The effects of sub-lethal concentrations of biocides copper, pyrethrins and atrazine on antibiotic tolerance of Escherichia coli.
Degree GrantorUniversity of Canterbury
Degree NameMaster of Science
Biocides are used in different stages of crop production as herbicides, insecticides and fungicides. The toxicological mechanisms of these chemicals on their target organism is known, however, their sub-lethal effects on microbes are not. Previous work from this laboratory had shown that commercial formulations of herbicides can change the susceptibility of bacteria to antibiotics upon concurrent exposure. This is most likely due to changes in the expression of genes involved in influx and efflux and hence a physiological effect, reversible without requiring mutation.
Out of the 12 combinations of biocide formulations and antibiotics tested, nine exhibited statistically significant differences in the minimum inhibitory concentration (MIC) between treatments in the presence and absence of biocides. The strongest effect was a greater than 30- fold change in MIC following exposure to copper and tetracycline. Where changes in susceptibility due to biocide exposure were observed, minimum inducing concentrations were identified from dose response curves. All observed effects were induced by biocide concentrations lower than the recommended application rate.
The formulation had no observed effect on mutation frequency and the change in susceptibility was displayed uniformly by the population. Thus the effects on phenotype are consistent with adaptive resistance. To test whether efflux pumps were indeed responsible for the observed effects, different pump mutant strains were tested for the effect of copper on tetracycline resistance. The tolerance to tetracycline and copper in the pump mutant strains were significantly lower than the wildtype, indicating that the pump contributes to intrinsic resistance to both substances. In the mutant strains, copper exposure increased the tetracycline MIC 3 to 5 folds. This indicates that the AcrAB-TolC efflux system is not the only contributors to adaptive resistance.
The persistence of the elevated tetracycline tolerant phenotype in the absence of copper fungicide was quantified. Although reversible, the induced phenotype was heritable for approximately 1 generation and the population uniformly reverted to a susceptible phenotype in the second generation post exposure.
Continuous exposure to copper and tetracycline induced a morphological change in most or all exposed bacteria. The morphology changes included filamentation and then return to single cells, depending on exposure time. The morphological changes did not correlate with changes in genotype because the frequency of acquired resistance did not change. Dilution and re-growth experiments were used to determine whether the single cell form was outgrowth of a minority population or arose from filaments. The source of single cells was determined to be filaments.