In vitro co-culture model of Trichomonas vaginalis, Candida albicans, and Lactobacillus crispatus: a system for assessing antimicrobial activity and microorganism interactions in vaginitis

<i>Trichomonas vaginalis</i> 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 <i>Candida albicans</i>, 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. <i>Lactobacillus crispatus</i>, a dominant species in the vaginal microbiota, produces antimicrobial compounds that help protect the vaginal mucosa. This study establishes an <i>in vitro</i> co-culture of <i>T. vaginalis</i>, <i>C. albicans</i>, and <i>L. crispatus</i> 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: <i>T. vaginalis</i> at 1.0 × 10⁶ trophozoites/mL (counted using a hemocytometer), 3.33 × 10⁴ CFU/mL for <i>C. albicans</i>, and either 5.53 × 10⁶ CFU/mL (for co-culture with the ATCC isolate) or 5.53 × 10⁷ CFU/mL (for co-culture with a fresh clinical isolate) for <i>L. crispatus</i>. The cell densities of <i>C. albicans</i> and <i>L. crispatus</i> 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, <i>L. crispatus</i> 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 <i>T. vaginalis</i> (100 µM) and <i>C. albicans</i> (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 <i>C. albicans</i> (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 <i>T. vaginalis</i>, <i>C. albicans</i>, and <i>L. crispatus</i>, including isolate-specific size variations in the protozoan. These findings suggest that this <i>in vitro</i> 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.