Assessment of anti-urease and consequential inhibitory potential of South African honey extracts on the multiplication of drug-resistant, vacA and cagA positive helicobacter pylori strains under acidic conditions

Helicobacter pylori, a neutralophile chronically infects the gastric stomach of more than half of the world’s population. Infection with the organism is associated with acute or chronic duodenal/gastric ulcer disease, gastritis, gastric adenocarcinoma, mucosa-associated tissue lymphoma (MALT) and primary B-cell gastric lymphoma and has been grouped as a class one carcinogen by the World Health Organisation (WHO). Prevalence of this organism is very high in developing countries especially in Africa, including South Africa. H. pylori treatment using the common first and second line regimens, triple therapy with two antibiotics and a proton pump inhibitor (PPI) is showing inefficiency due to increasing drug resistance. However, newly developed treatment regimens seem to be more expensive and are accompanied by more side effects. Honey contains phytochemicals which are a wealthy source of biologically active compounds some of which have been put into good use in the pharmaceutical industry. Pathogenesis of H. pylori infection in the human stomach relies on several virulence factors which include the urease enzyme, cagA and vacA. The urease enzyme actively hydrolyses urea to produce ammonia an important by-product involved in pH regulation favouring the survival of the organism in the acidic human stomach. This study therefore focuses on screening for anti-urease solvent extracts of South African honey, and evaluate whether inhibition of urease offsets the growth of H. pylori under acidic conditions. Locally produced natural honeys; Bush honey, Raw honey, Gold Crest honey, Q Bee honey, Little Bee honey, Fleures honey-radurised, Siyakholwa pure honey and Manuka honey; an import from New Zealand were purchased and the method by Syazana et al. (2010) was used for the extraction of compounds in honey. A standard strain ATCC 43526 (American Type Culture Collection, Manassas, VA, USA) and 48 pure cultures obtained from clinical isolates cultured from gastric corpus biopsy specimen of patients with gastric morbidities who were ix visiting the endoscopy unit in Livingstone Hospital, Port Elizabeth between June 2008 to December 2008 were initially used as source of urease enzyme as per extraction method done by Amin et al. (2013), but with modifications. Prior to urease extraction, H. pylori strains were identified by biochemical tests (urease, catalase, oxidase, Gram stain), confirmed by PCR targeting the glmM gene (140 bp) and drug resistance profiling was done on all the 48 strains according to Seanego et al. (2012). The screening for anti-urease active compounds was done according to Kaltwasser et al. (1966), a method relying on the reduction of NADH in a coupled urease dehydrogenase (GDH) system. Acetohydroxamic acid was used as a standard inhibitor. Prevalence of cytotoxin-associated gene A (cagA) gene and vacuolating cytotoxin gene A (vacA) gene was determined among all 48 clinical samples. The standard strains of H. pylori, X47 (cagA positive), J99 (vacA s1m1) and Tx30a (s2m2) were used as positive controls. H. pylori’s growth was then monitored under acidic pH in a cocktail spiked with anti-urease compounds (test samples) and in a cocktail without anti-urease compounds (negative control). Acetohydroxamic acid was used as a standard urease inhibitor. H. pylori multiplication was monitored in Brain Heart Infusion Broth (BHIB) adjusted to pH of 2, 3, 4, 5, 6 and 7. The strain MP01 was used as a standard urease negative strain while X47 and J99 were used as positive standards for cagA and vacA s1m1 respectively. The compounds that had anti-urease activity and were successful towards suppressing the multiplication of H. pylori under acidic environment, all other factors optimised, were subjected to gas chromatograph mass spectrometry (GC-MS) and liquid chromatograph spectrometry (LCMS) to determine volatile compounds and drugs in honey extracts respectively. The findings of this study revealed that at a concentration of 50 mg/mL, urease inhibition by petroleum ether extracts of Gold Crest and Fleures honey, hexane extracts of Little Bee and Manuka honey, and chloroform extracts of Bush honey and Q Bee honey had a range above or equal to 50 percent and there was no significance difference in urease inhibition percentage (I percent) of urease from different sources including that extracted from drug resistant H. pylori (p >0.05). Virulence factors are important for the pathogenesis of H. pylori. All the 48 clinical isolates were glmM (140 bp) positive and cagA was detected in 97.9 percent of the test isolates. The vacA gene was detected in all isolates but with different subtypes. The vacA allelic combination s1m1 was detected in 75 percent of the test isolates and s1m2 allelic combination was detected in 16.7 percent of the test isolates while the combination s2m2 was detected in 8.3 percent of the test isolates. None of the test isolates possessed the allelic combination s2m1. When H. pylori multiplication was monitored under acidic conditions in the presence of anti-urease active compounds, it was revealed that anti-urease active compounds in honey are capable of inhibiting the normal multiplication of H. pylori strains that are cagA positive, vacA positive and drug resistant. The GC-MS analysis showed that Fleures honey (urease I percent = 67.8 – 68.5 percent) and Gold Crest honey (urease I percent = 50.9 percent – 53.3 percent), all petroleum ether extracts had 27 and 26 volatile compounds. The hexane extract of Manuka honey (urease I percent = 50.0 – 53.2) had 43 compounds detected. The chloroform extract of Q Bee (urease I percent = 64.2 – 66.2 percent) had 13 volatile compounds detected. All the volatile compounds considered as representative samples of GC-MS analysis had a spectral matching ≥ 90 percent with the NIST11 library. However, the majority of compounds that were detected by LC-MS in representative honey extracts include vardenafil, urapidil, hydrocortisone, e.t.c which are drugs commonly used in the treatment of different ailments or infections that affect human beings. In addition, two xi drugs, sulfaquinoxaline and hydroxyquinoline which are used in veterinary medicine and antiseptic, disinfectant and pesticide applications in agricultural activities were detected in Little Bee honey. We therefore conclude that inhibition of urease has a bactericidal effect on drug resistant, cagA positive and vacA positive H. pylori strains growing under acidic environment

Prevalence and risk factors for Helicobacter pylori transmission in the Eastern Cape Province application of immunological molecular and demographic methods

Helicobacter pylori (H. pylori) is a microaerophilic, Gram-negative motile curved rod that inhabits the gastric mucosa of the human stomach. The organism chronically infects billions of people worldwide and is one of the most genetically diverse of bacterial species. Infection with the organism potentially induces chronic gastritis and peptic ulcer disease. In addition, H. pylori plays a role in the etiology of gastric cancer and gastric MALT lymphoma. The risk of infection is increased in those living in the developing world, which has been ascribed to precarious hygiene standards, crowded households, and deficient sanitation common in this part of the world. Thus, the aim of this study was to identify the risk factors in the transmission of H. pylori in our environment, i.e. in Nkonkobe Municipality in the Eastern Cape Province, South Africa. Faecal samples were collected from 356 apparently healthy subjects, consisting of 168 males and 188 females aged from 3 months to 60 years (Mean = 31 years). A standardized questionnaire was applied, it described demographic characteristics including age, sex, household hygiene, socioeconomic status, area of residence, duration of stay in the area, sharing bath water, sharing tooth brush, habit of sucking thumb, medication currently being taken or medication taken within the past three months, source of water, type of toilet used, education and occupation. A sandwich-type enzyme immunoassay amplification technology (Amplified IDEIA TM Hp StAR TM , Oxoid, UK) was used to analyze the faecal samples for the detection of H. pylori antigens using monoclonal antibodies specific for H. pylori antigens. To assess the possibility of faecal oral route with tap water as an intermediary link, PCR targeting the ureC (glmM), a highly conserved gene in H. pylori ii was carried out to detect H. pylori DNA in faecal samples of already positive samples by HpSA test as well as in direct tap water used by the H. pylori positive subjects. QIAamp DNA stool mini kit was used to extract DNA from faecal samples. Tap water samples were then obtained using sterile bottles from areas inhabited by H. pylori positive subjects as determined by HpSA test and PCR. DNA extraction from water samples was done using UltraCleanTM Water DNA Isolation Kit (0.22μm) according to the manufacturer’s instructions. PCR with primers specific for H. pylori glmM gene was carried out with both positive and negative controls incorporated. Fisher’s exact test was used to assess the univariate association between H. pylori infection and the possible risk factors. Odds ratio (OR) and the corresponding 95 percent confidence interval (CI) were calculated to measure the strength of association using EPI INFO 3.41 package. P values of .05). Interestingly, H pylori antigen was detected more often (100 percent) in the high socioeconomic group than in those of low socioeconomic group (85.9 percent). Sixteen (66.7 percent) of twenty four faecal samples that had previously tested positive for the organism by HpSA test were confirmed positive by PCR. However none of the treated tap water samples tested positive for the organism by PCR. The present iii study revealed a high prevalence of H. pylori in faecal samples of asymptomatic individuals in the Nkonkobe Municipality, an indication of active infection. The obtained results also revealed that direct treated tap water might not be playing a crucial role in the oral transmission of H. pylori in the studied population