Microevolution And Correlation Analysis Of Helicobacter Pylori Within A Nephritis Patient

Helicobacter pylori (H. pylori), a common human pathogen, has been associated with a variety of gastroduodenal diseases. Some extragastric diseases, for example, idiopathic thrombocytopenic purpura and iron deficiency anemia, possibly linked to H. pylori infection, have also been largely investigated in recent years.We have recently demonstrated the glomerular deposition of H. pylori antigen in some glomerulonephritis patients. The correlation between H. pylori infection and the onset and/or progression of immune-mediated nephritis need further investigation. Evaluation of the mixed infection and identification of particular strain will be important in the studies that try to explain the relationship between potential H. pylori pathogenic factors and disease outcomes.Recently, the continuous decreasing in the eradication rate of H. pylori infection, especially in the therapy based on amoxicillin (AMO), could not be well explained by clarithromycin resistance or proton pump inhibitor selection. Although the AMO has been one of the principle antibiotics used in H. pylori therapy because of a lower resistance, there is still lack of study on the effect of AMO on the sensitivity of other antibiotics used in H. pylori treatment.Part 1:Microevolution and correlation analysis of H. pylori within a nephritis patientIn the present study,18 H. pylori isolates were obtained from a Chinese patient with chronic gastritis and nephritis, and their genetic features and evolutionary history were investigated in detail using MLST and whole genomic sequencing. Antibiotic susceptibility testing was performed to indicate their phenotypic differences.Results showed all isolates fall into two clonal lineages, deriving from two different parent H. pylori strains, and they have been co-infecting the host for at least 12 years. These lineages have accumulated diversity during an estimated 2.8 and 4.2 years of evolution. Recombination between the lineages was a continuous ongoing process and was detected on both clades but the effect of recombination in one clade was nearly an order of magnitude higher than in the other. The two clades had obvious differences in restriction-modification systems. Recombination tracts containing the interspersion of recipient sequences were observed in the clinical isolates for the first time. The complex evolutionary history of a phage-related protein provided evidence for frequent re-infection of both clades by a single phage lineage during the past four years, indicating that phage-mediated recombination may play a role in the evolution of H. pylori.Although the two clonal lineages shared complete Cag pathogenicity island and VacA, whole genomic comparison showed them 50 kb region containing a type 2 TnPZ differentiation, which will provide a clue in the deeper understanding of the role of type 2 TnPZ in the pathogensis of H. pylori, especially in the development of nephritis.Part 2:The expression and DNA sequence analysis of amoxicillin resistant-related genes of H. pyloriH. pylori isolates have two kinds of AMO resistant, transient and not stable phenotype tolerance and stable resistance, and the latter is rare in the clinical isolates. The study is focus on the mRNA expression and DNA sequence analysis of AMO resistant-related genes of H. pylori. AMO resistant isolates induced in our lab were obtained from AMO sensitive H. pylori 26695. The relative qRT-PCR and sequence analysis were used to study the expression of eight AMO resistant-related genes, including five pbps encoding PBP1-PBP5, hopB and hopC encoding two Hop porin protein and hefA which encodes one efflux pump protein.H. pylori under different selective pressure of AMO (0.5,1.0,2.0,4.0 and 8.0 mg/1) were obtained. The relative qRT-PCR results showed that the expression patterns of these eight genes are different among AMO resistant and susceptible H. pylori 26695. The relative mRNA expression of genes we detected is higher in H. pylori respond to AMO below 4.0 mg/1, the mRNA expression of PBP1 and PBP2 is 2 fold of that of H, pylori 26695, and that of HefA under AMO 0.5 mg/1 and AMO 1.0 mg/1 is also 2 fold increased. Lower level mRNA expression of HopB and HopC, and higher expression of PBP1-PBP3 were observed in the H. pylori under the pressure of AMO 4.0 mg/1. The expression of hopC was also decreased under AMO 8.0 mg/1. These results show that these genes play different roles under different AMO selective pressure. In this study we also obtained four stable AMO resistant isolates and found T593K and 594G+two new mutations in PBP1. Besides, the higher MICs of levofloxacin, moxifloxacin and tetracycline were observed among all these stable AMO resistant isolates, which indicates that these isolates have already become multi-drug resistant. Our results shows the expression of all the genes we detected may play different roles in the unstable AMO resistant which often observed in clinical isolates. Both AMO tolerance and resistance are important factors affecting the efficacy of the current H. pylori eradicating therapies.In this research we studied on the population structure and microevolution of H. pylori within a single patient, as well as mechanism of both unstable and stable AMO resistance, which make better understanding the evolution and mechanism of genetic diversity of H. pylori and mechanism of AMO resistance. The present study will be helpful for knowing H. pylori deeply, and will be meaningful on guiding the following research on H. pylori pathogenesis and treatment.