Multi-omic dereplication of antibiotic production by diffusion chamber isolated bacteria from Australian soils

Soil bacteria are a major source of clinically useful antibiotics, yet the majority of soil-dwelling microorganisms remain uncultivable by standard laboratory methods. To access this untapped microbial diversity, we employed microbial diffusion chambers to isolate bacteria from ten Australian soil samples. A total of 1,218 bacterial isolates were recovered, representing a diverse collection spanning 61 genera from 32 families, covering the major known phyla of soil bacteria. Antibiotic activity screening revealed that 16% of isolates inhibited the growth of at least one of E. coli or S. aureus, with 120 isolates displaying activity against multidrug-resistant pathogens including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE). Mass spectrometry-based dereplication using GNPS identified known antibiotics in 33% of bioactive strains, including actinomycin D, nonactins, and valinomycin. Genomic analysis confirmed the presence of corresponding biosynthetic gene clusters (BGCs), while targeted analysis of selected strains uncovered production of additional antibiotics such as nigericin and streptothricin that were not initially detected by mass spectrometry. Our results demonstrate that diffusion chambers enhance bacterial recovery from soil and show the benefits of a combined pipeline including bioactivity screening, mass spectrometry, and genomics for effective antibiotic dereplication and discovery. Impact StatementThis study delivers a significant advance in natural product discovery by demonstrating that microbial diffusion chambers can dramatically improve the recovery of diverse soil bacteria with antibiotic-producing potential from Australian soil samples. By integrating in situ cultivation with high-throughput screening, mass spectrometry-based dereplication and genome mining, this study yielded more than 1,200 bacterial isolates--including many with activity against multidrug-resistant pathogens--and confirms the production of both known and previously undetected antibiotics. The discovery of streptothricin production via genomics, despite its absence from mass spectrometry data, underscores the power of a multi-layered dereplication strategy. This work not only validates the utility of diffusion chambers for unlocking the "rare biosphere" of uncultivable microbes but also highlights critical methodological refinements to diversify antibiotic-producing strains beyond Streptomyces. The findings will inform and accelerate future efforts to discover urgently needed antibiotics from environmental microbiomes. Data SummaryThe whole-genome sequences were deposited in the National Center for Biotechnology Information National Library of Medicine under BioProject accession number PRJNA1272227.