Prevalence, Antimicrobial Susceptibility and Molecular Characterization of Helicobacter pylori

Abstract

Helicobacter pylori colonizes in almost half of the population of developed and nearly all inhabitants of developing countries. We reported seroprevalence of H pylori infection in Islamabad (2008-09), the capital city of Pakistan. Subjects were analyzed in two study groups; Dyspeptic patients on the basis of Rome III criteria (n=196) and non-dyspeptic controls (n=118). H pylori status was determined using a commercial ELISA kit and its association was determined with risk factors such as age, sex, body mass index, marital status, educational level, residence, income group, use of NSAIDs and tobacco. Our results showed similar seroprevalence of -53% in both the groups. Low education (p<0.03) and lower socioeconomic status (p<0.03) were predictors of H pylori seropositivity in dyspeptic patients, whereas, increasing age (p<O.OOI), lesser education (p<0.03) and married subjects (p<0.02) were significantly associated with H pylori seropositivity in non-dyspeptic controls. One hundred and nine cases and controls each were matched for gender and age. The presence or absence of depression was based on Beck Depression Inventory (BDI-II). Univariate analyses did not reveal significant differences in marital status, residence, income level, and presence of H pylori antibody in the case versus control groups. Dyspeptic patients had a significantly lower level of education as compared to controls [641109 (60%) versus 44/109 (40%), p<O.OOI]. Forty-two percent (461109) dyspeptic patients were positive for depression as compared to 11 % (l211 09) of controls (p<O.OOI). A higher BMI predicted dyspepsia (25.03 for cases and 23.51 for controls, p<0.05). Two hundred and sixty nine dyspeptic patients were analyzed for determining the frequency and predictors of depression among dyspeptic patients. Socio-demographic variables as well as dyspeptic symptoms and important causes of dyspeptic symptoms were analyzed. On univariate analysis, mild depression was found associated with lower education status (p<O.OOl), a lesser income (p<0.02), and lower socioeconomic status (p<O.Ol) as well as rural residence (p<0.03). Smoking, alcohol-use, H pylori infection status, gender and dyspepsia were not found to be associated with depression. On multivariate analysis, education and income group remained significantly associated with mild depression. Clinically significant depression was found to be associated with lower education and rural residence on the multivariate analysis The effects of transport media, transportation time, culture media, blood from different sources, and effectiveness of a low cost atmosphere generation system (AGS) on isolation and culturing of H pylori was studied. Biopsy samples from 56 dyspeptic patients were cultured. Normal saline and Brain Heart infusion (BHI) broth were effective as transport media and showed isolation rate of 87.5% when samples were cultured within 2 hours of sampling. Isolation rate decreased when culturing was delayed upto 8 hours. Columbia blood agar and BHI agar showed 100% and 94% efficiency on subculturing, respectively. Thirty six (73.5%) out of 49 isolates were successfully subcultured under microaerophilic atmosphere generated by low-cost envelope system as compared to that produced by standard Campygen gas pack in anaerobic jar. Human or sheep blood in 5% concentration was found optimal for growth. In our study, antimicrobial susceptibility profile of H pylori isolates indicated a highest resistance for metronidazole (>90%), clarithromycin (38-40%), amoxycillin (40-49%), tetracycline (25-28%) and levofloxacin (18-20%) when analyzed using disc diffusion and agar dilution methods. Agar dilution method gave slightly higher resistance rates for all antibiotics tested as compared to disc diffusion method. The isolates were confirmed to be H pylori on the basis of 16S rRNA amplification and sequencing, and presence of tsaA gene, coding for AhpC, once reported a specie specific protein for H pylori. Fusion gene for H pylori tsaA in plasmid vector pGEX-6p-2 (pGEX-tsaA) and its prokaryotic expression system (pGEX-tsaA-E. cloi BL21DE3) was constructed and immunogenicity of induced recombinant fusion protein (rGST-AhpC) was determined. Target recombinant fusion protein was detected by SDS-PAGE. Western blot analysis demonstrated effective immunoreactivity against commercial anti-H pylori antibody. BLAST for nucleotide sequence analysis revealed 100% sequence homology of cloned pGEX-tsaA gene with corresponding sequence of the published gene. Reverse transcription (RT)-PCR for genes downstream to each of three cheV (cheVl, che V2, che V3) and fli genes (jliM, fliN and fliY) from H pylori specific deletion mutant strains was carried out to verify whether these mutants (catmut insertions) affect transcription of downstream genes or not. These analyses were part of two studies conducted to understand functional analysis of H pylori flagellar switch proteins and differential effects of cheV genes on motility of the bacterium. RT-PCR results verified that gene downstream to each of cheV andjli genes expressed as expected, confirming that genes deletion did not affect transcription of their respective downstream genes.