The interaction-mediated induction of antimicrobials in S. coelicolor by Aspergillus spp. under novel nutrient-deplete conditions.

Abstract

There is an ever-increasing need to identify novel antimicrobials, with Public Health England estimating 10 million deaths globally, owing to antimicrobial resistance by the year 2050 if left unchecked. Streptomyces, also known as nature’s pharmacists, has been a diverse mining source for novel antimicrobials. However, current literature reveals that Streptomyces’ true capacity to produce novel medically useful compounds can only be unleashed in co-culturing experiments that replicate the competitive and challenging microcosm seen in nature. This study aims to produce novel antimicrobial compounds by co-culturing Streptomyces coelicolor wild-type (WT) and mutant strains (M1146, M1152, M1154) with Aspergillus flavus and Aspergillus parasiticus under nutrient-deficient conditions. Minimal liquid medium (NMMP) and a modified version of NMMP, i.e., Weak Strength NMMP (WS NMMP) were utilized to create nutrient-deficient growth media as an environmental challenge with the aims of inducing novel secondary metabolites production in the co-cultured species. WS NMMP, developed for this study contains one-tenth of the carbon source and phosphate buffer solution as compared to regular NMMP. For all quantifiable data obtained in the study, co-cultures were grown on WS NMMP agar plates, at 30°C where Streptomyces was inoculated on Day 0, Aspergillus on Day 2, and plates were observed on Day 8 from bacterial inoculation. Cell-free supernatants from conditions causing Aspergillus growth inhibition were subsequently found to inhibit growth of ESKAPE pathogens (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 43300, Klebsiella pneumoniae ATCC 700603, Acinetobacter baumanii ATCC 19606, Pseudomonas aeruginosa ATCC 27853 & Enterococcus faecalis 51299) and were analysed by Ultra High-Performance Liquid Chromatography- Mass Spectrometry (UHPLC-MS) to identify specific novel metabolites associated with anti-microbial activity. The growth and physical interactions of the co-cultivated species were also monitored using Fluorescence and Scanning Electron Microscopy. Antimicrobial activity was profoundly observed under nutrient-depleted conditions. Inhibition to Aspergillus flavus sporulation around the seeded Streptomyces colony was most significant in mutants, M1146 and M1152 (p = <0.0001, n = 3). The ZOI for A. flavus growth around the seeded Streptomyces colony was 30.67mm ± 1.53 and 22.33mm ± 6.66 when co-cultured with M1152 and M1146 respectively, in contrast to 4.33mm ± 0.58 when co-cultured with WT. Aspergillus parasiticus radial growth was inhibited, with diameter of colony at 43.25mm ± 0.32 when co-cultured with all strains of S. coelicolor, which is a significant reduction from Aspergillus monocultures (51.67mm ± 0.58). Fluorescence images and scanning electron microscopy followed the same trend as the antimicrobial assays and showed a swelling and bursting of fungal hyphae with increased co-culturing periods. UHPLC-MS data was analysed using Compound Discoverer 3.3 Analytical Software. An abundance of known and novel secondary metabolites was produced by the co-cultured pairs (>45,000 compounds per co-culture). Chemical analyses of Log₂Fold changes and Peak Rating comparisons presented an upregulation of medically useful antibiotics and antifungals in mutant co-cultures in comparison with wildtype co-cultures. These data argue that S. coelicolor mutants competing with Aspergillus under nutrient-depleted conditions secrete secondary metabolites with broad range antimicrobial activity.

Keywords: Antimicrobial Resistance; Co-cultured; S. coelicolor mutants; A. flavus; A. parasiticus; Secondary Metabolites; Novel Antimicrobials

Date of Award	26 Feb 2025
Original language	English
Awarding Institution	Edge Hill University
Supervisor	P A Ashton (Director of Studies), CLARE STRODE (Supervisor), RAJEEV SHRIVASTAVA (Supervisor) & CHER-PHENG OOI (Supervisor)