Antagonistic effects of lipids against the anti-Escherichia coli and anti-Salmonella activity of thymol and thymol-ß-D-glucopyranoside in porcine gut and fecal cultures in vitro

Authors: Anderson, Robin; LEVENT, GIZEM - Texas A&M University; PETRUJKIC, BRANKO - University Of Belgrade; Harvey, Roger; Hume, Michael; He, Louis; Genovese, Kenneth - Ken; BEIER, ROSS - Retired ARS Employee; Poole, Toni; Crippen, Tawni - Tc; NISBET, DAVID - Retired ARS Employee

Submitted to: Frontiers in Veterinary Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/20/2021
Publication Date: 9/23/2021
Citation: Anderson, R.C., Levent, G., Petrujkic, B., Harvey, R.B., Hume, M.E., He, L.H., Genovese, K.J., Beier, R.C., Poole, T.L., Crippen, T.L., Nisbet, D.J. 2021. Antagonistic effects of lipids against the anti-Escherichia coli and anti-Salmonella activity of thymol and thymol-ß-D-glucopyranoside in porcine gut and fecal cultures in vitro. Frontiers in Veterinary Science. 8. Article 751266. https://doi.org/10.3389/fvets.2021.751266.
DOI: https://doi.org/10.3389/fvets.2021.751266

Interpretive Summary: New strategies are needed to reduce the carriage and spread of foodborne bacteria within food-producing animals and their production environment. Thymol is an essential oil shown to be a potent inhibitor of foodborne pathogens in the laboratory, but the demonstration of antimicrobial effectiveness when fed to animals has been inconsistent. We hypothesized that the inconsistency may be largely due to the fat-loving character of this chemical, which makes it more absorbable and more susceptible to being complexed by fat molecules in the gut. Chemically-linking thymol to glucose to form a new chemical called thymol-ß-D-glucopyranoside can decrease absorption. However, considering that contemporary swine diets often contain 5% or more added fat (to increase dietary energy content and reduce dustiness), we hypothesized that there may be sufficient residual undigested lipid in the large intestinal tract to soak up the new thymol-containing chemical, thereby limiting its availability and subsequent effectiveness. In support of this hypothesis, the antimicrobial effects of the free or new chemically-linked thymol were diminished by as much as 90% in gut contents containing added vegetable oil when compared to antimicrobial effects achieved in the absence of added oil. Inclusion of fat dispersing chemicals, such as bile acids, had little effect on vegetable oil-caused inhibition of the free thymol or new chemically-linked thymol. Based on these results, it seems reasonable to suspect that undigested lipid in the distal gut may limit the effectiveness of the free or new chemically-linked thymol. This research provides important information needed to overcome obstacles limiting the effectiveness of the free and chemically-linked thymol when fed to animals.

Technical Abstract: Strategies are sought to reduce the carriage and dissemination of zoonotic pathogens and antimicrobial resistant microbes within food-producing animals and their production environment. Thymol is an essential oil shown to be a potent bactericide in vitro, but demonstration of its efficacy when fed to animals has been inconsistent largely due to its lipophilicity which limits its passage and subsequent availability in the distal gastrointestinal tract. Conjugation of thymol to glucose to form thymol-ß-D-glucopyranoside can decrease absorption of the conjugate but in vivo passage of effective concentrations to the lower gut remains suboptimal. Considering contemporary swine diets often contain 5% or more added fat (to increase caloric density and reduce dustiness), we hypothesized that at 60 to 80% apparent digestibility there may be sufficient residual undigested lipid in the distal intestinal tract to sequester free or conjugated thymol, thereby limiting the availability and subsequent effectiveness of this biocide. In support of this hypothesis, the anti-Salmonella Typhimurium effects of 6 mM free or conjugated thymol, expressed as log10-fold reductions of colony forming units (CFU) mL-1, were diminished 90 and 58%, respectively, following 24-h in vitro anaerobic fecal incubation (at 39C) with 3% added vegetable oil compared to reductions achieved during culture without added oil (6.1 log10 CFU/mL). The antagonistic effect of vegetable oil on the bactericidal effect of free and conjugated thymol against Escherichia coli K88 tested similarly were diminished 86 and 84%, respectively, compared to reductions achieved in cultures incubated without added vegetable oil (5.7 log10 CFU/mL). Inclusion of taurine (8 mg/mL), bile acids (0.6 mg/mL), or emulsifiers, such as polyoxyethylene (40) stearate (0.2%), Tween 20, or Tween 80 (each at 1%) in the fecal incubations had little effect on vegetable oil-caused inhibition of free or conjugated thymol. Based on these results, it seems reasonable to suspect undigested lipid in the distal gut may limit the effectiveness of free or conjugated thymol. Accordingly, additional research is warranted to learn how to overcome obstacles diminishing bactericidal activity of free and conjugated thymol in the lower gastrointestinal tract of food-producing animals.