In vitro effect of amoxicillin and clarithromycin on the 3' region of cagA gene in Helicobacter pylori isolates

AIM:To evaluate the in vitro effect of amoxicillin and clarithromycin on the cag pathogenicity island(cag PAI).METHODS:One hundred and forty-nine clinical isolates of Helicobacter pylori(H.pylori)cultured from gastric biopsies from 206 Colombian patients with dyspeptic symptoms from a high-risk area for gastric cancer were included as study material.Antimicrobial susceptibility was determined by the agar dilution method.Resistant isolates at baseline and in amoxicillin and clarithromycin serial dilutions were subjected to genotyping(cagA,vacA alleles s and m),Glu-Pro-Ile-Tyr-Ala(EPIYA)polymerase chain reaction and random amplified polymorphic DNA(RAPD).Images of the RAPD amplicons were analyzed by Gel-Pro Analyzer 4.5program.Cluster analyses was done using SPSS 15.0statistical package,where each of the fingerprint bands were denoted as variables.Dendrograms were designed by following Ward’s clustering method and the estimation of distances between each pair of H.pylori isolates was calculated with the squared Euclidean distance.RESULTS:Resistance rates were 4%for amoxicillin and 2.7%for clarithromycin with 2%double resistances.Genotyping evidenced a high prevalence of the genotype cagA-positive/vacA s1m1.The 3’region of cagA gene was successfully amplified in 92.3%(12/13)of the baseline resistant isolates and in 60%(36/60)of the resistant isolates growing in antibiotic dilutions.Upon observing the distribution of the number of EPIYA repetitions in each dilution with respect to baseline isolates,it was found that in 61.5%(8/13)of the baseline isolates,a change in the number of EPIYA repetitions lowered antibiotic pressure.The gain and loss of EPIYA motifs resulted in a diversity of H.pylori subclones after bacterial adjustment to changing conditions product of antibiotic pressure.RAPD PCR evidenced the close clonal relationship between baseline isolates and isolates growing in antibiotic dilutions.CONCLUSION:Antibiotic pressure does not induce loss of the cag pathogenicity island,but it can leadin most cases-to genetic rearrangements within the3’region cagA of the founding bacteria that can affect the level of tyrosine phosphorylation impacting on its cellular effects and lead to divergence of cagA-positive subclones.

Genomic variability of Helicobacter pylori isolates of gastric regions from two Colombian populations

AIMTo compare the genomic variability and the multiple colonization of Helicobacter pylori (H. pylori) in patients with chronic gastritis from two Colombian populations with contrast in the risk of developing gastric cancer (GC): Túquerres-Nariño (High risk) and Tumaco-Nariño (Low risk).METHODSFour hundred and nine patients from both genders with dyspeptic symptoms were studied. Seventy-two patients were included in whom H. pylori was isolated from three anatomic regions of the gastric mucosa, (31/206) of the high risk population of GC (Túquerres) and (41/203) of the low risk population of GC (Tumaco). The isolates were genotyped by PCR-RAPD. Genetic diversity between the isolates was evaluated by conglomerates analysis and multiple correspondence analyses.RESULTSThe proportion of virulent genotypes of H. pylori was 99% in Túquerres and 94% in Tumaco. The coefficient of similarity of Nei-Li showed greater genetic diversity among isolates of Túquerres (0.13) than those of Tumaco (0.07). After adjusting by age, gender and type of gastritis, the multiple colonization was 1.7 times more frequent in Túquerres than in Tumaco (P = 0.05).CONCLUSIONIn Túquerres, high risk of GC there was a greater probability of multiple colonization by H. pylori. From the analysis of the results of the PCR-RAPD, it was found higher genetic variability in the isolates of H. pylori in the population of high risk for the development of GC.

Effect of treatment failure on the Cag A EPIYA motif in Helicobacter pylori strains from Colombian subjects

AIM To evaluate effect of treatment failure on cag A and vac A genotypes in Helicobacter pylori(H. pylori) isolates from Colombia.METHODS One hundred and seventy-six participants infected with H. pylori from Colombia were treated during 14 d with the triple-standard therapy. Six weeks later, eradication was evaluated by 13C-Urea breath test. Patients with treatment failure were subjected to endoscopy control; biopsies obtained were used for histopathology and culture. DNA from H. pylori isolates was amplified using primers specific for cag A and vac A genes. The phylogenetic relationships among isolates obtained before and after treatment were established by conglomerate analysis based on random amplified polymorphic DNA(RAPD) fingerprinting.RESULTS Treatment effectiveness was at 74.6%. Of the par-ticipants with treatment failure, 25 accepted subjected to a second endoscopy. Prevalence of posttreatment infection was 64%(16/25) and 40%(10/25) by histology and culture, respectively. Upon comparing the cag A and vac A genotypes found before and after therapy, multiple cag A genotypes(cag A-positive and cag A-negative) were found before treatment; in contrast, cag A-negative genotypes decreased after treatment. vac A s1m1 genotype was highly prevalent in patients before and after therapy. The 3'cag A region was successfully amplified in 95.5%(21/22) of the isolates obtained before and in 81.8%(18/22) of the isolates obtained after treatment. In the isolates obtained from patients with treatment failure, it was found that 72.7%(16/22) presented alterations in the number of EPIYA motifs, compared to isolates found before treatment.CONCLUSION Unsuccessful treatment limits colonization by lowvirulence strains resulting in partial and selective eradication in mixed infections, and acts on the cag A-positive strains inducing genetic rearrangements in cag A variable region that produces a loss or gain of EPIYA repetitions.