Screening for bioactive metabolite-producing bacterial endophytes from Elephantorrhiza elephantina

Abstract

Background: Medicinal plants are important source of natural compounds which are useful in the treatment of various diseases. The rich and diverse bioactive compounds in medicinal plants play a significant role in the commercial production of drugs. The escalating demand for medicinal plants in both developing and developed nations for primary healthcare, coupled with the exploration of pharmacologically novel drugs, has rendered these botanical species susceptible to extinction due to excessive harvesting and habitat degradation. Bacterial endophytes linked to medicinal plants are considered a viable strategy for generating bioactive compounds similar to those found in plants. This is due to their symbiotic association with the host plant and their possession of medicinal properties analogous to those of the plant hosts. The present study aimed to screen, isolate, identify and characterize metabolite-producing bacterial endophytes from Elephantorrhiza elephantina, extract bioactive metabolites, investigate the metabolites' biological activities and identify them using Liquid chromatography/mass spectrometry (LC/MS) analysis.

Methods: Screening of bacterial endophytes was done using the 16S rRNA metagenomics approach. Furthermore, bacterial endophytes were isolated from the surface sterilized rhizome of E. elephantina. To characterize and identify endophytic bacteria, the Gram staining technique and 16S rRNA gene sequencing were employed. Subsequent growth analysis and metabolite production was done, afterwards the extracts of selected endophytes underwent testing for antimicrobial activities against seven pathogenic strains. Additionally, their antioxidant potential was assessed through quantitative radical scavenging activity using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Liquid chromatography-quadruple time-of-flight tandem mass spectrometry (LCQToF-MS) was used to identify bioactive secondary metabolites.

Results: The microbial diversity of the leaves and rhizome consisted of phyla: Proteobacteria, Bacteroidota, Gemmatimonadota, Actinobacteriota, Verrucomicrobiota, Dependentiae, Firmicutes, and Armatimonodata. Five bacterial endophytes were isolated from the rhizome and identified using 16S rRNA, which were classified into three genera (Stenotrophomonas, Microbacterium and Pseudomonas). The crude extract of bacterial endophytes displayed antimicrobial activity against seven pathogenic strains and with the minimum inhibition concentrations ranging from 62.5 - 250 μg/mL. The endophytic bacteria's extract showed DPPH scavenging activity with the inhibition activity ranging from 43 % to 76%. Furthermore, the LC-QToF-MS revealed that the isolates produce secondary metabolites such as, avobenzone, dibutyl Phthalate, 9-Octadecenamide, cis-11-eicosenamide and beauvericin G2, that are known for antimicrobial, antioxidants and anti-inflammatory properties etc.

Conclusion: In this study, the 16S rRNA-based metagenome analysis of the bacterial populations in E. elephantina revealed that the roots have a higher density of bacteria when compared to leaves and that phylum Proteobacteria is the most dominant in both plant samples. The bacterial isolates correlated with the genera that were detected with the 16S rRNA-based metagenomics. In vitro activity assays revealed significant antimicrobial and antioxidant activity, which demonstrated the pharmaceutical potential for the isolates. Metabolites with known antioxidant and antimicrobial properties were identified from all isolates, which was in support of the activity assays. To our knowledge, this represents the initial documentation of bacterial endophytes linked to E. elephantina.