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.

Keywords: Pseudomonas aeruginosa; RNA-Seq; antibiotic susceptibility; azithromycin; host-mimicking media.

Fig. 1.

P. aeruginosa PAO1 demonstrated increased susceptibility to macrolide antibiotics under growth conditions that were physiologically relevant to human infection. (A) MICs of macrolides, expressed as micrograms per milliliter, vs. WT P. aeruginosa grown in MHB, RPMI + 5% MHB (RPMI), and RPMI + 5% MHB + 20% human serum (RPMI/serum). Values are the mode of a minimum of n = 3. (B) Time kill curves were performed against WT P. aeruginosa with 8 µg/mL AZM treatment in all three media. ***P < 0.0001 using an unpaired t test (n = 3).

Fig. 2.

AZM strongly synergized with antimicrobial peptides under physiologically relevant conditions and in a cutaneous P. aeruginosa infection model. (A) In vitro, AZM was combined with DJK-5 and IDR-1018 in checkerboard titration assays in MHB and under physiologically relevant conditions: RPMI + 5% MHB (RPMI) and RPMI + 5% MHB + 20% human serum (RPMI/serum). FICI values below 0.5 indicate high synergistic activity, 0.5 to 1 indicate weak synergy or an additive effect, values greater than 1 indicate antagonism or no synergy. (B and C) In vivo, synergy was observed in a murine abscess model. Mice were infected with bacteria and treated with 1 mg/kg AZM, 0.1 mg/kg DJK-5, 14 mg/kg IDR-1018, or a combination of AZM and peptide. Synergy occurred when the effect of the combination of drugs on abscess size (B) and bacterial CFU (C) recovered was significantly (P < 0.01) more pronounced than the sum of the effects of each agent alone in comparison to the saline-treated controls after 18 h. Statistical analysis was performed using one-way ANOVA, Kruskal–Wallis test with Dunn’s correction (two-sided). The asterisks indicate significant differences to the WT: *P < 0.05; **P < 0.01; ***P < 0.001. The open boxes indicate significant differences of the combination therapy over the sum of the effects of each agent alone: *P < 0.05; **P < 0.01.

Fig. 3.

P. aeruginosa PAO1 grown in RPMI + 5% MHB (RPMI) and RPMI + 5% MHB + 20% human serum (RPMI/serum) displayed distinct transcriptional changes in genes previously proposed to be involved in resistance and susceptibility to antibiotics when compared to P. aeruginosa grown in MHB. (A) Heatmap shows those resistome genes with a log₂ fold change ≥ ±2. Genes are grouped by PseudoCap functional class and mutant type, where mutant type indicates whether a mutant in that gene was predicted to be resistant, susceptible, or to have mixed effects to antibiotics. The arn operon (B) and two-component systems (C) were highly dysregulated in host conditions compared to MHB.

Fig. 4.

P. aeruginosa PAO1 grown in RPMI + 5% MHB (RPMI) and RPMI + 5% MHB + 20% human serum (RPMI/serum) had more genes differentially expressed after AZM treatment cf. cells grown in MHB. (A) Venn diagram of numbers of genes and overlap. (B) Functional classes significantly enriched by AZM treatment under physiologically relevant conditions but not in MHB, as determined using a hypergeometric distribution calculation with a false-discovery rate-corrected P value of <0.05. * indicates functional classes enriched after AZM treatment in either RPMI or RPMI/serum but not MHB. (C) Heatmap showing log₂ fold change (Log₂FC) of genes in some functional classes that were significantly enriched after AZM treatment in host-like media vs. MHB (Log₂FC ≥ ±2 to enable capture of the most significantly dysregulated genes).