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Evaluation of quantification methods to determine photodynamic action on mono- and dual-species bacterial biofilms

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Abstract

The effect of photodynamic inactivation (PDI) sensitized by 5,10,15,20-tetra(4-N,N,N-trimethylammoniophenyl)porphyrin (TMAP4+) on different components of mono- and dual-species biofilms of Staphylococcus aureus and Escherichia coli was determined by different methods. First, the plate count technique showed that TMAP4+-PDI was more effective on S. aureus than E. coli biofilm. However, crystal violet staining revealed no significant differences between before and after PDI biofilms of both bacteria. On the other hand, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method indicated a reduction in viable cells as the light exposure time increases in both, mono- and dual-species biofilms. Furthermore, it was determined that as the irradiation time increases, the amount of extracellular polymeric substances present in the biofilms decreased. This effect was presented in both strains and in the mixed biofilm, being more evident in S. aureus mono-specie biofilm. Finally, scanning electron microscopy analysis showed a decrease in the number of cells forming the biofilm after photosensitization treatments. This information makes it possible to determine whether the photodynamic action is based on damage to metabolic activity, extracellular matrix and/or biomass, which may be useful in establishing a fully effective PDI protocol for the treatment of microorganisms growing as biofilms.

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References

  1. Orazi, G., & O’Toole, G. A. (2020). “It takes a village”: Mechanisms underlying antimicrobial recalcitrance of polymicrobial biofilms. Journal of Bacteriology, 202, e00530-e619. https://doi.org/10.1128/jb.00530-19

    Article  CAS  Google Scholar 

  2. Anju, V. T., Busi, S., Imchen, M., Kumavath, R., Mohan, M. S., Salim, S. A., Subhaswaraj, P., & Dyavaiah, M. (2022). Polymicrobial infections and biofilms: Clinical significance and eradication strategies. Antibiotics, 11, 1731. https://doi.org/10.3390/antibiotics11121731

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Sauer, K., Stoodley, P., Goeres, D. M., Hall-Stoodley, L., Burmølle, M., Stewart, P. S., & Bjarnsholt, T. (2022). The biofilm life cycle—Expanding the conceptual model of biofilm formation. Nature Reviews Microbiology, 20(10), 608. https://doi.org/10.1038/s41579-022-00767-0

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Kranjec, C., Morales Angeles, D., Torrissen Mårli, M., Fernández, L., García, P., Kjos, M., & Diep, D. B. (2021). Staphylococcal biofilms: Challenges and novel therapeutic perspectives. Antibiotics, 10, 131. https://doi.org/10.3390/antibiotics10020131

  5. Reynoso, E., Ferreyra, D. D., Durantini, E. N., & Spesia, M. B. (2019). Photodynamic inactivation to prevent and disrupt Staphylococcus aureus biofilm under different media conditions. Photodermatology, Photoimmunology & Photomedicine, 35, 322. https://doi.org/10.1111/phpp.12477

    Article  CAS  Google Scholar 

  6. Pinto, R. M., Soares, F. A., Reis, S., Nunes, C., & Van Dijck, P. (2020). Innovative strategies toward the disassembly of the EPS matrix in bacterial biofilms. Frontiers in Microbiology, 11, 952. https://doi.org/10.3389/fmicb.2020.00952

    Article  PubMed  PubMed Central  Google Scholar 

  7. Banerjee, S., Ghosh, D., Vishakha, K., Das, S., Mondal, S., & Ganguli, A. (2020). Photodynamic antimicrobial chemotherapy (PACT) using riboflavin inhibits the mono and dual species biofilm produced by antibiotic resistant Staphylococcus aureus and Escherichia coli. Photodiagnosis and Photodynamic Therapy, 32, 102002. https://doi.org/10.1016/j.pdpdt.2020.102002

  8. Hu, X., Huang, Y.-Y., Wang, Y., Wang, X., & Hamblin, M. R. (2018). Antimicrobial photodynamic therapy to control clinically relevant biofilm infections. Frontiers in Microbiology, 9, 1299. https://doi.org/10.3389/fmicb.2018.01299

    Article  PubMed  PubMed Central  Google Scholar 

  9. Kim, S.-H., Park, S.-H., Kim, S.-S., & Kang, D.-H. (2019). Inactivation of Staphylococcus aureus biofilms on food contact surfaces by superheated steam treatment. Journal of Food Protection, 82(9), 1496. https://doi.org/10.4315/0362-028X.JFP-18-572

    Article  CAS  PubMed  Google Scholar 

  10. Toté, K., Berghe, D. V., Maes, L., & Cos, P. (2007). A new colorimetric microtitre model for the detection of Staphylococcus aureus biofilms. Letters in Applied Microbiology, 46(2), 249. https://doi.org/10.1111/j.1472-765X.2007.02298.x

    Article  PubMed  Google Scholar 

  11. Simonetti, O., Rizzetto, G., Radi, G., Molinelli, E., Cirioni, O., Giacometti, A., & Offidani, A. (2021). New perspectives on old and new therapies of staphylococcal skin infections: The role of biofilm targeting in wound healing. Antibiotics, 10, 1377. https://doi.org/10.3390/antibiotics10111377

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Lin, S., Yang, L., Chen, G., Li, B., Chen, D., Li, L., & Xu, Z. (2017). Pathogenic features and characteristics of food borne pathogens biofilm: Biomass, viability and matrix. Microbial Pathogenesis, 111, 285. https://doi.org/10.1016/j.micpath.2017.08.005

    Article  CAS  PubMed  Google Scholar 

  13. Peeters, E., Nelis, H. J., & Coenye, T. (2008). Comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates. Journal of Microbiological Methods, 72, 157. https://doi.org/10.1016/j.mimet.2007.11.010

    Article  CAS  PubMed  Google Scholar 

  14. Achinas, S., Keimpe Yska, S., Charalampogiannis, N., Krooneman, J., & JanWillem Euverink, G. (2020). A technological understanding of biofilm detection techniques: A review. Materials, 13, 3147. https://doi.org/10.3390/ma13143147

  15. Theuretzbacher, U., Baraldi, E., Ciabuschi, F., & Callegari, S. (2023). Challenges and shortcomings of antibacterial discovery projects. Clinical Microbiology and Infection, 29, 610. https://doi.org/10.1016/j.cmi.2022.11.027

    Article  PubMed  PubMed Central  Google Scholar 

  16. Spesia, M. B., & Durantini, E. N. (2022). Evolution of phthalocyanine structures as photodynamic agents for bacteria inactivation. The Chemical Record, 22(4), e202100292. https://doi.org/10.1002/tcr.202100292

    Article  CAS  PubMed  Google Scholar 

  17. Ravazzi, R., Neves, J. G., Santamaria, M.P., Pereira Rosa, L., Silva Rosa, F. C., & Santamaria, M. Jr. (2023). Porphyrin-associated fluorescence spectroscopy (Photogen®) for the optical diagnosis of dental biofilm in orthodontic treatment: An observational clinical trial. Photodiagnosis and Photodynamic Therapy, 42, 103580. https://doi.org/10.1016/j.pdpdt.2023.103580

  18. Li, Y., Du, J., Huang, S., Wang, S., Wang, Y., Cai, Z., Lei, L., & Huang, X. (2022). Hydrogen peroxide potentiates antimicrobial photodynamic therapy in eliminating Candida albicans and Streptococcus mutans dual-species biofilm from denture base. Photodiagnosis and Photodynamic Therapy, 37, 102691. https://doi.org/10.1016/j.pdpdt.2021.102691

    Article  CAS  PubMed  Google Scholar 

  19. Songca, S. P., & Adjei, Y. (2022). Applications of antimicrobial photodynamic therapy against bacterial biofilms. International Journal of Molecular Sciences, 23, 3209. https://doi.org/10.3390/ijms23063209

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Vinagreiro, C. S., Zangirolami, A., Schaberle, F. A., Nunes, S. S. C. C., Blanco, K. C., Inada, N. M., da Silva, G. J., Pais, A. C. C., Bagnato, V. S., Arnaut, L. G., & Pereira, M. (2020). Antibacterial photodynamic inactivation of antibiotic-resistant bacteria and biofilms with nanomolar photosensitizer concentrations. ACS Infectious Diseases, 6, 1517. https://doi.org/10.1021/acsinfecdis.9b00379

    Article  CAS  PubMed  Google Scholar 

  21. Sen, P., & Nyokong, T. (2021). Enhanced Photodynamic inactivation of Staphylococcus aureus with Schiff base substituted Zinc phthalocyanines through conjugation to silver nanoparticles. Journal of Molecular Structure, 1232, 130012. https://doi.org/10.1016/j.molstruc.2021.130012

    Article  CAS  Google Scholar 

  22. Spesia, M. B., & Durantini, E. N. (2023). Photosensitizers combination approach to enhance photodynamic inactivation of planktonic and biofilm bacteria. Photochemical and Photobiological Sciences, 22(10), 2433. https://doi.org/10.1007/s43630-023-00461-x

    Article  CAS  PubMed  Google Scholar 

  23. Caminos, D. A., Spesia, M. B., & Durantini, E. N. (2006). Photodynamic inactivation of Escherichia coli by novel meso-substituted porphyrins by 4-(3-N, N, N-trimethylammoniumpropoxy)phenyl and 4-(trifluoromethyl)phenyl groups. Photochemical and Photobiological Sciences, 5, 56. https://doi.org/10.1039/b513511g

    Article  CAS  PubMed  Google Scholar 

  24. Gsponer, N. S., Spesia, M. B., & Durantini, E. N. (2015). Effects of divalent cations, EDTA and chitosan on the uptake and photoinactivation of Escherichia coli mediated by cationic and anionic porphyrins. Photodiagnosis and Photodynamic Therapy, 12, 67. https://doi.org/10.1016/j.pdpdt.2014.12.004

    Article  CAS  PubMed  Google Scholar 

  25. Merchat, M., Spikes, G., Bertoloni, G., & Jori, G. (1996). Studies on the mechanism of bacteria photosensitization by meso-substituted cationic porphyrins. Journal of Photochemistry and Photobiology B: Biology, 35, 149. https://doi.org/10.1016/s1011-1344(96)07321-6

    Article  CAS  PubMed  Google Scholar 

  26. Kragh, K. N., Alhede, M., Kvich, L., & Bjarnsholt, T. (2019). Into the well—A close look at the complex structures of a microtiter biofilm and the crystal violet assay. Biofilm, 1, 100006. https://doi.org/10.1016/j.bioflm.2019.100006

    Article  PubMed  PubMed Central  Google Scholar 

  27. Barbosa, I. (2003). Improved and simple micro assay for sulfated glycosaminoglycans quantification in biological extracts and its use in skin and muscle tissue studies. Glycobiology, 13(9), 647. https://doi.org/10.1093/glycob/cwg082

    Article  CAS  PubMed  Google Scholar 

  28. Spesia, M. B., Caminos, D. A., Pons, P., & Durantini, E. N. (2009). Mechanistic insight of the photodynamic inactivation of Escherichia coli by a tetracationic zinc(II) phthalocyanine derivative. Photodiagnosis and Photodynamic Therapy, 6, 52. https://doi.org/10.1016/j.pdpdt.2009.01.003

    Article  CAS  PubMed  Google Scholar 

  29. Novaira, M., Cormick, M. P., & Durantini, E. N. (2012). Spectroscopic and time-resolved fluorescence emission properties of a cationic and an anionic porphyrin in biomimetic media and Candida albicans cells. Journal of Photochemistry and Photobiology A: Chemistry, 246, 67. https://doi.org/10.1016/j.jphotochem.2012.06.024

    Article  CAS  Google Scholar 

  30. Ferreyra, D. D., Reynoso, E., Cordero, P., Spesia, M. B., Alvarez, M. G., Milanesio, M. E., & Durantini, E. N. (2016). Synthesis and properties of 5,10,15,20-tetrakis[4-(3-N, N-dimethylaminopropoxy) phenyl] chlorin as potential broad-spectrum antimicrobial photosensitizers. Journal of Photochemistry & Photobiology, B: Biology, 158, 243. https://doi.org/10.1016/j.jphotobiol.2016.02.021

    Article  CAS  Google Scholar 

  31. Flemming, H.-C., Wingender, J., Szewzyk, U., Steinberg, P., Rice, S. A., & Kjelleberg, S. (2016). Biofilms: An emergent form of bacterial life. Nature Reviews Microbiology, 14(9), 563. https://doi.org/10.1038/nrmicro.2016.94

    Article  CAS  PubMed  Google Scholar 

  32. Wilson, C., Lukowicz, R., Merchant, S., Valquier-Flynn, H., Caballero, J., Sandoval, J., Okuom, M., Huber, C., Durham Brooks, T., Wilson, E., Clement, B., Wentworth, C. D., & Holmes, A. E. (2017). Quantitative and qualitative assessment methods for biofilm growth: A mini-review. Research & Reviews: Journal of Engineering and Technology, 6(4), 1.

  33. Millezi, F. M., Pereira, M. O., Batista, N. N., Camargos, N., Auad, I., Cardoso, M. D. G., & Piccoli, R. H. (2012). Susceptibility of monospecies and dual-species biofilms of Staphylococcus aureus and Escherichia coli to essential oils. Journal of Food Safety, 32(3), 351. https://doi.org/10.1111/j.1745-4565.2012.00387.x

    Article  CAS  Google Scholar 

  34. Pompermayer, D. M., & Gaylarde, C. C. (2000). The influence of temperature on the adhesion of mixed cultures of Staphylococcus aureus and Escherichia coli to polypropylene. Food Microbiology, 17, 361. https://doi.org/10.1006/fmic.1999.0291

  35. Martinez, S. R., Ibarra, L. E., Ponzio, R. A., Forcone, M. V., Wendel, A. B., Chesta, C. A., Spesia, M. B., & Palacios, R. E. (2020). Photodynamic inactivation of ESKAPE group bacterial pathogens in planktonic and biofilm cultures using metallated porphyrin-doped conjugated polymer nanoparticles. ACS Infectious Diseases, 6(8), 2202. https://doi.org/10.1021/acsinfecdis.0c00268

    Article  CAS  PubMed  Google Scholar 

  36. Grela, E., Kozłowska, J., & Grabowiecka, A. (2018). Current methodology of MTT assay in bacteria—A review. Acta Histochemica, 120(4), 303. https://doi.org/10.1016/j.acthis.2018.03.007

    Article  CAS  PubMed  Google Scholar 

  37. Ghasemi, M., Turnbull, T., Sebastian, S., & Kempson, I. (2021). The MTT assay: Utility, limitations, pitfalls, and interpretation in bulk and single-cell analysis. International Journal of Molecular Sciences, 22, 12827. https://doi.org/10.3390/ijms222312827

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Tan, L., Li, H., Chen, B., Huang, J., Li, Y., Zheng, H., Liu, H., Zhao, Y., & Wang, J. J. (2021). Dual-species biofilms formation of Vibrio parahaemolyticus and Shewanella putrefaciens and their tolerance to photodynamic inactivation. Food Control, 125, 107983. https://doi.org/10.1016/j.foodcont.2021.107983

    Article  CAS  Google Scholar 

  39. Wang, H., Cheng, H., Wang, F., Wei, D., & Wang, X. (2010). An improved 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay for evaluating the viability of Escherichia coli cells. Journal of Microbiological Methods, 82(3), 330. https://doi.org/10.1016/j.mimet.2010.06.014

    Article  CAS  PubMed  Google Scholar 

  40. Misba, L., Abdulrahman, H., & Khan, A. U. (2019). Photodynamic efficacy of toluidine blue O against mono species and dual species bacterial biofilm. Photodiagnosis and Photodynamic Therapy, 26, 383. https://doi.org/10.1016/j.pdpdt.2019.05.001

    Article  CAS  PubMed  Google Scholar 

  41. Beirão, S., Fernandes, S., Coelho, J., Faustino, M. A. F., Tomé, J. P. C., Neves, M. G. P. M. S., Tome, A. C., Almeida, A., & Cunha, A. (2014). Photodynamic inactivation of bacterial and yeast biofilms with a cationic porphyrin. Photochemistry and Photobiology, 90, 1387. https://doi.org/10.1111/php.12331

  42. Pereira, C. A., Romeiro, R. L., Borges Pereira Costa, A. C., Silva Machado, A. K., Campos Junqueira, J., & Olavo Cardoso Jorge, A. (2011) Susceptibility of Candida albicans, Staphylococcus aureus and Streptococcus mutans biofilms to photodynamic inactivation: an in vitro study. Lasers in Medical Science, 26, 341. https://doi.org/10.1007/s10103-010-0852-3

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Acknowledgements

Authors are grateful to Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) of Argentina and Agencia Nacional de Promoción Científica y Tecnológica (FONCYT PICT N°1482/19 and 2391/19) for financial support. R.B.A. thanks CONICET for the research fellowship. E.N.D. and M.B.S. are Scientific Members of CONICET. Special thanks to D.F.B.P.

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Fondo para la Investigación Científica y Tecnológica, PICT N°1482/19, Mariana B. Spesia, PICT N° 2391/19, Edgardo N. Durantini.

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  1. IDAS-CONICET, Departamento de Química, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Ruta Nacional 36 Km 601, X5804BYA, Río Cuarto, Córdoba, Argentina

    Rocío B. Acosta, Edgardo N. Durantini & Mariana B. Spesia

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Acosta, R.B., Durantini, E.N. & Spesia, M.B. Evaluation of quantification methods to determine photodynamic action on mono- and dual-species bacterial biofilms. Photochem Photobiol Sci (2024). https://doi.org/10.1007/s43630-024-00586-7

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