Thymol tolerance in Escherichia coli induces morphological, metabolic and genetic changes

Abstract

Background: Thymol is a phenolic compound used for its wide spectrum antimicrobial activity. There is a limited understanding of the antimicrobial mechanisms underlying thymol activity. To investigate this, E. coli strain JM109 was exposed to thymol at sub-lethal concentrations and after 16 rounds of exposure, isolates with a 2-fold increased minimal inhibitory concentration (MIC) were recovered (JM109-Thy^r). The phenotype was stable after multiple sub-cultures without thymol.

Results: Cell morphology studies by scanning electron microscopy (SEM) suggest that thymol renders bacterial cell membranes permeable and disrupts cellular integrity. ¹H Nuclear magnetic resonance (NMR) data showed an increase in lactate and the lactic acid family amino acids in the wild type and JM109-Thy^r in the presence of thymol, indicating a shift from aerobic respiration to fermentation. Sequencing of JM109-Thy^r defined multiple mutations including a stop mutation in the acrR gene resulting in a truncation of the repressor of the AcrAB efflux pump. AcrAB is a multiprotein complex traversing the cytoplasmic and outer membrane, and is involved in antibiotic clearance.

Conclusions: Our data suggests that thymol tolerance in E. coli induces morphological, metabolic and genetic changes to adapt to thymol antimicrobial activity.

Keywords: Acriflavine resistance regulator; Efflux pump; Escherichia coli; Resistance; Thymol.

Fig. 1

The effects of increasing concentrations of thymol on the growth of the wildtype JM109 E. coli (A), and JM109-Thy^r (B)

Fig. 2

Scanning electron image of E. coli JM109 cells. (A) Thymol untreated wildtype JM109 cells; (B) JM109-Thy^r thymol untreated cells; (C) wildtype JM109 thymol treated cells; (D) JM109-Thy^r thymol treated cells

Fig. 3

PCA-score plot illustrating the effect of different solvents on metabolic footprints derived from E. coli JM109 wildtype and JM109-Thy^r untreated and treated with a sub-lethal concentration of thymol (50μg l^− 1). N = 6 for each sample (JM109thy: wildtype JM109 with thymol; JM109M: JM109 thymol tolerant derivative; JM109Mthy: JM109 tolerant derivative with thymol)

Fig. 4

NMR spectra of JM109 wild-type and JM109-Thy^r strain grown with and without thymol. (A) S-line plot of wild-type JM109 (bottom) and JM109-Thy^r (top) grown without thymol, (B) partially assigned 700 MHz 1D spectra of wild-type (black) and JM109-Thy^r (red). (C) S-line plot of wild-type JM109 grown without thymol (top) and thymol treated (bottom), (D) partially assigned 700 MHz 1D-spectra of wild-type JM109 without thymol (black) and thymol treated (red). (E) S-line plot of JM109-Thy^r grown without thymol (top) and thymol treated (bottom), (F) partially assigned 700 MHz 1D-spectra of JM109-Thy^r grown without thymol (black) and thymol treated (red). Heat map indicates product concentration