Jaishree Paul

Real-time analysis of mucosal flora in patients with inflammatory bowel disease in India.

ABSTRACT Mucosa-associated bacterial flora from control individuals and inflammatory bowel disease (IBD) patients were evaluated by real-time analysis using 16S rRNA-based genus-specific primers. Our data show a clear delineation in concentration of bacteria between the predominating and subdominating genera under disease conditions, indicating that the subsets of bacteria participating in the pathogenesis of ulcerative colitis (UC) and Crohns disease (CD) are likely to be different.

Gene discovery in the Entamoeba invadens genome

Entamoeba invadens, a parasite of reptiles, is a model for the study of encystation by the human enteric pathogen Entamoeba histolytica, because E. invadens form cysts in axenic culture. With approximately 0.5-fold sequence coverage of the genome, we were able to get insights into E. invadens gene and genome features. Overall, the E. invadens genome displays many of the features that are emerging from ongoing genome sequencing efforts in E. histolytica. At the nucleotide level the E. invadens genome has on average 60% sequence identity with that of E. histolytica. The presence of introns in E. invadens was predicted with similar consensus (GTTTGT em leader A/TAG) sequences to those identified in E. histolytica and Entamoeba dispar. Sequences highly repeated in the genome of E. histolytica (rRNAs, tRNAs, CXXC-rich proteins, and Leu-rich repeat proteins) were found to be highly repeated in the E. invadens genome. Numerous proteins homologous to those implicated in amoebic virulence, (Gal/GalNAc lectins, amoebapores, and cysteine proteinases) and drug resistance (p-glycoproteins) were identified. Homologs of proteins involved in cell cycle, vesicular trafficking and signal transduction were identified, which may be involved in en/excystation and cell growth of E. invadens. Finally, multiple copies of a number of E. invadens genes coding for predicted enzymes involved in core metabolism and the targets of anti-amoebic drugs were identified.

Biosorption of arsenite (As(+3)) and arsenate (As(+5)) from aqueous solution by Arthrobacter sp. biomass.

In this study we investigated the role of arsenic-resistant bacteria Arthrobacter sp. biomass for removal of arsenite as well as arsenate from aqueous solution. The biomass sorption characteristics were studied as a function of biomass dose, contact time and pH. Langmuir, Freundlich and Dubinin-Radushkevich (D-R) models were applied to describe the biosorption isotherm. The Langmuir model fitted the equilibrium data better than the Freundlich isotherm. The biosorption capacity of the biomass for As+3 and As+5 was found to be 74.91 mg/g (pH 7.0) and 81.63 mg/g (pH 3.0), respectively using 1 g/L biomass with a contact time of 30 min at 28°C. The mean sorption energy values calculated from the D-R model indicated that the biosorption of As+3 and As+5 onto Arthrobacter sp. biomass took place by chemical ion-exchange. The thermodynamic parameters showed that the biosorption of As+3 and As+5 ions onto Arthrobacter sp. biomass was feasible, spontaneous and exothermic in nature. Kinetic evaluation of experimental data showed that biosorption of As+3 and As+5 followed pseudo-second-order kinetics. Fourier transform infrared spectroscopy (FT-IR) analysis indicated the involvement of possible functional groups (‒OH, ‒C\dbond O and ‒NH) in the As+3 and As+5 biosorption process. Bacterial cell biomass can be used as a biosorbent for removal of arsenic from arsenic-contaminated water.

Fluctuations in butyrate-producing bacteria in ulcerative colitis patients of North India.

AIM To study the interplay between butyrate concentration and butyrate-producing bacteria in fecal samples of ulcerative colitis (UC) patients vs control individuals. METHODS Fecal samples were collected from 14 control individuals (hemorrhoid patients only) and 26 UC patients (severe: n = 12, moderate: n = 6, remission: n = 8), recruited by the gastroenterologist at the Department of Gastroenterology, All India Institute of Medical Sciences, New Delhi, India. Disease activity in UC patients was determined by clinical colitis activity index. We employed fluorescent in situ hybridization in combination with flow cytometry to enumerate the clostridium cluster population targeted by 16S rRNA gene probe. Major butyrate-producing species within this cluster were quantified to see if any change existed in control vs UC patients with different disease activity. This observed change was further validated by quantitative polymerase chain reaction. In addition to this, we carried out gas chromatography to evaluate the changes in concentration of major short chain fatty acids (SCFAs), namely acetate, n-butyrate, iso-butyrate, in the above samples. Student t test and Graph pad prism-6 were used to compare the data statistically. RESULTS There was a significant decrease of Clostridium coccoides (control, 25.69% ± 1.62% vs severe, 9.8% ± 2.4%, P = 0.0001) and Clostridium leptum clusters (control, 13.74% ± 1.05% vs severe, 6.2% ± 1.8%, P = 0.0001) in fecal samples of UC patients. Furthermore, we demonstrated that some butyrate-producing members of the clostridial cluster, like Fecalibacterium prausnitzii (control, 11.66% ± 1.55% vs severe, 6.01% ± 1.6%, P = 0.0001) and Roseburia intestinalis (control, 14.48% ± 1.52% vs severe, 9% ± 1.83%, P = 0.02) were differentially present in patients with different disease activity. In addition, we also demonstrated decreased concentrations of fecal SCFAs, especially of n-butyrate (control, 24.32 ± 1.86 mmol/μL vs severe, 12.74 ± 2.75 mmol/μL, P = 0.003), iso-butyrate (control, 1.70 ± 0.41 mmol/μL vs severe, 0.68 ± 0.24 mmol/μL, P = 0.0441) and acetate (control, 39.51 ± 1.76 mmol/μL vs severe, 32.12 ± 2.95 mmol/μL, P = 0.047), in the fecal samples of UC patients. The observed decrease of predominant butyrate producers of clostridial clusters correlated with the reduced SCFA levels in active UC patients. This was further confirmed by the restoration in the population of some butyrate producers with simultaneous increase in the level of SCFA in remission samples. CONCLUSION Our observations indicate that decreases in members of the clostridial cluster resulting in reduced butyrate levels contribute to the etiology of UC.

Real-time analysis of gut flora in Entamoeba histolytica infected patients of Northern India

BackgroundAmebic dysentery is caused by the protozoan parasite Entamoeba histolytica and the ingestion of quadrinucleate cyst of E. histolytica from fecally contaminated food or water initiates infection. Excystation occurs in the lumen of small intestine, where motile and potentially invasive trophozoites germinate from cysts. The ability of trophozoites to interact and digest gut bacteria is apparently important for multiplication of the parasite and its pathogenicity; however the contribution of resident bacterial flora is not well understood. We quantified the population of Bacteroides, Bifidobacterium, Ruminococcus, Lactobacillus, Clostridium leptum subgroup, Clostridium coccoides subgroup, Eubacterium, Campylobacter, Methanobrevibacter smithii and Sulphur reducing bacteria using genus specific primers in healthy (N = 22) vs amebic patients (E. histolytica positive, N = 17) stool samples by Real-time PCR.ResultsAbsolute quantification of Bacteroides (p = .001), Closrtridium coccoides subgroup (p = 0.002), Clostridium leptum subgroup (p = 0.0001), Lactobacillus (p = 0.037), Campylobacter (p = 0.0014) and Eubacterium (p = 0.038) show significant drop in their population however, significant increase in Bifdobacterium (p = 0.009) was observed where as the population of Ruminococcus (p = 0.33) remained unaltered in healthy vs amebic patients (E. histolytica positive). We also report high prevalence of nimE gene in stool samples of both healthy volunteers and amebic patients. No significant decrease in nimE gene copy number was observed before and after the treatment with antiamebic drug.ConclusionsOur results show significant alteration in predominant gut bacteria in E. histolytica infected individuals. The frequent episodes of intestinal amoebic dysentery thus result in depletion of few predominant genera in gut that may lead to poor digestion and absorption of food in intestine. It further disturbs the homeostasis between gut epithelium and bacterial flora. The decrease in beneficial bacterial population gives way to dysbiosis of gut bacteria which may contribute to final outcome of the disease. Increase in the copy number of nimE gene harboring bacteria in our population reflects possible decrease in the availability of metronidazole drug during treatment of amoebiasis.

Biosorption of As(III) Ion on Rhodococcus sp. WB-12: Biomass Characterization and Kinetic Studies

Biomass obtained from arsenic resistant gram positive bacteria Rhodococcus sp. WB-12 was studied for the removal of arsenite from aqueous solution. The biomass sorption characteristic was investigated as a function of biomass doses, contact time, and pH. The Langmiur Freundlich, and Dubinin-Radushkevich (D-R) models were applied to describe the biosorption isotherm. The biosorption capacity of the biomass for As(III) was found to be 77.3 mg/g (pH 7.0) using 1 g/L biomass with the contact time of 30 min at 30°C. Kinetic evaluation of experimental data showed biosorption of As (III) followed pseudo-second-order kinetics. The Fourier transform infrared spectroscopy (FT-IR) analysis indicated the involvement of possible functional groups (-OH, -C=O, -NH) in the arsenite biosorption process. Thus, biomass derived from Rhodococcus sp. WB-12 cells has potential for use as biosorbent for the removal of arsenic from contaminated water.

Influence of sugars on endoglucanase and β-xylanase activities of a Bacillus strain.

SummaryABacillus sp. screened from termite infested soils produced significant amount of endoglucanase and xylanase enzymes when grown on a lignocellulosic substrate, rice husk. Biosynthesis of these enzymes was significantly enhanced by the addition of 0.2% cellobiose or glucose for endoglucanase and xylose for β-xylanase activities. In the actual hydrolyses, glucose and cellobiose at low concentrations acted as activitors of endoglucanase activity whereas cellobiose and xylose acted as inhibitors of β-xylanase activity.

TLR4 D299G polymorphism modulates cytokine expression in ulcerative colitis.

Background: Toll-like receptor 4 (TLR4) is a key cell surface receptor which recognizes lipopolysaccharide that leads to activation of innate immune system. Association of single nucleotide polymorphisms (SNPs) in TLR4 gene with the inflammatory bowel disease is influenced by ethnicity of the study population. Goal: To study association of SNPs in TLR4 gene in inflammatory bowel disease patients and to explore the influence of these SNPs on the level of mRNA expression of targeted cytokines in the ulcerative colitis (UC) biopsies. Methods: Two polymorphisms of TLR4 (D299G, T399I) gene were genotyped by PCR-RFLP in 199 UC, 46 Crohn’s disease (CD) patients, and 201 healthy controls. Expression of inflammatory cytokines was measured by RT-PCR in UC biopsies. Genotypes and allele frequencies were calculated by the Pearson &khgr;2 test, Fisher exact test, Student t test, and ANOVA. Results: TLR4 variant D299G showed significant association, with UC (P=0.009) and CD (P=0.039). T399I showed significant association with UC (P=0.006) but not with CD patients. Transcription of TLR4 (P=0.0006), tumor necrosis factor-&agr; (P=0.0009), interferon-&ggr; (P=0.028), interleukin (IL)-17 (P=0.01), IL-23 (P=0.0034), and IL-10 (P=0.018) were found to be significantly elevated in UC patients as compared to controls. Among UC patients, AG genotype of D299G was associated with decreased mRNA level of TLR4 (P=0.0069), tumor necrosis factor-&agr; (P=0.018), IL-17 (P=0.017), and IL-23 (P=0.011) as compared to AA genotype patients. In GG genotype interferon-&ggr; expression (P=0.014) was significantly decreased as compared to AA genotype. Conclusion: Polymorphisms in TLR4 gene were significantly associated with inflammatory bowel disease in North Indian population and they contribute in modulating transcription of inflammatory cytokines during UC leading to aberrant immune response.

Frequency of single nucleotide polymorphisms in NOD1 gene of ulcerative colitis patients: a case-control study in the Indian population

BackgroundEpidemiological studies have provided enough evidence that genetic factors have an important role in determining susceptibility to IBD. The most significant finding in the IBD research has been identification of mutations in the gene that encodes Nod2 (nucleotide-binding oligomerization domain 2) protein in a subgroup of patients with Crohns disease. However, a very similar gene encoding Nod1 protein still has not been well documented for its association with Ulcerative colitis patients. Detection of polymorphism in NOD1 gene using SNP analysis has been attempted in the present study. We evaluated frequency and significance of mutations present in the nucleotide-binding domain (NBD) of NOD1 gene in context to Indian population.MethodsA total of 95 patients with ulcerative colitis and 102 controls enrolled in the Gastroenterology department of All India Institute of Medical Sciences, New Delhi were screened for SNPs by DHPLC and RFLP techniques. Exon 6 locus in the NBD domain of NOD1 gene was amplified and sequenced. Genotype and allele frequencies of the patients and controls were calculated by the Pearsons χ2 test, Fishers exact test and ANOVA with Bonferronis correction using SPSS software version 12.ResultsWe have demonstrated DHPLC screening technique to show the presence of SNPs in Exon 6 locus of NBD domain of NOD1 gene. The DHPLC analysis has proven suitable for rapid detection of base pair changes. The data was validated by sequencing of clones and subsequently by RFLP analysis. Analyses of SNP data revealed 3 significant mutations (W219R, p = 0.002; L349P, p = 0.002 and L370R, p = 0.039) out of 5 in the Exon 6 locus of NBD domain of the gene that encompasses ATP and Mg2+binding sites. No significant association was observed within different sub phenotypes.ConclusionWe propose that the location of mutations in the Exon 6 spanning the ATP and Mg2+ binding site of NBD in NOD1 gene may affect the process of oligomerization and subsequent function of the LRR domain. Further studies are been conducted at the protein level to prove this possibility.

Molecular Epidemiology of Amoebiasis: A Cross-Sectional Study among North East Indian Population

Background Epidemiological studies carried out using culture or microscopy in most of the amoebiasis endemic developing countries, yielded confusing results since none of these could differentiate the pathogenic Entamoeba histolytica from the non-pathogenic Entamoeba dispar and Entamoeba moshkovskii. The Northeastern part of India is a hot spot of infection since the climatic conditions are most conducive for the infection and so far no systemic study has been carried out in this region. Methodology/Principal Findings Following a cross-sectional study designed during the period 2011–2014, a total of 1260 fecal samples collected from the Northeast Indian population were subjected to microscopy, fecal culture and a sensitive and specific DNA dot blot screening assay developed in our laboratory targeting the Entamoeba spp. Further species discrimination using PCR assay performed in microscopy, culture and DNA dot blot screening positive samples showed E. histolytica an overall prevalence rate of 11.1%, 8.0% and 13.7% respectively. In addition, infection rates of nonpathogenic E. dispar and E. moshkovskii were 11.8% (95% CI = 10.2, 13.8) and 7.8% (95% CI = 6.4, 9.4) respectively. The spatial distributions of infection were 18.2% (107/588) of Assam, 11.7% (23/197) of Manipur, 10.2% (21/207) of Meghalaya, and 8.2% (22/268) of Tripura states. Association study of the disease with demographic features suggested poor living condition (OR = 3.21; 95% CI = 1.83, 5.63), previous history of infection in family member (OR = 3.18; 95% CI = 2.09, 4.82) and unhygienic toilet facility (OR = 1.79; 95% CI = 1.28, 2.49) as significant risk factors for amoebiasis. Children in age group <15 yr, participants having lower levels of education, and daily laborers exhibited a higher infection rate. Conclusions/Significance Despite the importance of molecular diagnosis of amoebiasis, molecular epidemiological data based on a large sample size from endemic countries are rarely reported in the literature. Improved and faster method of diagnosis employed here to dissect out the pathogenic from the nonpathogenic species would help the clinicians to prescribe the appropriate anti-amoebic drug.