Mahendra Gaur

Comparative Analysis of 16S rRNA Gene Illumina Sequence for Microbial Community Structure in Diverse Unexplored Hot Springs of Odisha, India

ABSTRACT Odisha (East India) is home to several hot springs, of which Atri and Taptapani are the two with variation in temperature and located in the Mahanadi geothermal province having altitude 120 and 1800 ft., respectively, above sea level. Average temperature of Atri hot spring is as higher as 58 ± 5°C as compared to 48 ± 5°C of Taptapani. In-depth analysis of the microbial diversity of these hot springs through 16S rRNA deep sequencing analysis, targeting V3 region was performed using Illumina bar-coded sequencing platform. Existence of higher microbial diversity in Atri hot spring (1662 OTUs; 2708 species) as compared to Taptapani (1561 Operational Taxonomic Units [OTUs]; 2045 species) is supported by higher value of diversity indices for Atri (6.24, Shannon; 0.95, Simpson) than Taptapani (4.03, Shannon; 0.79, Simpson), probably due to favorable influence of environmental parameters around it. Irrespective of the four databases (GREENGENE, M5RNA, Ribosomal Database Project [RDP], and Small Subunit [SSU] databases) used for understanding community structure, the dominant phyla in the Atri hot spring were different from the predominant populations in the Taptapani in terms of percentage representation in different databases. From Principal Coordinates Analysis [PCoA] analysis, Atri and Taptapani metagenome, on comparison with other three metagenomes, were found to be matching with the community structure of hot springs of Gujarat, India, but differed from that of saline desert. Furthermore, predicted functional analysis in both the hot springs were found to be affiliated with carbohydrate, amino acids, energy, vitamins and cofactor, nucleotide, membrane transport metabolic pathways, and the genes involved in them, although their intensity of occurrence was varying as analyzed through PICRUSt and Tax4Fun probably due to physicochemical parameters prevailing around each hot spring. The present study for the first time has revealed the differential microbial community structure and predicted functional diversity of Atri and Taptapani hot springs of Odisha in such a great detail.

De Novo transcriptome assembly of Zingiber officinale cv. Suruchi of Odisha

Zingiber officinale Rosc., known as ginger, is an Asian crop, popularly used in every household kitchen and commercially used in bakery, beverage, food and pharmaceutical industries. The present study deals with de novo transcriptome assembly of an elite ginger cultivar Suruchi by next generation sequencing methodology. From the analysis 10.9 GB raw data was obtained which can be available in NCBI accession number SAMN03761185. We identified 41,969 transcripts using Trinity RNA-Seq from ginger rhizome of Suruchi variety from Odisha. The transcript length varied from 300 bp to 8404 bp with a total length of 3,96,40,526 bp and N50 of 1251 bp. To the best of our knowledge, this is the first transcriptome data of an elite ginger cultivar Suruchi released for Odisha state of India which will help molecular biologists to develop genetic markers for identification of cultivars.

Molecular characterization of extended spectrum β-lactamase-producing Enterobacteriaceae strains isolated from a tertiary care hospital

Infectious diseases caused by ESBL producing Enterobacteriaceae are an emerging problem worldwide which increases the empirical treatment failure, hospital cost, rate of morbidity and mortality. This also leads to the Hospital infection outbreak. Present study was undertaken to determine the frequency of blaTEM, blaCTX-M and blaSHV genes among Enterobacteriaceae. A total of 751 non-repeated clinical isolates of Enterobacteriaceae family were included in this study. Antibiotic susceptibility test was done and minimal inhibitory concentration (MIC) against four antibiotics was carried out. Five hundred fifteen multi drug resistant isolates were tested for ESBL by CLSI confirmatory method. Isolates showing ESBL positive by phenotypic method were screened for blaTEM, blaCTX-M and blaSHV genes by monoplex PCR. Two blaTEM and two blaCTX-M amplified products were selected randomly for sequencing. Sequencing data was submitted to NCBI data base. Of the 515 MDR isolates, 140 showed ESBL production by phenotypic method. All the ESBL producing isolates showed resistant to ceftazidime (100%). IMP, TGC and CL drugs could be preferred for the treatment of ESBL producers as these drugs showed a lower rate of resistance. blaTEM gene was the predominant (96.42%) followed by blaCTX-M (75%) and blaSHV (17.85%). All the three bla genes were occurred in 22 (17.14%) isolates. All the phenotypically confirmed ESBL producers were found contain any one of the three bla genes. It is concluded from the study that the blaTEM was predominantly found in Enterobacteriaceae and blaCTX-M gene also seemed to emerging.

Shift in Cyanobacteria Community Diversity in Hot Springs of India

ABSTRACT Two ecologically distinct tropical sulfur-rich alkaline hot springs, Taptapani at 48°C harboring mesophiles and Atri at 58°C comprising thermophiles situated in the Eastern Ghats foothills of India, differ in their geochemical conditions, and provide an interesting platform to unravel the eco-physiological reasons behind the differential cyanobacterial diversity. The predominance of mesophilic Arthronema (83.81%) in Taptapani and shifting predominance of thermophilic Leptolyngbya (96.25%) in Atri as discovered through 16S rRNA gene Illumina sequencing of their metagenomics DNA as a function of temperature are the intriguing features of the present study. Differential presence of the cyanobacterial community at the phylum level in these two hot springs was found to be correlated with the unequal coexistence of Chloroflexi, Taptapani the non-cyanobacteria members and the possible influence of physiochemical parameters including temperature. Variation in cyanobacterial diversity and composition of these hot springs as revealed through sequence analysis were also evinced by respective differences in richness, evenness, and Shannon diversity indices.

Investigation of the microbial community in the Odisha hot spring cluster based on the cultivation independent approach.

Deulajhari hot spring is located in the Angul district of Odisha. The significance of this hot spring is the presence of the hot spring cluster adjacent to the cold spring which attracts the attention of microbiologists to understand the role of physio-chemical factors of these springs on bacterial community structure. Next-generation sequencing technology helps us to depict the pioneering microflora of any ecological niche based on metagenomic approach. Our study represents the first Illumina based metagenomic study of Deulajhari hot spring DH1, and DH2 of the cluster with temperature 65 °C to 55 °C respectively establishing a difference of 10 °C. Comprehensive study of microbiota of these two hot springs was done using the metagenomic sequencing of 16S rRNA of V3‐V4 region extracting metagenomic DNA from the two hot spring sediments. Sequencing community DNA reported about 28 phyla in spring DH1 of which the majority were Chloroflexi (22.98%), Proteobacteria (15.51%), Acidobacteria (14.51%), Chlorobi (9.52%), Nitrospirae (8.54%), and Armatimonadetes (7.07%), at the existing physiochemical conditions like; temperature 65 °C, pH 8.06, electro conductivity 0.020 dSm− 1, and total organic carbon (TOC) 3.76%. About 40 phyla were detected in cluster DH2 at the existing physiochemical parameters like temperature 55 °C, pH 8.10, electro conductivity 0.019 dSm− 1, and total organic carbon (TOC) 0.58% predominated with Chloroflexi (41.98%), Proteobacteria (10.74%), Nitrospirae (10.01%), Chlorobi (8.73%), Acidobacteria (6.73%) and Planctomycetes (3.73%). Approximately 68 class, 107 order, 171 genus and 184 species were reported in cluster DH1 but 102 class, 180 order, 375 genus and 411 species in cluster DH2. The comparative metagenomics study of the Deulajhari hot spring clusters DH1, and DH2 depicts the differential profile of the microbiota. Metagenome sequences of these two hot spring clusters are deposited to the SRA database and are available in NCBI with accession no. SRX1459734 for DH1 and SRX1459735 for DH2.

Isolation of Cellulase Genes From Thermophilies

Abstract Microbial cellulases, a major group of industrial enzymes, have a wide range of applications as far as biotechnological, environmental issues, industrial products, and processes are concerned. Thermophilic bacteria act as a good source for industrial cellulase because of its compatibility during industrial harsh processes, for example, deinking of paper, softening of fabric material, pulp and paper, biostoning and biopolishing of fabric materials, juice, and animal feed, etc. Genome mediated application of thermophilic bacteria in various fields has become a rising fantasy among biotechnologists in the current scenario. The genetic manipulation of thermophilic bacteria may lead to enhanced cellulase production through recombinant technology, an emerging technology towards cellulase gene isolation of thermophilic bacteria and their expression in suitable hosts for enhanced cellulase production. Cellulase gene isolation from various thermophilic bacteria could be done through various approaches, such as a classical approach, whole genome isolation, whole metagenome isolation, and bioinformatics. In this chapter, various methods toward the isolation of cellulase genes from thermophilic bacteria are described which may lead to novel cellulase gene discovery as well as enhanced cellulase enzyme production.

Genetic diversity analysis of 60 ginger germplasm core accessions using ISSR and SSR markers

Abstract Molecular techniques play a critical role in studies of phylogeny and, thus, have been applied to understand the distribution and extent of genetic variation within and between species. In the present study, a genetic analysis was undertaken using molecular markers (9 ISSR and 13 SSR) on 60 ginger cultivars from different regions of the eastern coast of India (Odisha). The data obtained with 22 polymorphic markers revealed moderate to high diversity in the collection. Both ISSR and SSR markers were efficient in distinguishing all the 60 ginger cultivars. A total of 42 and 160 polymorphic bands were observed with ISSR and SSR markers, respectively. However, SSR markers were observed to be better at displaying average polymorphism (63.29%) than ISSR markers (55%). Analysis of molecular variance results showed that 52 and 66% of the variation occurred among different ginger populations, whereas 48 and 34% of the variation was found within populations, respectively, using ISSR and SSR markers, indicating that ginger cultivars display significant genetic diversity at the population level. Principal coordinates analysis and the dendrogram constructed out of combined data of both markers showed grouping of ginger accessions to their respective area of collection, indicating geographical closeness due to genetic similarity irrespective of the relationship that exists at the morphological level.

Genetic diversity study of various β-lactamase-producing multidrug-resistant Escherichia coli isolates from a tertiary care hospital using ERIC-PCR

Background & objectives: The prevalence of multidrug-resistant (MDR) Escherichia coli isolates producing β-lactamase enzyme is a growing problem across the globe. Strain typing is an epidemiologically important tool not only for detecting the cross transmission of nosocomial pathogens but also for determining the source of infection. The present study was conducted to understand the clonal relationship among various β-lactamase-producing MDR E. coli isolates using enterobacterial repetitive intergenic consensus (ERIC) polymerase chain reaction (PCR). Methods: A total of 41 MDR E. coli isolates were randomly collected from various clinical samples and processed. Isolated organisms were tested for antibiotics resistance pattern. Phenotypic detection of metallo β-lactamases (MBL) was carried out by the imipenem-ethylenediaminetetraacetic acid disc diffusion/double-disc synergy test. AmpC enzyme production was tested by a modified three-dimensional extract test. Results: Almost all isolates were found sensitive to colistin. A high percentage of drug resistance was observed in these isolates against ceftazidime (100%), cefotaxime (100%), cefepime (100%), ofloxacin (97.56%), amoxicillin/clavulanic acid (97.56%) and norfloxacin (85.36%). Of the 41 isolates, ESBL producers were found to be predominant, i.e., 22 (53.65%), followed by AmpC (6, 14.63%) and MBL (5, 12.19%). Interpretation & conclusions: At 60 per cent similarity cut-off value, the dendrogram analysis showed that there were a total of 14 unique clusters of ERIC (CL-1 - CL-14) within the 41 E. coli isolates, which revealed the genetic diversity existing between them.

Deciphering the structural community from the Deulajhari hot spring using the next-generation sequencing

Deciphering the microbial diversity of the Deulajhari thermal springs is the major goal of our study. In our study the taxonomic description of the bacterial community structure was deduced from the 1.52 Gb metagenomics sequence size from the Deulajhari hot spring located in the Angul district of Odisha. Covered with the dense foliage, it has a high temperature, i.e. 69°C and alkaline pH i.e. 8.09. Various physiochemical parameter analysis of the sediment like; electro conductivity (0.025dSm-1), total organic carbon content (0.356%), nitrogen (125 kg/ha), phosphorus (7.88 kg/ha) and potassium (169.34 kg/ha) were done. Sediment sample of the Deulajhari hot spring was further processed for the 16S rRNA V3-V4 region by the amplicon metagenome sequencing from community DNA. Approximately 88.12% of the total microbial diversity was represented by the Proteobacteria followed by Bacteroidetes 10.76%, Firmicutes 0.35%, Spirochetes 0.18%, Thermi 0.13% and chloroflexi 0.11% at the phylum level. Thus through the metagenomics sequencing of the Deulajhari hot spring using IIllumina platform, we represent the complete microbial community structure present there in respective of their allocation, profusion and diverse coexisting microbiota.

Comparative transcriptome analysis of ginger variety Suprabha from two different agro-climatic zones of Odisha

Ginger (Zingiber officinale Rosc.), a well-known member of family Zingiberaceae, is bestowed with number of medicinal properties which is because of the secondary metabolites, essential oil and oleoresin, it contains in its rhizome. The drug yielding potential is known to depend on agro-climatic conditions prevailing at the place cultivation. Present study deals with comparative transcriptome analysis of two sample of elite ginger variety Suprabha collected from two different agro-climatic zones of Odisha. Transcriptome assembly for both the samples was done using next generation sequencing methodology. The raw data of size 10.8 and 11.8 GB obtained from analysis of two rhizomes S1Z4 and S2Z5 collected from Bhubaneswar and Koraput and are available in NCBI accession number SAMN03761169 and SAMN03761176 respectively. We identified 60,452 and 54,748 transcripts using trinity tool respectively from ginger rhizome of S1Z4 and S2Z5. The transcript length varied from 300 bp to 15,213 bp and 8988 bp and N50 value of 1415 bp and 1334 bp respectively for S1Z4 and S2Z5. To the best of our knowledge, this is the first comparative transcriptome analysis of elite ginger cultivars Suprabha from two different agro-climatic conditions of Odisha, India which will help to understand the effect of agro-climatic conditions on differential expression of secondary metabolites.