In Vitro Effects of Thymol-beta-D-Glucopyranoside on Salmonella enterica Serovar Typhimurium and Escherichia coli K88

Abstract

Although thymol is bactericidal against many pathogens in vitro, its in vivo effectiveness against pathogens in the lower gastrointestinal tract is limited because of its rapid absorption in the proximal gut. Thymol-beta-D-glucopyranoside (beta-thymol), a conjugated form of thymol, can deliver thymol to the lower gastrointestinal tract and has shown antibacterial effects. In the present study, we examined the in vitro effects of beta-thymol on Salmonella enterica serovar Typhimurium (ST) and Escherichia coli K88 (K88). We inoculated one-half strength Mueller-Hinton broth with 5.8 +/- 0.09 log CFU/ml novobiocin- and naladixic acid-resistant (NN) ST (NVSL 95-1776) and 5.1 +/- 0.09 log CFU ml(-1) NN-resistant K88, with or without porcine feces (0.1% [wt/vol]) (fecal incubations). The resultant bacterial suspensions were distributed under N-2 to triplicate sets of tubes to achieve initial concentrations of 0, 3, 6, and 12 mM for ST treatments and 0, 3, 12, and 30 mM for K88 treatments. Samples were incubated at 39 degrees C and then plated onto NN-containing brilliant green agar and NN-containing MacConkey agar; ST and K88 CFU concentrations were determined via 10-fold dilutions, and viable cell counts were performed at 0, 6, and 24 h. No differences in ST CFU counts were observed in beta-thymol treated tubes without the added porcine feces (i.e., pure culture) at 6 or 24 h. However, in tubes that contained fecal incubations, ST CFU counts were reduced (P < 0.05) from controls at 6 h in tubes treated with 6 and 12 mM beta-thymol, whereas in tubes treated with 3, 6, and 12 mM beta-thymol the CFU counts were reduced (P < 0.05) at 24 h. No differences were observed in K88 CFU counts in pure culture or in fecal incubations at 6 h, but K88 CFU counts were reduced (P < 0.05) in both pure and fecal incubations at 24 h. The results from this study demonstrate that beta-thymol, in the presence of fecal suspensions, has anti-Salmonella and anti-E. coli effects, suggesting a role of beta-glycoside-hydrolyzing microbes for the release of bactericidal thymol from beta-thymol.