Figure 1: Doxycycline potentiates selection biases for and against tetracycline resistance by diverse otherwise neutrally selective antibiotics.

(a) YFP-labelled, tetracycline-sensitive (Green) and CFP-labelled, tetracycline-resistant (Red) E. coli are mixed and grown together over a diffusing toxin gradient in agar either absent or containing a uniform level of the tetracycline antibiotic, doxycycline (−Dox, +Dox, respectively). Tetracycline resistance is induced by doxycycline in +Dox plates and, without growth defect, by anhydrotetracycline added to −Dox plates. Final strain ratios reveal deviations from background inhibition (Yellow lawn with dark zone of clearing) that bias selection towards tetracycline resistance (Red ring) or sensitivity (Green ring). (b,c) Certain compounds such as erythromycin (b, Ery) and ciprofloxacin (c, Cpr) do not directly select on tetracycline resistance alone (−Dox plates), yet combine with a sub-inhibitory background level of a tetracycline (doxycycline) to select for resistance (Ery, +Dox, Red ring) or against resistance (Cpr, +Dox, Green ring). d, Nearly identical growth responses of tetracycline-sensitive (Tet^S, Green plots Strain Wyl,) and -resistant (Tet^R, Red plots, Strain t17cl) strains by ciprofloxacin alone (−Dox), diverge significantly in the presence of a uniform, sub-inhibitory level of doxycycline (+Dox), generating a region of strong ‘threshold selection’ between the MICs (grey vertical lines) where only the sensitive strain can grow (Green shading). Smoothing splines R²: 0.998 (Tet^R, −Dox), 0.997 (Tet^S, −Dox), 0.999 (Tet^R, +Dox), 0.995 (Tet^S, +Dox). e, Competing tetracycline-resistant (Red) and -sensitive (Green) bacteria are equally inhibited, and experience no change in relative growth along diffusing gradients of diverse non-tetracycline antibiotics acting alone (−Dox panels). Combining these antibiotic gradients with a uniform, sub-inhibitory level of doxycycline in the agar (+Dox panels) typically biases selection near the MIC (see Supplementary Fig. 2 for an exception) either towards tetracycline resistance (Red rings) or tetracycline sensitivity (Green rings).