Irshad Ahmed

Antimicrobial activities of Eugenol and Cinnamaldehyde against the human gastric pathogen Helicobacter pylori

BackgroundEradication of Helicobacter pylori is an important objective in overcoming gastric diseases. Many regimens are currently available but none of them could achieve 100% success in eradication. Eugenol and cinnamaldehyde that are commonly used in various food preparations are known to possess antimicrobial activity against a wide spectrum of bacteria.AimThe present study was performed to assess the in vitro effects of eugenol and cinnamaldehyde against indigenous and standard H. pylori strains, their minimum inhibitory concentrations (MICs) and time course lethal effects at various pH.MethodsA total of 31 strains (29 indigenous and one standard strain of H. pylori ATCC 26695, one strain of E. coli NCIM 2089) were screened. Agar dilution method was used for the determination of drug sensitivity patterns of isolates to the commonly used antibiotics and broth dilution method for the test compounds.ResultsEugenol and cinnamaldehyde inhibited the growth of all the 30 H. pylori strains tested, at a concentration of 2 μg/ml, in the 9th and 12th hours of incubation respectively. At acidic pH, increased activity was observed for both the compounds. Furthermore, the organism did not develop any resistance towards these compounds even after 10 passages grown at sub-inhibitory concentrations.ConclusionThese results indicate that the two bioactive compounds we tested may prevent H. pylori growth in vitro, without acquiring any resistance.

Ancestral European roots of Helicobacter pylori in India

BackgroundThe human gastric pathogen Helicobacter pylori is co-evolved with its host and therefore, origins and expansion of multiple populations and sub populations of H. pylori mirror ancient human migrations. Ancestral origins of H. pylori in the vast Indian subcontinent are debatable. It is not clear how different waves of human migrations in South Asia shaped the population structure of H. pylori. We tried to address these issues through mapping genetic origins of present day H. pylori in India and their genomic comparison with hundreds of isolates from different geographic regions.ResultsWe attempted to dissect genetic identity of strains by multilocus sequence typing (MLST) of the 7 housekeeping genes (atp A, efp, ure I, ppa, mut Y, trp C, yph C) and phylogeographic analysis of haplotypes using MEGA and NETWORK software while incorporating DNA sequences and genotyping data of whole cag pathogenicity-islands (cag PAI). The distribution of cag PAI genes within these strains was analyzed by using PCR and the geographic type of cag A phosphorylation motif EPIYA was determined by gene sequencing. All the isolates analyzed revealed European ancestry and belonged to H. pylori sub-population, hpEurope. The cag PAI harbored by Indian strains revealed European features upon PCR based analysis and whole PAI sequencing.ConclusionThese observations suggest that H. pylori strains in India share ancestral origins with their European counterparts. Further, non-existence of other sub-populations such as hpAfrica and hpEastAsia, at least in our collection of isolates, suggest that the hpEurope strains enjoyed a special fitness advantage in Indian stomachs to out-compete any endogenous strains. These results also might support hypotheses related to gene flow in India through Indo-Aryans and arrival of Neolithic practices and languages from the Fertile Crescent.

Comparative genomics of Helicobacter pylori isolates recovered from ulcer disease patients in England

BackgroundGenomic diversity of H. pylori from many different human populations is largely unknown. We compared genomes of 65 H. pylori strains from Nottingham, England. Molecular analysis was carried out to identify rearrangements within and outside the cag-pathogenicity-island (cag PAI) and DNA sequence divergence in candidate genes. Phylogenetic analysis was carried out based on various high-resolution genotyping techniques.ResultsAnalyses of virulence genes (cag T, cag E, cag A, vac A, ice A, oip A and bab B) revealed that H. pylori strains from England are genetically distinct from strains obtained from other countries. The toxigenic vac A s1m1 genotype was found to be less common and the plasticity region cluster was found to be disrupted in all the isolates. English isolates showed a predominance of ice A1 alleles and a functional proinflammatory oip A gene. The English H. pylori gene pool revealed several Asian/oriental features. This included the predominance of cag A – glr (cag A right junction) motif types III and II (up to 42%), presence of vac A m1c alleles and phylogenetic affinity towards East Asian / Amerindian gene pools based on fluorescent amplified fragment length polymorphism (FAFLP) analysis and glm M sequence analysis.ConclusionOverall, our results demonstrated genetic affinities of H. pylori in England with both European and the Asian gene pools and some distinctive genetic features of virulence genes that may have evolved in this important European population.

Comparing Genomes of Helicobacter pylori Strains from the High-Altitude Desert of Ladakh, India

ABSTRACT The genomic diversity of Helicobacter pylori from the vast Indian subcontinent is largely unknown. We compared the genomes of 10 H. pylori strains from Ladakh, North India. Molecular analysis was carried out to identify rearrangements within and outside the cag pathogenicity island (cag PAI) and DNA sequence divergence in candidate genes. Analyses of virulence genes (such as the cag PAI as a whole, cagA, vacA, iceA, oipA, babB, and the plasticity cluster) revealed that H. pylori strains from Ladakh are genetically distinct and possibly less virulent than the isolates from East Asian countries, such as China and Japan. Phylogenetic analyses based on the cagA-glr motifs, enterobacterial repetitive intergenic consensus patterns, repetitive extragenic palindromic signatures, the glmM gene mutations, and several genomic markers representing fluorescent amplified fragment length polymorphisms revealed that Ladakhi strains share features of the Indo-European, as well as the East Asian, gene pools. However, the contribution of genetic features from the Indo-European gene pool was more prominent.

Polymerase chain reaction based analysis of the cytotoxin associated gene pathogenicity island of Helicobacter pylori from saliva: an approach for rapid molecular genotyping in relation to disease status.

Background and Aims: The genetic composition of the intricate cytotoxin associated gene pathogenicity island (cag PAI) of Helicobacter pylori is known to significantly influence the outcome of the disease. Hence, analysis of complete cag PAI of H. pylori isolated from saliva would be of immense importance in standardizing saliva as a reliable non‐invasive diagnostic specimen and also to evaluate the type of H. pylori infection. The aim of the present study was to analyze the genes of cag PAI of H. pylori for their presence and correlating them with the disease status of the patients.

Microevolution of Helicobacter pylori Type IV Secretion Systems in an Ulcer Disease Patient over a Ten-Year Period

ABSTRACT Helicobacter pylori cagA and vacA genotypes have been used for almost a decade as stable entities to link the severity of gastritis and ulcer disease. We describe here microevolution of the two genomic islands, cag pathogenicity island (cagPAI; 40 kb) and tfs3 (16 kb) from isolates obtained at inclusion (one subclone) and after a 10-year period (two subclones) from a duodenal ulcer patient. Our results indicate microevolution in cagA, cagE, and cag7 genes of the cagPAI and open reading frames G, P, and L in tfs3, which possibly leads to inactivation or pseudogenization of these genes. Interestingly, no significant reduction in the severity of gastroduodenal pathology was found. These results point to an obvious difficulty in correlating the continuously evolving virulence factors such as the cagPAI genes with disease characteristics that appear to remain stable.

Isocitrate dehydrogenase of Helicobacter pylori potentially induces humoral immune response in subjects with peptic ulcer disease and gastritis.

Background H. pylori causes gastritis and peptic ulcers and is a risk factor for the development of gastric carcinoma. Many of the proteins such as urease, porins, flagellins and toxins such as lipo-polysaccharides have been identified as potential virulence factors which induce proinflammatory reaction. We report immunogenic potentials of isocitrate dehydrogenase (ICD), an important house keeping protein of H. pylori. Methodology/Principal Findings Amino acid sequences of H. pylori ICD were subjected to in silico analysis for regions with predictably high antigenic indexes. Also, computational modelling of the H. pylori ICD as juxtaposed to the E. coli ICD was carried out to determine levels of structure similarity and the availability of surface exposed motifs, if any. The icd gene was cloned, expressed and purified to a very high homogeneity. Humoral response directed against H. pylori ICD was detected through an enzyme linked immunosorbent assay (ELISA) in 82 human subjects comprising of 58 patients with H. pylori associated gastritis or ulcer disease and 24 asymptomatic healthy controls. The H. pylori ICD elicited potentially high humoral immune response and revealed high antibody titers in sera corresponding to endoscopically-confirmed gastritis and ulcer disease subjects. However, urea-breath-test negative healthy control samples and asymptomatic control samples did not reveal any detectable immune responses. The ELISA for proinflammatory cytokine IL-8 did not exhibit any significant proinflammatory activity of ICD. Conclusions/Significance ICD of H. pylori is an immunogen which interacts with the host immune system subsequent to a possible autolytic-release and thereby significantly elicits humoral responses in individuals with invasive H. pylori infection. However, ICD could not significantly stimulate IL8 induction in a cultured macrophage cell line (THP1) and therefore, may not be a notable proinflammatory agent.