Azithromycin represses evolution of ceftazidime/avibactam resistance by translational repression of rpoS in Pseudomonas aeruginosa

Antibiotic combinations can slow down resistance development and/or achieve synergistic therapeutic effects. In this study, we observed that a combined use of ceftazidime-avibactam (CZA) with azithromycin effectively repressed CZA resistance development in Pseudomonas aeruginosa. Transcriptome analysis revealed that subinhibitory concentrations of azithromycin reduced the expression of genes involved in stress-induced mutagenesis, including the stress response sigma factor rpoS. Interestingly, ribosome profiling revealed global redistribution of ribosomes by azithromycin, among which ribosome stalling was significantly intensified near the 5´ terminus of the rpoS mRNA. Further DNA mutational analysis revealed that azithromycin represses the translation of rpoS through its 5´-terminal rare codons, which in turn reduced its transcription. These in vitro observations have been recapitulated in vivo where azithromycin-repressed CZA resistance development when P. aeruginosa was passaged in mice. Overall, our study revealed the molecular mechanism of azithromycin-mediated repression of antibiotic resistance development, providing a promising antibiotic combination for the treatment of P. aeruginosa infections.