Wagstaff, CarolAldossari, Huda M2025-08-042025-07-31https://hdl.handle.net/20.500.14154/76094While plant-derived antimicrobial agents are increasingly recognized, the bioactivity of glucosinolates (GLS) from underutilized Brassicaceae species such as Eruca sativa (rocket) remains poorly characterized. E. sativa contains unique GLS, including Diglucothiobeinin, Glucosativin, and Dimeric 4-mercaptobutyl glucosinolate, which are structurally distinct from those in well-characterized Brassicaceae vegetables. This study aimed to elucidate the antimicrobial efficacy and mechanistic pathways of E. sativa-derived GLS against bacterial pathogens, with implications for natural food safety interventions. Methodologically, a series of protocols combining analytical chemistry and molecular microbiology were employed. High-performance liquid chromatography (HPLC) was optimised to isolate four glucosinolate (GLS) compounds (Diglucothiobeinin, Glucosativin, DMB, and 4-Methoxyglucobrassicin), with purity and structural identity confirmed via Ultra High-Performance Liquid Chromatography UHPLC-MS. Antimicrobial assays revealed concentration-dependent inhibition of Escherichia coli K-12, with Diglucothiobeinin exhibiting the highest potency (MIC: 450 ng/μL), followed by glucosativin and DMB (MIC: 800 ng/μL). Glucosativin also demonstrated selective efficacy against antibiotic-resistant Listeria monocytogenes strains. Further studies showed E. coli K-12 metabolized GLS via endogenous β-glucosidases, delaying growth inhibition. Deletion mutants lacking ascB or bglA genes (encoding 6-phospho-β-glucosidases) exhibited impaired GLS consumption and enhanced susceptibility, confirming bacterial enzymatic activation, their involvement in GLS metabolism and subsequent generation of antimicrobial compounds. Screening of the KEIO collection (single-gene knockout strains in the K12 BW25113 strain background) identified genes involved in metabolising GLS. The ΔemrA mutant, which lacks a functional EmrAB-TolC efflux system, exhibited hypersensitivity to both intact GLSs and their hydrolysis products, underscoring the role of this efflux system in mediating resistance. In contrast, the ΔccmE mutant, deficient in cytochrome c maturation, displayed increased resistance, likely due to alterations in redox homeostasis. These findings provide novel insights into the antimicrobial potential of E. sativa-derived GLSs, which combine direct inhibitory effects with bacterial metabolism-dependent activation. The results highlight their potential as natural alternatives against antibiotic-resistant pathogens, particularly in food preservation and sanitation contexts where microbial enzymatic activity may enhance efficacy.108enGlucosinolatesEruca sativaAntimicrobial activityascB or bglAΔemrAΔccmEBacterial β-glucosidasesKEIO collection.Thesis