Pseudomonas aeruginosa accounts for numerous severe acute and chronic infections, exhibiting high morbidity and mortality rates. This phenomenon is linked to their ability to form biofilms, as well as, their inherent and acquired resistance to a broad spectrum of existing antibiotics, which is regulated by their quorum sensing (QS) systems [1]. The commonest QS signal molecules that orchestrate the virulence machinery and biofilm production within P. aeruginosa are the N-acyl homoserine lactones (AHL or HSL), such as C4-HSL, C6-HSL and C12-HSL. Morgan et al. (2025) have documented the extraction of longer fatty acid chain AHLs (>C12-HSL) from the ethyl acetate extract of a clinical strain of P. aeruginosa, which was linked to elevated pyocyanin production and emulsification index [2]. Carbapenems such as imipenem, meropenem, and doripenem are the antibiotics of choice particularly in instances of multidrug resistance P. aeruginosa strains. However, the surging occurrence of carbapenem-resistant P. aeruginosa (CRPA) globally presents a significant challenge, resulting in the designation of carbapenems as a last-resort treatment [3]. Consequently, the inhibition of QS signaling molecules can be employed to prevent the formation of biofilm and the emergence of multiple antibiotic resistances in these bacteria.

Quorum quenching enzymes produced by several environmental bacterial species, including lactone ring cleavers lactonases, amide bond hydrolyzers’ amidases or acylases, and acyl chain reducing oxidoreductases, represent effective strategies for countering P. aeruginosa resistance mechanisms and biofilm formation [4]. The N-acyl homoserine lactonase aiiA from Bacillus species showcased their hydrolyzing capabilities against HSL signals ranging from short to long fatty acid chains, including C4, C6, C8, and C12-HSL [5], [6], [7]. However, the effectiveness of Bacillus cereus QQ enzymes such as aiiA lactonase and penicillin acylase II in combating longer chain HSL (>C12-HSL), which are associated with elevated virulence profiles and antimicrobial resistance, as well as the influence of side chain length on the QQ activity of these enzymes, has seldom been explored [7]. Furthermore, the combination of QQ enzymes with antibiotics and antibacterial peptides offers a promising strategy to address multidrug resistant bacterial infections [8]. However, Bacillus species penicillin acylase can hydrolyze the amide bond found in the side chain of the penicillin antibiotic, while maintaining the integrity of the β-lactam amide bond in penicillin resulting in the formation of 6-aminopenicillanic acid (6-APA) [9]. Wherefore, potential interactions with antibiotics and antibacterial agents may exist and must be investigated before the initiation of combination therapies. On the other hand, the application of gamma irradiation for the decontamination of crude bacterial and plant extracts prior to their use in in vitro and in vivo assays has been investigated in various studies [10]. Nevertheless, the effects of radiation for decontamination purposes on enzymatic activity of QQ enzymes remain inadequately studied. It has been previously reported that optimal enzymatic activities for both AiiA and acylase QQ enzymes was recorded over the pH range of 6–12 and a temperature range of 20–60 °C [11], [12].

This research sought to investigate the QQ activity of the crude bacterial extract derived from the environmental strain ISM25 against clinical P. aeruginosa isolates, one of which is known to produce longer chain HSLs. Initially, we will screen the environmental isolate for the presence of both QQ enzyme genes, aiiA and acylase, and employ In silico methods to create a 3D protein model for both QQ enzymes and assess their physiochemical properties and enzymatic activities against HSLs ligands (both short and long chained (>C12 HSLs) through In silico docking studies. The potential interactions between the penicillin acylase II and carbapenem antibiotics will also be investigated during the In silico docking studies. Afterwards, in vitro biofilm index, GC-MS, and meropenem MIC determination will be conducted on pathogenic P. aeruginosa isolates utilizing the crude ISM25 protein extract to confirm the expression of both QQ enzymes by the ISM25 strain as well as their in silico activities. Finally, we will examine the impact of gamma radiation on the QQ activity of the crude bacterial extract derived from ISM25, which could provide valuable insights into the advantages and disadvantages associated with the decontamination of QQ enzymes through gamma radiation.