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 -cinnamaldehyde are essential oil (EO) compounds with broad-spectrum antimicrobial activities against foodborne pathogens, including O157:H7. However, little is known regarding direct resistance and cross-resistance development in 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 O157:H7 was able to grow in the presence of sublethal thymol (1/2T), carvacrol (1/2C), or -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 HO) 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 value [ ], <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 O157:H7 adapted to sublethal thymol, carvacrol, or-cinnamaldehyde. The data suggested that caution should be exercised when using EO compounds as food antimicrobials, due to the potential stress resistance development in O157:H7. The present study was designed to understand transcriptomic changes and the potential development of direct and cross-resistance in essential oil (EO)-adapted O157:H7. The results demonstrated altered growth behaviors of O157:H7 during adaptation in sublethal thymol, carvacrol, and -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.

Effects of Sublethal Thymol, Carvacrol, and trans -Cinnamaldehyde Adaptation on Virulence Properties of Escherichia coli O157:H7

“…Similarly, BssS was reported to reduce bacterial biofilm formation by influencing cell signaling, and the deletion of the bssS gene was found to increase biofilm formation in E. coli (29). Consistent with these observations, our previous RNA sequencing study (14) revealed that multiple points of biofilm formation pathways were affected, including genes for the biosynthesis of flagella (flhCD, fliAZ, and flgM), poly-N-acetylglucosamine (csrB), colonic acid (wza), and curli (csgD).…”

“…Our previous study (14) using RNA sequencing revealed altered expression of various virulence genes in E. coli O157:H7 grown to early stationary phase in the presence of sublethal Thy, Car, or TC, including genes related to flagellar biosynthesis and function, chemotaxis, biofilm development, type III secretion systems (T3SS), and efflux pumps. However, the biological significance of these transcriptional changes has not yet been verified.…”

Antimicrobial Adaptation in Uropathogenic Escherichia coli

“…It was hypothesised that induced uptake of iron would enhance microbial survival under stress conditions and contribute to microbial oxidative resistance (by sequestering free irons, which generate ROS). This study also reported decreased expression of genes associated with motility and biofilm formation [226]. We have also observed downregulation of flagella Biofilm formation in the cinnamaldehyde exposed strain increased when grown on a plastic surface (Table 5.2).…”

“…A study by Yuan et al [226] showed that exposure to cinnamaldehyde caused an upregulation in genes associated with metabolism but virulence genes associated with biofilm formation were repressed in E.coli. This could explain the decrease in biofilm formation after exposure to cinnamaldehyde.…”

“…RNA sequencing of the cinnamaldehyde exposed isolate revealed tonB expression was upregulated, the promoter region is located within the tonB gene [289] so it is possible that the mutation has affected the promotor regioncausing the upregulation. Proteins associated with iron transport have been shown to be upregulated in cinnamaldehyde exposed E.coli previously [226]. It was hypothesised that induced uptake of iron would enhance microbial survival under stress conditions and contribute to microbial oxidative resistance (by sequestering free irons, which generate ROS).…”