P. aeruginosa and C. albicans are human opportunistic pathogens frequently associated with nosocomial infections. Pathogenic interactions between them have already been identified and it has been reported that C. albicans’ morphology and virulence are significantly affected by the presence of P. aeruginosa. In this work, the interaction between these 2 microorganisms in mixed biofilms was studied. The behavior of C. albicans cells after applying P. aeruginosa-specific phages, which lyse P. aeruginosa biofilm cells in the mixed biofilms, was analyzed as well as the ability of C. albicans biofilm formation in the presence of P. aeruginosa phage-resistant phenotypes.
Biofilms were formed in 24-well microplates, containing 1 ml of YPD medium and 10 µl of each cellular suspension (OD600nm = 1), during 24 and 48 h. Phages (phiIBB-PAA2 and phiIBB-PAP21) were applied in 24 h old mixed biofilms and samples were taken after 2, 6 and 24 h of phage biofilm infection for CFU counts.
The results revealed that C. albicans proliferation was clearly inhibited by the presence of wildtype P. aeruginosa ATCC 10145 and PAO1 strains. Conversely, the proliferation of P. aeruginosa was not influenced by the presence of C. albicans. After the infection, it was observed that both phages were effective in depleting Pseudomonas biofilm cells from the mixed biofilms. This reduction resulted in an increase in the amount of C. albicans cells during phage infection. However, at 24 h post-infection of mixed biofilms, an increase of P. aeruginosa biofilm cells was also observed as compared to the numbers at 6 h post-infection. This suggests that P. aeruginosa cells acquired resistance to the phages between 6 and 24 h of infection. Surprisingly, this increase in P. aeruginosa at 24 h post-infection did not interfere with C. albicans biofilm growth and accordingly, an increase in C. albicans cells was observed. These results suggest that the surviving P. aeruginosa cells after phage attack have changes in their phenotype resulting in a diminished ability to inhibit C. albicans biofilm growth. To evaluate if the regrowth of C. albicans cells in the infected biofilms was caused by the emergence of phage-resistant P. aeruginosa phenotypes, which did not inhibit C. albicans biofilm growth, several P. aeruginosa colonies were isolated after 24 h of mixed biofilm infection with phage phiIBB-PAA2 and tested for their susceptibility to the phages used in this study. Most of these phenotypes showed resistance to the phage phiIBB-PAA2 and 1 of these strains was also resistant to phage phiIBB-PAP21. Mixed biofilms with C. albicans were once again performed with these P. aeruginosa ATCC 10145-phenotype variants and revealed that most of them could co-inhabit better with C. albicans than their wildtype strain
