Effects of azithromycin in Pseudomonas aeruginosa burn wound infection

Abstract

Background: Cutaneous thermal injuries (i.e., burns) remain a common form of debilitating trauma, and outcomes are often worsened by wound infection with environmental bacteria, chiefly Pseudomonas aeruginosa.

Materials and methods: We tested the effects of early administration of a single dose of azithromycin, with or without subsequent antipseudomonal antibiotics, in a mouse model of standardized thermal injury infected with P aeruginosa via both wound site and systemic infection. We also tested the antimicrobial effects of these antibiotics alone or combined in comparative biofilm and planktonic cultures in vitro.

Results: In our model, early azithromycin administration significantly reduced wound and systemic infection without altering wound site or circulating neutrophil activity. The antimicrobial effect of azithromycin was additive with ciprofloxacin but significantly reduced the antimicrobial effect of tobramycin. This pattern was reproduced in biofilm cultures and not observed in planktonic cultures of P aeruginosa.

Conclusion: These data suggest that early administration of azithromycin following burn-related trauma and infection may reduce P aeruginosa infection and potential interactions with other antibiotics should be considered when designing future studies.

Keywords: Azithromycin; Biofilm; Burn; Burn wound; Ciprofloxacin; Cystic fibrosis; Drug–drug interaction; Pseudomonas aeruginosa; Thermal injury; Tobramycin.

Figure 1

Mice challenged with combined cutaneous burn and infection with PAO1 had significantly greater weight loss compared with mice not burned, with or without PAO1 challenge (Figure 1a, mean +/− SEM, *P<0.01). Weight loss was maximal at 48 hours after challenge. Skin tissue myeloperoxidase content, reflecting neutrophil accumulation, steadily increased in mice challenged with burn and PAO1, peaking by 48 hours (Figure 1b, mean +/− SEM). Mice without thermal injury but challenged with PAO1 did not have increased MPO content in the skin through 72 hours, indicating that there was no significant neutrophil recruitment to the wound without thermal injury. *P<0.001. Burden of bacterial infection was significantly greater at the skin wound and inoculation site, but consistently had spread to both the lungs and spleen by 72 hours after inoculation (Figure 1c, mean +/− 95% CI).

Figure 2. Effect of Azithromycin on Local and Systemic Bacterial Infection

A single administration of azithromycin (20 mg/mL i.p.) 4 hours following inoculation of the burn wound significantly reduced P. aeruginosa infection both at the burn site and systemically in the lung and spleen (Figure 2a, mean +/− 95% CI. ∞P=0.05, #P<0.001, *Spleen P<0.05). Graphs reflect N=15 from 5 replicate experiments. (NS, normal saline control; AZM, azithromycin; *P<0.05, #P<0.01). Azithromycin group trended toward greater weight recovery at 72 hours following challenge but this was not statistically significant (Figure 2b). Azithromycin did not significantly affect the number of circulating neutrophils in the peripheral blood or neutrophil accumulation at the site of injury (2c) measured at 72 hours.

Figure 3. Effect of Antibiotic Monotherapy

Mice treated with each of the three antibiotics tested as monotherapy showed similar weight loss to saline control at 72 hours after challenge (Figure 3a, mean +/− SEM, N=10–16). Azithromycin, ciprofloxacin, or tobramycin administration each reduced both wound site (Figure 3b, #P<0.01) and systemic infection of PAO1 (Figures 3c, 3d, ∞P<0.001, #P<0.01) when compared with saline control. The comparison with saline control was statistically significant and tobramycin appeared to be superior as monotherapy in concentrations tested, but no statistically significant difference was observed between antibiotics. NS, normal saline; AZM, azithromycin; CPRO, ciprofloxacin; TBRA, tobramycin.

Figure 4. Divergent Effects of Azithromycin when Added to Ciprofloxacin or Tobramycin

We observed a difference in effect of early administration of azithromycin with subsequent ciprofloxacin or tobramycin on P. aeruginosa infection at the skin. A trend toward additive antimicrobial effect with ciprofloxacin was noted while apparent antagonism with tobramycin was observed. Significantly greater bacteria were cultured from the wound when azithromycin preceded tobramycin administration compared with tobramycin administration alone (Figure 4a; #P<0.01, N=8–10 from 3 replicate experiments). Early administration of azithromycin appeared to increase bacterial spread to the lung in animals treated with tobramycin compared with tobramycin alone (*P<0.02, N=12–15). This was not observed with azithromycin and ciprofloxacin where the combination resulted in a reduction in lung bacterial density that was not statistically significant compared to ciprofloxacin alone. Splenic P. aeruginosa growth showed similar trends but was relatively low in all antibiotic treatment groups and no significant differences were observed (data not shown).

Figure 4. Divergent Effects of Azithromycin when Added to Ciprofloxacin or Tobramycin

We observed a difference in effect of early administration of azithromycin with subsequent ciprofloxacin or tobramycin on P. aeruginosa infection at the skin. A trend toward additive antimicrobial effect with ciprofloxacin was noted while apparent antagonism with tobramycin was observed. Significantly greater bacteria were cultured from the wound when azithromycin preceded tobramycin administration compared with tobramycin administration alone (Figure 4a; #P<0.01, N=8–10 from 3 replicate experiments). Early administration of azithromycin appeared to increase bacterial spread to the lung in animals treated with tobramycin compared with tobramycin alone (*P<0.02, N=12–15). This was not observed with azithromycin and ciprofloxacin where the combination resulted in a reduction in lung bacterial density that was not statistically significant compared to ciprofloxacin alone. Splenic P. aeruginosa growth showed similar trends but was relatively low in all antibiotic treatment groups and no significant differences were observed (data not shown).

Figure 5. In Vitro Biofilm Culture

P. aeruginosa biomass reductions (i.e. antimicrobial effect) after 22 hours flow compared with baseline were: no antibiotic 9%, azithromycin 32%, ciprofloxacin 10%, tobramycin 68%. Ciprofloxacin (4 mcg/mL) had no statistically significant effect on P. aeruginosa biofilm biomass when compared with no antibiotic exposure. Azithromycin and especially tobramycin (40 mcg/mL) were more effective than no treatment or the other antibiotics tested (*P<0.05, ∞P <0.001). The addition of azithromycin to ciprofloxacin was significantly more effective at reducing P. aeruginosa biomass than ciprofloxacin alone (*P<0.05). This combined treatment resulted in 34% P. aeruginosa biomass reduction compared with 10% observed with ciprofloxacin alone. When azithromycin was added to tobramycin, the effective biomass reduction was lost (68% to 28%, P<0.001) and was no longer statistically significant when compared with no treatment or azithromycin alone.

Figure 5. In Vitro Biofilm Culture

P. aeruginosa biomass reductions (i.e. antimicrobial effect) after 22 hours flow compared with baseline were: no antibiotic 9%, azithromycin 32%, ciprofloxacin 10%, tobramycin 68%. Ciprofloxacin (4 mcg/mL) had no statistically significant effect on P. aeruginosa biofilm biomass when compared with no antibiotic exposure. Azithromycin and especially tobramycin (40 mcg/mL) were more effective than no treatment or the other antibiotics tested (*P<0.05, ∞P <0.001). The addition of azithromycin to ciprofloxacin was significantly more effective at reducing P. aeruginosa biomass than ciprofloxacin alone (*P<0.05). This combined treatment resulted in 34% P. aeruginosa biomass reduction compared with 10% observed with ciprofloxacin alone. When azithromycin was added to tobramycin, the effective biomass reduction was lost (68% to 28%, P<0.001) and was no longer statistically significant when compared with no treatment or azithromycin alone.

Figure 5. In Vitro Biofilm Culture

P. aeruginosa biomass reductions (i.e. antimicrobial effect) after 22 hours flow compared with baseline were: no antibiotic 9%, azithromycin 32%, ciprofloxacin 10%, tobramycin 68%. Ciprofloxacin (4 mcg/mL) had no statistically significant effect on P. aeruginosa biofilm biomass when compared with no antibiotic exposure. Azithromycin and especially tobramycin (40 mcg/mL) were more effective than no treatment or the other antibiotics tested (*P<0.05, ∞P <0.001). The addition of azithromycin to ciprofloxacin was significantly more effective at reducing P. aeruginosa biomass than ciprofloxacin alone (*P<0.05). This combined treatment resulted in 34% P. aeruginosa biomass reduction compared with 10% observed with ciprofloxacin alone. When azithromycin was added to tobramycin, the effective biomass reduction was lost (68% to 28%, P<0.001) and was no longer statistically significant when compared with no treatment or azithromycin alone.