Quorum Sensing Inhibition by Sponge-Associated Bacillus Species: Suppressing Pseudomonas aeruginosa Virulence Factors

<b>Background/Objectives</b>: The growing threat of antimicrobial resistance has intensified the search for alternative therapeutic approaches. Quorum sensing (QS) inhibition, which disrupts bacterial communication and virulence, represents a promising approach to mitigating infection. Given the complexity of the sponge holobiont, sponge-associated microorganisms may demonstrate QS inhibitory properties and serve as potential sources of novel anti-virulence agents. This study aimed to investigate the QS inhibitory potential of sponge-associated <i>Bacillus</i> species against <i>Pseudomonas aeruginosa</i>, a multidrug-resistant pathogen that relies on QS for virulence regulation. <b>Methods</b>: Ninety-eight bacterial isolates were obtained from seven intertidal South African sponges. Biosensor-based sandwich assays using <i>Chromobacterium violaceum</i> identified 15 isolates with putative QS inhibition (QSI) activity, including five classified as <i>Bacillus</i> species via 16S rRNA gene sequencing. Crude extracts from these isolates, cultivated in medium Mannitol (Mann) and medium 5294, were screened for their ability to inhibit QS-regulated virulence factors in <i>P. aeruginosa</i>. <b>Results</b>: Extracts, particularly from medium 5294, exhibited significant QSI activity without cytotoxic effects. The five most potent extracts, i.e., <i>Bacillus mobilis</i> SP2-AB7 (5294), <i>Bacillus wiedmannii</i> SP5-AB7 (Mann), <i>B. mobilis</i> SP2-AB7 (Mann), and <i>Bacillus cereus</i> SP1-AB4 (Mann and 5294), inhibited both Las- and Rhl-regulated virulence factors, including pyocyanin, pyoverdine, elastase, protease, rhamnolipid production, motility, and initial adhesion, achieving inhibition rates of up to 93% (<i>p</i> < 0.05). Molecular analysis confirmed the presence of the <i>aiiA</i> lactonase gene in key isolates, while GC-MS and FTIR profiling revealed medium-specific differences in metabolite production. <b>Conclusions</b>: Sponge-associated <i>Bacillus</i> species from KwaZulu-Natal exhibit robust QSI activity against <i>P. aeruginosa</i>, highlighting their potential as sources of alternative anti-virulence agents. Further characterization and in vivo validation are needed to assess their therapeutic application in combatting resistant infections.