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. 2024 Oct;16(5):e70027.
doi: 10.1111/1758-2229.70027.

Investigating the antimicrobial and antibiofilm properties of marine halophilic Bacillus species against ESKAPE pathogens

Monica M Murphy  1 , Eamonn P Culligan  1 , Craig P Murphy  1
Affiliations

Investigating the antimicrobial and antibiofilm properties of marine halophilic Bacillus species against ESKAPE pathogens

Monica M Murphy et al. Environ Microbiol Rep. 2024 Oct.

Abstract

Antimicrobial resistance (AMR), known as the "silent pandemic," is exacerbated by pathogenic bacteria's ability to form biofilms. Marine compounds hold promise for novel antibacterial drug discovery. Two isolates from preliminary saltwater environment screening demonstrated antimicrobial activity and were subsequently identified as Bacillus subtilis MTUA2 and Bacillus velezensis MTUC2. Minimum inhibitory concentrations (MICs), minimum biofilm inhibition concentrations (MBICs) and minimum biofilm eradication concentrations (MBECs) required to prevent and/or disrupt bacterial growth and biofilm formation were established for MRSA, Staphylococcus aureus, Acinetobacter baumannii and Escherichia coli. The metabolic activity within biofilms was determined by the 2,3,5-triphenyltetrazolium chloride assay. Both Bacillus species exhibited unique antimicrobial effects, reducing MRSA and S. aureus planktonic cell growth by 50% and sessile cell growth for S. aureus and E. coli by 50% and 90%, respectively. No effect was observed against A. baumannii. Significant MBIC and MBEC values were achieved, with 99% inhibition and 90% reduction in MRSA and S. aureus biofilms. Additionally, 90% and 50% inhibition was observed in E. coli and A. baumannii biofilms, respectively, with a 50% reduction in E. coli biofilm. These findings suggest that the mode of action employed by B. subtilis MTUA2 and B. velezensis MTUC2 metabolites should be further characterized and could be beneficial if used independently or in combination with other treatments.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

FIGURE 1
FIGURE 1
Diagrammatic representation of putative bacteriocin gene clusters of interest from B. subtilis MTUA2 (subtilosin_(SboX) and subtilomycin) and B. velezensis MTUC2 (plantazolicin, mersacidin and amylocyclin).
FIGURE 2
FIGURE 2
Antimicrobial activity of the cell free supernatant (CFS; 50% concentration) of B. subtilis MTUA2 (left) and B. velezensis MTUC2 (right) against (A) Gram‐positive and (B) Gram‐negative test strains was measured at A600. All experiments were performed in triplicate and the results are expressed as mean ± standard error (SEM). Significant difference is denoted at ***p < 0.001, **p < 0.01, *p < 0.05, compared to the untreated control (denoted as 100% bacterial growth).
FIGURE 3
FIGURE 3
Erradication effect of B. subtilis MTUA2 (left) and B. velezensis MTUC2 (right) CFS on pre‐formed biofilms of (A) Gram‐positive and (B) Gram‐negative test strains. Following exposure to CFS, the pre‐formed biofilms were incubated at 37°C for 24 h. The pre‐formed biofilms of both Gram‐positive and Gram‐negative test strains were stained with 1% crystal violet and assessed by measuring at A595. Results are expressed as mean compared to the untreated control denoted as 100% bacterial growth. Values are the means of four biological repeats (n = 3 technical repeats in each biological repeats).
FIGURE 4
FIGURE 4
Effect of B. subtilis MTUA2 and B. velezensis MTUC2 CFS on the metabolic activity of (A) Gram‐positive and (B) Gram‐negative biofilm cells. The established biofilms of Gram‐positive and Gram‐negative test strains were incubated in the presence of CFS concentrations at 37°C for 24 h. The metabolic activity of Gram‐positive and Gram‐negative biofilm cells were a‐lysed using the TTC reduction assay and assessed by measuring as A500. The results are expressed as mean compared to the untreated control denoted as 100% bacterial growth. Values are the means of four biological repeats (n = 3 technical repeats in each biological repeat).
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