Abstract
Aim: Visualization of Staphylococcus aureus biofilm using histochemical staining and combined histochemistry (HC) and immunohistochemistry (IHC).Methods: The ability of S. aureus S54F9 to form biofilm was tested in vitro. Hereafter, infected bone tissue was collected from two different porcine models of osteomyelitis inoculated with S. aureus strain S54F9. The infection time was five and fifteen days, respectively. Twenty-five different histochemical staining protocols were tested in order to find the stains that could identify extracellular biofilm matrix. Protocols with an optimal visualization of biofilm extracellular matrix were combined with an immunohistochemical protocol based on a specific antibody against S. aureus. The combined protocols were applied to the tissue from the porcine models and to infected bone tissue from a child suffering from chronic staphylococcal osteomyelitis for more than a year.Results: S. aureus S54F9 showed an ability to form biofilm in vitro. Visualization of biofilm, i.e. bacterial cells and extracellular matrix in different colours, was seen when the immunohistochemical protocol was combined with Alcian Blue pH3, Luna and Methyl-pyronin green. The bacterial cells were red to light brown and the extracellular matrix either light blue, blue or orange depending on the histochemical stain. In the porcine models and the human case 10 and 90 percent, respectively, of the bacterial aggregates in a 100x magnification field displayed both the extracellular matrix and the bacterial cells simultaneously in two different colours.Conclusions: A combination of HC and IHC can be used to diagnose and characterise biofilm infections on a routine basis.
Highlights
Bacterial biofilms are microbial derived communities, characterized by aggregates of bacterial cells that are attached to a substratum/interface or to each other and embedded in a matrix of extracellular polymeric substances [1,2,3]
The extracellular matrix of a biofilm is composed of molecules produced both by the bacteria and the host and includes polysaccharides, structural proteins, enzymes, nucleic acids and lipids [4]
Despite extensive research focusing on bacterial biofilms, a comprehensive analysis of which histochemical stains that can identify the components of the extracellular biofilm matrix is lacking
Summary
Bacterial biofilms are microbial derived communities, characterized by aggregates of bacterial cells that are attached to a substratum/interface or to each other and embedded in a matrix of extracellular polymeric substances [1,2,3]. In most in-vitro biofilms, the bacteria account for less than 10 % of the dry mass, whereas the extracellular matrix can account for more than 90 % [4]. The extracellular matrix of a biofilm is composed of molecules produced both by the bacteria and the host and includes polysaccharides, structural proteins, enzymes, nucleic acids and lipids [4]. All the different molecules have specific roles in favour of the http://www.jbji.net survival and growth of the embedded bacteria (Table 1) [4]. Pathologists are often not aware of the presence of biofilm formation when examining slides for diagnosing bacterial infection in routine diagnostic laboratories
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