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Molecular Basis of Pathogenicity in Helicobacter pylori

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Molecular Basis of Pathogenicity in Helicobacter pylori

     JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 2010, p. 37763778 Vol. 48, No. 10 0095-1137/10/$12.00 doi:10.1128/JCM.00472-10 Copyright ? 2010, American Society for Microbiology. All Rights Reserved.

    Molecular Basis of Pathogenicity in Helicobacter pylori

    Clinical Isolates

    Ivy Bastos Ramis, Tesieˆ Leopoldo Fonseca, Ernani Pinho de Moraes, Ma?rcia Silveira Fernandes,

    Raul Mendoza-Sassi, Obirajara Rodrigues, Carlos Renan Varela Juliano,

    Carlos James Scaini, and Pedro Eduardo Almeida da Silva* Laborato?rio de Biologia Molecular, Universidade Federal do Rio Grande, Rua General Oso?rio, S/N, Rio Grande, RS, Brazil

    Received 5 March 2010/Returned for modication 17 June 2010/Accepted 27 July 2010

    This study identi;ed pathogenicity genes in 40 Helicobacter pylori clinical isolates. The cagA, vacA, and iceA

    genes were detected in 65%, 97.5%, and 97.5% of the isolates, respectively. The cagA, iceA1, and vacAs1a/m1

    genes were related to erosive gastritis, whereas the vacAs2/m2 and iceA2 genes were associated with enanthe- matous gastritis.

     Helicobacter pylori is considered the major etiologic agent of dium Columbia agar (Oxoid, United Kingdom), supplemented chronic active gastritis, an essential catalyst in the emergence with 7% sheep blood and with a selective mixture for Helico- of peptic ulcer, and a risk factor for the development of gastric bacter species isolation (Cefar, Brazil). The agar plates were cancer (17). Studies indicate that the evolution of the infection incubated under microaerophilic conditions (5 to 15% Oand 2 depends in part on the expression of specic bacterial patho- 10% CO) at 37?C for 4 to 10 days (14). The identication of 2 genicity genes, such as cagA (cytotoxin-associated gene A), H. pylori was performed using catalase, oxidase, and urease vacA (vacuolating cytotoxin), and iceA (induced by contact tests, microscopy, and ureA gene detection (12, 19). with epithelium) (2). The DNA extraction was performed after 48 h of bacterial The cagA gene is considered to be a marker for the presence growth. Colonies were collected and resuspended in 500 l of of a cagA pathogenicity island (8). The cagA-positive H. pylori 1 TE buffer. The suspension was centrifuged at 10,000 g strains increase interleukin-8 production and gastric inamma- for 5 min, and the supernatant was thereafter discarded. The tion (5). The vacA gene encodes a vacuolating cytotoxin able to DNA from the clinical isolates was then extracted with DNAzol induce the formation of cytoplasmic vacuoles in epithelial cells reagent (Invitrogen, United States) by the method of the (11). This gene comprises two variable regions: the signal re- manufacturer. gion, with two alleles, s1 (subtypes s1a, s1b, and s1c) and s2, The presence of the ureA, cagA, vacA, and iceA genes in the and the middle region, with the alleles m1 and m2 (3, 28). In isolates was investigated by PCR using the primers described general, the s1/m1 strains produce large amounts of vacuo- previously (6, 10, 21, 31). The PCR was performed as described lating cytotoxin, the s1/m2 strains produce moderate by Rota et al. (for the ureA and cagA genes) and by Benenson amounts, and the s2/m2 strains produce little or none (3). et al. (for the alleles of the vacA and iceA genes) (4, 27). The iceA gene has two alleles: iceA1 and iceA2. The iceA1 The statistical analysis was performed by using Fishers exact allele is associated with peptic ulcer, and iceA2 is related to test, a chi-squared test, and a chi-squared test for linear trend. asymptomatic gastritis (24, 29). P values of less than 0.05 were considered statistically signi- This study analyzed the presence of cagA, vacA, and iceA cant. genes in clinical isolates and correlated these ndings with the The presence of the pathogenicity genes was studied in 40 endoscopic diagnosis. Forty isolates of H. pylori were obtained clinical isolates of H. pylori. From those, 50% (20 of 40) were from biopsy specimens of the gastric antrum collected from obtained from patients with endoscopic diagnosis of enan- dyspeptic patients admitted to the upper gastrointestinal en- thematous gastritis and 50% (20 of 40) were obtained from doscopic ward in the Hospital of the Federal University of Rio patients with erosive gastritis.

    Grande, Rio Grande do Sul, Brazil. This study was approved The cagA gene was identied in 65% (26 of 40) of the by the ethics committee of our university. Informed consent isolates. This frequency is similar to that found in previous was obtained from all patients. studies of cagA in Brazil (14, 16, 18). The vacA and iceA genes After collection, the biopsy specimens were kept in brain were detected in 97.5% (39 of 40) of the samples. The vacAs1b heart infusion broth (Acumedia, United States) with 20% glyc- (43.6%) and vacAm2 (53.9%) alleles were the most frequently erol and refrigerated (4 to 8?C) for a maximum of 4 h (22). This detected in the 39 isolates, as well as the iceA2 allele (71.8%). broth was thereafter vortexed, and 200 l was added to me- This is an expected result, because these alleles have been reported in other studies (7, 18, 26). Moreover, 12.8% of the isolates veried the presence of the m1 and m2 alleles of the * Corresponding author. Mailing address: Laborato?rio de Microbio- vacA gene, and 5.1% of the isolates had both iceA alleles. logia Molecular, Universidade Federal do Rio Grande, Rua General The detection of more than one allele of the middle region of Oso?rio, S/N, Rio Grande, RS, Brazil. Phone: 55 53 32338895. Fax: 55 vacA, as well as the identication of both iceA alleles in the 53 32338863. E-mail: pedre@furg.com.br. same isolate, suggests coinfection of two different strains of H. Published ahead of print on 4 August 2010.

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    TABLE 1. Association between the cagA gene and the allelic tous gastritis, a nding that suggests that such alleles are re- combinations of the vacA gene in isolates of H. pylori lated to minor damage in gastric mucosa (1). However, a sta-

    tistically signicant difference was not found in the association % with or without cagA gene (no. with gene astatus/total no. of samples) between either cagA or vacA and the clinical manifestations. Genotype The iceA1 allele was detected in 66.7% of isolates from pa- cagA positive cagA negative tients with erosive gastritis, while iceA2 was identied in 57.1% vacAs1a/m1 100.0 (5/5) of isolates from patients with enanthematous gastritis. The vacAs1b/m1 87.5 (7/8) 12.5 (1/8) iceA1 allele may be associated with a more severe form of vacAs1a/m2 100.0 (5/5) gastritis because iceA1-positive strains produce more inam- vacAs1b/m2 66.7 (4/6) 33.3 (2/6) vacAs2/m2 10.0 (1/10) 90.0 (9/10) mation-inducing cytokines, such as interleukin-8, which are vacAs1b/m1m2 100.0 (3/3) potent chemotactic factors that activate polymorphonuclear 50.0 (1/2) vacAs2/m1m2 50.0 (1/2) leukocytes that contribute to enhanced inammatory re- vacA negative 100.0 (1/1) sponses (13, 30). This nding agrees with those of previous a0.001. Pstudies (24, 25). In this work, a statistically signicant associa-

    tion was observed between iceA and the endoscopic diagnosis

    (P 0.047). pylori. Cases of patients being infected with multiple strains of Based on the data presented above, we conclude that the H. pylori are not uncommon, being more frequent in areas of detection of cagA, vacA, and iceA genes allows an improved high H. pylori prevalence (9, 15, 23). evaluation of the pathogenic potential from clinical isolates. In The association between the cagA and vacA genes is de- this study, the cagA gene, the combination vacAs1a/m1, and scribed in Table 1. All cagA-positive isolates conrmed the the iceA1 allele were related to erosive gastritis; similarly, the presence of vacA. The combinations vacAs1a/m1, vacAs1b/m1, combination vacAs2/m2 and the iceA2 allele were related to an vacAs1a/m2, vacAs1b/m2, and vacAs1b/m1m2 were present attenuated form of gastritis. Therefore, the genotyping of the mainly in cagA-positive samples. A statistically signicant as- microorganism appears to be a clinically relevant procedure sociation was observed between cagA and vacA (P 0.001). and can contribute to the prognosis of H. pylori infection. The relationship of pathogenicity genes with gastric disor- ders is described in Table 2. The cagA gene and the combina- REFERENCES tion vacAs1a/m1 were frequently detected in isolates from pa- 1. Araya, J. C., L. Anabalo?n, I. Roa, M. Bravo, M. A. Villaseca, P. Guzma?n, and tients with erosive gastritis. Similar ndings were reported by J. C. Roa. 2004. Relacio?n de la genotipicacio?n de Helicobacter pylori con la other authors (14, 20). These genes are directly related to the forma e intensidad de la gastritis en poblacio?n adulta portadora de patología ga?strica benigna. Rev. Med. Chil. 132:13451354. inltration of polymorphonuclear cells, which causes severe 2. Arents, N. L. A., A. A. Van Zwet, J. C. Thijs, A. M. D. Kooistra-Smid, K. R. epithelial damage. 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