High throughput sequencing based detection of SARS-CoV-2 prevailing in wastewater of Pune, West India

Given a large number of SARS-CoV-2 infected individuals, clinical detection has proved challenging. The wastewater-based epidemiological paradigm would cover the clinically escaped asymptomatic individuals owing to the faecal shedding of the virus. We hypothesised using wastewater as a valuable resource for analysing SARS-CoV-2 mutations circulating in the wastewater of Pune region (Maharashtra; India), one of the most affected during the covid-19 pandemic. We conducted a case study in open wastewater drains from December 2020-March 2021 to assess the presence of SARS-CoV-2 nucleic acid and further detect mutations using ARTIC protocol of MinION sequencing. The analysis revealed 108 mutations across six samples categorised into 40 types of mutations. We report the occurrence of mutations associated with B.1.617 lineage in March-2021 samples, simultaneously also reported as a Variant of Concern (VoC) responsible for the rapid increase in infections. The study also revealed four mutations; S:N801, S:C480R, NSP14:C279F and NSP3:L550del not currently reported from wastewater or clinical data in India but reported in the world. Further, a novel mutation NSP13:G206F mapping to NSP13 region was observed from wastewater. Notably, S:P1140del mutation was observed in December 2020 samples while it was reported in February 2021 from clinical data, indicating the instrumentality of wastewater data in early detection. This is the first study in India to conclude that wastewater-based epidemiology to identify mutations associated with SARS-CoV-2 virus from wastewater as an early warning indicator system.

Production and partial purification of alpha-amylase from a novel isolate Streptomyces gulbargensis.

Extracellular amylase production by a newly isolated alkali-thermotolerant strain Streptomyces gulbargensis DAS 131 was optimized and characterized. The highest amylase production was achieved by growing S. gulbargensis DAS 131 in media with 1% starch. Strain exhibited maximal activity at pH 9.0 and 45 degrees C and relatively stable in alkaline conditions (pH 11). Starch and peptone were found to be the good source of carbon and nitrogen with a yield of 2,216.6 and 2,156.1 U, respectively. Maltose and maltotriose were the main end products of starch hydrolysis, indicating alpha-amylase activity. SDS-PAGE analysis revealed a monomeric form with a molecular weight of 55 kDa.

Production, characterization and application of keratinase from Streptomyces gulbargensis.

A Streptomyces gulbargensis newly isolated, thermotolerant feather-degrading bacterial strain was investigated for its ability to produce keratinase enzyme. Maximum keratinolytic activity was observed at 45 degrees C and pH 9.0 at 120 h of incubation. Activity was completely stable (100%) between 30 and 45 degrees C and pH 7.0-9.0, respectively. Addition of starch to the growth medium affects the activity by means of increase in keratinase secretion. After seven days of cultivation, 10-fold increase (14.3 U ml(-1)) in keratinase activity was observed in the presence of 3g starch (per liter) of the medium. The enzyme was monomeric and had a molecular mass of 46 kDa. The enzyme activity was significantly inhibited by CaCl(2) and partly inhibited by EDTA, whereas, Na(2)SO(3) enhance the enzyme activity by 2.9 times more. In addition, native chicken feather was completely degraded at 96 h of incubation. The results obtained showed that newly isolated strain S. gulbargensis could be a useful in biotechnology in terms of valorization of keratin-containing wastes or in the leather industry.

Saccharomonospora saliphila sp. nov., a halophilic actinomycete from an Indian soil.

A Gram-positive, moderately halophilic actinomycete strain, designated YIM 90502(T), was isolated from a sample of muddy soil collected from Gulbarga, Karnataka Province, India, and subjected to a polyphasic taxonomical analysis. The isolate grew optimally at 28 degrees C and in the presence of 10 % (w/v) NaCl. The isolate was characterized chemotaxonomically as having meso-diaminopimelic acid in the cell-wall peptidoglycan and galactose and arabinose as whole-cell sugars. The predominant menaquinone was MK-9(H(4)), while MK-8(H(4)) was found in smaller amounts. The phospholipids were phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine. The major fatty acids were iso-C(16 : 0) (49.2 %) and C(17 : 1)omega6c (9.1 %). The DNA G+C content of the isolate was 71.8 mol%. A phylogenetic tree based on 16S rRNA gene sequences showed that the isolate fell within the evolutionary radiation encompassed by the genus Saccharomonospora. 16S rRNA gene sequence similarity between strain YIM 90502(T) and the type strains of Saccharomonospora species ranged from 92.42 % (with Saccharomonospora xinjiangensis CCTCC AA 97021(T)) to 97.45 % (with Saccharomonospora azurea KCTC 9693(T)). Levels of DNA-DNA relatedness between strain YIM 90502(T) and S. azurea KCTC 9693(T), Saccharomonospora halophila DSM 44411(T) and Saccharomonospora paurometabolica DSM 44619(T) were 46.0, 41.0 and 42.5 %, respectively. On the basis of phenotypic, phylogenetic and genotypic data, strain YIM 90502(T) was classified in the genus Saccharomonospora as a member of a novel species, for which the name Saccharomonospora saliphila sp. nov. is proposed, with YIM 90502(T) (=KCTC 19234(T) =DSM 45087(T)) as the type strain.

Streptomyces tritolerans sp. nov., a novel actinomycete isolated from soil in Karnataka, India.

A Gram-positive, nonmotile, moderately halophilic, alkali and thermotolerant strain designated DAS 165(T), was isolated from a dry land soil sample from the Gulbarga region, Karnataka province, India. The isolate produced yellow substrate mycelia and gray aerial mycelia on most tested media. Strain DAS 165(T) showed growth in the presence of 5 to 7% NaCl and at 45 degrees C. The DNA G + C content was 69.7%. 16S rRNA gene sequence analysis together with these characteristics consistently assigned strain DAS 165(T) to the genus Streptomyces. The 16S rRNA gene sequence analysis revealed that strain DAS 165(T) was most closely related to S. tendae ATCC 19812(T) (D 63873) with a sequence similarity of 99.6% (three nucleotide differences out of 1,517). Strain DAS 165(T) formed a distinct clade based on analysis of the almost complete sequence and 120-nucleotide variable gamma region of the 16S rRNA gene. Despite the high sequence similarity, strain DAS 165(T) was phenotypically different from S. tendae ATCC 19812(T). DNA-DNA hybridization between these strains was 47% showing that strain DAS 165(T) is a distinct genomic species. Phenetic and genetic results support the classification of strain DAS 165(T) as a new species, for which the name S. tritolerans is proposed, with strain DAS 165(T) as the type strain (=DSM 41899(T )= CCTCCAA 206013(T)).