COMPARISON OF THE EFFECT OF ANTIBIOTICS AND BACTERIOPHAGE PHAGE SAVB14 ON BIOFILMS FORMED BY STAPHYLOCOCCUS AUREUS VARIANT BOVIS

Authors

  • Ю. В. Горюк State Agrarian and Engineering University in Podilya
  • М. Д. Кухтин Ternopil Ivan Puluj National Technical University
  • В. В. Горюк State Agrarian and Engineering University in Podilya
  • С. П. Керничний State Agrarian and Engineering University in Podilya

DOI:

https://doi.org/10.37406/2706-9052-2020-1-19

Keywords:

mastitis, biofilms, antibiotics, bacteriophage Phage SAvB14, S. aureus var. bovis

Abstract

During the development of mastitis in cows, the formation of a biofilm pathogen is an effective way to preserve it in the microenvironment of mammary gland. Biofilm infections are difficult to treat with antimicrobials, and bacterial resistance to antibiotics increases to 1000-fold level, compared with what is observed when grown in planktonic conditions. The aim of study – to determine and compare the effect of antimicrobial drugs and bacteriophage Phage SAvB14 in the destruction of biofilms formed by S. aureus var. bovis. Isolation and species identification of staphylococci were performed according to conventional methods using BD Baird-Parker Agar medium (HiMedia, India). Determination of ability of staphylococci to form biofilms and the number of viable bacteria was determined by the Stepanovic method. The study of sensitivity of microorganisms in biofilm form was performed on daily microbial biofilms grown in plastic Petri dishes. After 24 hours of incubation of cultures, the dishes were washed three times from planktonic (unattached) microorganisms with sterile phosphate buffer and introduced the studied antibacterial agents. After exposure, the dishes were washed three times with sterile phosphate buffer, introduced 5 cm3 of sterile 0.9% sodium chloride solution and washed the biofilm, took 1.0 cm3 of suspension, prepared a series of ten-fold dilutions, inoculated 1.0 cm3 of each dilution in Petri dishes, poured MPA and incubated at temperature of 370C for 24–48 hours to determine the number of bacteria. In determining the effect of antibiotics on bacterial biofilms, it was found that of the studied antibiotics, enrofloxacin worked best probably due to its low molecular weight and ability to penetrate the pores and channels of the biofilm to microbial cells. After the action of enrofloxacin on biofilms, staphylococcal bacteria were completely inactivated. Also, the antibiotics ceftriaxone and doxycycline were effective against bacteria in biofilms. After the action of ceftriaxone, the number of surviving bacteria was lg 1.9 ± 1.1 CFU/cm2 of biofilm area, and doxycycline lg 2.5 ± 1.2 CFU/cm2. At the same time, under the action of antibiotics penicillins, aminoglycosides and macrolides, the number of surviving microbial cells was about lg 5.3 CFU/cm2 of biofilm area. In studies on the effect of bacteriophage Phage SAvB14 on biofilms formed by S. aureus var. bovis, there was their degradation. At this, viable microbial cells from the biofilm were not isolated. In this case, we can say that the phages penetrated and reached the staphylococcal cells throughout the thickness of biofilm and bacteria were susceptible to this phage. That is, there was a passive treatment of biofilm with phages, in which lysis depended on the rate of virus uptake. Therefore, the obtained results of laboratory studies indicate the prospects of effective use of our selected specific staphylococcal bacteriophage Phage SAvB14 for the destruction of biofilm formed by S. aureus var. bovis – in mastitis of cows.

Author Biographies

Ю. В. Горюк, State Agrarian and Engineering University in Podilya

Horiuk Y. V. 
Ph.D (Veterinary Sciences)

М. Д. Кухтин, Ternopil Ivan Puluj National Technical University

Kukhtyn M. D.
Dr. Sc. (Veterinary Sciences), Professor

В. В. Горюк, State Agrarian and Engineering University in Podilya

Horiuk V. V. 
Ph.D. (Veterinary Sciences), Associate Professor

С. П. Керничний, State Agrarian and Engineering University in Podilya

Kernychnyi S. P. 
Ph.D. (Veterinary Sciences), Associate Professor

References

Azeredo, J., & Sutherland, I. W. (2008). The use of phages for the removal of infectious biofilms. Current Pharmaceutical Biotechnology, 9(4), 261–266. doi: 10.2174/138920108785161604

Zimmerli, W., & Sendi, P. (2019). Role of rifampin against staphylococcal biofilm infections in vitro, in animal models, and in orthopedic-device-related infections. Antimicrobial Agents and Chemotherapy, 63(2), e01746–18. doi: 10.1128/AAC.01746-18

Felipe, V., Breser, M. L., Bohl, L. P., da Silva, E. R., Morgante, C. A., Correa, S. G., & Porporatto, C. (2019). Chitosan disrupts biofilm formation and promotes biofilm eradication in Staphylococcus species isolated from bovine mastitis. International Journal of Biological Macromolecules, 126, 60–67. doi: 10.1016/j.ijbiomac.2018.12.159

Horiuk, Y. V., Kukhtyn, M. D., Strayskyy, Y. S., Havrylianchyk, R. Y., Horiuk, V. V., & Fotina, H. A. (2018). Comparison Of The Minimum Bactericidal Concentration Of Antibiotics On Planktonic And Biofilm Forms Of Staphylococcus Aureus: Mastitis Causative Agents. Research Journal of Pharmaceutical, Biological and Chemical Sciences, 9(6), 616–622.

Iglesias, Y. D., Wilms, T., Vanbever, R., & Van Bambeke, F. (2019). Activity of antibiotics against Staphylococcus aureus in an in vitro model of biofilms in the context of cystic fibrosis: influence of the culture medium. Antimicrobial Agents and Chemotherapy, 63(7), e00602-19. doi: 10.1128/AAC.00602-19

Stewart, P. S., & Franklin, M. J. (2008). Physiological heterogeneity in biofilms. Nature Reviews Microbiology, 6(3), 199. doi: 10.1038/nrmicro1838

Hymes, S. R., Randis, T. M., Sun, T. Y., & Ratner, A. J. (2013). DNase inhibits Gardnerella vaginalis biofilms in vitro and in vivo. The Journal of Infectious Diseases, 207(10), 1491–1497. doi: 10.1093/infdis/jit047

Dias, R. S., Eller, M. R., Duarte, V. S., Pereira, A. L., Silva, C. C., Mantovani, H. C., Oliveira, L. L., Silva, E. de A. M., & Paula, S. O. (2013). Use of phages against antibiotic-resistant Staphylococcus aureus isolated from bovine. J. Anim. Sci., 91, 3930–3939. doi: 10.2527/jas.2012-5884

Tkhilaishvili, T., Lombardi, L., Klatt, A. B., Trampuz, A., & Di Luca, M. (2018). Bacteriophage Sb-1 enhances antibiotic activity against biofilm, degrades exopolysaccharide matrix and targets persisters of Staphylococcus aureus. International Journal of Antimicrobial Agents, 52(6), 842–853. doi: 10.1016/j.ijantimicag.2018.09.006

Vos, P., Garrity, G., Jones, D., Krieg, N. R., Ludwig, W., Rainey, F. A., & Whitman, W. B. (Eds.). (2011). Bergey's manual of systematic bacteriology: Volume 3:The Firmicutes. Springer Science & Business Media.

Stepanović, S., Vuković, D., Dakić, I., Savić, B., & Švabić-Vlahović, M. (2000). A modified microtiter-plate test for quantification of staphylococcal biofilm formation. Journal of Microbiological Methods, 40(2), 175–179. doi: 10.1016/S0167-7012(00)00122-6

Wills, Q. F., Kerrigan, C., & Soothill, J. S. (2005). Experimental bacteriophage protection against Staphylococcus aureus abscesses in a rabbit model. Antimicrobial Agents and Chemotherapy, 49(3), 1220–1221. doi: 10.1128/AAC.49.3.1220-1221.2005

Bahamondez-Canas, T. F., Zhang, H., Tewes, F., Leal, J., & Smyth, H. D. (2018). PEGylation of tobramycin improves mucus penetration and antimicrobial activity against Pseudomonas aeruginosa biofilms in vitro. Molecular Pharmaceutics, 15(4), 1643–1652. doi: 10.1021/acs.molpharmaceut.8b00011

Kukhtyn, M., Berhilevych, О., Kravcheniuk, K., Shynkaruk, O., Horiuk, Y., & Semaniuk, N. (2017). Formation of biofilms on dairy equipment and the influence of disinfectants on them. Eastern-European Journal of Enterprise Technologies, 5/11(89), 26–33. doi: 10.15587/1729-4061.2017.110488

Horiuk, Y. V. (2018). Fagotherapy of cows mastitis as an alternative to antibiotics in the system of obtaining environmentally safe milk. Scientific Messenger of Lviv National University of Veterinary Medicine and Biotechnologies, 20(88), 42–47. doi: 10.32718/nvlvet8807

Gutierrez, D., Vandenheuvel, D., Martínez, B., Rodríguez, A., Lavigne, R., & García, P. (2015). Two phages, phiIPLA-RODI and phiIPLA-C1C, lyse mono-and dual-species staphylococcal biofilms. Applied and Environmental Microbiology, 81(10), 3336–3348. doi: 10.1128/AEM.03560-14

Lopetuso, L., Giorgio, M., Saviano, A., Scaldaferri, F., Gasbarrini, A., & Cammarota, G. (2019). Bacteriocins and Bacteriophages: Therapeutic Weapons for Gastrointestinal Diseases? International Journal of Molecular Sciences, 20(1), 183-193. doi: 10.3390/ijms20010183

Published

2020-05-29

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