Molecular and phenotypic characterization of Helicobacter pylori isolates from patients with gastroduodenal pathologies in the Eastern Cape Province of South Africa.

Abstract

Helicobacter pylori is an important human pathogen known to chronically infect billions of people worldwide, causing a number of gastric related diseases. Prevalence of this organism is very high in Africa and has been reported to vary between and even within countries. H.pylori eradication using the two antibiotics regimen and a proton pump inhibitor often fails due to increasing drug resistance. Studies in South Africa have demonstrated the presence of this organism in the study area. This study investigated the prevalence of H. pylori in the Eastern Cape Province of South Africa; determined the antimicrobial susceptibility patterns of isolates; the molecular basis of the resistance pattern of isolates; the most prevalent genotype and the sequence diversity of the genes involved in virulence. We examined 254 consecutive patients who were referred to Livingstone Hospital, Port Elizabeth with gastric related morbidities between June 2008 to December 2008 for endoscopy and determined the prevalence of infection with respect to age, sex, endoscopic diagnosis, ethnic background and lifestyle. Two gastric biopsies were collected (one from the antrum and the other from the corpus) and the organism was isolated on Columbia agar base. Determination of antimicrobial susceptibility/resistant patterns of the isolates to the current antibiotics employed in treatment were executed. Genotyping was carried out using PCR based approach to determine the prevalence of virulence genes (cagA, vacA and iceA) while the molecular basis of resistance and diversity of virulence genes were determined by sequencing the amplified products. Presumptive isolates were further confirmed by PCR targeting the glmM gene. The overall prevalence of H. pylori was 66.14% (168/254). Prevalence was highest (100%) amongst patients with duodenitis (1/1), gastric cancer (GC) (4/4) and gastric erosion (5/5) as they were all positive for the organism. However, patients with gastritis (75%; 6/8), gastric ulcer (GU) (70.8%; 17/24), duodenal ulcer (DU) (65%; 26/40), non-ulcer dyspepsia (NUD) (64.7%%; 55/85), and gastro- oesophageal reflux disease (GERD) (64%; 29/45) also had high prevalence rates of the organism. The organism was not isolated (0%) from patients with gastro-duodenitis and atypical oesophageal reflux disease respectively. The prevalence of infection was highest amongst the coloured (66.92%; 87/130) and lowest in whites (59.52%; 25/ 42). Susceptibility was determined using the Kirby-Bauer disc diffusion and agar dilution methods. Data was analyzed and marked susceptibility of isolates was observed for ciprofloxacin (100%) and amoxicillin (97.5%). Isolates also demonstrated good activity to clarithromycin (80%) and gentamicin (72.5%). However, marked resistance (95.5%) was observed for metronidazole. The MIC ranged from 0.0625–8 μg/mL. The lowest MIC with a range of 0.0625 - 1μg/mL was recorded for ciprofloxacin while the highest (5–8μg/mL) was noted for gentamicin. Multidrug resistance was a common phenomenom encountered in this study. Thirty-two (17.02%) isolates showed multidrug-resistance to metronidazole and erythromycin (METRERTR). The least resistance pattern was CLARTETRAMXRMETRGENRERTR (O.53%) and ERTR (O.53%). Polymerase Chain Reaction using specific primer sequences was used to identify the presence of virulence genes. cagA was identified in 90% of the strains investigated. Fifty-eight of the 100 strains had the vacA signal sequence genotype s1 and 26 had subtype s2. Combined vacA s1/s2 was detected in 16 of the strains. vacA middle region analysis showed that 8(8%) strains were m1 while 50 were m2. Combined vacA m1/m2 was detected in 36 of the strains. s1m2 (20%) and s2m2 (20%) genotypes were the most common allelic combinations of the vacA gene among our strains. Multiple vacA genotypes were detected in this study, amongst which the most prevalent was s1m1m2 (61%) 28/46. IceA1 was present in 2 (2%) of our strains while iceA2 was present in 58 of all the samples analyzed. Sequenced data indicated that rdxA and frxA truncation was found only in metronidazoleresistant strains. Mutation in the rdxA gene may contribute more significantly than frxA gene to the high level of resistance to metronidazole. Two point mutations (A2142G and A2143G) in the 23SrRNA genes of clarithromycin- resistant strains were detected. The findings from this study support the need to continue monitoring the antibiotic susceptibility of H. pylori in the Eastern Cape Province of South Africa to guide empiric treatment of such infection.