Non-antibiotic pharmaceuticals are toxic against Escherichia coli with no evolution of cross-resistance to antibiotics

Abstract

Antimicrobial resistance can arise in the natural environment via prolonged exposure to the effluent released by manufacturing facilities. In addition to antibiotics, pharmaceutical plants also produce non-antibiotic pharmaceuticals, both the active ingredients and other components of the formulations. The effect of these on the surrounding microbial communities is less clear. We aimed to assess whether non-antibiotic pharmaceuticals and other compounds produced by pharmaceutical plants have inherent toxicity, and whether long-term exposure might result in significant genetic changes or select for cross-resistance to antibiotics. To this end, we screened four non-antibiotic pharmaceuticals (acetaminophen, ibuprofen, propranolol, metformin) and titanium dioxide for toxicity against Escherichia coli K-12 MG1655 and conducted a 30 day selection experiment to assess the effect of long-term exposure. All compounds reduced the maximum optical density reached by E. coli at a range of concentrations including one of environmental relevance, with transcriptome analysis identifying upregulated genes related to stress response and multidrug efflux in response ibuprofen treatment. The compounds did not select for significant genetic changes following a 30 day exposure, and no evidence of selection for cross-resistance to antibiotics was observed for population evolved in the presence of ibuprofen in spite of the differential gene expression after exposure to this compound. This work suggests that these compounds, at environmental concentrations, do not select for cross-resistance to antibiotics in E. coli.

Fig. 1. Toxicity screen of non-antibiotic pharmaceuticals against E. coli.

Compounds a acetaminophen (+A), b ibuprofen (+I), c titanium dioxide (+T), d propranolol (+P), and e metformin (+M) were screened at various concentrations against E. coli over a 24 incubation in a 96-well plate in a microplate reader. A no-compound control (‘No compound’, purple) was included for all screens. f Area under the curve (AUC) values for the following concentrations are given against their representative no-compound control (-); 1 ng/mL acetaminophen, 5 ng/mL ibuprofen, 1 µg/mL titanium dioxide, 0.5 ng/mL propranolol, 0.5 ng/mL metformin. *p < 0.05, one-way ANOVA. All AUC values are shown in Supplementary Fig. 1. Measurements in triplicate, error bars depict standard deviation.

Fig. 2. Genes differentially expressed in the presence of ibuprofen.

Genes significantly upregulated (yellow) (false discovery rate [FDR] threshold of p < 0.05 and an absolute log fold change [FC] of at least one) in the presence of ibuprofen. Genes which had an absolute log FC of at least one but did not reach the FDR threshold are shown in grey and are considered to not be significantly differentially expressed. Selected genes are labelled.

Fig. 3. Growth of ancestor and evolved populations.

Growth of evolved populations (yellow, six independent biological replicates P1-P6) in the presence of the compound in which their selection experiment was conducted in comparison to growth of the ancestral lineage (blue, Anc); a media-only control, b acetaminophen, c ibuprofen, d titanium dioxide, e propranolol, and f metformin. Measurements in triplicate, error bars depict standard deviation.