Helicobacter pylori Diagnosis, Virulence Genes, Resistance to Conventional antibiotics and Antimicrobial activity of selected Medicinal plants on Isolates obtained from Patients undergoing Endoscopy at Aga Khan University Hospital, Kenya

Abstract

Helicobacter pylori colonizes the gut of persons of all ages, and is the etiologic agent for acute and chronic gastritis and a predisposing factor in peptic ulcer disease, gastric carcinoma and B-cell mucosa-associated lymphoid tissue lymphoma. It is a class 1 carcinogen. Prompt detection of H. pylori is imperative for effective treatment. Rapid urease test is the most routinely used diagnostic method. Culture, histology and molecular methods are the available alternatives. Diagnostic performances of these methods have never been evaluated in Kenya. The growing resistance of H. pylori to triple therapy antibiotics imposes an exploration for alternative treatment which could be naturally occurring, readily available, and cost effective. Hence, antimicrobial activity of aqueous and ethanol extracts of naturally occurring Aloe secundiflora, Bridelia micrantha and Lippia javanica herbal medicines to H. pylori was determined, while also identifying any known potential bacteriocidal active phytochemicals. To achieve these objectives, gastric biopsies were obtained from 274 patients suffering from gastritis. The biopsies were subjected to rapid urease test, histology and culture diagnostic procedures for diagnosis and for comparison of performance of the methods. Whole genome sequence for H. pylori isolates was performed to determine the presence of cagA, vacA and dupA virulence genes. These enabled the possible association between these genes and gastro-duodenal diseases to be determined. Phenotypic antimicrobial resistance profile of H. pylori isolates to five antibiotics such as clarithromycin, metronidazole, tetracycline, levofloxacin and amoxicillin, which are the commonly used conventional antibiotics, was determined through E-test strips. Antimicrobial susceptibility testing was also performed using agar well diffusion technique for H. pylori isolates on the aqueous and methanol extracts of Aloe secundiflora, Brideria micrantha and Lippia javanica medicinal plants. Zone of inhibition, minimum inhibitory concentration and minimum bactericidal concentration was determined. Post hoc comparisons using the Tukey HSD test was used to compare score, while independent-samples t-test was used to compare zones of inhibition. Presence of phytochemical in plant extract was determined through Gas Chromatography-Mass Spectrometry. A total of 274 antrum biopsy specimens were collected from patients, fifty two percent (52%) were female and the age 15-82) years. The Pronto Dry Rapid Urease® test out performed culture in sensitivity and NPV measures. Out of 274 gastric biopsies, 147(54%) were positive for Pronto dry rapid urease® test, 122(44%) for histology and 91(33%) for culture. Virulence genes cagA, dupA, vacA s1/m1 were detected in all disease conditions. Detection of vacA s1 in 23 (55%) in isolates with gastric ulcer disease was significantly high (P = 0.036). All (100%) H. pylori isolates expressed no antimicrobial resistance to Tetracycline. This study demonstrated that the total resistance rates of H. pylori to metronidazole, amoxycillin and clarithromycin were 93,63, and 23%, respectively, There was a significant difference in the zones of inhibition for A. secundiflora p<0.0001), B. mirantha, p<0.0001) and L. javanica, p<0.0001). Post hoc comparisons using the Tukey HSD test indicated that the mean score for A. secundiflora was significantly different than B. mirantha, L. Javanica, p<0.0001 and ciprofloxacin p<0.0001). The aqueous extracts of A. secundiflora leaves had the highest MIC at 1.56 mg/ml. Key bioactive compounds by GC-MS identified included Butylated bydroxytoulene Phenol, 2,4-bis(1,1-dimethylethyl) Bibutyl bhthalate benzyl butyl phthalate. The performance of Pronto dry rapid urease® was commendable as it was comparable to the ‘gold standard’. cagA, vacA and dupA virulent genes could possibly be associated with gastro-duodenal diseases. H. pylori faces serious resistance to most of the triple therapy antibiotics, except tetracycline, necessitating explorations that can lead to new anti-H. pylori agent . As such there is hope since extracts of A. secundiflora, B. micrantha and L. javanica have compounds with antimicrobial activities and possess inhibitory prospects to H. pylori.