Antibiotics limit adaptation of drug resistant Staphylococcus aureus to hypoxia

Bacterial pathogens are confronted with a range of challenges including exposure to antibiotic treatment and harsh physiological conditions at the site of infection that can alter the fitness benefits and costs of acquiring antibiotic resistance. Here we develop an experimental system to recapitulate resistance gene acquisition by Staphylococcus aureus and test how subsequent evolution of the resistant bacterium is modulated by antibiotic treatment and oxygen levels, both of which are known vary extensively at the site of infection. We show that acquiring tetracycline resistance was costly for S. aureus in hypoxic but not in normoxic conditions. Treatment with tetracycline or doxycycline drove the emergence of enhanced resistance through mutations in an RluD-like protein encoding gene and through duplications of tetL, encoding the acquired tetracycline-specific efflux pump. By contrast, evolutionary adaptation by S. aureus to hypoxic conditions, which evolved in the absence of antibiotics through mutations affecting gyrB, was impeded by antibiotic treatment. Together these data suggest that horizontal acquisition of a new resistance mechanism is merely a starting point for the emergence of high-level resistance under antibiotic selection, but that antibiotic treatment constrains pathogen adaptation to other important environmental selective forces, such as hypoxia, which in turn could limit the survival of these highly resistant but poorly adapted genotypes after the antibiotic treatment is ended.