Daniel Berdejo, William L Kelley, Rafael Pagán, Diego García-Gonzalo, Adriana Renzoni, Beatriz Chueca
Food preservation by the use of essential oils (EOs) is being extensively studied, because of the antimicrobial properties of their individual constituents (ICs). Three resistant mutants (termed CAR, CIT and LIM) of Escherichia coli MG1655 were selected by subculturing with the ICs carvacrol, citral and (+)-limonene oxide, respectively. These derivative strains showed increased MIC values to ICs and concomitant enhanced resistance to various antibiotics (ampicillin, trimethoprim, chloramphenicol, tetracycline, kanamycin, novobiocin, norfloxacin, cephalexin and nalidixic acid) compared with the parental strain (WT).Whole genome sequencing (WGS) of these hyper-resistant strains permitted the identification of single nucleotide polymorphisms (SNPs) and deletions in comparison with WT.In order to analyze the contribution of these mutations to the increased antimicrobial resistance detected in hyper-resistant strains, derivative strains were constructed by allelic reversion. A role of SoxR D137Y missense mutation in CAR was confirmed in growth under the presence of some ICs and antibiotics, and in its tolerance to ICs, but not to heat lethal treatments. In CIT, increased resistance relied on a contribution between several detected SNPs resulting in a frameshift in MarR and an in-frame GyrB ΔG157 mutation. Finally, both the insertion resulting in an AcrR frameshift and large chromosomal deletions found in LIM, correlated with the hyper-resistant phenotype of this strain. The nature of the obtained mutants suggests intriguing links to cellular defense mechanisms previously implicated in antibiotic resistance.IMPORTANCE The antimicrobial efficacy of ICs has been proven over the years together with their potential to improve traditional heat treatments by reducing treatment intensity and consequently adverse effects on food quality. However, the mechanisms of bacterial inactivation by ICs are still not well understood, in contrast to antibiotics. We performed WGS of three ICs hyper-resistant E. coli strains. The information provided a detailed insight into the mechanisms of bacterial resistance arising from exposure to carvacrol, citral, and (+)-limonene oxide. Future experiments will undoubtedly yield additional insights into genes and pathways contributing to the acquisition of endogenous ICs resistance.