Analysis Of Culturable Bacteria Flora In The Stomach Of Patients With Gastric Disease And Its Clinical Significance

Objective To investigate the influence of culturable bacteria in the stomach of patients with disease of upper digestive tract.Methods 1.Isolation bacteria from stomach biopsies of patients:Gastric biopsies were taken from patients who were suffering from stomach discomfort under painless endoscopy.Rapid urease test(RUT),bacterial isolation and H.pylori specific 16S rRNA PCR amplification were performed on these biopsies.H.pylori and non-H.pylori isolates were differentiated by colonial morphology,Gram's staining,RUT and PCR by H.pylori specific 16S rRNA primers.Urease activity of non-H.pylori strains were further identified by RUT and conventional urease test.20 urease positive non-H.pylori strains were selected for species identification by 16S rRNA PCR and sequencing with universal primers.2.Bacterial culture and antibiotic susceptibilities of H.pylori and non-H.pylori in the stomach of children:A total of 346 gastric biopsy specimens from children affected by gastric diseases were selected for bacterial culture.H.pylori and non-H.pylori were identified by specific PCR and 16S rRNA sequencing,respectively.Antibiotic susceptibilities of H.pylori and non-H.pylori were tested by the E-test and disk diffusion methods,respectively.Rapid diagnosis of H.pylori was also performed by H.pylori-specific PCR.3.Animal experiments:H.pylori NCTC11637 and two of the non-H.pylori strains exhibiting multidrug resistant(MDR)had been selected.One MDR strain,Pseudomonas putida,was urease positive,and the other MDR strain,Streptococcus mitis,is urease negative.Mice,3-week-old,were infected with H.pylori and non-H.pylori successively or only infected with one strain via intragastric gavage.Two months later,mice were anaesthetized and serum IL-6,IL-8 levels were tested by ELISA.The stomach of mice was dissected,half of them were underwent histologic analysis,and the other half was homogenized.One hundred microliter homogenates were used for bacterial isolation.Result 1.In 606adults cases,the positive rate of RUT of gastric biopsies was 58.4%(354/606),and the positive rate of H.pylori isolation was 29.7%(180/606).113 biopsies were negative in H.pylori isolation but positive in H.pylori specific PCR.The rate of H.pylori infection(positivity in H.pylori isolation or specific PCR)was 48.3%(293/606).The positive rate of RUT was higher than the rate of H.pylori infection(?~2=12.337,P<0.05).61 H.pylori negative biopsies were RUT positive,and 80 strains with urease activity determined by RUT and conventional urease test were isolated from these samples.All those 20 species identified by 16Sr RNA sequencing were urease positive species.2.In 346 children,22 H.pylori strains were obtained from culture,The positive rate of H.pylori was 32.9%(114/346).The positive rate was greater in boys 40.3%than in girls 23.30%(?~2=11.080,P=0.001);The H.pylori infection rate was higher in the school age group(5-14y)35.1%,than in the pre-school age group(1-4y)19.1%(?~2=4.688,P=0.030).22 H.pylori strains were obtained from culture.Resistance rates of 22 H.pylori strains by the E-text were as follows:metronidazole,86.4%;tetracycline,13.6%;amoxicillin,22.7%;levofloxacin,31.8%;clarithromycin,31.8%.366 non-H.pylori strains were obtained,including 193 with urease activity.Non-H.pylori resistance rates by the kirby-bauer method were as follows:metronidazole,94.8%;tetracycline,26.2%;amoxicillin,42.6%;levofloxacin,15.3%;clarithromycin,46.7%.3.Animal experiments:(1)Results of serum IL-6 and IL-8 measured:The levels of serum IL-6 compared:The content of serum IL-6 in each group was different and the difference was significant(analysis of variance F=3.867,P=0.004),H.pylori infection group,H.pylori subsequent P.putida infection group,P.putida group and S.mitis group IL-6 were higher than that of normal group,and the difference were significant(t values were 2.297,3.511,2.673,3.194,the orders of P values were 0.031?0.002?0.018?0.009);There was no significant difference in serum IL-6 leves between H.pylori subsequent S.mitis infection group and normal group(t=1.261,P=0.223).The levels of serum IL-8 compared:The levels of serum IL-8 compared:The content of serum IL-8 in each group was different and the difference was significant(analysis of variance F=3.080,P=0.014),H.pylori infection group,H.pylori subsequent P.putida infection group,P.putida group,H.pylori subsequent S.mitis infection group IL-8 were higher than that of normal group,and the difference were significant(t values were4.644?3.544?2.402?2.635?3.567,the orders of P values were 0.000?0.002?0.027?0.017?0.003).(2)Results of mice gastric mucosa bacteria isolation:The experiment bacteria were used to infect the gastric mucosa of mice for 2 months,experiment bacteria were cultured positive from infected mice,normal mice were negetive.(3)Histologicl analysis of gastric mucosa in mice demonstrated leukocyte infiltration in H.pylori group,H.pylori subsequent P.putida infection group,P.putida group,H.pylori subsequentS.mitis infection group;the inflammatory reaction in S.mitis group and normal groupwere not obvious.Conclusion There are other bacteria including those urease positive non-H.pylori and may disturb clinical diagnosis of H.pylori infection.Resistant even MDR strains of non-H.pylori could colonize in the stomach and cause certain degree inflamatory reaction,further studies on the effects of non-H.pylori in stomach are worthy to be carried out.