Purification and mechanistic characterisation of bacillomycin D homologues from Bacillus velezensis PD9 with antibiofilm activity against methicillin-resistant Staphylococcus aureus.

Antimicrobial resistance continues to undermine treatment of multidrug-resistant infections, necessitating new antimicrobial agents with distinct mechanisms of action. Bacillus velezensis PD9 (BvPD9), isolated from Heterotrigona itama propolis, produces antibacterial lipopeptides; however, the active bacillomycin D homologues and their mechanisms remain insufficiently characterised. Here, an optimised extraction and chromatographic purification workflow was developed, enabling selective enrichment of two bioactive fractions putatively assigned by LC-TOF-MS/MS as C14-enriched bacillomycin D and a C14/C15-bacillomycin D homologue mixture. The C14-enriched bacillomycin D fraction exhibited selective activity against Staphylococcus spp. and vancomycin-sensitive Enterococcus faecium (VSEF), with minimum inhibitory concentrations (MICs) of 16-32 µg/mL, whereas the C14/C15 homologue mixture demonstrated broader antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and selected ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) (MICs 16-512 µg/mL), with the lowest MIC (16 µg/mL) observed against VSEF. Both compounds retained activity following heat exposure (40-80 °C), wide pH variation (4-12), and proteolytic enzymes. The C14/C15 homologue mixture was prioritised for further evaluation because of its broader antibacterial spectrum and displayed concentration-dependent inhibition of biofilm formation, membrane permeabilisation, and ultrastructural alterations, together with changes in bacterial DNA-content distribution without detectable direct DNA binding. Preliminary safety evaluation demonstrated concentration-dependent haemolysis and cytotoxicity at higher concentrations, while zebrafish embryos showed concentration-dependent mortality with an LC50 of 109.54 µg/mL at 120 hpf. Serial passaging resulted in a four-fold MIC increase only during the final passages, whereas chloramphenicol showed no detectable MIC change under the same conditions. Overall, the findings demonstrate antibacterial and membrane-associated activities of PD9-derived bacillomycin D-enriched fractions and provide a basis for further investigation of individually purified homologues.