Anti-biofilm action of ozonized sunflower oil against bacteria isolated from the uterus of mares susceptible to endometritis

Abstract

Biofilm formation in the equine reproductive tract is thought to be involved with chronic endometritis, inhibiting any response to antibiotic therapy. No publications correlate the action of ozonized oils with the breakdown of biofilm mass from bacterial isolates. This work aimed to evaluate the efficiency of ozonated sunflower oil in breaking biofilm produced in vitro by bacteria isolated from the uterus of susceptible mares. Uterine samples from 41 mares with a history of subfertility (negative embryo collections or pregnancy for three consecutive cycles) were used. Samples were collected using a cytological brush and stored in BHI broth and 0.9% saline, then submitted for microbial culture and identification and in vitro sensitivity at LABRAPE-UFRPE, Brazil. Biofilm production was evaluated by the crystal violet method using 96-well polystyrene microplates. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were determined by the microdilution method, where concentrations of ozonated sunflower oil ranging from 12.71 to 203.43 mEq of peroxide were used. To evaluate the action of the ozonated oil in breaking the biofilm, bacteria were subjected to a concentration of 203.43 mEq of peroxide, with the use of extra virgin sunflower oil as a negative control. The results were submittedto Student's T-Test for paired samples, and the significance level was P<0.05. The test was performed using the statistical software BioEstat 5.0®. Overall, 75.6% of the samples had positive microbiological isolation, while 24.4% were negative. Of the 21 isolates subjected to biofilm production, 76.2% (16/21) showed positive production, with 43.75% (7/16) weak producers, 31.25% (5/16) moderate producers, and 25% (4/16) strong producers. After the action of the ozonized oil on the previously formed biofilm, of the six samples of Gram-negative bacilli tested, only one sample from the genus Klebsiella spp. showed no statistical difference in biofilm formation, while the others showed biofilm breakage after treatment, which corresponded to a reduction in biofilm mass to 0.098 (0.209 ± 0.054). However, in assessment of biofilm mass after treatment, for Gram-positive cocci, five samples showed no change, three samples showed a reduction, and two samples showed an increase. In conclusion, ozonized sunflower oil (concentration of 203.43 mEq of peroxide) demonstrated satisfactory results in breaking the in vitro biofilm produced by Gram-negative bacteria. It proved to be inefficient in breaking the mass of biofilm produced by Gram-positive bacteria. The efficiency of ozonized sunflower oil is directly linked to its peroxide concentration.