Development and antimicrobial susceptibility studies of in vitro monomicrobial and polymicrobial biofilm models with Aspergillus fumigatus and Pseudomonas aeruginosa

<jats:title>Abstract</jats:title><jats:sec><jats:title>Background</jats:title><jats:p>Mixed microbial infections of the respiratory tracts with<jats:italic>P. aeruginosa</jats:italic>and<jats:italic>A. fumigatus</jats:italic>capable of producing biofilms are commonly found in cystic fibrosis patients. The primary objective of this study was to develop an<jats:italic>in vitro</jats:italic>model for<jats:italic>P. aeruginosa</jats:italic>and<jats:italic>A. fumigatus</jats:italic>polymicrobial biofilm to study the efficacy of various antimicrobial drugs alone and in combinations against biofilm-embedded cells. Simultaneous static cocultures of<jats:italic>P. aeruginosa</jats:italic>and sporelings were used for the development of in vitro<jats:italic>P. aeruginosa</jats:italic>-<jats:italic>A. fumigatus</jats:italic>polymicrobial biofilm in SD broth in 24-well cell culture plates at 35°C, and the biofilm formation was monitored microscopically and spectrophotometrically. Using<jats:italic>P. aeruginosa</jats:italic>-<jats:italic>A. fumigatus</jats:italic>sporelings cocultures we examined the effects of various antimicrobial drugs alone and in combination against polymicrobial biofilm by CFU and tetrazolium reduction assays.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>In simultaneous static cocultures<jats:italic>P. aeruginosa</jats:italic>cells killed<jats:italic>A. fumigatus</jats:italic>conidia, whereas the bacterial cells showed no substantial fungicidal effect on sporelings grown for 12 h or longer at 35°C. Monospecies cultures of<jats:italic>P. aeruginosa</jats:italic>produced loosely adhered monomicrobial biofilm and addition of 10% bovine serum to the growth medium inhibited the formation of monomicrobial biofilm by<jats:italic>P. aeruginosa</jats:italic>whereas it produced tightly adhered polymicrobial biofilm in the presence of<jats:italic>A. fumigatus</jats:italic>mycelial growth.<jats:italic>A. fumigatus</jats:italic>produced firmly adherent monomicrobial and polymicrobial biofilms. A comparison of CFU and MTT assays showed that the latter is unsuitable for studying the effectiveness of antimicrobial treatment against polymicrobial biofilm. Tobramycin alone and in combination with posaconazole was highly effective against monomicrobial and polymicrobial biofilms of<jats:italic>P. aeruginosa</jats:italic>whereas cefepime alone and in combination with posaconazole showed excellent activity against monomicrobial biofilm of<jats:italic>P. aeruginosa</jats:italic>but was less effective against polymicrobial biofilm. Monomicrobial and polymicrobial biofilms of<jats:italic>A. fumigatus</jats:italic>showed similar susceptibility to posaconazole with and without the antibacterial drug.</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>Simultaneous static coculture of<jats:italic>A. fumigatus</jats:italic>sporelings grown for 12 h or longer was superior to ungerminated conidia with<jats:italic>P. aeruginosa</jats:italic>for the development of<jats:italic>A. fumigatus</jats:italic>-<jats:italic>P. aeruginosa</jats:italic>biofilm.<jats:italic>P. aeruginosa</jats:italic>-<jats:italic>A. fumigatus</jats:italic>polymicrobial biofilm shows differential susceptibility to antimicrobial drugs whereas the susceptibility of<jats:italic>A. fumigatus</jats:italic>to antimicrobial drugs was unchanged.</jats:p></jats:sec>