Whole-Genome Sequencing and Genetic Analysis Reveal Novel Stress Responses to Individual Constituents of Essential Oils in Escherichia coli

Abstract

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 of ICs and concomitantly enhanced resistance to various antibiotics (ampicillin, trimethoprim, chloramphenicol, tetracycline, kanamycin, novobiocin, norfloxacin, cephalexin, and nalidixic acid) compared to those for the parental strain (wild type [WT]). Whole-genome sequencing (WGS) of these hyperresistant strains permitted the identification of single nucleotide polymorphisms (SNPs) and deletions in comparison to the WT. In order to analyze the contribution of these mutations to the increased antimicrobial resistance detected in hyperresistant strains, derivative strains were constructed by allelic reversion. A role of the SoxR D137Y missense mutation in CAR was confirmed by growth in the presence of some ICs and antibiotics and by its tolerance to ICs but not to lethal heat treatments. In CIT, increased resistance relied on contributions by 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 were correlated with the hyperresistant 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 E. coli strains that are hyperresistant to ICs. The information provided 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 resistance to ICs.

Keywords: carvacrol; citral; heat treatment; limonene; mechanisms of resistance; pulsed electric fields; whole-genome sequencing.

FIG 1

Genomic maps of the chromosomes of the Escherichia coli MG1655 derivative strains CAR, CIT, and LIM.

FIG 2

Inactivation of Escherichia coli WT strain MG1655 (●) and its derivative strain CAR (A), CIT (B), or LIM (C) (○); a derivative strain from ICs with the reverted genotype, BC010 (A), BC020 (B), or BC030 (C) (▲); and the WT strain with a mutant genotype, BC022 (B) or BC032 (C) (△), after treatment with 200 μl/liter of carvacrol. Data are means ± standard deviations (error bars). Horizontal gray dotted lines represent the limit of detection (5 log₁₀ cycles).

FIG 3

Inactivation of Escherichia coli WT strain MG1655 (●) and its derivative strain CAR (A), CIT (B), or LIM (C) (○); a derivative strain from ICs with the reverted genotype, BC010 (A), BC020 (B), or BC030 (C) (▲); and the WT strain with a mutant genotype, BC022 (B) or BC032 (C) (△), after treatment with 300 μl/liter of citral. Data are means ± standard deviations (error bars). Horizontal gray dotted lines represent the limit of detection (5 log₁₀ cycles).

FIG 4

Inactivation of Escherichia coli WT strain MG1655 (●) and its derivative strain CAR (A), CIT (B), or LIM (C) (○); a derivative strain from ICs with the reverted genotype, BC010 (A), BC020 (B), or BC030 (C) (▲); and the WT with a mutant genotype, BC022 (B) or BC032 (C) (△), after treatment with 500 μl/liter of (+)-limonene oxide. Data are means ± standard deviations. Horizontal gray dotted lines represent the limit of detection (5 log₁₀ cycles).

FIG 5

Inactivation of Escherichia coli WT strain MG1655 (●) and its derivative strain CAR (A), CIT (B), or LIM (C) (○); a derivative strain from ICs with a reverted genotype, BC010 (A), BC020 (B), or BC030 (C) (▲); and the WT strain with a mutant genotype, BC022 (B) or BC032 (C) (△), after heat treatment at 55°C. Data are means ± standard deviations (error bars). Horizontal gray dotted lines represent the limit of detection (5 log₁₀ cycles).