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ABSTRACT: Antimicrobial resistance is one of the largest and most pressing global health threats. This is not only a huge burden on the global economy but also a growing threat to animal, environmental, plant, and human health, and new strategies are needed to avoid resistance and improve treatment. Novel antimicrobial resistance genes are normally first detected once they cause problems in clinical infections, and we have limited knowledge on their evolutionary trajectories. Current antimicrobial susceptibility testing and research have a limited focus on key environmental factors in pathogen-reservoir-host interactions, possibly leading to inaccurate results that do not reflect the in vivo conditions. Focusing on differences in pH, we determined the MIC of a panel of isogenic strains expressing CTX-M-15 and CMY-2 β-lactamases. We found that pH has a large impact on the activity of β-lactamases, and treatment of these resistant isolates could be possible if the pH of the environment is modified. We verified this using enzyme kinetics, co-cultures, and growth experiments, suggesting that exposure to different environmental conditions may lead to distinct evolutionary trajectories for specific β-lactamases. Exploring the effect of different temperatures, we also observed a differential effect of avian and mammal host temperatures. Environmental factors such as pH and temperature may have a large unnoticed effect on antimicrobial resistance, and we might use this knowledge to renew and extend the use of old antibiotics for certain infections.