Effects of Antimicrobial Combinations on Pseudomonas aeruginosa-Aspergillus fumigatus Mixed Microbial Biofilm

Abstract

Objectives: Co-infection with P. aeruginosa and A. fumigatus is frequently seen in patients with cystic fibrosis. These microorganisms are known to produce biofilm both in vitro and in vivo. The biofilm-embedded microbial cells are frequently refractory to conventional antimicrobial therapy. The primary objective of this study was to evaluate the efficacy of several anti-pseudomonal antimicrobials such as cefepime, imipenem and ciprofloxacin individually and in pair-wise combinations with antifungal drugs on P. aeruginosa-A. fumigatus polymicrobial biofilm and compare the results with those obtained in monomicrobial biofilm. Methods: Biofilms of P. aeruginosa and A. fumigatus isolates were grown in 24-well cell culture plates in Sabouraud’s dextrose broth at 35 ˚C. The activities of cefepime, imipenem and ciprofloxacin alone and in two-drug combination with voriconazole, posaconazole, amphotericin B and anidulafungin on monomicrobial and polymicrobial biofilms, as well as on planktonic cells were examined by CFU assay. Results: Scanning electron microscopic studies showed that A. fumigatus produced firmly adherent mixed microbial biofilm with P. aeruginosa on Thermanox plastic coverslips with increased synthesis of extracellular matrix in the presence of the bacterial cells. The fungal hyphae in monomicrobial and polymicrobial biofilms were realigned during biofilm growth forming parallel-packed bundles with no apical or dichotomous branching. Typically, P. aeruginosa produced firmly adherent mixed microbial biofilm with increased synthesis of extracellular matrix in the presence of A. fumigatus hyphae, but tended to form loosely adherent monomicrobial biofilm. Overall, the susceptibility of P. aeruginosa to cefepime and imipenem alone and in pair-wise combination with antifungal drugs was significantly decreased in polymicrobial biofilms (≈0 to 0.5 logs CFU reduction at 16µg/ml) when compared to monomicrobial biofilms (≈1.5 to 4.5 logs CFU reduction at 16µg/ml) (p values ranged from 0.0076 to 0.0509). On the other hand, the efficacy of ciprofloxacin in monomicrobial and polymicrobial biofilms was similar (≈2.5 to 3.5 logs CFU reduction at 16µg/ml). A. fumigatus monomicrobial and polymicrobial biofilms were similarly susceptible to antifungal drugs with and without the antibacterial in the combination. Time-kill experiments performed at 4 times the MICs of the drugs (0.5µg/ml to 4µg/ml) showed that the planktonic cells of P. aeruginosa (≈4 to 4.5 logs CFU reduction) and A. fumigatus (≈2.5 to 3 logs CFU reduction) in monocultures and mixed microbial cultures were similarly susceptible to antimicrobial drugs. Conclusions: In our model, the P. aeruginosa cells associated with P. aeruginosa-A. fumigatus polymicrobial biofilms were recalcitrant to certain antibacterial drugs compared to monomicrobial biofilms, whereas the planktonic cell monocultures and mixed microbial cultures showed no significant difference in their antimicrobial drug susceptibility profiles.