Frequency of vacA, cagA and babA2 virulence markers in Helicobacter pylori strains isolated from Mexican patients with chronic gastritis

Annals of Clinical Microbiology and Antimicrobials Frequency of vacA, cagA and babA2 virulence markers in Helicobacter pylori strains isolated from Mexican patients with chronic gastritis Gloria Luz Paniagua 1 Eric Monroy 1 Raymundo Rodrguez 0 Salvador Arroniz 1 Cristina Rodrguez 1 Jos Luis Corts 0 Ausencio Camacho 0 Erasmo Negrete 1 Sergio Vaca 1 0 Hospital General Regional 72 del Instituto Mexicano del Seguro Social , Av. G. Baz s/n, Tlanepantla, 54000, Estado de Mexico , Mexico 1 Facultad de Estudios Superiores Iztacala, Universidad Nacional Autonoma de Mexico, Avenida de los Barrios 1, Los Reyes Iztacala , Tlalnepantla, 54090, Estado de Mexico , Mexico Background: Helicobacter pylori has been strongly associated with chronic gastritis, peptic and duodenal ulcers, and it is a risk factor for gastric cancer. Three major virulence factors of H. pylori have been described: the vacuolating toxin (VacA), the cytotoxin-associated gene product (CagA) and the adhesion protein BabA2. Since considerable geographic diversity in the prevalence of H. pylori virulence factors has been reported, the aim of this work was to establish the H. pylori and vacA, cagA and babA2 gene status in 238 adult patients, from a marginal urban area of Mexico, with chronic gastritis. Methods: H. pylori was identified in cultures of gastric biopsies by nested PCR. vacA and cagA genes were detected by multiplex PCR, whereas babA2 gene was identified by conventional PCR. Results: H. pylori-positive biopsies were 143 (60.1%). All H. pylori strains were vacA+; 39.2% were cagA+; 13.3% were cagA+ babA2+ and 8.4% were babA2+. Mexican strains examined possessed the vacA s1, m1 (43.4%), s1, m2 (24.5%), s2, m1 (20.3%) and s2, m2 (11.9%) genotypes. Conclusion: These results show that the Mexican patients suffering chronic gastritis we have studied had a high incidence of infection by H. pylori. Forty four percent (63/143) of the H. pylori strains analyzed in this work may be considered as highly virulent since they possessed two or three of the virulence markers analyzed: vacA s1 cagA babA2 (9.8%, 14/143), vacA s1 babA2 (4.9%, 7/143), and vacA s1 cagA (29.4%, 42/143). However, a statistically significant correlation was not observed between vacAs1, cagA and babA2 virulence markers (2 test; P > 0.05). - Background Helicobacter pylori is a spiral-shaped Gram-negative bacterium that has been strongly associated with chronic gastritis and peptic ulcer disease [1,2], and it is a risk factor for gastric cancer [3-5] Three major virulence factors of H. pylori have been described: the cytotoxin-associated gene product (CagA), the vacuolating toxin (VacA) and the adhesion protein BabA2. The cytotoxin-associated gene A (CagA) is a protein with a molecular mass of approximately 125140 kDa, encoded by the cagA gene, [6,7], that is translocated into gastric epithelial cells by a type IV secretion system, encoded by the cag pathogenicity island (cag PAI) [8]. Inside epithelial cells CagA is phosphorylated on tyrosine residues by host cell Src kinases and stimulates cell-signaling pathways [9], which in turn causes elongation of the cell [10] and activation of proto-oncogenes [11]. The vacuolating cytotoxin gene vacA is polymorphic, varying in the signal and middle regions. The main signal region alleles are s1 and s2, whereas the middle region alleles are m1 and m2 [12,13]. VacA is a toxin that binds to several epithelial receptors [14-16] and forms hexameric pores [17], which later are endocytosed and converted in large vacuoles [18]. The BabA adhesin of H. pylori is an outer membrane protein that binds to the fucosylated histo-blood group antigens on the surface of gastric epithelial cells [19,20]. It has been reported that H. pylori strains possessing babA2 gene, which encodes active BabA adhesin, are associated with IFdiegnutrifeica1tion of H. pylori isolated from gastric biopsy samples and genotyping of its main virulence genes by PCR Identification of H. pylori isolated from gastric biopsy samples and genotyping of its main virulence genes by PCR. Images shown are from representative gel electrophoresis. A: Lane 1, MWM 50 bp-ladder; Lanes 2 and 9 negative control without DNA; Lanes 38, 10 and 11, H. pylori isolated from gastric biopsy samples (417-bp amplicon); Lane 12, reference strain H. pylori ATCC 43629. B: Lane 1, negative control without DNA, Lane 2, MWM 50 bp-ladder; Lanes 36, H. pylori isolated from gastric biopsy samples (230-bp amplicon); Lane 7, H. pylori ATCC 43629. C: Lane 1, MWM 50-bp-ladder; Lane 2, H. pylori isolated from gastric biopsy sample (vacA s1, m1 cagA); Lane 3, negative control without DNA; Lanes 45, H. pylori isolated from gastric biopsy samples (vacA s1, m1); Lane 6, ATCC 43629. D: Lane 1, MWM 200 bp-ladder; Lane 2, ATCC 43629; Lanes 3,4,6 and 8, babA2-positive H. pylori isolated from gastric biopsy samples; Lane 5, control without DNA; Lane 7, babA2negative H. pylory isolated from gastric biopsy sample. increased gastric inflammation [21] and increased risk for duodenal ulcer and adenocarcinoma [22]. H. pylori virulence factors frequency varies widely. For instance, vacAs1 prevalence fluctuates from 48% [23] to 100% [24] whereas cagA prevalence fluctuates from 66.9% [23] to 83.6% [25] and 100% [26]. babA2 reported frequencies vary from 46% [27] to 82.3% [28] in SouthAmerican countries. Since considerable geographic diversity in the prevalence of H. pylori virulence factors has been reported, the aim of this work was to establish the H. pylori and vacA, cagA and babA2 gene status in 238 adult patients, from a marginal urban area of Mexico, with chronic gastritis. Materials and methods Subjects and clinical samples Two hundred and thirty eight patients, endoscopic diagnosed with chronic gastritis (154 women and 84 men) with an average age of 52.24 years (range 16 to 83), who had undergone endoscopy in Hospital General Regional 72 of the Instituto Mexicano del Seguro Social at Tlalnepantla, Estado de Mexico, Mexico, were included in this study. Written informed consent for participation was obtained from every patient before the study. The ethics committee at Hospital General Regional 72 approved the study protocol in advance. Antral biopsy specimens were evaluated for the presence of H. pylori by culture. The genotype profiles of H. pylori isolates were determined by PCR. H. pylori culture For bacterial culture, biopsy specimens were macerated and homogenized in Brucella Broth and a 100 L aliquot was inoculated on Casman Agar (Difco) containing 5% horse blood and H. pylori selective supplement (Oxoid-SR 147E). Agar plates were incubated in 6% CO2, for up to four days. Colonies were identified as H. pylori according to standard criteria including negative Gram staining, typical cell morphology, and positive reactions to catalase, oxidase, and urease. Identification of H. pylori by nested PCR H. pylori DNA was extracted from colonies collected in microcentrifuge tubes containing (...truncated)