Assessment of Streptococcus pyogenes microcolony formation in infected skin by confocal laser scanning microscopy
Introduction
Bacterial glycocalyx is a polysaccharide-containing material produced by bacteria [1]. Bacteria sometimes adhere to certain surfaces in vitro and in vivo and multiply there, forming a thin bacterial mass. Such bacterial colonization, in the form of a thin film-like structure, has been referred to as biofilm [2]. It has been reported that biofilm-forming bacteria are more resistant to antibiotics and even resistant to immunologic attack [2], [3]. Electron microscope (EM) techniques required that biofilm specimens be dehydrated, a process known to significantly reduce the total volume of exocellular matrix material (glycocalyx) and lead to collapse the matrix, compression of the cells, and distortion of the architecture. This has been obviated by the use of the confocal laser scanning microscopy (CLSM), which allows horizontal and vertical optical thin sectioning of hydrated biofilms [4]. CLSM analysis is non-destructive, and CLSM makes it possible to analyze live biofilm structures. Concanavalin A (ConA) is used as a marker for detecting glycocalyx on staphylococcal cells and its mechanism of action involves the reaction of ConA and mannose [5], [6], [7], [8]. Previous studies using CLSM have shown that Staphylococcus aureus cells in the stationary growth phase alone attached on coverslips produced glycocalyx in vitro [6]. Another study using CLSM revealed that S. aureus cells isolated from bullous impetigo, furuncle, and atopic dermatitis lesions formed microcolonies encircled by glycocalyx on damaged mouse skin tissues, which conventional antimicrobial agents could not eradicate without the help of polymorphonuclear leukocytes (PMNs) [7]. Recent study using CLSM revealed that S. aureus cells generally produced glycocalyx in skin lesions of bullous impetigo, atopic dermatitis, and pemphigus foliaceus, and the glycocalyx might collapse during dehydration and most of the S. aureus cells might be removed in a routine light microscope section [8]. In the present study, we used CLSM to investigate the status of microcolonization by attached Streptococcus pyogenes and S. aureus cells in streptococcal impetigo lesions, S. pyogenes cells in infected skin lesions, and the influence of an antimicrobial agent in croton-oil inflamed mouse skin. Production of materials that stained with fluorescein isothiocyanate-conjugated (FITC)-ConA by S. pyogenes cells in vitro were also assessed.
Section snippets
Patients
Specimens were obtained from one patient (a 16-year-old man) with streptococcal impetigo (2 days after onset) and atopic dermatitis. The patient's face and upper extremity were covered with thick-walled pustules on an erythematous base with crusts, and he had regional adenopathy with fever (>38.5 °C). S. pyogenes (T-2 type) and S. aureus (oxacillin susceptibility 0.5 μg/ml; coagulase type II producer and toxic shock syndrome toxin-1 non-producer) strains were codetected in pustules on the upper
Appearance of S. pyogenes in pustules of streptococcal impetigo
In pustule lesions of streptococcal impetigo, light microscopy using H&E staining showed necrosis with the infiltrations of numerous PMNs (Fig. 1A), and a few microcolonies were located on the lesion's outer walls that PMNs did not infiltrate (Fig. 1A–C). In this method (Appendix A) [6], [7], [8], S. aureus cells without glycocalyx were found to stain only with safranine (red), S. aureus covered with glycocalyx to stain with safranine and FITC-ConA (yellow; Fig. 1F), and S. aureus to stain with
Discussion
S. pyogenes is an important organism of bacterial infections in both adults and children [9]. Impetigo is the most common bacterial skin infection in children [10]. All cases of bullous impetigo are caused by S. aureus and non-bullous impetigo are usually due to S. pyogenes [11], although recently in Japan S. pyogenes and S. aureus are often simultaneously detected from many cases of non-bullous impetigo with atopic dermatitis. Almost all lesions of streptococcal impetigo begin with vesicles,
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