Rajesh Kumar Sahoo

Molecular Identification of Acidophilic Manganese (Mn)-Solubilizing Bacteria from Mining Effluents and Their Application in Mineral Beneficiation

ABSTRACT The study of the microbial ecology in extreme acidic environments has provided an important foundation for the development of mineral biotechnology. The present investigation reports the isolation, identification and molecular characterization of indigenous manganese (Mn) solubilizing acidophilic bacterial strains from mine water samples from Odisha, India. Four morphologically distinct bacterial strains showing visible growth on Mn-supplemented plates of varying pH were isolated and identified. Mn solubilizing ability of the isolates was tested by growing them on Mn-supplemented agar plates. The appearance of lightening around the growing colonies of all the isolates demonstrated their Mn solubilizing ability in the medium. 16 S rRNA sequencing was carried out and the bacterial isolates were taxonomically classified as Enterobacter sp. AMSB1, Bacillus cereus AMSB3, Bacillus nealsonii AMSB4 and Staphylococcus hominis AMSB5. The evolutionary timeline was studied by constructing neighbor-joining phylogenetic trees. The ability of acidophilic microorganisms to solubilize heavy metals is supported by five basic mechanisms which include: enzymatic conversion, metal effluxing, reduction in sensitivity of cellular targets, intra- or extracellular sequestration, and permeability barrier exclusion. Such ecological studies undoubtedly will provide insights into Mn biogeochemical processes occurring in leaching environments. The application of acidophilic microbiology in mineral biorecovery and beneficiation has a large future potential.

Quantitative approach to track lipase producing Pseudomonas sp. S1 in nonsterilized solid state fermentation.

Proliferation of the inoculated Pseudomonas sp. S1 is quantitatively evaluated using ERIC–PCR during the production of lipase in nonsterile solid state fermentation an approach to reduce the cost of enzyme production. Under nonsterile solid state fermentation with olive oil cake, Pseudomonas sp. S1 produced 57·9 IU g−1 of lipase. DNA fingerprints of unknown bacterial isolates obtained on Bushnell Haas agar (BHA) + tributyrin exactly matched with that of Pseudomonas sp. S1. Using PCR‐based enumeration, population of Pseudomonas sp. S1 was proliferated from 7·6 × 104 CFU g−1 after 24 h to 4·6 × 108 CFU g−1 after 96 h, which tallied with the maximum lipase activity as compared to control. Under submerged fermentation (SmF), Pseudomonas sp. S1 produced maximum lipase (49 IU ml−1) using olive oil as substrate, while lipase production was 9·754 IU ml−1 when Pseudomonas sp. S1 was grown on tributyrin. Optimum pH and temperature of the crude lipase was 7·0 and 50°C. Crude enzyme activity was 71·2% stable at 50°C for 360 min. Pseudomonas sp. S1 lipase was also stable in methanol showing 91·6% activity in the presence of 15% methanol, whereas 75·5 and 51·1% of activity were retained in the presence of 20 and 30% methanol, respectively. Thus, lipase produced by Pseudomonas sp. S1 is suitable for the production of biodiesel as well as treatment of oily waste water.

Investigation of bacterial diversity of hot springs of Odisha, India.

16S rRNA deep sequencing analysis, targeting V3 region was performed using Illumina bar coded sequencing. Sediment samples from two hot springs (Atri and Taptapani) were collected. Atri and Taptapani metagenomes were classified into 50 and 51 bacterial phyla. Proteobacteria (45.17%) dominated the Taptapani sample metagenome followed by Bacteriodetes (23.43%) and Cyanobacteria (10.48%) while in the Atri sample, Chloroflexi (52.39%), Nitrospirae (10.93%) and Proteobacteria (9.98%) dominated. A large number of sequences remained taxonomically unresolved in both hot springs, indicating the presence of potentially novel microbes in these two unique habitats thus unraveling the importance of the current study. Metagenome sequence information is now available at NCBI, SRA database accession no. SRP057428.

Phylogenetic study of metallo-β-lactamase producing multidrug resistant Pseudomonas aeruginosa isolates from burn patients

The present study was carried out to understand the clonal relationship using enterobacteriaceae repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) among metallo-β-lactamase (MBL) producing multidrug resistant Pseudomonas aeruginosa isolates from burn victims and their susceptibility to commonly used anti-pseudomonal agents. In the present study 94 non-duplicate P. aeruginosa strains from the wound samples of burn patients were included. Identification of the isolates was done by biochemical methods and antibiotic sensitivity was done by disc diffusion method following CLSI (Clinical Laboratory Standard Institute) guidelines. By using imipenem (IPM)-EDTA disk diffusion/double disc synergy method carbapenem resistant organisms were tested for MBL. To define the clonal relationship ERIC-PCR was used. Of the 94 isolates, 18 (19.14%) were found resistant to IPM and MBL production was shown 11 (11.70%) by the IPM-EDTA disc diffusion method. From dendrogram of the ERIC-PCR profile four major clusters were obtained (A, B, C and D). Cluster B contained the majority of the isolates (6 strains 1, 4, 8, 9, 10 and 11). This study using ERIC-PCR of randomly collected isolates exhibits high genetic diversity which rules out cross contamination frequency.

Prevalence of TEM, SHV, and CTX-M genes of extended-spectrum β-lactamase-producing Escherichia coli strains isolated from urinary tract infections in adults

Urinary tract infections (UTIs) are one of the major sources of widespread infectious diseases in the community as well as in the hospitals which increase the cause of morbidity and mortality. Prevalence of extended-spectrum-β-lactamase (ESBL)-producing uropathogenic E. coli isolates has been found to be increased rapidly across the world. The present study was undertaken to find out the frequency of blaTEM, blaCTX-M, and blaSHV genes among E. coli isolates from UTI and detect their sensitivity pattern. A total of 112 non-repeated E. coli isolates obtained from urine samples of UTI diagnosed patients were included in this study. Antibiotic susceptibility test was done by disc diffusion method. Seventy seven (68.75%) isolates were MDR and tested for ESBL. ESBL-positive isolates were screened for blaTEM, blaCTX-M, and blaSHV genes by monoplex PCR (polymerase chain reaction). Among 46 ESBL-producing E. coli isolates, 8.69% harboured all the three bla genes. The blaTEM was the predominant (93.47%) gene followed by blaCTX-M (82.6%) and blaSHV (4.34%). It can be concluded that the prevalence of MDR (multidrug resistance) ESBL-producing E. coli appears to be high and the highest identified gene was blaTEM. The knowledge of resistance pattern can help physician’s select suitable empirical antibiotic regimens, so that antibiotics showing high-resistance pattern can be avoided.

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.

Functional Genome Screening to Elucidate the Colistin Resistance Mechanism.

Antibiogram profile of 1590 clinical bacterial isolates based on thirteen different antimicrobial compounds showed that 1.6% of the bacterial isolates are multidrug resistant. Distribution pattern based on 16S rRNA sequence analysis showed that Pseudomonas aeruginosa constituted the largest group (83.6%) followed by Burkholderia pseudomallei sp. A191 (5.17%), Staphylococcus sp. A261 (3.45%). Among the various antibiotics used, colistin appeared to be the most effective against the Gram negative bacteria. Burkholderia pseudomallei sp. A191 and Pseudomonas aeruginosa sp. A111 showed resistance to 1500 μg/ml and 750 μg/ml of colistin respectively which constitutes 7.7% of the bacterial population. A functional genomics strategy was employed to discover the molecular support for colistin resistance in Burkholderia pseudomallei sp. A191. A pUC plasmid-based genomic expression library was constructed with an estimated library size of 2.1 × 107bp. Five colistin resistant clones were obtained after functional screening of the library. Analysis of DNA sequence of five colistin resistant clones showed homology to two component regularity systems (TCRS) encoding for a histidine kinase (mrgS) and its regulatory component (mrgR). Cross complementation assay showed that mutations in mrgS were sufficient enough to confer colistin resistant phenotype in a sensitive strain.

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.

Statistical optimization for lipase production from solid waste of vegetable oil industry

ABSTRACT The production of biofuel using thermostable bacterial lipase from hot spring bacteria out of low-cost agricultural residue olive oil cake is reported in the present paper. Using a lipase enzyme from Bacillus licheniformis, a 66.5% yield of methyl esters was obtained. Optimum parameters were determined, with maximum production of lipase at a pH of 8.2, temperature 50.8°C, moisture content of 55.7%, and biosurfactant content of 1.693 mg. The contour plots and 3D surface responses depict the significant interaction of pH and moisture content with biosurfactant during lipase production. Chromatographic analysis of the lipase transesterification product was methyl esters, from kitchen waste oil under optimized conditions, generated methyl palmitate, methyl stearate, methyl oleate, and methyl linoleate.

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.