Abstract
The predicament of antimicrobial resistance necessitates the urgent discovery of novel antimicrobial agents. The marine ecosystem represents a valuable reservoir of new antimicrobial molecules, making it an important focus in the search for alternative therapeutic agents. This research explored the antimicrobial potential of marine bacterial samples, obtained from the Sodwana Bay region, KwaZulu Natal, South Africa, through an integrated genomic and metabolomic approach. Bacteria was isolated and initially evaluated for antimicrobial efficacy against clinically relevant strains; Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Mycobacterium smegmatis using the cross-streak method.
Two potent isolates, subjected to whole genome sequencing (WGS) using Illumina technology followed by de novo assembly with Unicycler, and bioinformatic analyses for antimicrobial resistance genes, biosynthetic gene clusters (BGCs), mobile genetic elements, CRISPR-Cas systems, and prophages. Secondary Metabolites (SM) were produced from the active isolates and extracted using the solvent extraction method. Crude extracts were evaluated by minimum inhibitory concentration (MIC) and disc/agar diffusion assays. They were further characterized by gas chromatography–mass spectrometry (GC–MS). Initial cross-streaking assays confirmed the antimicrobial activity of both isolates against the test microorganisms.
Whole genome sequencing and phylogenetic analyses confirmed the taxonomic classification of both isolates within the Bacillus genus. Multiple antimicrobial resistance (AMR) genes were identified, though both strains were predicted to be non-pathogenic by PathogenFinder. B. rugosus EG02 possessed multiple antibiotic resistance genes, including qacG, qacJ, vanY/vanT (vanG cluster), FosBx1, and mphK. Correspondingly, B. safensis EG03 carried resistance genes including qacG, qacJ, and vanY/vanT from the vanG cluster, alongside a Bacillus pumilus cat86 homolog.
Biosynthetic gene cluster (BGC) analysis identified 15 clusters in B. rugosus EG02 encompassing NRPS, PKS, RiPPs (e.g., azole- and RRE-containing), and terpenes. B. safensis EG03 was found to have 16 clusters, including nonribosomal peptide synthetases (NRPS), polyketides (PKS), RiPPs, terpenes, and betalactones, suggesting strong biosynthetic potential for diverse secondary metabolites. Multiple
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MGEs detected in B. rugosus EG02 include Tn4401, IS26, Sul1, Tn21 with high sequence identities, whereas B. safensis EG03 contained an ISBsu1 element (90.16% identity).
CRISPR analyses indicated genomic adaptability. B. rugosus EG02 exhibited two CRISPR arrays but lacked associated cas genes. CRISPR-Cas systems were detected in B. safensis EG03 (Type IIIB with a conserved 32 bp DR sequence). B. rugosus EG02 contained one intact prophage (PHASTER score 100, GC 41.44%) homologous to Bacillus phage SPP1. Prophage analysis for B. safensis EG03 revealed two regions: an intact prophage homologous to Brevib_Jimmer1_NC_029104 (PHASTER score 110, GC 41.24%), and an incomplete prophage homologous to PHAGE_Thermu_OH2_NC_021784 (PHASTER score 20, GC 40.45%).
The two isolates, Bacillus rugosus EG02 (B. rugosus) and Bacillus safensis EG03 (B. safensis), displayed differential antimicrobial activity. B. rugosus EG02 showed MICs of 2.5 mg/mL against S. aureus, 5.0 mg/mL against E. coli, 10.0 mg/mL against K. pneumoniae, and 20 mg/mL against M. smegmatis. Disc diffusion confirmed S. aureus as most susceptible (19.5 mm). B. safensis EG03 exhibited MICs of 0.3125 mg/mL against E. coli, 0.625 mg/mL against S. aureus, 10.0 mg/mL against K. pneumoniae, and 5.0 mg/mL against M. smegmatis. Disc diffusion showed high efficacy against E. coli (21.0 mm) and S. aureus (20.4 mm), comparable to ciprofloxacin.
Gas chromatography–mass spectrometry of crude extracts identified numerous SM, including dimethyl sulfone, carbamic acid phenyl ester, nonadecanoic acid, and 2-aminonicotinic acid, compounds associated with antimicrobial properties. This research underscores the genomic potential of Bacillus species derived from marine environment as sources of new antimicrobial compounds and provides a foundational understanding of their resistance mechanisms, genomic plasticity, and the bioactive metabolites they produce.