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

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 <i>Pseudomonas aeruginosa</i>. Transcriptome analysis revealed that subinhibitory concentrations of azithromycin reduced the expression of genes involved in stress-induced mutagenesis, including the stress response sigma factor <i>rpoS</i>. Interestingly, ribosome profiling revealed global redistribution of ribosomes by azithromycin, among which ribosome stalling was significantly intensified near the 5´ terminus of the <i>rpoS</i> mRNA. Further DNA mutational analysis revealed that azithromycin represses the translation of <i>rpoS</i> through its 5´-terminal rare codons, which in turn reduced its transcription. These <i>in vitro</i> observations have been recapitulated <i>in vivo</i> where azithromycin-repressed CZA resistance development when <i>P. aeruginosa</i> 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 <i>P. aeruginosa</i> infections.IMPORTANCEAntibiotic resistance, a global public health challenge, demands the development of novel antibiotics and therapeutic strategies. Ceftazidime-avibactam (CZA) is a combination of a β-lactam antibiotic with a β-lactamase inhibitor that is effective against various gram-negative bacteria such as <i>Pseudomonas aeruginosa</i>. However, clinical CZA-resistant isolates have been reported. Here, we found that combining CZA with azithromycin can effectively suppress the development of resistance in <i>P. aeruginosa in vitro</i> and <i>in vivo</i>. Moreover, we found that azithromycin represses the translation initiation of <i>rpoS</i> through its 5´-terminal rare and less frequent codons, thereby subsequently reducing the mutational frequency of CZA resistance. Therefore, our work provides a promising antibiotic combination for the treatment of <i>P. aeruginosa</i> infections.