co-culture model of , , and : a system for assessing antimicrobial activity and microorganism interactions in vaginitis.

is a flagellated protozoan causing trichomoniasis, the most common non-viral sexually transmitted infection. It is associated with various complications, particularly in asymptomatic carriers. Another major cause of vaginitis is , a normal member of the vaginal microbiota, which causes vulvovaginal candidiasis when immune imbalances occur, leading to recurrent infections. Treatment-resistant strains of these pathogens pose a significant challenge. , a dominant species in the vaginal microbiota, produces antimicrobial compounds that help protect the vaginal mucosa. This study establishes an co-culture of , , and to simulate the vaginal microenvironment at the site of infection. MRS medium was chosen for the co-culture, with initial cell densities determined as follows: at 1.0 × 10 trophozoites/mL (counted using a hemocytometer), 3.33 × 10 CFU/mL for , and either 5.53 × 10 CFU/mL (for co-culture with the ATCC isolate) or 5.53 × 10 CFU/mL (for co-culture with a fresh clinical isolate) for . The cell densities of and were quantified as colony-forming units (CFU) on selective agar. The incubation period for co-culture, ensuring optimal growth of all microorganisms, was 24 hours. In co-culture, at both tested densities acidified the medium. The co-culture system demonstrated lower MIC values for metronidazole (50 µM in the ATCC isolate co-culture and 25 µM with the fresh clinical isolate) and lower MFC values for fluconazole (6.25 µM), compared to monocultures of (100 µM) and (12.50 µM). Furthermore, the triple co-culture increased the cytotoxicity to vaginal cell and erythrocytes for the ATCC isolate while significantly inhibited both biofilm formation and metabolic activity of (by up to 92% and 90%, respectively), as well as its yeast-to-hyphae transition (by up to 70%). SEM analyses highlighted the morphological differences among , , and , including isolate-specific size variations in the protozoan. These findings suggest that this co-culture system is a valuable tool for evaluating the antimicrobial efficacy of novel compounds against vaginitis pathogens and for studying interactions within the vaginal microenvironment.