Analysis Of Gastric Non-Helicobacter Pylori Helicobacter (NHPH) Species In Patients With Gastric Diseases

Helicobacter pylori (H. pylori) is not the only bacterium which can colonizes the stomach of human. Gastric non-Helicobacter pylori Helicobacters (NHPH) is a group of long spiral-shaped bacteria which naturally colonizes the stomach of animals. And the infection of NHPH has been associated with the development of gastritis, gastric ulcerationand gastric MALT lymphoma in human. As previous studies described, these bacteria have been detected in 0.2-6% of human patients. But there is no research data of NHPH infection rate in patients in China.Therefore, Helicobacter species specific PCR were performed to assess the prevalence of NHPH infection in the patients suffering from gastric diseases in this study. And the molecular typing of H. suis in human and pigs was performed by using multilocus sequence typing (MLST).Part One:Prevalence of gastric non-Helicobacter pylori Helicobacter (NHPH) species in patients with gastric diseasesTo gain insight into the prevalence of NHPH infections in patients suffering from gastric diseases in China, urease gene based specific PCR of five NHPH species (H. felis, H. bizzozeronii, H. heilmannii sensu stricto (s.s.) and H. salomonis)were performed on DNA extracts from rapid urease test (RUT) positive gastric biopsies of 1517 patients followed by nucleotide sequencing. H. pylori cultivation and specific PCR were performed to assess H. pylori coinfection in the patients. Besides, H. suis specific PCR were performed on 202 H. pylori-negative DNA extracts of gastric biopsies which were confirmed by real-time PCR and specific PCR.The results revealed that NHPH infection was detected in 11.87%(178/1499) of H. pylori positive patients. And the number is significantly higher than what has been described previously in other countries. H. suis (6.94%) was the most prevalent NHPH species. The prevalence of H. felis, H. bizzozeronii, H. heilmannii s.s. and H. salomonis in the patients was 2.20%,0.13%,0.07% and 2.54% respectively. Results revealed some patients were co-infected with three different Helicobacter species. Besides, eight (3.96%) H.suis-positive DNA extracts were detected in the 202 H. pylori-negative DNA extracts. And the result revealed that there was a high rate of H. suis infection in the H. pylori-negative patients.This study is the first to investigate the prevalence of NHPH infections of large samples of patients with gastric diseases in China. NHPH infections are fairly common in Chinese patients. H. suis was shown to be by far the most prevalent NHPH species. This should be kept in mind when diagnosing the cause of gastric pathologies in patients.Part Two:Multilocus sequence typing of the porcine and human gastric pathogen H. suisH. suis is a gram-negative bacterium which naturally colonizes the stomach of pigs and non-human primates. The infection of H. suis is considered to be associated with gastric diseases in human. Direct contact with pigs and consumption of contaminated pork may constitute the route of transmission for H. suis infections in humans. In China, H. suis infection, which is associated with pig breeding industry and the dietary habit (eating pork tripes), is concerned, but without related research. In this study, H. suis specific PCR was performed on 60 fresh stomach samples of pigs to assess the infection, and the genetic polymorphism and evolutionary relationships of H. suis in Chinese patients and pigs, as well as the foreign H. suis strains, were performed by using MLST.Results revealed that 20 (33.33%) of the 60 stomach samples were H. suis positive.14 new ST types of H, suis were assigned by MLST. The 22 H. suis strains were distributed into three groups.Two human H. suis strains showed a very close relationship to the 20 porcine strains. MLST revealed Chinese and foreign H. suis strains were distributed into three groups, and the evolutionary relationship was very close. Most of Chinese H. suis strains in groups could tend to a cluster.Part Three:Detection of 23S rRNA gene mutations associated with clarithromycin resistance in H. pyloriCurrently, eradication failure of therapy regimens with CLA has been increasing because of the increasing of CLA resistant H. pylori. CLA resistance of H. pylori is mainly caused by point mutations of the genomic 23 S rRNA, mostly at position 2142/43 (A2142 to G/C; A2143 to G). However, there is no sufficient data to evaluate the fraction of coverage of these three point mutations in CLA resistant strains in China. In this study, PCR followed by sequencing was used to detect 23 S rRNA gene mutations in DNA extracts of 1086 gastric biopsies.Results revealed that the mainly point mutation of 23S rRNA was A2143G (66.12%), followed by A2142G (1.48%) and A2142C (0.16%). About 67.76%of CLA-resistant strains have the three point mutations, but 32.24%of CLA-resistant strains without them. These results revealed that the fraction of coverage of the three point mutations in strains would not reflect the real situation of CLA resistance in H. pylori. Besides, two point mutations (C1951T and G1933A), which were only found in some CLA-resistant strains, may involved in CLA resistance.Part Four:Isolation of H. pylori strains and analysis of CYP2C19 genetic polymorphisms with the same gastric biopsy samplesIf it is available to perform H. pylori cultivation and analysis CYP2C19 genetic polymorphisms by using the same one gastric biopsy, the individualized treatment of H. pylori would be more consummate and used extensively. In this study, PCR followed by sequencing was used to analyze the CYP2C19 genetic polymorphisms in patients. The possibility of using the same gastric biopsy sample for the isolation of H. pylori and the detection of CYP2C19 genetic polymorphisms was evaluated.Results revealed that the CYP2C19 specific PCR of all DNA extracts was successful and there were no statistical differences with the rate of HP cultivation by using the half and the whole gastric biopsy. Results reveal that it is available for using one gastric biopsy to perform H. pylori cultivation and the detection of CYP2C19 genetic polymorphisms.