Monoespecies and mixed biofilms of Trichophyton and Staphylococcus: nutrient influence and presence of persisters cells

Author(s)

Jenyffie Araujo Belizario

• Document type: Master's Dissertation
• Press: Araraquara. 2023-05-24.
• Institution: Universidade Estadual Paulista (Unesp). Faculdade de Ciências Farmacêuticas. Araraquara
• Defense date: 2023-04-28
• Advisor: Maria José Soares Mendes Giannini; Regina Helena Pires

Abstract

Dermatophytes are fungi that cause dermatophytosis, a fungal disease that affects keratinized tissues, and the association of dermatophyte fungi with bacteria tends to cause an exacerbated infectious condition that is difficult to treat. Both microorganisms can form biofilms, and the availability of nutrients can influence their adhesion capacity and architecture. Furthermore, dermatophytes' microbiological and clinical resistance may be associated not only with the biofilm formation but also with the persistent cells (persister cells) presence, which could contribute to the emergence of chronic, recalcitrant infections. In this scenario, the study objective was to characterize the formation of a mixed biofilm between Trichophyton rubrum and Staphylococcus aureus or Staphylococcus epidermidis in different culture media. The presence of persistent cells in these biofilms alone will also be evaluated. Biofilms were formed at different incubation times and culture media and quantified by crystal violet (biomass) and metabolic activity (tetrazolium salt reduction) methodologies. Colony Forming Units (CFU) were determined by agar plating methodology. Biofilms were also photo-documented by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). The presence of persistent cells was determined by UFC test after exposure to antimicrobials. Microbial communities showed higher production of biomass and metabolically active cells in Brain Heart Infusion (BHI) medium compared to Roswell Park Memorial Institute (RPMI-1640) and Muller-Hinton Broth (MHI). Both bacterial species, when combined with T. rubrum, were able to form mature biofilms in 72h in the three tested media; however, differences between populations were observed. A more significant amount of biomass and metabolic activity was observed when the bacterial inoculum was added to the fungal biofilm in the final stages of formation. The CFU assay showed that the T. rubrum development in mixed biofilms was dependent on the time of addition of the bacteria to the fungal biofilm in formation, and the lack of pre-adhesion directly affected the evolution of the fungus in the biofilm. Electron micrographs of monospecies biofilms of T. rubrum showed a coordinated network of robust and intertwined hyphae, while in biofilms of Staphylococcus spp., a vast number of agglomerated cocci was observed. When combined, the predominance of T. rubrum or Staphylococcus spp. depended on the interaction conditions, with bacterial communities prevailing in the early stages, and hyphae was not observed. On the contrary, the fungal community dominated the biofilm in the final stages. CLSM data showed that the T. rubrum biofilm had a thickness of 363 μm, with septate, intertwined and well developed, while the biofilms of S. aureus (177 μm) and S. epidermidis (178 μm) showed an ample number of cocci throughout the field. The thicknesses of the mixed biofilms, under the final interaction conditions, were similar to the T. rubrum monospecies biofilm, mainly when associated with S. epidermidis (369 μm). However, few hyphae with irregular shapes and sizes were observed in the initial stages. The study of the presence of persistent cells in biofilms suggested that antibiotic therapeutic failure in chronic infections may be related to the presence of this subpopulation and plays a significant role in biofilm tolerance. The selection of effective antimicrobials in non-toxic concentrations is necessary to ensure success in these infections. Knowledge of the interaction between T. rubrum and Staphylococcal species and the assessment of persistent cells in these biofilms are unpublished and may contribute to advancing knowledge of the host immune response, antimicrobial resistance, and disease progression, in addition to subsidizing new therapeutic targets. (AU)

• FAPESP's process: 21/03805-6 - Monospecies and mixed biofilms of Trichophyton and Staphylococcus: influence of nutrients and presence of persister cells
• Grantee: Jenyffie Araujo Belizario
• Support Opportunities: Scholarships in Brazil - Master