title: Development and antimicrobial susceptibility studies of in vitro monomicrobial and polymicrobial biofilm models with Aspergillus fumigatus and Pseudomonas aeruginosa.
abstract: BACKGROUND
  Mixed microbial infections of the respiratory tracts with P. aeruginosa and A. fumigatus capable of producing biofilms are commonly found in cystic fibrosis patients. The primary objective of this study was to develop an in vitro model for P. aeruginosa and A. fumigatus polymicrobial biofilm to study the efficacy of various antimicrobial drugs alone and in combinations against biofilm-embedded cells. Simultaneous static cocultures of P. aeruginosa and sporelings were used for the development of in vitro P. aeruginosa-A. fumigatus polymicrobial biofilm in SD broth in 24-well cell culture plates at 35°C, and the biofilm formation was monitored microscopically and spectrophotometrically. Using P. aeruginosa-A. fumigatus sporelings cocultures we examined the effects of various antimicrobial drugs alone and in combination against polymicrobial biofilm by CFU and tetrazolium reduction assays.
  
  
  RESULTS
  In simultaneous static cocultures P. aeruginosa cells killed A. fumigatus conidia, whereas the bacterial cells showed no substantial fungicidal effect on sporelings grown for 12 h or longer at 35°C. Monospecies cultures of P. aeruginosa produced loosely adhered monomicrobial biofilm and addition of 10% bovine serum to the growth medium inhibited the formation of monomicrobial biofilm by P. aeruginosa whereas it produced tightly adhered polymicrobial biofilm in the presence of A. fumigatus mycelial growth. A. fumigatus 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 P. aeruginosa whereas cefepime alone and in combination with posaconazole showed excellent activity against monomicrobial biofilm of P. aeruginosa but was less effective against polymicrobial biofilm. Monomicrobial and polymicrobial biofilms of A. fumigatus showed similar susceptibility to posaconazole with and without the antibacterial drug.
  
  
  CONCLUSIONS
  Simultaneous static coculture of A. fumigatus sporelings grown for 12 h or longer was superior to ungerminated conidia with P. aeruginosa for the development of A. fumigatus-P. aeruginosa biofilm. P. aeruginosa-A. fumigatus polymicrobial biofilm shows differential susceptibility to antimicrobial drugs whereas the susceptibility of A. fumigatus to antimicrobial drugs was unchanged.
authors.forename: Elias K
authors.initials: EK
authors.lastname: Manavathu
authors.forename: Dora L
authors.initials: DL
authors.lastname: Vager
authors.affiliation: Henry Ford Hospital, 2799 West Grand Boulevard, Detroit, Michigan 48202, USA. jvazquez@gru.edu.
authors.forename: Jose A
authors.initials: JA
authors.lastname: Vazquez
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001232:Aspergillus fumigatus / Q000187:drug effects* / Q000254:growth & development / Q000378:metabolism / Q000502:physiology*; D018441:Biofilms / Q000187:drug effects* / Q000254:growth & development*; D015169:Colony Count, Microbial; D008826:Microbial Sensitivity Tests; D050296:Microbial Viability / Q000187:drug effects; D008853:Microscopy; D011550:Pseudomonas aeruginosa / Q000187:drug effects* / Q000254:growth & development / Q000378:metabolism / Q000502:physiology*; D013053:Spectrophotometry; D013194:Staining and Labeling; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D013778:Tetrazolium Salts; D013844:Thiazoles; C022616:thiazolyl blue