Stress Resistance Development and Genome-Wide Transcriptional Response of Escherichia coli O157:H7 Adapted to Sublethal Thymol, Carvacrol, and trans-Cinnamaldehyde

Abstract

Thymol, carvacrol, and trans-cinnamaldehyde are essential oil (EO) compounds with broad-spectrum antimicrobial activities against foodborne pathogens, including Escherichia coli O157:H7. However, little is known regarding direct resistance and cross-resistance development in E. coli O157:H7 after adaptation to sublethal levels of these compounds, and information is scarce on microbial adaptive responses at a molecular level. The present study demonstrated that E. coli O157:H7 was able to grow in the presence of sublethal thymol (1/2T), carvacrol (1/2C), or trans-cinnamaldehyde (1/2TC), displaying an extended lag phase duration and a lower maximum growth rate. EO-adapted cells developed direct resistance against lethal EO treatments and cross-resistance against heat (58°C) and oxidative (50 mM H₂O₂) stresses. However, no induction of acid resistance (simulated gastric fluid, pH 1.5) was observed. RNA sequencing revealed a large number (310 to 338) of differentially expressed (adjusted P value [P_adj ], <0.05; fold change, ≥5) genes in 1/2T and 1/2C cells, while 1/2TC cells only showed 27 genes with altered expression. In accordance with resistance phenotypes, the genes related to membrane, heat, and oxidative stress responses and genes related to iron uptake and metabolism were upregulated. Conversely, virulence genes associated with motility, biofilm formation, and efflux pumps were repressed. This study demonstrated the development of direct resistance and cross-resistance and characterized whole-genome transcriptional responses in E. coli O157:H7 adapted to sublethal thymol, carvacrol, or trans-cinnamaldehyde. The data suggested that caution should be exercised when using EO compounds as food antimicrobials, due to the potential stress resistance development in E. coli O157:H7.IMPORTANCE The present study was designed to understand transcriptomic changes and the potential development of direct and cross-resistance in essential oil (EO)-adapted Escherichia coli O157:H7. The results demonstrated altered growth behaviors of E. coli O157:H7 during adaptation in sublethal thymol, carvacrol, and trans-cinnamaldehyde. Generally, EO-adapted bacteria showed enhanced resistance against subsequent lethal EO, heat, and oxidative stresses, with no induction of acid resistance in simulated gastric fluid. A transcriptomic analysis revealed the upregulation of related stress resistance genes and a downregulation of various virulence genes in EO-adapted cells. This study provides new insights into microbial EO adaptation behaviors and highlights the risk of resistance development in adapted bacteria.

Keywords: E. coli O157:H7; RNA sequencing; cross-resistance; direct resistance; essential oil adaptation.

FIG 1

Growth curves of E. coli O157:H7 grown in TSB containing 1.6% (vol/vol) ethanol (control), 0.16 mg/ml thymol (1/2T), carvacrol (1/2C), or trans-cinnamaldehyde (1/2TC) for 24 h at 37°C.

FIG 2

Heat map of genes whose mRNA level significantly changed in Escherichia coli O157:H7 growing in tryptic soya broth containing 1.6% (vol/vol) ethanol (control), 0.16 mg/ml thymol (1/2T), carvacrol (1/2C), or trans-cinnamaldehyde (1/2TC). Three biological replicates were included for each sample.

FIG 3

Expression levels of selected genes related to membrane (pspD, pspG), heat (ibpB), and oxidative (grxA, soxS) stress responses and iron uptake (ECs0632, feoA), in E. coli O157:H7 nonadapted (control) or adapted to a sublethal concentration of thymol (1/2T), carvacrol (1/2C), or trans-cinnamaldehyde (1/2TC), determined by RT-qPCR.