Selective advantage of resistant strains at trace levels of antibiotics: a simple and ultrasensitive color test for detection of antibiotics and genotoxic agents

Abstract

Many studies have examined the evolution of bacterial mutants that are resistant to specific antibiotics, and many of these focus on concentrations at and above the MIC. Here we ask for the minimum concentration at which existing resistant mutants can outgrow sensitive wild-type strains in competition experiments at antibiotic levels significantly below the MIC, and we define a minimum selective concentration (MSC) in Escherichia coli for two antibiotics, which is near 1/5 of the MIC for ciprofloxacin and 1/20 of the MIC for tetracycline. Because of the prevalence of resistant mutants already in the human microbiome, allowable levels of antibiotics to which we are exposed should be below the MSC. Since this concentration often corresponds to low or trace levels of antibiotics, it is helpful to have simple tests to detect such trace levels. We describe a simple ultrasensitive test for detecting the presence of antibiotics and genotoxic agents. The test is based on the use of chromogenic proteins as color markers and the use of single and multiple mutants of Escherichia coli that have greatly increased sensitivity to either a wide range of antibiotics or specific antibiotics, antibiotic families, and genotoxic agents. This test can detect ciprofloxacin at 1/75 of the MIC.

FIG. 1.

Scoring resistant mutants by colony color. Strains J93 (Lac⁻ Cip^s) and J93140C (Lac⁺ Cip^r) were grown for 25 generations in LB medium (left) and LB medium containing Cip at <1/5 of the MIC (right). Cip^s colonies are white (Lac⁻), and Cip^r colonies are blue (Lac⁺).

FIG. 2.

Competition experiments between Cip^s and Cip^r strains. Strains J93 (Lac⁻ Cip^s) and J93140C (Lac⁺ Cip^r) were grown together in LB medium with different concentrations of Cip, and parallel cultures were scored at intervals.

FIG. 3.

Competition experiments between tetracycline-sensitive (Tet^s) and Tet^r strains. Cultures of J93140 (Lac⁺ Tet^s) and J93140TZ (Lac⁻ Tet^r) were grown together in LB medium with different concentrations of Tet, and parallel cultures were scored at intervals. The 31-ng/ml data series was obtained from a different starting mix, which is why the zero time point is slightly different than those of the other concentrations.

FIG. 4.

Chromogenic proteins. Colonies and cultures of BW25113 carrying pGEM-T-11 or pGEM-T-14 are yellow or purple, respectively.

FIG. 5.

Concept of color test for detection of antibiotics and genotoxic agents. WT, wild type.

FIG. 6.

Color test for Cip. The color test depicted here includes a mixture of the wild type (yellow) and a tolC derivative (purple) grown in LB medium with the indicated concentrations of Cip.

FIG. 7.

Color test for Cip. The same test described in the legend to Fig. 6 but with a tolC recC derivative used in the mixture with the wild type.

FIG. 8.

Plate test. A mixture of the wild type (yellow) and a tolC recC derivative (purple) is spread on an LB plate, and a drop of a solution containing an antibiotic, in this case, Cip, is applied. See the text for details. The photograph (using a Canon 8.1-megapixel camera) was subjected to density analysis, which illustrated an ∼87% difference in grayscale intensity between the inner yellow ring and the rest of the plate.