Investigation of rare actinomycetes for novel antimicrobials

Abstract

Nocardia are a genus of ubiquitous environmental bacteria belonging to the phylum Actinobacteria. Genomics has revealed that Nocardia species are endowed with extensive and varied arrays of secondary metabolite biosynthetic gene clusters with the potential to produce natural products that have antibiotic properties. Furthermore, the abundance of such gene clusters within the Nocardia rivals that of Streptomyces, the signature genus among the Actinobacteria, owed to the fact that Streptomyces species have yielded many clinically used antibiotics. This project aimed to address the current antibiotic resistance crisis and the shortfall in new compounds within the drug discovery pipeline. A range of natural product discovery techniques were utilised amongst different Actinobacteria with a particular focus on a collection of species within the generally overlooked genus Nocardia. This study had three primary objectives, the first was to use a traditional, high-throughput, empirical screen of 169 pathogenic actinomycetes predominantly from the genus Nocardia. These isolates were screened for antibiotic activity on 19 distinct growth media against a panel of five highly prevalent, multidrug resistant pathogens (Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecium and Acinetobacter baumannii). Secondly, whole genome sequencing and bioinformatic interrogation of 100 Nocardia species was conducted to assess their genetic potential to biosynthesise natural products. This facilitated the selection of a single Nocardia isolate which possessed a non-ribosomal peptide synthetase locus that appeared to be unique amongst other Nocardia species. The locus was also transcriptionally silent. Bioengineering using promoter refactoring was employed to activate expression of this gene cluster, the product of which might have potential as a novel antimicrobial. Thirdly, by utilisation of liquid chromatography-mass spectrometry (LC-MS), bioinformatics and molecular networking, a metabolomic approach was employed to gain a global secondary metabolic footprint of ten predicted “biosynthetically talented" Nocardia species grown on five distinct media types. This project identified:

(i) A Nocardia sp. with activity against multidrug resistant Acinetobacter baumannii.

(ii) Two Streptomyces isolates (Streptomyces cacaoi and Streptomyces sp.) which exhibited antimicrobial activity against multidrug resistant Escherichia coli and Acinetobacter baumannii respectively. Secondary metabolite extracts from each of these producing isolates were investigated by LC-MS/MS and the resulting spectra was assessed for uniqueness through a dereplication data platform developed specifically for bacterial natural product identification. No hits for previously discovered metabolites were obtained suggesting that the antimicrobials discovered within this project appear to be unique and have potential as new drug leads for today’s ever-decreasing antibiotic discovery pipeline.

(iii) Four distinct families of bioactive secondary metabolites that were produced by multiple Nocardia species following LC-MS/MS and molecular network analysis. The identified secondary metabolites were correlated with genome sequence data to identify their probable biosynthetic origin in Nocardia species.