High-Pressure Processing’s (HPP) non-thermal inactivation of cells has been largely incompatible with food products in which the activity of selected cultures is intended (e.g., bio-preservation). This work aims to overcome this limitation using a cocoa butter encapsulation system for freeze-dried cultures that can be integrated with HPP technology with minimal detrimental effects on cell viability or activity capabilities. Using commercially available freeze-dried protective cultures, the desiccated cells survived HPP (600 MPa, 5 °C, 3 min) and subsequently experienced a 0.66-log increase in cell counts during 2 h of incubation. When the same culture was rehydrated prior to HPP, it underwent a >6.07-log decrease. Phosphate-buffered saline or skim milk inoculated with cocoa butter-encapsulated culture up to 24 h before HPP displayed robust cell counts after HPP and subsequent plating (8.37–9.16 CFU/mL). In addition to assessing viability following HPP, the study sought to test the applicability in a product in which post-HPP fermentation is desired While HPP-treated encapsulated cultures initially exhibited significantly delayed fermentative processes compared to the positive controls, by 48 h following inoculation, the HPP samples’ pH values bore no significant difference from those of the positive controls (encapsulated samples: pH 3.83 to 3.92; positive controls: pH 3.81 to 3.85). The HPP encapsulated cultures also maintained high cell counts throughout the fermentation (≥8.95 log CFU/mL).

This work presents a multi-hurdle approach that addresses antimicrobial resistance by minimizing the selective pressure of antimicrobials using a novel colicinogenic–phage system. We have created two synthetic T7 phages (T7-E1 and T7-M) by inserting the gene of colicin E1 (Cea) or colicin M (Cma) into the genome of the T7 phage, thereby adding an additional colicin-based hurdle to the T7 lytic cycle. The colicin–phages' efficacy in suppressing the outgrowth of a T7-resistant sub-population within a mixed culture of Escherichia coli was demonstrated using a challenge matrix design under planktonic and structured conditions. When T7-resistant cells were present at 1% of the total planktonic population, T7-E1 delayed the outgrowth. At 0.1% resistance, T7-M delayed resistant outgrowth, whereas T7-E1 suppressed the resistant sub-population. When T7-E1 and T7-M were combined into a triple-hurdle treatment, the T7-E1/T7-M cocktail completely suppressed a mixed planktonic population of 50% resistance cell concentrations. In structured environments, the colicin–phage treatments formed clear and confluent plaque-like zones of clearing in the mixed populations of 50% resistant cells with a lawn density of 1 × 10 ⁶ CFU/mL. Reducing the lawn density to 1 × 10 ⁵ CFU/mL diminished the multi-hurdle treatments' effectiveness, as demonstrated by localized zones of clearing within turbid bacterial lawns, highlighting the relationship between bacterial lawn density and phage effectiveness in structured environments. Fluctuation assays revealed persistence as the predominant mechanism for overcoming the treatments by T7-sensitive E. coli . Results indicate that T7-M treatment significantly reduces persister formation compared to WT-T7, while T7-E1 unexpectedly increases persister formation significantly. This suggests a complex relationship between antimicrobial stress and persister formation. IMPORTANCE: Antimicrobial resistance (AMR) poses a significant challenge in treating bacterial infections. To address this, we present a multi-hurdle approach that combines the power of different antimicrobials to target resistance. We have weaponized the natural predator of Escherichia coli , the T7-phage, by engineering it to produce toxins called colicins, resulting in a colicin–phage antimicrobial. This multi-hurdled approach aims to decrease resistance risk because survival requires different tactics to overcome the phage and colicin activity, thus adding a hurdle in a bacterium's pathway to resistance. In cases of pre-existing resistance, the colicin effectively controlled the sub-population resistant to the phage. When investigating the emergence of resistance, we discovered that antimicrobial persistence was the predominant survival strategy. These findings reveal an essential slice of the AMR pie by emphasizing bacterial survival tactics that are not based on resistance genes. By expanding our AMR lens to include persistence, we can more effectively address treatment failure.