Impact of antibiotic concentration gradients on nitrate reduction and antibiotic resistance in a microfluidic gradient chamber

Highlights: • Antibiotic and nitrate concentration gradients were established in microfluidics. • Shewanella migrated to obtain nutrients in habitats with bactericidal antibiotics. • Acutely lethal concentrations of antibiotics limited Shewanella access to nitrate. • Emergence of antibiotic resistance was not detected. In order to explore the impact of antibiotics on the bacterial metabolic cycling of nitrate within contaminated soil and groundwater environments, we compared the effects of polymyxin B (PMB) and ciprofloxacin (CIP) concentration gradients on the distribution and activity of a wild type (WT) and a flagella deficient mutant (Δflag) of Shewanella oneidensis MR-1 in a microfluidic gradient chamber (MGC). Complementary batch experiments were performed to measure bacteriostatic versus bactericidal concentrations of the two antibiotics, as well as their effect on nitrate reduction. Prior work demonstrated that PMB disrupts cell membranes while CIP inhibits DNA synthesis. Consistent with these modes of action, batch results from this work show that PMB is bactericidal at lower concentrations than CIP relative to their respective minimum inhibitory concentrations (MICs) (≥5× MIC_PMB vs. ≥20× MIC_CIP). Concentration gradients from 0 to 50× the MIC of both antibiotics were established in the MGC across a 2-cm interconnected pore network, with nutrients injected at both concentration boundaries. The WT cells could only access and reduce nitrate in regions of the MGC with PMB at PMB, whereas this occurred with CIP up to 50× MIC_CIP; and cells extracted from these MGCs showed no antibiotic resistance. The distribution of Δflag cells was further limited to lower antibiotic concentrations (≤1× MIC_PMB, ≤43× MIC_CIP) due to inability of movement. These results indicate that S. oneidensis access and reduce nitrate in bactericidal regions via chemotactic migration without development of antibiotic resistance, and that this migration is inhibited by acutely lethal bactericidal levels of antibiotics.