Evaluation of different methods for detecting helicobacter pylori isolated from human and the effect of probiotics on the bacterial growth. +CD.

This study included two different grouping samples (human and some domestic animals). In human group, (130) dyspeptic patients were subjected to esophageal gastroduodenoscopy and gastric biopsy specimens were taken from the antrum and body were applied for microbiological analysis which included: urease test, bacterial culture using different culture media, direct biopsy smear examination using Giemsa and Gram stain as well as histological test for detecting Helicobacter pylori using Hematoxyline, Eosin (H E) and Giemsa stains. Venous blood samples were also collected randomly from (75) patients for enzyme linked immunosorbant assay (ELISA test) and (15) healthy persons with no history of dyspepsia, or peptic ulcer disease were used as control. Results showed that occurrence of H.pylori was (81.5%) among dyspeptic patients, and there was a significant correlation between H.pylori infection and duodenitis (89%), gastric erosion (83%), duodenal ulcer (81%) and gastritis disease (75.6 %). Results also showed that using modified selective medium was better than the nonselective for primary isolation of bacteria, with a recovery rate of (24%) by using brain-heart infusion agar and (6.2%) for the nonselective blood agar. Histological investigation using Giemsa and (H&E) stains were proved to be the most sensitive method for detecting H.pylori
infection with a sensitivity of (98%), while the sensitivity of bacterial identification by the biopsy urease test, direct biopsy smear examination and culture of biopsy specimens were (95%), (80%) and (29%) respectively. Statistical analysis of the ELISA results showed that there were significance differences in the mean of immunoglobulin G (IgG) specific antibodies concentration observed among males and females of the gastric cancer group compared with the controls, while no significant differences were revealed among males and females of other groups. When the isolates of H.pylori were subjected to the sensitivity test against (12) antibiotics, results showed that ciprofloxacin was the most effective antibiotic against the isolates when the sensitivity percentage was (62%). Penicillin G, ampicillin, cloxicillin and amoxicillin on the other hand, were the least effective antibiotics when bacterial sensitivity ranged between (8%) and (37%). Depending on the antibiotic sensitivity results, five isolates of H.pylori were selected (due to their multiple resistances to antibiotics) for determining the minimal inhibitory concentration (MIC) of some antibiotics. Results showed that bacterial isolates were able to grow in high concentration (512µg/ml) of penicillin and amoxicillin, and moderate concentration (265µg/ml) of metronidazole, with slight resistance for clarithromcin and tetracycline (32µg/ml). When inhibitory activity of lactic acid bacteria (LAB) isolates against H.pylori was tested on solid medium, less inhibitory activity against it, was detected compared to the liquid medium, the inhibitory activity increased after incubation periods of (48) and (72) hours. Minimum inhibitory concentration (MIC) of LAB concentrated filtrate was estimated. Results showed that (50%) and (60%) concentrations of such filtrates were effective in the inhibition of H.pylori growth. MICs were determined for the four - fold concentrated filtrates of Lactobacillus acidophilus against adhesion property of H.pylori HP1, HP2 and HP3. Results showed that filtrates were able to lower adhesion of pathogenic bacteria H.pylori to the epithelial cells when the average of adherence decreased to (5-15 bacteria/ cells) instead of (60-75 bacterial/ cell) recorded by HP1, (70-85 bacterial/ cell) by HP2 and (80-95 bacterial/ cell) obtained by HP3 before treatment. Also an interesting finding upon probiotic treatment against H.pylori isolates with decreased in urease activity was observed when a color change was detected after (10, 18 and 24) hr; rather than few minutes before treatment. This could be due to lactic acid and other inhibitory substances produced in concentrated filtrate by LAB isolates. A transformational change was detected when a coccoid form of H.pylori was observed after treatment with LAB isolate. In the animal group, a total of (170) samples of animal (20 bovine and 100 sheep gastric biopsies as well as 50 raw sheep milk samples) were collected from Sheikh Maroof and Al-Doora slaughtering houses in Baghdad and investigated for the presence of H. pylori. Different culture media, procedures and histological examinations were used. Results showed that H.pylori was not present in any of these samples.