Top-Down Characterization of an Antimicrobial Sanitizer, Leading From Quenchers of Efficacy to Mode of Action

Abstract

We developed a top-down strategy to characterize an antimicrobial, oxidizing sanitizer, which has diverse proposed applications including surface-sanitization of fresh foods, and with benefits for water resilience. The strategy involved finding quenchers of antimicrobial activity then antimicrobial mode of action, by identifying key chemical reaction partners starting from complex matrices, narrowing down reactivity to specific organic molecules within cells. The sanitizer electrolyzed-water (EW) retained partial fungicidal activity against the food-spoilage fungus Aspergillus niger at high levels of added soils (30-750 mg mL −1), commonly associated with harvested produce. Soil with high organic load (98 mg g −1) gave stronger EW inactivation. Marked inactivation by a complex organics mix (YEPD medium) was linked to its protein-rich components. Addition of pure proteins or amino acids (≤1 mg mL −1) fully suppressed EW activity. Mechanism was interrogated further with the yeast model, corroborating marked suppression of EW action by the amino acid methionine. Pre-culture with methionine increased resistance to EW, sodium hypochlorite, or chlorine-free ozonated water. Overexpression of methionine sulfoxide reductases (which reduce oxidized methionine) protected against EW. Fluoroprobe-based analyses indicated that methionine and cysteine inactivate free chlorine species in EW. Intracellular methionine oxidation can disturb cellular FeS-clusters and we showed that EW treatment impairs FeS-enzyme activity. The study establishes the value of a top-down approach for multi-level characterization of sanitizer efficacy and action. The results reveal proteins and amino acids as key quenchers of EW activity and, among the amino acids, the importance of methionine oxidation and FeS-cluster damage for antimicrobial mode-of-action.

Composition of Challenge Substance in Standardized Antimicrobial Efficacy Testing of Wound Antimicrobials Is Essential to Correctly Simulate Efficacy in the Human Wound Micro-Environment

“…Most likely, the addition of peptides to the challenge substance is the reason for the improved simulation of the clinical conditions by the low burden of the modified peptide-challenge (TSB + LB), which in terms more adequately approximates the acute and chronic wound micro-environment with its higher degree of proteolytic degradation. Peptides, such as peptones, have shown a significant influence on the efficacy of wound irrigation products in earlier studies [ 20 , 21 , 22 ]. Therefore, the highly reactive chlorine-based species are more likely to interact with the exposed amino acid aspects of peptides than the more complex structure of macro proteins, resulting in a faster consumption of active agents by side-interactions rather than wanted interaction with microorganisms [ 23 , 24 ].…”

Microbial Metal Resistance within Structured Environments Is Inversely Related to Environmental Pore Size

“…The beads were briefly washed and then transferred to fresh medium to allow surviving cells to grow into the medium, with back-extrapolation of resultant growth parameters serving to estimate that survival. The growth curves were analyzed by exponential regression to estimate the y -intercepts (after bead outgrowth) by extrapolation from the exponential phase of growth ( 22 ). These values were compared between treatments to estimate relative viable cell numbers in structures after the original treatments.…”

“…An aliquot (300 μl) of the medium, now containing cells, was transferred to a 48-well microtiter plate, and further growth was monitored during incubation at 24°C with shaking in a BioTek Powerwave XS microplate spectrophotometer and OD 600 measurement every 30 min. Resulting growth curves were used to infer back to a theoretical starting OD using multiple regression ( 22 ).…”

Evolving challenges and strategies for fungal control in the food supply chain

“…EW has been in use for a number of years, but its potential for treating fresh produce is still being explored and developed ( Youssef and Hussien, 2020 ). The resilience of EW activity to the presence of incidental soil, often associated with fresh produce, has recently been established ( Wohlgemuth et al. , 2020 ).…”