The biochemistry and genetics of herbicide-induced changes in antibiotic resistance in Salmonella enterica and Escherichia coli.
Thesis DisciplineCellular and Molecular Biology
Degree GrantorUniversity of Canterbury
Degree NameMaster of Science
Antibiotic resistance is a leading global health concern and is no longer a threat of the future (World Health Organization, 2014). In order to combat resistance effectively, the different factors that influence its development need to be understood. Previous work from this lab showed that exposure of Escherichia coli and Salmonella enterica serovar Typhimurium to three commercial herbicide formulations caused a change in the antibiotic response including resistance to antibiotics from different classes, with up to a 6-fold change in susceptibility being observed (Kurenbach et al., 2015). Formulations have both active ingredients and adjuvants. To investigate which components of the herbicide formulations could be responsible for the observed effects, S. Typhimurium was separately exposed to either the pure active ingredients, dicamba, 2,4-dichlorophenoxyacetic acid (2,4-d) or glyphosate, or two surfactants, carboxymethyl cellulose (CMC) or Tween80. The pattern of antibiotic resistance phenotypes caused by the active ingredients varied in direction and intensity, but was similar to the pattern observed for the formulations. Tween80 and CMC either increased or had no effect on resistance to the tested antibiotics. These results suggested that the direction of the change in resistance to different antibiotics is determined by the active ingredient but may be modulated by other components in the formulation. The observed effects occurred within recommended application rate concentrations, and some were within the maximum residue limits (MRLs) set by the Codex Alimentarius Commission (Codex Alimentarius Commission, 2012). In addition, the surfactants tested are also found in processed food products and both induced increases in resistance to some antibiotics at concentrations within those recommended for food (Codex Alimentarius Commission, 2015). The effects of different chemicals that induce similar changes in antibiotic resistance were also shown to be additive, which suggests that bacteria exposed to low concentrations of multiple compounds may still experience the observed changes in antibiotic susceptibility. Two approaches were taken to determine the mechanism by which herbicides alter the antibiotic resistance phenotype of bacteria. Firstly, the broad-spectrum efflux pump inhibitor phenylalanine-arginine-β-naphthylamide (PAβN) (Lomovskaya & Bostian, 2006) was used to demonstrate that the effects were likely caused by changes in the levels of antibiotic efflux. Then, to test specific influx and efflux components, a selection of E. coli knockout strains from the Keio collection were used. The change in response of these strains to antibiotics upon exposure to the herbicides was determined and compared to the wild-type. Although the data was highly variable, it showed that the multidrug efflux pump AcrAB-TolC was likely involved in the phenomenon.