EDTA suppresses bacterial perseverance to 2-phenoxyethanol.

Perseverance is a phenotype in which a small subpopulation of bacteria continues to divide for hours in the presence of antimicrobial concentrations that inhibit bulk population growth, without acquiring genetic resistance. Perseverance has previously been reported only in antibiotic-treated bacteria, but not in preservative-treated bacteria. However, perseverance could limit preservative efficacy, and we investigated this phenomenon in preservative-treated bacteria. Cosmetic products rely on preservatives to control microbial growth; however, preservative types and concentrations are limited for safety reasons, and boosters are often added to enhance efficacy. Some formulations also contain nutrients, such as plant extracts, that promote heterogeneous microbial behavior. Therefore, single-cell analyses are needed to understand these responses and assess preservation risk. In this study, we combined time-lapse microscopy and a microdevice to perform single-cell analysis of Staphylococcus ureilyticus, a potential cosmetic contaminant, when exposed to the preservative 2-phenoxyethanol (PE) and booster ethylenediaminetetraacetate (EDTA). Conventional time-kill assays confirmed that EDTA enhanced the bactericidal effect of PE. Single-cell analysis revealed a subpopulation of cells that underwent up to five divisions over 23.4 h of PE exposure during viable count decline. Pedigree analysis showed that dividing lineages did not share cell fates with their ancestors, supporting a non-mutational perseverance phenotype. Compared with PE alone, the addition of EDTA markedly reduced single-cell division, with most cells dividing once or not at all despite similar population-level kill curves. These results demonstrate that single-cell analysis can reveal microbial behaviors masked in population-based assays and that EDTA may suppress persevering subpopulations that threaten product preservation.IMPORTANCEThis study highlights the significance of investigating the behavior of individual microbes in cosmetic products that employ limited microbial control strategies but may contain ingredients capable of supporting microbial proliferation. Through single-cell analysis of dynamic microbial responses to the preservative phenoxyethanol (PE) and booster ethylenediaminetetraacetate (EDTA), we demonstrate bacterial perseverance in the presence of PE and its suppression by EDTA. By enhancing the bactericidal effects of PE and suppressing perseverance, EDTA reinforces its role as a preservative booster. These findings advance our understanding of preservative performance and provide insights that may improve both the safety and effectiveness of preservatives in cosmetic products.