Gene expression plasticity under multiple stresses drives higher tolerance to a macrolide in saline and warmer environments.

The widespread presence of antibiotics in the environment at sub-inhibitory concentrations imposes a selective pressure that promotes the spread of resistance. In the field, antibiotics interact with diverse physicochemical parameters that can attenuate or intensify their fitness effects. Gene expression is a central plastic trait that governs phenotypes at a higher level of integration and modulates the strength of selection, yet how synergistic or antagonistic fitness effects arise from interactions among transcriptional responses remains poorly understood. Here, we characterized gene-expression interactions underlying fitness-level interactions previously identified between a macrolide, temperature and salinity, and proposed a general methodological framework for assessing the impact of multiple stressors on gene expression. We analyzed the transcriptional response of Escherichia coli to azithromycin (AZI) across two salinity and temperature conditions. De novo and antagonistic interactions were prevalent, with evidence of cross-regulations between salt and AZI. High salinity increased tolerance by two orders of magnitude and, similarly to AZI, induced a downregulation of carbon metabolism. Reduced temperature, which canceled the salinity protective effect, enhanced carbon metabolism and counteracted this shift. Salinity additionally restored stress-response pathways, largely repressed by AZI. Third-order interactions attenuated the contribution of salinity relative to AZI, but the number of affected genes declined exponentially with interaction order, suggesting that higher-order interactions at the gene-expression level should play a minor role in the responses to multiple stressors. By modulating transcriptional responses to AZI, simple environmental parameters could reshape the adaptive landscape of antibiotic resistance, potentially altering the spectrum of resistance mutations likely to spread.