Unravelling the mechanisms of gradient of antibiotic sensing by bacteria.*

Motile bacteria may exhibit chemotactic responses by detecting chemical gradients using chemoreceptors. These receptors relay signals that guide bacterial movement towards or away from certain compounds. Bacteria are often exposed to the gradient of antibiotics, which can create selective pressure. To combat this situation, bacteria develop resistance to the antibiotic. Although many bacterial species exhibit surface motility and/or swimming motility, our understanding of how these antibiotic gradients influence their movement is surprisingly limited. In this work, we evaluate how the bacteria respond in the presence of antibiotic gradient. For this experiment, we use Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis and different fluoroquinolone groups of antibiotics (Levofloxacin, Ofloxacin, and Norfloxacin). To understand the chemotactic behaviour of the population of bacteria, we perform the soft agar plate assay, and for single bacteria, we use the capillary assay. Chemotaxis of wild-type bacterial strains on a soft agar plate was analysed in terms of expansion speed, which is computed by taking the outer ring radius and time of incubation. Wildtype susceptible strains were repelled by antibiotics discs, thereby forming a zone of clearance, whereas resistant strains traversed towards the discs. In capillary assay, the bacteria are exposed to a stable linear gradient, and the result depicted that the antibiotics act as chemoattractants. Upon doing the gradient quantification, it was very evident that the bacteria showed bi-phasic sensing behaviour, both attraction and repulsion to the same chemical depending on its concentration when exposed to the antibiotic gradient. At low concentrations of an antibiotic, E. coli exhibits a positive chemotactic response, where the bacteria are attracted towards the source of the chemical. At high concentrations, the antibiotic triggers a negative chemotactic response, causing the bacteria to move away from the source. This is due to saturation of the chemoreceptors or activation of different signalling pathways that lead to repulsion.