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

摘要

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.