic in Latin America.

[Identification of enterotoxins and cytotoxins of Escherichia coli by Vero cell culture and solid-phase hybridization (colony blot)]. / Identificación de enterotoxinas y citotoxinas de Escherichia coli por cultivo de células Vero e hibridación en fase sólida (colony blot).

We studied 40 E. coli strains from meat and hamburger and 64 strains from stools of people: 14/64 from traveler's diarrhea and 50/64 from infants with and without diarrhea. We want to know if they could be producing of cytotoxin and enterotoxin in Vero culture cells as well as phenotypic and genotypic relationships. The serotypes that we isolated were different than O:157H:7. We found 3 cytotoxic strains, 77 heat labile enterotoxic strains on Vero culture cells, and 19 E. coli strains with cytotoxin and LT enterotoxin. One strain isolated from infant with diarrhea was negative sorbitol but cytotoxic effect and it was O:55 serotype. The colony blot and cytotonic were found in 90 (86.5%) E. coli strains. The sensitivity of colony blot was 93.75%.

[Polymerase chain reaction (PCR) for the identification of toxigenic Vibrio cholerae O1 in oysters]. / La técnica de reacción de polimerización en cadena (PCR) para la identificación de Vibrio cholerae O1 toxigénico en ostiones.

PCR was made with ctx2 (CGG GCA GAT TCT AGA CCT CCT G) y ctx3 (CGA TGA TCT TGG AGC ATT CCC AC) primers for subunit A of cholera toxin, 30 cycles of temperature on samples of 50 g of oysters added in 450 ml of peptone alcaline water that were inoculated with 15 x 10(6), 0.75 x 10(6) and 0.15 x 10(6) CFU/ml of toxigenic 6707 V. cholerae O1 reference strain. The samples were tested by three microbiological methods: INDRE's method uses 1 x 10(-1) dilution of sample, two fold pass to peptone alcaline water pH 9 incubated 18 h and 6 h at 37 degrees C, the Food and Drugs Administration (FDA) method uses 10(-1) to 10(-6) dilutions of sample, 6 h incubation and reincubation for 18 h at 37 and 42 degrees C and the Mexican laboratories (LMD) with 10(-4) to 10(-3) dilutions, the samples were incubated for 6 h and then reincubated for 18 h at two temperatures 37 and 42 degrees C. The PCR by INDRE's method was positive with 3 x 10(2) CFU/ml/g oyster. In the FDA's method the PCR detected DNA in 10(-4) dilution with 3 x 10(1) CFU/ml/g oyster and in LMD's method the PCR was positive in 10(-3) with 3 CFU/ml/g oyster. The results of the PCR were obtained between 5-6 h, and later V. cholerae O1 was isolated by three microbiological methods. The PCR reproducibility was better on DNA sample diluted 1:4 and 10 microliters of sample increased from 1:1000 to 1:10000 the sensitivity of PCR.

[Principal serotypes of Salmonella identified in 10703 strains in Mexico from 1982 to 1993]. / Principales serotipos de Salmonella identificados en 10703 cepas en México entre 1982 y 1993.

From 1982 to 1993, 10703 Salmonella strains from The National Network of Diarroheal Laboratories of Mexico were sent to the Enteric Bacteriology Laboratory of INDRE. The strains were confirmed by serology and 119 different Salmonella serotypes were found. The most frequent serotypes were as follows: S. typhimurium, S, enteritidis, S. agona & S. typhi. The strains were classified according to the source of isolation as follows: 6671 strains (62.33%) from clinical samples, mainly of faecal origin; 2903 (27.1%) from food for human consumption; 425 from food for animal consumption, 665 (6.21%) from environment or fomites and 39 (0.36%) from animals. The most frequent serotype in clinical samples was S. typhimurium among 96 different serotypes. The main serotype from blood cultures was S. typhi although 27 other serotypes were found. Of thirteen serotypes related to diarrhoeal outbreaks the higher frequency of S. typhimurium was observed but S. typhi caused more outbreaks. A frequency of 119/> 2000 serotypes was observed, that means less than 5% of Salmonella known serotypes. A yearly variability on serotype predominance was observed as well as changes on source of isolation. This results suggest that epidemiological surveillance of salmonellosis should be continued and improved, looking for cases, asymptomatic carriers and contaminated food for human consumption.

[Phenotypic and genotypic characterization of Vibrio cholerae O1]. / Caracterizatión fenotípica y genotípica de Vibrio cholerae O1.

We made 52180 tests for isolation and identification of toxigenic V. cholerae O1 from rectal swabs and reference strains. We isolated 17.6% V. cholerae O1 strains in 1991, 43.5% in 1992 and 38.9% in 1993. The main serovar in 1991 was Inaba, whereas in 1993 a similar percentage was serovar Ogawa. The phenotype of V. cholerae strains was determined by hemolysis test, Voges-Proskauer test, polymyxin B resistance and phages 4 and 5 resistance. All of the mexican strains were El Tor. There were 2.9-0.75% hemolytic strains from 1991 to 1993, but they were negative when the test was made in tube with human erythrocytes. The resistotypes were performed in 24526 selected strains by Kirby-Bauer method and MIC tests. All of the strains were sensitive, except more than 100 strains isolated in Veracruz that were resistant to tetracycline and doxycycline. Detection of cholera toxin was made by ELISA and on culture of Vero and CHO cells. All the V. cholerae O1 strains were toxigenic. The genotype was determined by PCR and ribotyping. The PCR amplified one 564 pb fragment on V. cholerae O1. The ribotypes of mexican strains were 5 and 6a.

[Cytotonic and cytotoxic effect of cholera toxin on Vero cells and its relation to PCR]. / Efecto citotónico y citotóxico de la toxina colérica en células Vero y su relación con PCR.

We studied 40 Vibrio cholerae strains: 16 from stool, 16 from sewage and 8 from food. The serotypes were Inaba in 21 strains, 8 Ogawa strains and 11 V. cholerae non-O1. PCR was made with ctx2 and ctx3 primers with 25 cycles of temperature: 1 min at 94 degrees C, 1 min at 60 degrees C and 1 min at 72 degrees C. 24 V. cholerae strains were positive: 18/24 Inaba y 6/24 Ogawa. PCR was negative for 16 strains: 3 Inaba serotype, 2 Ogawa y 11 V. cholerae non-O1. In Vero culture cells 18 strains were cytotonic, 21 cytotoxic and 1 strain was negative. ELISA was positive for 11 strains with PCR positive. The PCR sensitivity was 95.83% compared with culture cells. V. cholerae O1 produced cytotoxic effect on Vero culture cells, maybe related to ACE factor. Colony blot was made with a specific probe labeled with digoxigenin and it could detect 4 Vibrio cholerae toxigenic strains with PCR negative. All V. cholerae Non O1 strains were PCR negative.

[Antibiotic resistance pattern of 24, 526 strains of Vibrio cholerae O1 isolated in Mexico from 1991 to 1993]. / Patrón de resistencia a los antibióticos de 24,526 cepas de Vibrio cholerae O1 aisladas en México de 1991 a 1993.

Profile of antimicrobial resistance by Kirby-Bauer method was performed on 24526 Vibrio cholerae O1 strains isolated in México (1991-1993) from fecal swabs in cholera cases and from asymptomatic carriers. Minimal inhibitory concentration (MIC) tests for tetracycline (Te) and doxycycline (D) were done on selected strains. Single antibiotic discs were used at concentrations of: Te, 30 micrograms; D, 30 micrograms; erythromycin (E), 15 micrograms; chloramphenicol (CM), 30 micrograms; ampicillin (AM), 10 micrograms; trimethoprim-sulfamethoxazole (SXT) 1.25 micrograms/23.75 micrograms. Strains whose halos were of a smaller diameter than the intermediate value were considered resistant. It is important to maintain surveillance on antimicrobial susceptibility as epidemiological marker on geographical selected areas in order to detect changes of resistant patterns.

[Epidemiology and etiology of infectious diarrheas. The case of Mexico]. / Epidemiología y etiología de las diarreas infecciosas. El caso de México.

Gastrointestinal infections represent a health problem. It is estimated that 1647 million cases of diarrhea and 3.2 million deaths due to this cause occur among children less than five years of age per year. Those belonging to this age group have 15 times more risk of dying because of diarrhea. Cases of liquid acute diarrhea with blood represent 80% of cases, diarrhea with blood represent 10%. Most frequent causes of liquid diarrhea are enterotoxigenic Escherichia coli and rotaviruses and most frequent causes of bloody diarrhea are Shigella, E. coli (EHEC and EPEC). Campylobacter jejuni and Entamoeba histolytica. Annually 15,000 cases of typhoid fever are reported that continue being a public health problem. A negative correlation has been observed between the use of oral rehydration and infant mortality due to diarrhea. After prevention and control measures for cholera, a decrease in morbidity and mortality due to diarrhea has been observed. However, to reduce mortality due to this cause, it is necessary to treat the cases of acute dysentery and persistent diarrhea as well as to increase coverage of health care, to standardize the studies of etiology of diarrhea in Mexico, to establish surveillance centers for the study of diarrhea that give information on the distribution, frequency and trends of microbial agents and to achieve standardized microbiological and parasitological studies of etiology of diarrhea that support public health interventions as vaccination and selective administration of antibiotics.

[Perspectives on cholera vaccines]. / Perspectivas de las vacunas contra el cólera.

Perspectives of cholera vaccines with emphasis in their possible usage in Latin American countries are discussed. Microbiology, antigenicity, relevant aspects of traditional serology, protective immune responses and epidemiological data up to December, 1991, are presented. Indications of parenteral vaccines are discussed. Finally, perspectives of usage of cholera vaccines in Latin America are analyzed.

[Preparation and evaluation of coagglutination reagents for the identification of Vibrio cholerae 01. Its trial during a cholera outbreak in Minatitlán, Veracruz]. / Preparación y evaluación del reactivo de coaglutinación para la identificación de Vibrio cholerae 01. Su ensayo durante un brote de cólera en Minatitlán, Veracruz.

This study was realized in Minatitlán, Veracruz during a cholera outbreak. 169 rectal swabs were taken from hilles and their contacts. They were transfer alkaline peptone water for enrichment to V. cholerae and incubated for 8 hrs to 37 degrees C, 70 were positive for V. cholerae in both techniques. The coagglutination was done with a reagent prepared at the Instituto de Diagnóstico y Referencia Epidemiológicos of México and the culture were also performed in the same Institute. We obtained 100% of sensitivity and specificity of co-agglutination in relation with culture. This results gave the possibility to use this kind or reagents for a rapid presumptive diagnosis of cholera.

Frequency of identification of cytotoxigenic strains of Escherichia coli in cases of diarrhea from rural and urban communities.

It has been suggested that strains of Escherichia coli producing Vero-Toxin (VTEC) may cause diarrhea or hemorrhagic colitis; however, there are not enough studies to support this hypothesis. We studied the frequency of isolation of VTEC strains in patients with acute diarrhea from rural and urban communities. A total of 1430 strains were analyzed, 361 coming from 118 patients from the rural community (Cadereyta, Qro.) and 1069 from the urban district (D.F.); 95 of these patients were asymptomatic, 213 suffered from watery diarrhea and 43 had bloody diarrhea. For production of toxins, strains were grown in tryptic soy broth for 24h and the culture supernatant was inoculated on HeLa cells; strains were considered cytotoxic when they caused lysis in at least 50% of the cells. In the rural community, VTEC strains were isolated in 20% of the asymptomatics, in 45% of the watery diarrhea patients and in 76% of patients with bloody diarrhea. Frequency of isolation was significantly higher in patients with diarrhea than in asymptomatics (P less than 0.05). The relative risk to present watery diarrhea was 3 and to present bloody diarrhea was 12. In the urban district, VTEC strains were isolated in 13, 7.9 and 4.5% from asymptomatics, watery diarrhea and bloody diarrhea patients, respectively; the relative risk for diarrhea was 1. Colonization by VTEC strains is significantly higher in patients from the rural community and these infected patients have an important risk to develop diarrhea.