The distribution of jhp0940, jhp0945, jhp0947, jhp0949 and jhp0951 genes of Helicobacter pylori in China

Background The plasticity region of Helicobacter pylori (H. pylori) is a large chromosomal segment containing strain-specific genes. The prevalence of the plasticity region genes of the H. pylori strains in China remains unknown. The aim of this study was to examine the status of these genes and to assess the relationship between the genes and the diseases caused by H. pylori infection. Methods A total of 141 strains were isolated from patients with chronic active gastritis (CAG), peptic ulcer disease (PUD) and gastric carcinoma (GC). The prevalence of jhp0940, jhp0945, jhp0947, jhp0949 and jhp0951 was determined using PCR, and the results were analyzed using the chi-squared test. Results The prevalence rates of jhp0940, jhp0945, jhp0947, jhp0949 and jhp0951 in the H. pylori strains were 42.55, 51.06, 20.57, 56.03 and 63.12 %, respectively. The prevalence rates of jhp0940 were similar in the isolates from the CAG, PUD and GC patients, and there was no association between the jhp0940 status and any of the diseases. In contrast, the prevalence rates of jhp0945, jhp0947, jhp0949 and jhp0951 were significantly higher in the PUD and GC isolates than in the CAG isolates (p < 0.01). A univariate analysis showed that jhp0945, jhp0947, jhp0949 and jhp0951 increased the risk of PUD, while only jhp0951 was significantly associated with PUD in the multivariate analysis (p = 0.0149). The jhp0945-positive isolates were significantly associated with an increased risk for GC (p = 0.0097). Conclusion The plasticity region genes are widely distributed in Chinese patients, and a high prevalence of these genes occurs in more serious diseases. Therefore, jhp0951 status is an independent factor associated with the development of PUD, and jhp0945 may predict the future development of GC in patients with CAG and is considered to be the best candidate disease marker for H. pylori-related diseases.


Conclusion:
The plasticity region genes are widely distributed in Chinese patients, and a high prevalence of these genes occurs in more serious diseases. Therefore, jhp0951 status is an independent factor associated with the development of PUD, and jhp0945 may predict the future development of GC in patients with CAG and is considered to be the best candidate disease marker for H. pylori-related diseases.
Background H. pylori is the major cause of chronic gastritis, peptic ulcer disease, gastric carcinoma and mucosa-associated lymphoid tissue lymphoma [1,2]. The majority of infected individuals remain asymptomatic throughout their lifetime, and only approximately 15 % develop gastroduodenal diseases. Variations in the clinical outcomes of these diseases have been attributed to differences in environmental factors, bacterial strains and host genetics [3,4]. A number of bacterial virulence factors that are associated with these diseases have been described for H. pylori, such as the cag pathogenicity island (cag PAI) [5,6].
A comparison of the genomes of two H. pylori strains revealed that in addition to the cag PAI, a large region of approximately 45 kb in strain J99 and 68 kb in strain 26695 is present in both strains and has been termed the "plasticity region" [7,8] . Up to 50 % of the strainspecific genes transferred from other species are located in the plasticity region [9]. Whether these strain-specific genes influence the severity of different diseases or the biological functions of the ORFs in the plasticity region still remains unknown. Recent studies have revealed that the jhp0940, jhp0945, jhp0947, jhp0949 and jhp0951 genes from H. pylori are associated with an increased risk for gastroduodenal diseases [3,[9][10][11]. In a Brazilian study, the jhp0947 gene was found to be involved in the development of duodenal ulcers (DUs) and GC [11]. In addition, Romo-González et al. found that jhp0951 is also associated with DUs [9]. In China, the number of H. pylori infections has exceeded 60 %, and the high-risk incidence of GC poses a serious health and economic burden. Moreover, there are no reports discussing the prevalence of these genes or the relationship between the genes and the severity of the clinical outcomes of the abovementioned diseases in China. The aim of this study was to assess the prevalence of jhp0940, jhp0945, jhp0947, jhp0949 and jhp0951 and to determine their association with H. pylori-related diseases.

Strains
A total of 141 H. pylori strains were selected from the H. pylori strain bank of China at the National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention. A total of 40 strains were isolated from patients in Heilongjiang (HLJ) Province (located in northern China), and another 101 strains were isolated from patients from Jiangxi (JX) Province (located in southeast China). These strains were related with CAG only (n = 58), PUD (n = 45) or GC (n = 38). Two strains with fully sequenced genomes, including strain 26695, which was isolated from a gastritis patient, and strain J99, which was isolated from a patient with a duodenal ulcer, were used as controls. This study was approved by the Ethics Committee of National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention. The written informed consent was obtained from all patients.

Culture and extraction of genomic DNA
The strains stored in brain heart infusion in −80°C were recovered on Columbia agar plates (Oxoid) supplemented with 5 % fresh defibrinated sheep blood and kept under microaerophilic conditions (5 % O2, 10 % CO2 and 85 % N2) at 37°C for 3 days. Colonies displaying typical H. pylori morphology were selected and identified by Gram staining and urease, oxidase, and catalase activity testing. The bacterial cells on chocolate agar plate were washed twice with phosphate buffer saline (PBS, pH7.5) and centrifuged at 5000 rpm for 10 min. The chromosomal DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Germany) according to the manufacturer's instructions.
Determination of the jhp0940, jhp0945, jhp0947, jhp0949 and jhp0951 status via PCR The status of the genes was determined via PCR using the primer pairs shown in Table 1. The amplification of the genes was performed in a volume of 25 μl containing 25 pmol of both forward and reverse primers. The PCR conditions were 95°C for 5 min, followed by 35 cycles of 95°C for 45 s, 52°C for 45 s, and 72°C for 45 s, and finally 72°C for 5 min. Two primer sets were used for the jhp0940 gene, and when the PCR using one pair was positive, the jhp0940 status was determined to be positive. The status of the genes was positive in strain J99 and negative in strain 26695.

Statistical analysis
The prevalence rates of all five genes were evaluated. The association between each genotype and the clinical outcomes were quantified using the chi-squared test. A probability (p) value equal to or less than 0.05 was considered to be statistically significant, and a value of 0.001 or less was considered to be highly significant. Univariate analysis and a multivariate logistic regression model were used to calculate the odds ratios (ORs) of the clinical outcomes using SAS 9.3.

Geographic variation of plasticity region genes
The results were divided into two groups based on their geographic variation. By comparing the results of the strains from HLJ Province and JX Province, we The relationship between jhp0940, jhp0945, jhp0947, jhp0949 and jhp0951 The status of all of the genes showed that they are significantly associated with each other ( Table 3). The status of jhp0945 was associated with jhp0949 (p = 0.009) and jhp0951 (p = 0.022). All of the jhp0947-positive isolates possessed jhp0949, and both were significantly associated with jhp0951 (p = 0.0008 and p < 0.0001, respectively). When combining the five genes together, the majority of the genotypes were all negative or all positive (the −/−/−/−/genotype or the +/+/+/+/+ genotype, 12.06 %, 17/141). The rates of the all-positive genotypes were 15.79 % (6/38), 20 % (9/45) and 3.45 % (2/58) in the isolates from the patients with GC, PUD and CAG only, respectively. In contrast, the rates of the all-negative genotypes were 5.26 % (2/38), 2.22 % (1/45) and 24.14 % (14/58) for GC, PUD, and CAG, respectively.

The relationship between the gene status and the clinical outcomes
A univariate analysis showed that there was no significant association between the jhp0940 status and the selected diseases (Table 4), but the status of jhp0945, jhp0947, jhp0949 and jhp0951 was significantly associated with a lower risk for CAG ( A multivariate analysis, including age and jhp0940, jhp0945, jhp0947, jhp0949 and jhp0951 status, was performed to determine the factors that were related to the  The coefficient value for assessing the association between each pair of genes. A p value of 0.001 or less was considered to be highly significant The coefficient value a was analyzed using Fisher's test clinical outcomes of the selected diseases (

Discussion
The plasticity region is a recently identified locus found in the chromosome of H. pylori strains J99 and 26695 that displays similar characteristics to pathogenicity islands [7,8]. The majority of the H. pylori strainspecific genes that are transferred from other species are located in the plasticity region [9,12,13]. The genes present in the plasticity region have been highlighted as potential pathogenic markers and may account for the differences in the H. pylori strain virulence, resulting in various clinical outcomes. The jhp0940, jhp0945, jhp0947, jhp0949 and jhp0951 genes, which are specific for strain J99, have recently been reported to be associated with H. pylori-related diseases and are potential markers for the risk of gastrointestinal diseases [10]. Because of their geographic variation, the relationship between these genes and the severity of certain diseases has been discussed, but the results are often not in agreement. The prevalence of these genes and their relationship with certain diseases in China is currently unknown; therefore, we examined the distribution of these virulence markers and their relationship to the clinical outcomes of patients infected with H. pylori.
Previous study found that there were no significant associations between the gastroduodenal diseases and the status of jhp0940, jhp0945 and jhp0949 in East Asia strains [10]. Yakoob et al. demonstrated that jhp0940 and jhp0947 in Pakistan strains were associated with GC and PUD [14]. In our study, there was no association between jhp0940 and diseases. The positive rates of jhp0945, jhp0947, jhp0949 and jhp0951 were much higher in PUD, and there was a significant association between the genes. However, the multivariate analysis showed that only jhp0951 was independently associated with the development of PUD in our study. Jhp0951, which encodes an integrase from the XerCD family, is involved in the response to acidic environments [15]. In DUs, acid secretion increases, causing the mucosa to be continuously exposed to a low pH, which is consistent with our results [9]. As the virulence genes were associated with each other in our study, the strong linkage of jhp0945, jhp0947 and jhp0949 with PUD may be due to the significant association between jhp0951 and PUD. We also speculate that all of these factors act synergistically in causing damage to the host.
It is well known that the development of GC is marked by a slow progression that begins with H. pylori-induced chronic superficial gastritis, which then progresses to atrophic gastritis, intestinal metaplasia, dysplasia and eventually GC [16,17]. In our study, the multivariate analysis showed that the only independent virulence gene that  increased the risk of GC was jhp0945, indicating a significant association between the two. However, the jhp0945 status showed a negative association with CAG (Table 5), which conflicts with the process of GC development. In our study, we speculate that the majority of the individuals with CAG do not carry the jhp0945 gene, while the few jhp0945-positive individuals potentially develop GC due to a combination of the bacteria, the host and other environmental factors, which is consistent with the fact that of the H. pylori-infected individuals, 80-90 % have clinically asymptomatic gastritis, while only 1-2 % develop GC [18]. Jhp0945-positive isolates may be more likely to develop severe diseases, and jhp0945 status may be a risk indicator for GC development. Additional prospective studies are still necessary to further confirm our speculations.

Conclusions
In conclusion, this was the first study in China to evaluate the relationship between plasticity region genes and clinical disease outcomes. We found that jhp0951 status is an independent factor for discriminating PUD and may influence the association of other virulence factors with certain diseases. Jhp0945 may predict the future development of GC in patients with CAG and is considered to be the best candidate disease marker for H. pylori-related diseases.