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author:("kanade, Dilip")
1.  A pmoA-based study reveals dominance of yet uncultured Type I methanotrophs in rhizospheres of an organically fertilized rice field in India 
3 Biotech  2016;6(2):135.
Rice fields are one of the important sources of anthropogenic methane. Methanotrophs can oxidize up to 30 % of the produced methane and thus have a pivotal environmental role in methane mitigation. India occupies the largest region under rice cultivation; however, most of the studies done on methanotrophic communities have focused on the Northern region. We studied methanotrophic community of a flooded, organically fertilized rice field using pmoA clone library approach. Organic rice fields impose a more serious threat as they produce more methane. pmoA gene is the main functional gene which is primarily used for taxonomical analysis of methanotrophs. Our results showed that the pmoA clone libraries from two growth stages of rice were dominated by pmoA sequences which were very distant from cultivated Type Ia methanotrophic genera (80–82 % nucleotide similarity) indicative of the presence of a putatively novel genus. We designated this group of clones as ‘rice field clones’ as this also includes many pmoA sequences originating from other rice fields. Thus, our current knowledge of methanotroph diversity from Indian rice fields has been expanded revealing that a substantial portion of methanotrophic diversity is unexplored.
Electronic supplementary material
The online version of this article (doi:10.1007/s13205-016-0453-3) contains supplementary material, which is available to authorized users.
PMCID: PMC4910840
Methanotrophs; Rice fields; Molecular approach; Clone libraries; pmoA: gene for particulate methane monooxygenase subunit
2.  Genome sequencing of multidrug resistant novel Clostridium sp. BL8 reveals its potential for pathogenicity 
Gut Pathogens  2014;6:30.
The human gut microbiome is important for maintaining the health status of the host. Clostridia are key members of the human gut microbiome, carrying out several important functions in the gut environment. Hence understanding the role of different Clostridium species isolated from human gut is essential. The present study was aimed at investigating the role of novel Clostridium sp. isolate BL8 in human gut using genome sequencing as a tool.
The genome analysis of Clostridium sp. BL8 showed the presence of several adaptive features like bile resistance, presence of sensory and regulatory systems, presence of oxidative stress managing systems and presence of membrane transport systems. The genome of Clostridium sp. BL8 consists of a wide variety of virulence factors like phospholipase C (alpha toxin), hemolysin, aureolysin and exfoliative toxin A, as well as adhesion factors, proteases, Type IV secretion system and antibiotic resistance genes. In vitro antibiotic sensitivity testing showed that Clostridium sp. BL8 was resistant to 11 different tested antibiotics belonging to 6 different classes. The cell cytotoxicity assay confirmed the cytotoxic effect of Clostridium sp. BL8 cells, which killed 40% of the Vero cells after 4 hrs of incubation.
Clostridium sp. BL8 has adapted for survival in human gut environment, with presence of different adaptive features. The presence of several virulence factors and cell cytotoxic activity indicate that Clostridium sp. BL8 has a potential to cause infections in humans, however further in vivo studies are necessary to ascertain this fact.
PMCID: PMC4115488  PMID: 25076986
Clostridium; Virulence; Antibiotic resistance; Ion Torrent PGM™
3.  Comparative Genome Analysis of Megasphaera sp. Reveals Niche Specialization and Its Potential Role in the Human Gut 
PLoS ONE  2013;8(11):e79353.
With increasing number of novel bacteria being isolated from the human gut ecosystem, there is a greater need to study their role in the gut ecosystem and their effect on the host health. In the present study, we carried out in silico genome-wide analysis of two novel Megasphaera sp. isolates NM10 (DSM25563) and BL7 (DSM25562), isolated from feces of two healthy individuals and validated the key features by in vitro studies. The analysis revealed the general metabolic potential, adaptive features and the potential effects of these isolates on the host. The comparative genome analysis of the two human gut isolates NM10 and BL7 with ruminal isolate Megasphaera elsdenii (DSM20460) highlighted the differential adaptive features for their survival in human gut. The key findings include features like bile resistance, presence of various sensory and regulatory systems, stress response systems, membrane transporters and resistance to antibiotics. Comparison of the “glycobiome” based on the genomes of the ruminal isolate with the human gut isolates NM10 and BL revealed the presence of diverse and unique sets of Carbohydrate-Active enzymes (CAZymes) amongst these isolates, with a higher collection of CAZymes in the human gut isolates. This could be attributed to the difference in host diet and thereby the environment, consequently suggesting host specific adaptation in these isolates. In silico analysis of metabolic potential predicted the ability of these isolates to produce important metabolites like short chain fatty acids (butyrate, acetate, formate, and caproate), vitamins and essential amino acids, which was further validated by in vitro experiments. The ability of these isolates to produce important metabolites advocates for a potential healthy influence on the host. Further in vivo studies including transcriptomic and proteomic analysis will be required for better understanding the role and impact of these Megasphaera sp. isolates NM10 and BL7 on the human host.
PMCID: PMC3832451  PMID: 24260205
4.  Changes in human gut flora with age: an Indian familial study 
BMC Microbiology  2012;12:222.
The gut micro flora plays vital role in health status of the host. The majority of microbes residing in the gut have a profound influence on human physiology and nutrition. Different human ethnic groups vary in genetic makeup as well as the environmental conditions they live in. The gut flora changes with genetic makeup and environmental factors and hence it is necessary to understand the composition of gut flora of different ethnic groups. Indian population is different in physiology from western population (YY paradox) and thus the gut flora in Indian population is likely to differ from the extensively studied gut flora in western population. In this study we have investigated the gut flora of two Indian families, each with three individuals belonging to successive generations and living under the same roof.
Denaturation gradient gel electrophoresis analysis showed age-dependant variation in gut microflora amongst the individuals within a family. Different bacterial genera were dominant in the individual of varying age in clone library analysis. Obligate anaerobes isolated from individuals within a family showed age related differences in isolation pattern, with 27% (6 out of 22) of the isolates being potential novel species based on 16S rRNA gene sequence. In qPCR a consistent decrease in Firmicutes number and increase in Bacteroidetes number with increasing age was observed in our subjects, this pattern of change in Firmicutes / Bacteroidetes ratio with age is different than previously reported in European population.
There is change in gut flora with age amongst the individuals within a family. The isolation of high percent of novel bacterial species and the pattern of change in Firmicutes /Bacteroidetes ratio with age suggests that the composition of gut flora in Indian individuals may be different than the western population. Thus, further extensive study is needed to define the gut flora in Indian population.
PMCID: PMC3511239  PMID: 23013146
Indian population; Firmicutes/Bacteroidetes ratio; Human gut microflora; YY-paradox
5.  Comparison of 16S rRNA gene sequences of genus Methanobrevibacter 
BMC Microbiology  2004;4:20.
The phylogeny of the genus Methanobrevibacter was established almost 25 years ago on the basis of the similarities of the 16S rRNA oligonucleotide catalogs. Since then, many 16S rRNA gene sequences of newly isolated strains or clones representing the genus Methanobrevibacter have been deposited. We tried to reorganize the 16S rRNA gene sequences of this genus and revise the taxonomic affiliation of the isolates and clones representing the genus Methanobrevibacter.
The phylogenetic analysis of the genus based on 786 bp aligned region from fifty-four representative sequences of the 120 available sequences for the genus revealed seven multi-member groups namely, Ruminantium, Smithii, Woesei, Curvatus, Arboriphilicus, Filiformis, and the Termite gut symbionts along with three separate lineages represented by Mbr. wolinii, Mbr. acididurans, and termite gut flagellate symbiont LHD12. The cophenetic correlation coefficient, a test for the ultrametric properties of the 16S rRNA gene sequences used for the tree was found to be 0.913 indicating the high degree of goodness of fit of the tree topology. A significant relationship was found between the 16S rRNA sequence similarity (S) and the extent of DNA hybridization (D) for the genus with the correlation coefficient (r) for logD and logS, and for [ln(-lnD) and ln(-lnS)] being 0.73 and 0.796 respectively. Our analysis revealed that for this genus, when S = 0.984, D would be <70% at least 99% of the times, and with 70% D as the species "cutoff", any 16S rRNA gene sequence showing <98% sequence similarity can be considered as a separate species. In addition, we deduced group specific signature positions that have remained conserved in evolution of the genus.
A very significant relationship between D and S was found to exist for the genus Methanobrevibacter, implying that it is possible to predict D from S with a known precision for the genus. We propose to include the termite gut flagellate symbiont LHD12, the methanogenic endosymbionts of the ciliate Nyctotherus ovalis, and rat feces isolate RT reported earlier, as separate species of the genus Methanobrevibacter.
PMCID: PMC415545  PMID: 15128464

Results 1-5 (5)