Capacity of biofilm production by filamentous fungi isolated from the uterus of susceptible mares

Abstract

Fungi are opportunistic microorganisms that adhere to surfaces. Their intrauterine growth is influenced by impaired immunity, indiscriminate use of antibiotics, and poor vulvar conformation. Many publications use yeast research models, but only some report biofilm production by filamentous fungi. This work aimed to biofilm capacity from isolates of filamentous fungi found in uterine samples from susceptible mares. Uterine saline lavage samples from 41 mares with a history of subfertility were collected and sent to LABRAPE-UFRPE, Brazil. Samples were cultured on Sabouraud Dextrose Agar plates with 10% chloramphenicol and incubated at 25°C for up to 15 days, inspecting every 24 hours. After development of the colonies, identification used the macro and micromorphological aspects of the somatic and reproductive structures, comparing them with the literature. To induce biofilm production, isolates were re-cultured on Sabouraud dextrose agar at 37°C for 72 h. Conidia were collected by washing the surface of the cultures using 5mL PBS, pH 7.2, supplemented with 0.025% (v/v) Tween 20. Subsequently, conidia were counted in a Neubauer chamber, and the inoculum was adjusted to the concentration of 1 × 105 cells in RPMI 1640and buffered to pH 7.0. Biofilms were produced in 96-well microtiter plates by adding 200µL of standardized cell suspension in MOPS-RPMI 1640 buffer in each well, at each determined period (24, 48, 72, and 96h) then incubated at 37°C. At each time interval, biofilms were washed three times with PBS pH 7.2 then quantified using crystal violet technique (absorbance 570nm). Of the samples, 12.2% were positive for fungal endometritis, with Cladosporium spp. isolated in 60% of the positive samples, Trichosporon spp. and Penicillium spp. in 20% of the positive samples each. After induction of biofilm production, fungi of the genus Cladosporium spp. and Penicillium spp. showed a high biofilm production capacity, with a growth peak at 48 hours after incubation and a gradual decrease in this production in the subsequent hours. Cladosporium spp. showed low biofilm production until 96h of incubation, and Penicillium spp zeroed its formation simultaneously. In conclusion filamentous fungi isolated from the uterus of mares have a greater biofilm production capacity after 48 hours incubation, which tends to decrease over time but can be maintained for up to 96 hours, depending on the gender involved in the infection, as well as the quantity and quality of the substrate available.