Abstract
BackgroundMixed 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.ResultsIn 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.ConclusionsSimultaneous 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.
Highlights
Mixed microbial infections of the respiratory tracts with P. aeruginosa and A. fumigatus capable of producing biofilms are commonly found in cystic fibrosis patients
Tetrazolium reduction assay In addition to Colony forming unit (CFU) assay, we evaluated the effects of antimicrobial drugs on monomicrobial and polymicrobial biofilms of A. fumigatus and P. aeruginosa by the tetrazolium reduction assay [47,48]
The formation of mixed microbial biofilm is considered to be a serious clinical problem in Cystic fibrosis (CF) patients as well as in other patient groups prone to airway infection with P. aeruginosa and A. fumigatus, we know very little about the antibiotic susceptibility of P. aeruginosa-A. fumigatus polymicrobial biofilm
Summary
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. A recent retrospective study of the respiratory tract microbiology of cystic fibrosis patients revealed that their airways were colonized by multiple microorganisms, in particular Pseudomonas aeruginosa (62% prevalence) in association with Aspergillus species [24]. Among the numerous Aspergillus isolates recovered from the respiratory tracts of cystic fibrosis patients, A. fumigatus is the most predominant species with a prevalence ranging from 11% to 14% in the United States [28] and as high as 60% to 78% in Europe [29,30], followed by A. terreus. Invasive aspergillosis can occur in persons with cystic fibrosis, after lung transplantation, the most common complication of Aspergillus infection is allergic bronchopulmonary aspergillosis [31,32,33,34], a condition that causes the deterioration of lung function associated with wheezing, shortness of breath, cough and chest pain
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