Identification of novel targets of azithromycin activity against Pseudomonas aeruginosa grown in physiologically relevant media

Abstract

Pseudomonas aeruginosa causes severe multidrug-resistant infections that often lead to bacteremia and sepsis. Physiologically relevant conditions can increase the susceptibility of pathogens to antibiotics, such as azithromycin (AZM). When compared to minimal-inhibitory concentrations (MICs) in laboratory media, AZM had a 16-fold lower MIC in tissue culture medium with 5% Mueller Hinton broth (MHB) and a 64-fold lower MIC in this tissue culture medium with 20% human serum. AZM also demonstrated increased synergy in combination with synthetic host-defense peptides DJK-5 and IDR-1018 under host-like conditions and in a murine abscess model. To mechanistically study the altered effects of AZM under physiologically relevant conditions, global transcriptional analysis was performed on P. aeruginosa with and without effective concentrations of AZM. This revealed that the arn operon, mediating arabinosaminylation of lipopolysaccharides and related regulatory systems, was down-regulated in host-like media when compared to MHB. Inactivation of genes within the arn operon led to increased susceptibility of P. aeruginosa to AZM and great increases in synergy between AZM and other antimicrobial agents, indicating that dysregulation of the arn operon might explain increased AZM uptake and synergy in host-like media. Furthermore, genes involved in central and energy metabolism and ribosome biogenesis were dysregulated more in physiologically relevant conditions treated with AZM, likely due to general changes in cell physiology as a result of the increased effectiveness of AZM in these conditions. These data suggest that, in addition to the arn operon, there are multiple factors in host-like environments that are responsible for observed changes in susceptibility.