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A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy
Published on: January 25, 2017
Assessment of Streptococcus pyogenes microcolony formation in infected skin by confocal laser scanning microscopy
Hisanori Akiyama1, Shin Morizane, Osamu Yamasaki
1Department of Dermatology, Okayama University Graduate School of Medicine and Dentistry, Shikata-cho 2-5-1, 700-8558, Okayama, Japan. akiyamah@cc.okayama-u.ac.jp
Background:
Streptococcus pyogenes and Staphylococcus aureus are often simultaneously detected from many cases of non-bullous impetigo with atopic dermatitis.
Objectives:
Using confocal laser scanning microscopy (CLSM), to investigate formation of S. pyogenes microcolonies in skin lesions.
Methods:
The S. pyogenes cells in the stationary growth phase alone were strongly stained with fluorescein isothiocyanate-concanavalin A (FITC-ConA), and this staining was reduced by pretreatment with amylase. Although the components of sugars in glycocalyx produced by S. pyogenes cells are unknown, we suggested that the materials stained by FITC-ConA were consistent with the presence of ConA-reactive sugars in glycocalyx produced by S. pyogenes cells.
Results:
S. pyogenes cells associated with streptococcal impetigo skin and croton-oil inflamed mouse skin formed microcolonies encircled by materials (glycocalyx) that stained strongly with FITC-ConA, and these findings were consistent with those in biofilms. In croton-oil inflamed mouse skin, polymorphonuclear leukocytes (PMNs) infiltrated to just below the epidermis in the cefdinir-treated group but only to the middle dermis in the cefdinir-non-treated group. In this case S. pyogenes and S. aureus cells formed separate microcolonies and existed independently in the outer walls of pustule lesions of streptococcal impetigo.
Conclusion:
In skin infections, S. pyogenes and S. aureus formed aggregates of microcolonies (similar to that in biofilms) encircled by glycocalyx, which can make the infection hard to eradicate using an antimicrobial agent alone. The effect of conventional antimicrobial agents against biofilm is mainly due to the increase of the invasion of PMNs into the biofilm.
Insights
Streptococcus pyogenes and Staphylococcus aureus form biofilms in skin infections, making them difficult to treat with antibiotics alone. These bacterial microcolonies are encased in a protective glycocalyx, hindering antimicrobial efficacy.
Area of Science:
- Microbiology
- Dermatology
- Infectious Diseases
Background:
- *Streptococcus pyogenes* and *Staphylococcus aureus* are frequently co-detected in non-bullous impetigo, particularly in patients with atopic dermatitis.
- Understanding the in vivo behavior of these bacteria in skin lesions is crucial for effective treatment strategies.
Purpose of the Study:
- To investigate the formation of *S. pyogenes* microcolonies in skin lesions using confocal laser scanning microscopy (CLSM).
- To characterize the structural organization of bacterial aggregates in impetigo lesions.
Main Methods:
- *S. pyogenes* cells were stained with fluorescein isothiocyanate-concanavalin A (FITC-ConA) to visualize glycocalyx components.
- Confocal laser scanning microscopy (CLSM) was employed to examine bacterial microcolony formation in both human skin samples and inflamed mouse skin models.
- The infiltration of polymorphonuclear leukocytes (PMNs) was assessed in response to antimicrobial treatment in a mouse model.
Main Results:
- *S. pyogenes* formed microcolonies surrounded by a FITC-ConA-reactive glycocalyx in impetigo skin lesions and inflamed mouse skin, resembling biofilm structures.
- *S. pyogenes* and *S. aureus* formed distinct microcolonies within pustule lesions of streptococcal impetigo.
- Antimicrobial treatment (cefdinir) in mice increased PMN infiltration into the skin lesions.
Conclusions:
- *S. pyogenes* and *S. aureus* form biofilm-like aggregates encased in glycocalyx in skin infections, contributing to treatment resistance.
- The efficacy of conventional antimicrobial agents against these bacterial communities may be enhanced by promoting host immune cell (PMN) infiltration.
