Studies On The Relationship Between Normal Flora In Stomach And Helicobacter Pylori Infection

Helicobacter pylori(Hp)was proved to be related with chronic gastritis, peptic ulcer, mucosa associated lymphoid tissue, gastric carcinoma, since the pathogen was first was identified by Austrian scientist Dr. Warren and Marshall. In vitro and in vivo studies and epidemiological investigations indicated that Hp was pathogenic and should be eliminated by antibiotics or combination of antibiotics with proton pump inhibitor (PPI) or H2 receptor antagonist when treat diseases associated with this bacteria. The drug resistance of Hp makes clearance of it problematic. In present, many Hp infection remain exclusive.Epidemiological investigations showed that the rate of colonizzation was different from that of infectious disease. Approximately about 25%-50%,70-90% and of people carry Hp in deceloped countries and developing countries respectively, however the the percentage of bacterial infection diseases was far lower than the colonizion rate. Does the normal carrier need the clearance of colonized bacteria? Why the outcome of infection differs greatly among peoples. The answers were controversial. Treatment of antibiotics is able to erade the Hp in about 80%-90% of patients. The rate of infected diseases does not decrease after clearance of bacteria. What is the reason? Obviously it is inappropriate to take the Hp infection as the sole factor causing digestive diseases, which is accepted by increasing investigators.The Hp infection is a complicated process, which is related with gastric microen -vironment, stressful, host genetic background, bacterial virulence etc. Currently the relationship betweent Hp and gastric microenvironment especially gastric microflora is of increasing interest.Hp-infection animol model studies showed that it is easier to colinize on immunodeficient and SPF BALB/c mice and cause typic pathological changes than normal mice. And mice with large amount of Lactobacillus acidophilus (La) was able to inhibit the growth Hp and the diseases occurred when the copies of La decreased. These data indicated that the bacteria colonized in stomach was closely related with the the state of Hp infection, and the gastric microenvironment affected the outcome of Hp infection.The appearance of Hp in stomach does not mean the gastric diseases. Science Hp is a conditional pathogen, it is of greater impotance to reconstruct the hemostasis of gastric microenvironment and inhibit the growth of Hp than to kill the bacteria completely. When treating gastric diseases with antibiotics, the antibiotics will kill the normal flora in the stomach at the same time and destroy the microenvironment, which help the re-infection of Hp and other bacteria. Therefore reconstruction the hemostasis of stomach microenviron -ment is able to inhibit the growth of Hp, to prevent the Hp associated diseases and the reinfection of Hp.Current studies treat the normal flora imbalance by oral uptake of La and cure the gastric disease. However, no data about the number of colonized bacteria and Hp is available. La isolates from the health people will provide an alternative approach for the treatment of` Hp infection, especially in combination with the PPI and other methods. Objectives:1 To determine the composition normal flora in people from different state of Hp infection and its corelation with the bacterial infection.2 To isolate a La strain that is able to inhibit the growth of La effectively for further treatment Hp infection.Methods1 Isolation and identification of bacteria from Hp negative people, Hp normal carrier and Hp patients.Homogenates of stomach fron patients by gastroscope detection were cultivated on the brain heart infusion agar plate, anaerobic bacteria agar plate, MRS plate, COBA plate, EMB plate and PDA plate in 37℃for 1-2 days. The visible colonies on plates were identified by gram stain and biochemical reactions.2 Isolation of La strains test their ability to inhibit the growth of Hp infection. La strains isolated from the health people were grown on MRS plate (pH=3) for 2 days at 37℃. Their ability to inhibit the growth of Hp was detected by disk diffusion method. The isolate with the most inhibitive activity was tested its capacity of acid resistance, bile resistance and antibiotics resistance.3 Analysis of La JK57 on the clearance of Hp infection in clean grade BALB/c mice model.Clean grade BALB/c mice were divided into 6 groups randomly and infected with Hp for 5 weeks. All mice were treated with JK57 and then the colonized bacteria in mice stomach were determined as describe before, and the pathologic manifestation was evaluated ata the same time.Results:1 Different compositions of gastric normal flora.1) Different number and positive rate of La colonized.The rate of La of Hp negative people is significantly higher than that of Hp infected patients (P<0.05). The total number of bacteria of Hp normal carrier is greater than that of Hp infected patients2) Different ratios of bacteria in stomach.The percentage of Lactobacillus detected in Hp negative people is the larger than that of Hp normal carrier and Hp infected patient (P<0.05). And the percentage and number of anaerobic bacteria from Hp infected patient is significantly higher than that from Hp negative people. In the case of enterobacter and streptococcus, their percentage of Hp infected patient are different from that of Hp negative people (P<0.05).2 Isolation of La strains for Hp infection treatment.1) 16 La strains were isolated and identified, and their ability to inhibit the growth of Hp by disk diffusion method. Three strains were identified as the most effective La for Hp inhibition, namely JK57, JK54 and JK78, The inhibition zone was greater than 15mm, which was similar to that of 0.25mg/ml of ampicillin and 0.05mg/ml of metronidazole.2) In acid resistance test, JK57, JK54 and JK78 were able to grow when pH>3. When pH is lower than 2, Only the JK57 survived (P<0.05). And in bile resistance test, almost all of the three strains showed good bile resistance while grown in MRS in the presence or absence of bilis bubata (P﹥0.05).3 Treatment of Hp infection in BALB/c mice by JK57.1) Clearance of Hp in mice.The clearance rate of Hp in mice treated with Jk57 combined with PPI and PPI only were 80% and 90% respectively, while that of JK57 treated mice is 60%. The clearance rate of mice treated with Jk57 combined with PPI and PPI only were effective (P<0.05). And there was no difference when comparing with antibiotics combined with PPI.2) The composition of stomach bacteria after treatment of JK57The total number of bacteria from mice treated with JK57 was about 105-106 CFU per gram, and that of JK57 combined with PPI and antibiotics combined with PPI were 106-107CFU per gram and 104-105CFU per gram respectively. The total number bacteria from mice treated with JK57 and JK57 combined with PPI is significantly higher than that of antibiotics combined with PPI (P<0.05).3) Pathologic manifestations changes in mice treated with JK57The recovery of inflammation from mice treated with JK57, JK57 combined with PPI and ntibiotics combined with PPI was greater than that with physiological saline (P<0.05). No significant difference of imflimation score was observed among mice treated with JK57, K57 combined with PPI and antibiotics combined with PPI (P﹥0.05).Conclusions1 Significant differents of normal flora composition were observed among Hp negative people, Hp normal carrier and Hp positive patients. The number of lactobacillus from Hp positive patients was significantly lower than that of health people. However, the anaerobic bacteria and other bacteria increased. These data indicated that the number of bacteria in stomach was corelated with that of Hp colonization.2 JK57 was isolated from normal Hp carrier and was shown to be able to inhibit the growth of Hp effectively. Moreover JK57 showed remarkable acid resistance, and bile resistance, which laid a solid foundation for the future use of JK57 in the treatment of Hp infection.3 Hp chronic infection model was constructed successfully. The pathologic reaction in mice was effectively inhibited by La JK57 treatment, the rate of which is similar to that of traditional triple therapies. This effect would be better when JK57 was combined with PPI. Meanwhile, treatment of JK57 was able to incease the number of lactobacillus, especially in combination with PPI.