16S rRNA Sequencing for Molecular Identification of Marine Actinomycetes from Marine Soil Sediments: An In-vitro Study

Introduction: Despite the existence of numerous antimicrobial drugs in the market, some pathogenic organisms are increasingly becoming resistant to them. Therefore, it is imperative to seek novel antibiotics that can combat resistant pathogenic organisms. Actinomycetes are an excellent source of secondary metabolites, including antibiotics. The marine ecosystem serves as a rich resource for many strains of actinomycetes, as it is an unexplored habitat teeming with millions of different types of microorganisms. In recent years, marine actinomycetes have gained recognition as one of the most abundant sources of unique and varied metabolites. Given that actinomycetes are a widely distributed population, studies on them have gained momentum in India and other nations due to their production of unique chemicals with bioactive properties. Aim: To isolate and molecularly identify novel actinomycete species through 16S rRNA sequencing from the Vellar estuary in the Cuddalore district, Tamil Nadu, India. Materials and Methods: The present study was an in-vitro study in which isolation and molecular identification were conducted using soil samples collected from the Vellar estuary in July 2020, with the study concluding in June 2024. Most bioactive compounds are primarily produced by actinomycete species. Hence, this study focused on isolating various actinomycete species; a serial dilution technique was employed and samples were inoculated using the pour plate method. Based on colony colour and morphology, the grown colonies were inoculated separately on starch casein agar medium, supplemented with 0.1% chloramphenicol and 0.1% griseofulvin to prevent contamination. Following the isolation of pure cultures, DNA was extracted using the cetyl trimethyl ammonium bromide method. Electrophoresis and polymerase chain reaction methods were performed for molecular identification. A 16S rRNA sequencing method was utilised for the molecular identification of marine actinomycetes. Isolating and cultivating actinomycetes may prove difficult due to their slow growth rate and complex nutritional requirements. Results: Four actinomycetes were isolated and designated as VM05, VM06, VM07 and VM08. Among the four isolates, VM05 and VM06 underwent molecular identification using 16S rRNA sequencing. Molecular characterisation revealed that VM06 was identified as Glutamicibacter mysorens KGM1 and VM05 as Streptomyces griseoflavus KRSG1. The accession numbers for the sequences, which were stored in a gene bank, were PP280550 and PP278023. Conclusion: Glutamicibacter myorens KGM1 and Streptomyces griseoflavus KRSG1 were identified using 16S rRNA sequencing molecular methods. Therefore, the marine environment is a promising source for novel microorganisms.