Antibiofilm action of ozonated sunflower oil against bacteria isolated from the uterus of mares susceptible to endometritis

Antonio Brito da Silva Filho; Laís Querino Barbosa Freire; Maria Eduarda Pedrosa Leite; Lamartine Rodrigues Martins; Gilvannya Gonçalves de Sobral; Marcelo Mendonça; Elizabete Rodrigues da Silva; Haroldo Vargas Leal Júnior; Gustavo Ferrer Carneiro

doi:10.1590/1984-3143-AR2025-0116

ORIGINAL ARTICLE

Antibiofilm action of ozonated sunflower oil against bacteria isolated from the uterus of mares susceptible to endometritis

Antonio Brito da Silva Filho; Laís Querino Barbosa Freire; Maria Eduarda Pedrosa Leite; Lamartine Rodrigues Martins; Gilvannya Gonçalves de Sobral; Marcelo Mendonça; Elizabete Rodrigues da Silva; Haroldo Vargas Leal Júnior; Gustavo Ferrer Carneiro

http://dx.doi.org/10.1590/1984-3143-AR2025-0116 Anim Reprod, vol.23, n3, e20250116, 2026

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Abstract

This study aimed to characterize the antibiofilm action of ozone-enriched sunflower oil (O₃) against pathogens associated with endometritis in mares. Initially, samples from 41 mares with a history of subfertility were stored in BHI broth and 0.9% NaCl solution. The samples underwent microbial culture, identification, and in vitro sensitivity testing at LABRAPE-UFRPE, Brazil. Biofilm production was evaluated by the crystal violet method. Subsequently, biofilm-forming bacteria were subjected to the action of different concentrations of ozonated sunflower oil. Of the 21 isolates tested for biofilm production, 76.2% (16/21) showed positive production, with 43.75% (7/16) being weak producers, 31.25% (5/16) moderate producers, and 25% (4/16) strong producers. After the action of ozonated sunflower oil at a concentration of 203.43 mEq/L, a reduction was observed in the mass of the gram-negative bacilli biofilm, however, no statistical difference was found for the gram-positive cocci biofilm. The results show that ozonated sunflower oil presented satisfactory results in breaking down gram-negative bacteria biofilm.

Keywords

ozone, endometritis, peroxide, reproduction, equines

References

Abbas S, Kiani H, Gohar F, Zahra S, Javed A, Khan S, Khan D. Understanding the role of bacterial biofilm in antibiotic resistance: defensive strategies and clinical challenges. In: Dincer S, Ozdenefe MS, Takci HAM, editors. Exploring bacterial biofilms. London: IntechOpen; 2025. https://doi.org/10.5772/intechopen.1009440.

Almatroudi A. Biofilm resilience: molecular mechanisms driving antibiotic resistance in clinical contexts. Biology. 2025;14(2):165. PMid:40001933.

Almeida BFM, Amatti LZ, de Souza GG, Garcia LV, Montechiesi DF, Ignácio FS, de Oliveira PL, Costa LR, Floriano BP, Bosculo MRM, Joaquim JGF, Rubio CP. Effect of uterine ozone therapy and anticoagulant sampling on oxidative stress parameters in mares. Res Vet Sci. 2021;136:503-11. PMid:33878612.

Araújo MS, Prado JCS, Aguiar FLL, Silva FA, Viana YN, Dantas JP, Anzolin AP, Bastos PL, Fontenelle ROS, Barbosa FCB. Antifungal and antibiofilm activities of ozonated sunflower oil against Candida albicans strains: a pilot study and clinical perspectives. Archives of Current Research International. 2024;24:494-502.

Bailey P. Ozonization in organic chemistry. New York: Academic Press; 1978.

Beehan DP, Wolfsdorf K, Elam J, Krekeler N, Paccamonti D, Lyle SK. The evaluation of biofilm-forming potential of Escherichia coli collected from the equine female reproductive tract. J Equine Vet Sci. 2016;35:935-9.

Cabrera T, Patorelo M, Alvarenga MA. Prevalência e perfil de sensibilidade bacteriana em éguas com endometrite. Encicl Biosf. 2016;13:1013.

Canisso IF, Stewart J, Coutinho da Silva MA. Endometritis in mares: current advances and future perspectives. Theriogenology. 2020;150:321-32.

Carter GR. Fundamentos da bacteriologia e micologia veterinária. São Paulo: Roca; 1988. 249 p.

Causey RC, Weber JA, Emmans EE, Stephenson LA, Homola AD, Knapp KR, Crowley IF, Pelletier DC, Wooley NA. The equine immune response to Streptococcus equi subspecies zooepidermicus during uterine infection. Vet J. 2006;172(2):248-57. PMid:15950504.

Clinical and Laboratory Standards Institute – CLSI. Performance standards for antimicrobial susceptibility testing. CLSI supplement M100. 34th ed. Wayne: CLSI; 2024.

Criegee R. Mechanism of ozonolysis. Angew Chem Int Ed Engl. 1975;14(11):745-52.

Darwish SF, Asfour HAE. Investigation of biofilm forming ability in staphylococci causing bovine mastitis using phenotypic and genotypic assays. ScientificWorldJournal. 2013;2013(1):378492. https://doi.org/10.1155/2013/378492. PMid:24298212.

Díaz MF, Hernandez R, Martinez G, Vidal G, Gómez M, Fernández H, Garcés R. Comparative study of ozonated olive oil and ozonated sunflower oil. J Braz Chem Soc. 2006;17(2):403-7.

Díaz-Bertrana L, García-Seco T, Sánchez-Margallo FM, Pérez-Marín CC. Prevalence and microbiological profile of infectious endometritis in subfertile mares. Vet Rec. 2021;189(6):e512. PMid:34057217.

Diel de Amorim M, Gartley CJ, Foster RA, Hill A, Scholtz EL, Hayes A, Chenier TS. Comparison of clinical signs, endometrial culture, endometrial cytology, uterine low volume lavage, and uterine biopsy, and combinations in the diagnosis of equine endometritis. J Equine Vet Sci. 2015;44:54-61.

Donlan RM, Costerton JW. Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev. 2002;15(2):167-93. https://doi.org/10.1128/CMR.15.2.167-193.2002. PMid:11932229.

Elvis AM, Ekta JS. Ozonoterapia: uma revisão clínica. J Nat Sci Biol Med. 2011;2(1):66-70. https://doi.org/10.4103/0976-9668.82319. PMid:22470237.

Ferreira JC, Amaral RS, Cardoso LM, Marchió SP, Rodrigues MA, Alves CEF, Meira C. Endometrial angiogenesis induced by uterine insufflation with an oxygen-ozone gas mixture in mares. Reprod Domest Anim. 2021;56(9):1176-83. https://doi.org/10.1111/rda.13958. PMid:34021934.

Ferris RA, McCue PM, Borlee GI, Glapa KE, Martin KH, Mangalea MR, Hennet ML, Wolfe LM, Broeckling CD, Borlee BR. Model of chronic equine endometritis involving a Pseudomonas aeruginosa biofilm. Infect Immun. 2017;85(12):e00332-17. https://doi.org/10.1128/IAI.00332-17. PMid:28970274.

Ferris RA, McCue PM, Borlee GI, Loncar KD, Hennet ML, Borlee BR. In vitro efficacy of nonantibiotic treatments on biofilm disruption of Gram-negative pathogens and an in vivo model of infectious endometritis utilizing isolates from the equine uterus. J Clin Microbiol. 2016;54(3):631-9. https://doi.org/10.1128/JCM.02861-15. PMid:26719448.

Ferris RA, Wittstock SM, McCue PM, Borlee BR. Evaluation of biofilms in Gram-negative bacteria isolated from the equine uterus. J Equine Vet Sci. 2014;34(1):121. https://doi.org/10.1016/j.jevs.2013.10.082.

Gentili V, Strazzabosco G, Salgari N, Mancini A, Rizzo S, Beltrami S, Schiuma G, Casciano F, Alogna A, Passarella D, Davinelli S, Scapagnini G, Medoro A, Rizzo R. Ozonated oil in liposome eyedrops reduces the formation of biofilm, selection of antibiotic-resistant bacteria, and adhesion of bacteria to human corneal cells. Int J Mol Sci. 2023;24(18):14078. https://doi.org/10.3390/ijms241814078. PMid:37762377.

Higa B, Cintra BS, Álvarez CM, Ribeiro AB, Ferreira JC, Tavares DC, Enriquez V, Martinez LR, Pires RH. Ozonated oil is effective at killing Candida species and Streptococcus mutans biofilm-derived cells under aerobic and microaerobic conditions. Med Mycol. 2022;60(8):55. https://doi.org/10.1093/mmy/myac055. PMid:35869980.

Kozdrowski R, Sikora M, Buczkowska J, Nowak M, Ras A, Dziecioł M. Effects of cycle stage and sampling procedure on interpretation of endometrial cytology in mares. Anim Reprod Sci. 2015;154:56-62. https://doi.org/10.1016/j.anireprosci.2015.01.009. PMid:25660623.

Leblanc MM, Magsig J, Stromberg AJ. Use of a low-volume uterine flush for diagnosing endometritis in chronically infertile mares. Theriogenology. 2007;68(3):403-12. https://doi.org/10.1016/j.theriogenology.2007.04.038. PMid:17543379.

Ledea O. Ozone treatment in medicine [thesis]. Havana: National Center for Scientific Research; 2003.

Liu HY, Prentice EL, Webber MA. Mechanisms of antimicrobial resistance in biofilms. Npj Antimicrob Resist. 2024;2(1):27. https://doi.org/10.1038/s44259-024-00046-3. PMid:39364333.

Melo PC, Ferreira LM, Nader A Fo, Zafalon LF, Vicente HIG, Souza V. Comparison of methods for the detection of biofilm formation by Staphylococcus aureus isolated from bovine subclinical mastitis. Braz J Microbiol. 2013;44(1):119-24. https://doi.org/10.1590/S1517-83822013005000031. PMid:24159293.

Merhi Z, Garg B, Moseley-LaRue R, Moseley AR, Smith AH, Zhang J. Ozone therapy: a potential therapeutic adjunct for improving female reproductive health. Med Gas Res. 2019;9(2):101-5. https://doi.org/10.4103/2045-9912.260652. PMid:31249259.

Nasr RA, Abushadady HM, Hussein SH. Biofilm formation and presence of icaAD gene in clinical isolates of staphylococci. Egypt J Med Hum Genet. 2012;13(3):269-74. https://doi.org/10.1016/j.ejmhg.2012.04.007.

Nielsen CK, Kjems J, Mygind T, Snabe T, Meyer Rl. Efeitos do Tween 80 no crescimento e formação de biofilme em meios de laboratório. Front Microbiol. 2016;7:1878.

Noel CC, Motta FS, Francisco NLS, Almeida NR, Soares LC. Perfil de suscetibilidade antimicrobiana e produção de “slime” de isolados de Staphylococcus spp. provenientes de casos de mastite bovina na região Sul-Fluminense. Rev Saúde. 2016;7(1):22-6.

Oliveira IB, Peixoto RM, Silva DR, Pinheiro JW Jr, Oliveira AAF, Mota RA. Análise comparativa entre o exame citológico e microbiológico no diagnóstico de endometrite equina. Vet Zootec. 2010;17(1):43-6.

O’Toole G, Kaplan HB, Kolter R. Biofilm formation as microbial development. Annu Rev Microbiol. 2000;54(1):49-79. https://doi.org/10.1146/annurev.micro.54.1.49. PMid:11018124.

Overbeck W, Witte TS, Heuwieser W. Comparison of three diagnostic methods to identify subclinical endometritis in mares. Theriogenology. 2011;75(7):1311-8. https://doi.org/10.1016/j.theriogenology.2010.12.002. PMid:21251703.

Patra S, Saha S, Singh R, Tomar N, Gulati P. Biofilm battleground: unveiling the hidden challenges, current approaches and future perspectives in combating biofilm-associated bacterial infections. Microb Pathog. 2025;198:107155. https://doi.org/10.1016/j.micpath.2024.107155. PMid:39586337.

Perez RR, Nunez SA, Baluja C, Otero ML. Ozonation kinetics of glucosamine and N-acetyl glucosamine in aqueous medium. Ozone Sci Eng. 1995;17(4):463-7. https://doi.org/10.1080/01919519508547349.

Quinn PJ, Carter ME, Markey BK, Carter GR. Clinical veterinary microbiology. London: Wolfe; 1994.

Repciuc CC, Vișan GAM, Teleky BE, Pintea A, Novac CȘ, Oros NV. Physico-chemical and antimicrobial evaluation of ozonated olive oil produced with a medical-grade generator for veterinary purposes. Microorganisms. 2025;13(8):1932. https://doi.org/10.3390/microorganisms13081932. PMid:40871436.

Schwartz A, Kontorschikova CN, Malesnikov OV, Sánchez GM, Re L, Gribkova IA. Guía para el uso médico del ozono: fundamentos terapêuticos e indicaciones. 1. ed. Madrid: Artes Gráficas; 2011.

Silva V, Peirone C, Amaral JS, Capita R, Alonso-Calleja C, Marques-Magallanes JA, Martins Â, Carvalho Á, Maltez L, Pereira JE, Capelo JL, Igrejas G, Poeta P. High efficacy of ozonated oils on the removal of biofilms produced by methicillin-resistant Staphylococcus aureus (MRSA) from infected diabetic foot ulcers. Molecules. 2020;25(16):3601. https://doi.org/10.3390/molecules25163601. PMid:32784722.

Thanomsub B, Anupunpisit V, Chanphetch S, Watcharachaipong T, Poonkhum R, Srisukonth C. Effects of ozone treatment on cell growth and ultrastructural changes in bacteria. J Gen Appl Microbiol. 2002;48(4):193-9. https://doi.org/10.2323/jgam.48.193. PMid:12469318.

Thomassian A. Enfermidades dos cavalos. 4. ed. São Paulo: Varela; 2007. p. 258–9.

Troedsson MHT. Uterine clearance and resistance to persistent endometritis in the mare. Theriogenology. 2011;75(2):375-81. PMid:10734380.

Tukia E, Hallman I, Penttilä M, Hänninen S, Kareskoski M. RNA in situ hybridization of Escherichia coli in equine endometrial biopsies. Front Vet Sci. 2023;10:1186051. https://doi.org/10.3389/fvets.2023.1186051. PMid:37360403.

Vargas H, Nascimento JAA Jr, Maciel GN Jr. Natural products derivatives are able to biofilm breakage in vitro of bacteria isolated from mare uterus. In: Congresso Latinoamericano de Microbiologia; 2019; Assunção, Paraguai,. São Paulo: Sociedade Brasileira de Microbiologia; 2019. p. 222.

Vieira MH, Boreski D, Franzen Matte B, Arias JLO, Martins C Jr, Bauab TM, Gabriel SA, Chin CM. Ozonized sunflower oil: standardization and mechanisms of the antimicrobial effect. Int J Mol Sci. 2025;26(18):9156. https://doi.org/10.3390/ijms26189156. PMid:41009715.

Woodward EM, Christoffersen M, Campos J, Betancourt A, Horohov D, Scoggin KE, Squires EL, Troedsson MHT. Endometrial inflammatory markers of the early immune response in mares susceptible or resistant to persistent breeding-induced endometritis. Reproduction. 2013;145(3):289-96. https://doi.org/10.1530/REP-12-0452. PMid:23580950.

Submitted date:
07/31/2025

Accepted date:
03/31/2026

Antibiofilm action of ozonated sunflower oil against bacteria isolated from the uterus of mares susceptible to endometritis

Antonio Brito da Silva Filho; Laís Querino Barbosa Freire; Maria Eduarda Pedrosa Leite; Lamartine Rodrigues Martins; Gilvannya Gonçalves de Sobral; Marcelo Mendonça; Elizabete Rodrigues da Silva; Haroldo Vargas Leal Júnior; Gustavo Ferrer Carneiro

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