It More than Adds Up: Interaction of Antibiotic Mixing and Temperature

Use of antibiotics for the treatment and prevention of bacterial infections in humans, agri- and aquaculture as well as livestock rearing leads to antibiotic pollution of fresh water and these antibiotics have an impact on free-living bacteria. While we know which antibiotics are most common in natural environments such as rivers and streams, there is considerable uncertainty regarding antibiotics' interactions with one another and the effect of abiotic factors such as temperature. Here, we used an experimental approach to explore the effects of antibiotic identity, concentration, mixing and water temperature on the growth of Pseudomonas fluorescens, a common, ubiquitous bacterium. We exposed P. fluorescens to the four antibiotics most commonly found in surface waters (ciprofloxacin, ofloxacin, sulfamethoxazole and sulfapyridine) and investigated antibiotic interactions for single and mixed treatments at different, field-realistic temperatures. We observed an overall dependence of antibiotic potency on temperature, as temperature increased efficacy of ciprofloxacin and ofloxacin with their EC₅₀ lowered by >75% with a 10 °C temperature increase. Further, we show that mixtures of ciprofloxacin and ofloxacin, despite both belonging to the fluoroquinolone class, exhibit low-temperature-dependent synergistic effects in inhibiting bacterial growth. These findings highlight the context dependency of antibiotic efficacy. They further suggest antibiotic-specific off-target effects that only affect the bacteria once they enter a certain temperature range. This has important implications as freshwater systems already contain multi-drug antibiotic cocktails and are changing temperature due to environmental warming. These factors will interact and affect aquatic food webs, and hence this creates an urgent need to adapt and improve laboratory testing conditions to closer reflect natural environments.