Impact of azithromycin on the clinical and antimicrobial effectiveness of tobramycin in the treatment of cystic fibrosis

Abstract

Background: Concomitant use of oral azithromycin and inhaled tobramycin occurs in approximately half of US cystic fibrosis (CF) patients. Recent data suggest that this combination may be antagonistic.

Methods: Test the hypothesis that azithromycin reduces the clinical benefits of tobramycin by analyses of clinical trial data, in vitro modeling of P. aeruginosa antibiotic killing, and regulation of the MexXY efflux pump.

Results: Ongoing administration of azithromycin associates with reduced ability of inhaled tobramycin, as compared with aztreonam, to improve lung function and quality of life in a completed clinical trial. In users of azithromycin FEV₁ (L) increased 0.8% during a 4-week period of inhaled tobramycin and an additional 6.4% during a subsequent 4-week period of inhaled aztreonam (P<0.005). CFQ-R respiratory symptom score decreased 1.8 points during inhaled tobramycin and increased 8.3 points during subsequent inhaled aztreonam (P<0.001). A smaller number of trial participants not using azithromycin had similar improvement in lung function and quality of life scores during inhaled tobramycin and inhaled aztreonam. In vitro, azithromycin selectively reduced the bactericidal effects tobramycin in cultures of clinical strains of P. aeruginosa, while up regulating antibiotic resistance through MexXY efflux.

Conclusions: Azithromycin appears capable of reducing the antimicrobial benefits of tobramycin by inducing adaptive bacterial stress responses in P. aeruginosa, suggesting that these medications together may not be optimal chronic therapy for at least some patients.

Keywords: Azithromycin; Clinical trial; Cystic fibrosis; Drug interaction; Inhaled antibiotics; MexXY; Pseudomonas aeruginosa; Tobramycin.

Figure 1. Relative change in FEV₁ (L) during 4-week use of inhaled tobramycin followed by 4-week use of inhaled aztreonam

Panel A: Mean (SEM) improvement in lung function for users of azithromycin (AZM, left) and non-users of azithromycin (No AZM, right). Panel B: Improvement in FEV₁ (L) during the 8-week study period reflecting inhaled tobramycin (TOB) immediately followed by inhaled aztreonam (ATM) in subjects completing both inhaled antibiotic study periods. AZM users; N = 108 during tobramycin and 71 during aztreonam. AZM non users; N = 40 during tobramycin and 22 during aztreonam.

Figure 2. Absolute change in CFQ-R Respiratory Symptom Scale

Mean (SEM) improvement in self-reported, disease-related quality of life for users of azithromycin (AZM, left) and non-users of azithromycin (No AZM, right). The dashed line represents the threshold for clinically significant improvement. AZM users; N = 102 during tobramycin (TOB) and 68 during aztreonam (ATM). AZM non users; N = 39 during tobramycin and 22 during aztreonam.

Figure 3. Change in sputum P. aeruginosa density

Both users (AZM) and non-users (No AZM) of azithromycin demonstrated a reduction in sputum P. aeruginosa density over 4-weeks use of inhaled tobramycin (TOB). A further reduction in P. aeruginosa density was present in both users and non-users of azithromycin during 4-weeks use of inhaled aztreonam (TOB+ATM). AZM users; N = 85 during tobramycin and 53 during aztreonam. AZM non-users; N = 27 during tobramycin and 16 during aztreonam.

Figure 4. P. aeruginosa density of biofilm aggregate cultures

Panel A: azithromycin (AZM), tobramycin (TOB); N = 30 isolates from unique persons with CF, each tested once; log₁₀ CFU graphed as mean + SEM. Panel B: Difference in CFU density when azithromycin is added to comparative anti-pseudomonal antibiotics, including: amikacin (AMK), colistin (COL), levofloxacin (LVX), and tobramycin (TOB) (N = 30, log₁₀ CFU graphed as mean + SEM). Statistical comparison is the change from zero X-axis, which represents the value from simultaneous cultures grown with the antibiotic of interest but without addition of azithromycin.

Figure 5. Gene expression of PA5471, mexX, and mexY

Panel A: maximum PA5471 gene expression across all time points tested in clinical P. aeruginosa isolates (mean and each value shown). Data represent fold induction vs. control cultures with no antibiotic added. Panel B: mexX expression. Panel C: mexY expression.

Figure 6. Correlation in Gene expression and P. aeruginosa killing after antibiotic challenge

Panel A: Correlation between PA5471 expression and mexX (left) or mexY (right) expression. Data represent fold induction vs. control cultures with no antibiotic added. P < 0.01 for correlation with both mexX and mexY. Azithromycin shown in blue, azithromycin plus tobramycin shown in red. Panel B: correlation in gene expression and bacterial killing with the combination of tobramycin and azithromycin.

Figure 6. Correlation in Gene expression and P. aeruginosa killing after antibiotic challenge

Panel A: Correlation between PA5471 expression and mexX (left) or mexY (right) expression. Data represent fold induction vs. control cultures with no antibiotic added. P < 0.01 for correlation with both mexX and mexY. Azithromycin shown in blue, azithromycin plus tobramycin shown in red. Panel B: correlation in gene expression and bacterial killing with the combination of tobramycin and azithromycin.

Figure 7. PAO1 density of biofilm aggregate cultures with MexX disruption

PAO1 parent strain vs. PAO1 generated strain with mexX disruption. Both strains achieved similar bacterial density without antibiotic challenge. Azithromycin reduced bacterial killing when added to tobramycin in the parent strain and, conversely, improved bacterial killing when added to tobramycin in the mexX disrupted strain (P < 0.01, N = 4, log₁₀ CFU graphed as mean + SEM). UNT: untreated, AZM: azithromycin 20 ug/mL, TOB: tobramycin (100 ug/mL).