Effects of sub-lethal concentrations of copper ammonium acetate, pyrethrins and atrazine on the response of Escherichia coli to antibiotics

Abstract

Background: Antibiotic resistance in human and animal pathogens is mainly the outcome of human use of antibiotics. However, bacteria are also exposed to thousands of other antimicrobial agents. Increasingly those exposures are being investigated as co-selective agents behind the rapid rise and spread of resistance in bacterial pathogens of people and our domesticated animals. Methods: We measured the sub-lethal effects on antibiotic tolerance of the human pathogen/commensal Escherichia coli caused by exposure to three common biocide formulations based on either copper, pyrethrins, or atrazine as active ingredients. The influence of the efflux pump AcrAB-TolC was investigated using deletion strains, and the persistence of observed effects was determined. Results: Some effects were seen for all biocides, but the largest effects were observed with copper in combination with the antibiotic tetracycline. The effect was caused by both the induction of the adaptive efflux system and by chelation of the antibiotic by copper. Finally, persistence of the adaptive response was measured and found to persist for about two generations. Conclusions: Through a combination of microbe-chemical and chemical-chemical interactions, humanity may be creating micro-environments in which resistance evolution is accelerated.

Keywords: antibiotic resistant bacteria; antibiotics; atrazine; biocides; copper; pyrethrins.

Figure 1.. Change in EoP when E. coli BW25113 is (grey) or is not (black) exposed to biocides.

The x-axes scale is antibiotic concentrations in µg/mL. Biocide concentrations used were 450 µg/mL for copper ammonium acetate, 140 µg/mL for pyrethrin, and 1000 µg/mL for atrazine. Values are means of at least three independent experiments; error bars are standard errors (SEM, with SEM=standard deviation/√n). Asterisks indicate P-values for antibiotic*herbicide interaction terms (see Materials and Methods). * P<0.05; ** P<0.01; *** P<0.001; ns, not significant.

Figure 2.. Dose response curves for E. coli BW25113.

Antibiotic concentrations used (in µg/mL) were as follows: Tet: 10 µg/mL for copper; Str: 2 µg/mL for copper and 10 µg/mL for pyrethrins. Values are means of at least 3 independent experiments; error bars are standard errors (SEM, with SEM=standard deviation/√n). Asterisks indicate the lowest biocide concentration for which a statistically significant change in EoP by at least 100-fold compared to the antibiotic only occurred. * P<0.05; ** P<0.01; *** P<0.001; ns, not significant.

Figure 3.. Change in EoP when E. coli deletion strains are (grey) and are not (black) exposed to Cu in the presence of Tetracycline.

The x-axis indicates antibiotic concentration in µg/mL. Copper was added at 450 µg/mL. Error bars are standard errors (SEM, with SEM=standard deviation/√n). Asterisks indicate P values for interaction terms (see Materials and Methods). * P<0.05; ** P<0.01; *** P<0.001; ns, not significant.

Figure 4.. Single-cell fluorescence intensity of E. coli BW25113 (pHJ101) expressing red fluorescent mScarlet protein under the control of the tolC promoter without (left) or with (right) copper exposure.

The violin plots show the distribution of the single-cell fluorescence within the cell population. The median is depicted by the bar in the box; the box represents the 25% and 75% quartiles.

Figure 5.. Chelation of tetracycline by copper.

X-axis is time in hrs after adding copper and tetracycline (flasks 1–4, solid lines), tetracycline (flasks 5 & 6, dashed lines), or copper (flask 7, dotted line). The first data point in each series indicates time of inoculation with E. coli BW25113. Values are means of three independent experiments ± SD.