The Prevalence And Role Of RdxA And FrxA Genes In Resistance Of Metronidazole In Helicobacter Pylori From Children

As one of the most common infectious agents, Helicobacter pylori (H.pylori) hasbeen established as a major cause of chronic gastritis and peptic ulcer diseases, andconsidered closely associated with some gastric carcinoma and Mucosa-AssociatedLymphoid Tissue (MALT) lymphoma while the eradication of H.pylori could not onlyefficaciously keep peptic ulcer diseases from initial invasion and recidivation, but alsoachieve complete regression of partial MALT lymphoma. Once comes the infection inchildren, the susceptible population for H.pylori, would probably cause not onlychronic gastritis and peptic ulcer diseases but also iron deficiency even anemia andgrowth retardation because of scarce spontaneous cure. As some findings havesuggested, the eradication of H.pylori in childhood was helpful to blockade or delaythe development of atrophy and intestinal metaplasia in gastric mucosa, and further todecrease incidence on gastric adenocarcinoma and MALT lymphoma. With stablehigh and nearly bacteriostatic-bactericidal concentration against gastric acid,metronidazole (MTZ) plays a important role in currently recommended therapy foreradication of H.pylori, which combines a proton pump inhibitor and two antibiotics.However, there are growing concerns about increasing MTZ resistance of H.pyloriand its impact on the efficacy of eradication therapy due to primary and secondaryresistant isolates (MTZ~R) since its widespread acceptance in kinds of therapy (evenin oral and gynecological diseases). While urgent and important, no documented datawas available concerning associated with either the monitoring prevalence or themechanism of MTZ resistance in H.pylori from Chinese children;What's more, therelie disputes about determination of minimal inhibitory concentration (MIC) for MTZbetween commonly used methods such as Epsilometer-test (E-test) and agar dilution(recommended by American National Committee for Clinical Laboratory Standards).Therefore, the purposes of our study are to investigate that disputes, and analyze theprevalence and mechanism of MTZ resistance in H.pylori from children.During the periods from 2002 Oct to 2004 Jan and 2004 Dec to 2005 Jul, one total 44 and another 83 H.pylori isolates were separated from gastric antrum biopsy of local child patients, who were submitted to gastroscopy because of symptoms in upper gastrointestinal tract and confirmed H.pylori -associated gastritis or peptic ulcer diseases in the Children's Hospital of Zhejiang University Medical School. All the biopsies were ground with a tissue homogenizer, and incubated on selective medium plates enriched 5% fresh sheep blood at 37 °C for 3 days under a microaerophilic (5%O2, 15%CC>2 and 80%N2) atmosphere. Colonies with simple needlepoint-like morphology were selected for Gram staining and biochemical activity tests including urease, oxidase and catalase, and then H.pylori were identified when all the biochemical activities were positive with Gram negative "S"-shape bacilli under light microscope. All the susceptibility of H.pylori for MTZ was determined by two-fold agar dilution method, simultaneously by E-test method for 55 H.pylori isolates. A strain was considered resistant to MTZ when the MIC>8mg/L by agar dilution method. Statistical analysis: the prevalence rates (%) of MTZ resistance were calculated respectively for the 44 and 83 isolates, and the MIC distributions were evaluated at MIC50 (the MIC point 50% isolates included) and MIC90 (the MIC point 90% isolates included). Analysis was performed by using STATA 8.0 program, and the difference between the prevalence rates was statistically significant when;?<0.05.Then 11 random-selected wild MTZ-sensitive H.pylori isolates (MTZS) and the corresponding acquired MTZR isolates were enrolled in genetic fingerprinting by randomly amplified polymorphic DNA based on PCR (RAPD-PCR) to detect general mutations in the genomes. 9 pairs of wild MTZS and acquired MTZR H.pylori isolates, together with 5 random-selected wild MTZR isolates (MIC^256mg/L), were enrolled in PCR to detect general mutations in rdxA and frxA genes. Sequencing analysis was performed by using program DNAMAN 5.2.2 according to the reported sequence of H:pylori 26695 in National Center of Biotechnology Information (NCBI), where rdxA gene was named HP0954 and frxA gene HP0642.The results showed these: (1) Between the 44-isolate group from 2002 Oct to 2004 Jan and the 83-isolate group from 2004 Dec to 2005 Jul, the MTZ prevalence rates were 31.82% (14/44) vs 51.81% (43/83) (P=0.031), the ranges of MICs from <0.125 to 128mg/L vs from 0.25 to >256mg/L, the MIC50 points at 0.5mg/L vs 16mg/L and the MIC90 points stable at 128mg/L, respectively. Obviously the prevalence rates of were growing while the MIC50 point increasing at 25 degrees. (2)According to two-fold agar dilution method, the sensitivity of E-test method was 73.08%, specificity 100%, agreement rate 87.27%, the perfect associativity (x2=32.38, P<0.001), while there exists a difference ranging from 22 to 26 degrees between 28 of 55 corresponding MICs simultaneously determined by E-test and agar dilution method. And those meant that agar dilution method should be simultaneously enrolled in to avoid missed diagnosis of MTZR isolate when H.pylori isolate was susceptible to MTZ by E-test method. (3) And overall the profile partners of genetic fingerprinting by RAPD-PCR as quality control, 8 of 11 seemed identical to each other while 3 of 11 distinct;whether identical profile partners or not, corresponding sized rdxA and frxA DNA fragments could always be amplified from the pairs of wild MTZS isolate and acquired MTZR isolate, with high identity (99.45% ± 0.30% for rdxAs and 99.24% ± 0.33% forfrxAs, respectively). Those probably suggested it's some original mutations in genomes of wild MTZS isolates due to in vitro induction with MTZ that made them acquired MTZR isolates, but the distinction of profile partners could not make for identification of resistance or not. Therefore it's necessary for further sequencing analysis of rdxA and frxA genes.Based on all the sequencing analysis of rdxA and frxA genes, we found these: According to H.pylori 26695 in NCBI, the identities for the rdxA andfrxA genes from wild MTZS isolates were 94.47%'95.89% (95.00% ± 0.16%) and 94.04%'95.54% (94.71% ± 0.16%) , gap (due to base insertion or deletion) 0 and 0" 7 (1.44 ± 0.78) locus, respectively. For the rdxA and frxA genes from acquired MTZR isolates according to wild MTZS isolates, the identities were 97.63%" 100% (99.45% ± 0.30%) and 97.14%' 100% (99.24% ± 0.33%), gap 0" 1 (0.33 ± 0.17) and 0~6 (1.78 ± 0.66) locus, respectively. Identical sequences were found 1 for rdxA gene and 2 for frxA genes, in detail. Among 34 locus of new point mutation in rdxA genes of acquired MTZR isolates, there was 1 base insertion, 2 base deletion, 31 base substitution while base transition (purine to purine or pyrimidine to pyrimidine) were prevalent in those base transition (15/31 for Y type and 11/31 for R type, in detail);no new multiple mutation was found. Among 52 locus of new point mutation in frxA genes of acquired MTZR isolates, there were 15 base insertion, 3 base deletion, 34 base transition while base transition were prevalent in those base transition (17/34 for Y type and 11/34 for R type, in detail);2 new multiple mutation were found. According to H.pylori 26695, the identities for rdxA and frxA genes from 5 random-selected wild MTZR isolates were 94.44%~ 95.42% (95.06% ± 0.20%) and 93.88%" 98.77% (95.29% ± 0.89%), gap 0" 3 (1.6 ± 0.6)and 0" 1 (0.40 ± 0.24) locus, respectively;among the 155 locus ofprimary point mutation, there were 2 base deletion, 153 base substitution while base transition were prevalent in those base transition (65/153 for R type and 58/153 for Y type, in detail);no primary multiple mutation was found. Among the 31 locus of new point mutation and base substitution in rdxA genes of acquired MTZR isolates, there were 12 substitutions identical to primary point mutation in wild MTZR isolates at locus and type, 1 substitution identical at locus but distinct at type, 1 base insertion corresponding to the locus of primary base substitution in rdxA gene of wild MTZR isolate, while all the rest base substitution, insertion or deletion were distinct at locus or type. Among the 34 locus of new point mutation and base substitution \nfrxA genes of acquired MTZR isolates, there were 16 base substitutions and 1 base deletion identical to primary point mutation in wild MTZR isolates at locus and type, 1 substitution identical at locus but distinct at type, 6 base insertion or deletion corresponding to the locus of primary base substitution in frxA genes of wild MTZR isolates, while all the rest base substitution, insertion or deletion were distinct at locus or type;what's more, 1 multiple mutation infrxA gene of acquired MTZR isolate was corresponding to the locus of primary base substitution in frxA genes of wild MTZR isolates. Above all the results suggested us these: The mutations were arbitrary at locus and types in rdxA and frxA genes of H.pylori isolates, but the dependability between the mutations and level of MTZ resistance needs more proofs by plenty of further researches. Something interesting, the finding of identical DNA sequences of rdxA and frxA genes from wild MTZS isolates and corresponding acquired MTZR isolates, suggested us the presence of additional MTZ resistance mechanisms other than mutations of the rdxA and/or frxA genes.In summary, the conclusions of our study are these: (1) The prevalence of MTZ resistance seems growing in H.pylori from local children while the MIC50 increasing at 25 degrees. (2) To avoid missed diagnosis of MTZR isolates, agar dilution method is in need when E-test method is used in microbial sensitivity test of MTZ for clinical H.pylori isolates. (3) The mutations were arbitrary at locus and types in rdxA and frxA genes of H.pylori isolates. (4) There might be additional MTZ resistance mechanisms other than mutations of the rdxA and/or frxA genes.