Untapped bacterial diversity and metabolic potential within Unkeshwar hot springs, India.

Hot springs support diverse and interesting groups of microorganisms adapted to extreme conditions and gaining attention in biotechnological applications. However, due to limitations of cultivation methods, a majority of such extremophiles remain uncultivated and unexplored. The advent of multiple cultivation conditions and specialized culture media could possibly aid to access the unexplored microbial portion of hot springs. In the present study, different media and isolation strategies were applied to isolate hitherto unexplored bacterial taxa in the water samples collected from Unkeshwar hot springs, India. Molecular, phylogenetic and predictive functional characterization of the isolated bacterial population was done using 16S rRNA sequencing coupled with Tax4Fun tools. Furthermore, representative isolates were screened for important enzymes (cellulase, xylanase, amylase, and protease) and heavy metal tolerance (chromium, arsenic) properties. A total of 454 bacterial isolates obtained were mapped into 57 unique bacterial genera and 4 different bacterial phyla. Interestingly, 37 genera not previously isolated from Indian hot springs, were isolated for the first time in the present study. However, most of these genera (23 out of 37) were reported only in metagenomics studies from Indian and global hot springs. Furthermore, around 14 genera not previously cultivated and not detected in metagenomics studies of hot springs are documented here. The metabolic potential was ascertained by determining the abundance of specific genes using in silico based Tax4Fun tool, which identified around 315 metabolic pathways for metabolism of carbohydrates, synthesis of secondary metabolites and degradation of xenobiotic compounds. Bioprospection study revealed that 33 and 25 bacterial genera were positive for enzyme production and resistance to the heavy metals, respectively. The present study revealed the advantages of cultivation methods using a comprehensive multiple isolation approach for exploring untapped and unique bacterial diversity, and also utilities for various biotechnological and environmental applications.

Current Developments and Challenges in Plant Viral Diagnostics: A Systematic Review.

Plant viral diseases are the foremost threat to sustainable agriculture, leading to several billion dollars in losses every year. Many viruses infecting several crops have been described in the literature; however, new infectious viruses are emerging frequently through outbreaks. For the effective treatment and prevention of viral diseases, there is great demand for new techniques that can provide accurate identification on the causative agents. With the advancements in biochemical and molecular biology techniques, several diagnostic methods with improved sensitivity and specificity for the detection of prevalent and/or unknown plant viruses are being continuously developed. Currently, serological and nucleic acid methods are the most widely used for plant viral diagnosis. Nucleic acid-based techniques that amplify target DNA/RNA have been evolved with many variants. However, there is growing interest in developing techniques that can be based in real-time and thus facilitate in-field diagnosis. Next-generation sequencing (NGS)-based innovative methods have shown great potential to detect multiple viruses simultaneously; however, such techniques are in the preliminary stages in plant viral disease diagnostics. This review discusses the recent progress in the use of NGS-based techniques for the detection, diagnosis, and identification of plant viral diseases. New portable devices and technologies that could provide real-time analyses in a relatively short period of time are prime important for in-field diagnostics. Current development and application of such tools and techniques along with their potential limitations in plant virology are likewise discussed in detail.

Biodegradation of mixed polycyclic aromatic hydrocarbons by pure and mixed cultures of biosurfactant producing thermophilic and thermo-tolerant bacteria.

Applicability of thermophilic and thermo-tolerant microorganisms for biodegradation of polycyclic aromatic hydrocarbons (PAHs) with low water solubility is an interesting strategy for improving the biodegradation efficiency. In this study, we evaluated utility of thermophilic and thermo-tolerant bacteria isolated from Unkeshwar hot spring (India) for biodegradation of four different PAHs. Water samples were enriched in mineral salt medium (MSM) containing a mixture of four PAHs compounds (anthracene: ANT, fluorene: FLU, phenanthrene: PHE and pyrene: PYR) at 37 °C and 50 °C. After growth based screening, four potent strains obtained which were identified as Aeribacillus pallidus (UCPS2), Bacillus axarquiensis (UCPD1), Bacillus siamensis (GHP76) and Bacillus subtilis subsp. inaquosorum (U277) based on the 16S rRNA gene sequence analysis. Degradation of mixed PAH compounds was evaluated by pure as well as mixed cultures under shake flask conditions using MSM supplemented with 200 mg/L concentration of PAHs (50 mg/L of each compound) for 15 days at 37 °C and 50 °C. A relatively higher degradation of ANT (92%- 96%), FLU (83% - 86%), PHE (16% - 54%) and PYR (51% - 71%) was achieved at 50 °C by Aeribacillus sp. (UCPS2) and mixed culture. Furthermore, crude oil was used as a substrate to study the degradation of same PAHs using these organisms which also revealed with similar results with the higher degradation at 50 °C. Interestingly, PAH-degrading strains were also positive for biosurfactant production. Biosurfactants were identified as the variants of surfactins (lipopeptide biosurfactants) based on analytical tools and phylogenetic analysis of the surfactin genes. Overall, this study has shown that hot spring microbes may have a potential for PAHs degradation and also biosurfactant production at a higher temperature, which could provide a novel perspective for removal of PAHs residues from oil contaminated sites.

Bioactivities and molecular networking-based elucidation of metabolites of potent actinobacterial strains isolated from the Unkeshwar geothermal springs in India.

The bioactive potential of Actinobacteria endemic to hot springs has rarely been investigated. This study highlights the cultivable diversity and bioactivities of Actinobacteria associated with the Unkeshwar hot springs, India. Potent strains were evaluated for their biosynthetic potentials and metabolite analysis was performed using effective dereplication molecular networking tools. A total of 86 actinobacterial strains were isolated and grouped into 21 distinct genera, based on 16S rRNA gene sequence analysis. These strains included rare members such as Micromonospora, Marmoricola, Actinomadura, Cellulomonas, Cellulosimicrobium, Janibacter, Rothia, Barrentisimonas, Dietzia and Glycomyces. In antimicrobial screening, Micromonospora sp. strain GH99 and Streptomyces sp. strain GH176 were found to be potent antimicrobial strains. The metabolic extracts of these strains exhibited strong antimicrobial activity against Staphylococcus epidermidis (NCIM 2493), Shigella flexneri (NCIM 5265), Klebsiella pneumonia (NCIM 2098), and Salmonella abony (NCIM 2257). The extracts also displayed strong anti-biofilm and anticancer activities against Pseudomonas aeruginosa (NCIM 5029), Acinetobacter junii (NCIM 5188) and breast cancer cell line MCF7, respectively. Both strains also tested positive for the presence of the PKS biosynthetic gene cluster in their genomes. To effectively delineate the secondary metabolites, the extracts were subjected to MS/MS-guided molecular networking analysis. Structurally diverse compounds including the polyketides 22-dehydroxymethyl-kijanolide (GH99 strain) and Abyssomicin I (GH176 strain) were detected in the extracts. Interestingly, Brevianamide F was detected in the extract of Micromonospora, which has previously been mostly found in fungal species. Other compounds such as cyclic tripeptides, Cyclo(l-Pro-d-Ile) and Cyclo(d-Pro-l-Phe), were also identified in this strain. In summary, for the first time, we explored the diversity of Actinobacteria and evaluated their bioactive potential from the Unkeshwar hot springs. The potent strains isolated in the study could be useful in drug discovery programs.

Mechanisms of Plant Tolerance to RNA Viruses Induced by Plant-Growth-Promoting Microorganisms.

Plant viruses are globally responsible for the significant crop losses of economically important plants. All common approaches are not able to eradicate viral infection. Many non-conventional strategies are currently used to control viral infection, but unfortunately, they are not always effective. Therefore, it is necessary to search for efficient and eco-friendly measures to prevent viral diseases. Since the genomic material of 90% higher plant viruses consists of single-stranded RNA, the best way to target the viral genome is to use ribonucleases (RNase), which can be effective against any viral disease of plants. Here, we show the importance of the search for endophytes with protease and RNase activity combined with the capacity to prime antiviral plant defense responses for their protection against viruses. This review discusses the possible mechanisms used to suppress a viral attack as well as the use of local endophytic bacteria for antiviral control in crops.

Investigation of Microbial Diversity in Geothermal Hot Springs in Unkeshwar, India, Based on 16S rRNA Amplicon Metagenome Sequencing.

Microbial diversity in geothermal waters of the Unkeshwar hot springs in Maharashtra, India, was studied using 16S rRNA amplicon metagenomic sequencing. Taxonomic analysis revealed the presence of Bacteroidetes, Proteobacteria, Cyanobacteria, Actinobacteria, Archeae, and OD1 phyla. Metabolic function prediction analysis indicated a battery of biological information systems indicating rich and novel microbial diversity, with potential biotechnological applications in this niche.

Complete metagenome sequencing based bacterial diversity and functional insights from basaltic hot spring of Unkeshwar, Maharashtra, India.

Unkeshwar hot springs are located at geographical South East Deccan Continental basalt of India. Here, we report the microbial community analysis of this hot spring using whole metagenome shotgun sequencing approach. The analysis revealed a total of 848,096 reads with 212.87 Mbps with 50.87% G + C content. Metagenomic sequences were deposited in SRA database with accession number (SUB1242219). Community analysis revealed 99.98% sequences belonging to bacteria and 0.01% to archaea and 0.01% to Viruses. The data obtained revealed 41 phyla including bacteria and Archaea and including 719 different species. In taxonomic analysis, the dominant phyla were found as, Actinobacteria (56%), Verrucomicrobia (24%), Bacteriodes (13%), Deinococcus-Thermus (3%) and firmicutes (2%) and Viruses (2%). Furthermore, functional annotation using pathway information revealed dynamic potential of hot spring community in terms of metabolism, environmental information processing, cellular processes and other important aspects. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of each contig sequence by assigning KEGG Orthology (KO) numbers revealed contig sequences that were assigned to metabolism, organismal system, Environmental Information Processing, cellular processes and human diseases with some unclassified sequences. The Unkeshwar hot springs offer rich phylogenetic diversity and metabolic potential for biotechnological applications.

Detection of Clostridium tetani in human clinical samples using tetX specific primers targeting the neurotoxin.

Tetanus resulting from ear injury remains an important health problem, particularly in the developing world. We report the successful detection of Clostridium tetani using tetX specific primers targeting the Cl. tetani neurotoxin. The sample was obtained from an ear discharge of a case of otogenic tetanus in a 2-year-old male child. Based on the culture results of the ear discharge, Gram staining and virulence testing by genotyping, a diagnosis of tetanus was confirmed. This is the first report from India on the successful detection of Cl. tetani in a human clinical sample using tetX specific primers targeting the Cl. tetani neurotoxin.

Isolation of Streptococcus tigurinus - a novel member of Streptococcus mitis group from a case of periodontitis.

Streptococcus tigurinus is a new member of the Streptococcus viridians group and is closely related to Streptococcus mitis, Streptococcus pneumoniae, Streptococcus pseudopneumoniae, Streptococcus oralis, and Streptococcus infantis. The type strain AZ_3a(T) of S. tigurinus was originally isolated from a patient with infective endocarditis. Accurate identification of S. tigurinus is facilitated only by newer molecular methods like 16S rRNA gene analysis. During the course of study on bacteraemia and infective endocarditis with reference to periodontitis and viridians group of streptococci, a strain of S. tigurinus isolated from subgingival plaque of a patient with periodontitis identified by 16S rRNA gene analysis, which was originally identified as Streptococcus pluranimalium by Vitek 2. Confirmation by 16S rRNA gene analysis showed 99.39% similarity (1476/1485 bp) with S. tigurinus AZ_3a(T) (AORU01000002). To the best of our knowledge, this is the first report of isolation of S. tigurinus from the oral cavity of a periodontitis patient.