Confocal scanning laser microscopy for the Study of Biofilms in tissues of the upper airway

PARAJE, MG

Microscopy: Science, Technology, Applications and Education

FORMATEX RESEARCH CENTER

Lugar: Badajoz, Spain ; Año: 2010; p. 590 - 596

INTRODUCTION: Bacteria exist in two principal forms, as free-floating planktonic replicating cells and in biofilms. The latter are defined as organized communities of bacteria collaborating among themselves and attached to an inert or living surface contained in a self-produced polymeric matrix, made principally of exopolysacharides, nucleic acids, and proteins.The planktonic form of the bacteria has been very useful in understanding acute infections. However, chronic forms are more related to the presence of biofilms, with current research indicating an important role for bacterial biofilms in recurrent or chronic infection, including those which are not responsive to a culture-appropriate antibiotic therapy. In particular, biofilms are involved in chronic otitis, chronic tonsillitis, cholesteatomas and other inflammatory and infectious disorders. The link between the concept of biofilms and chronic infectious disease is an ongoing subject of investigation. Confocal scanning laser microscopy (CSLM) allows the  non-destructive study of biofilms, through an examination all the layers at different depths from which it is possible to reconstruct a three-dimensional structure. OBJECTIVE: The aim of this work was to determine and to characterize the presence of these biofilms in tissues of the upper airway, using CSLM in comparative forms with optical and fluorescence microscopy. Also, quantification of the in vitro biofilm development of the isolated bacteria was carried out. MATERIALS AND METHODS: Adenoids and tonsils were studied in operated pediatric and adult patients with nasal polyps. The criteria for classifying bacterial biofilms in vivo by optical and fluorescence microscopy were: the presence of the characteristic bacterial morphology and micro and macro- colonies; and surrounding polysaccharide blush with tower formation for CSLM. The presences of biofilms in the tissue obtained by surgery were identified by Gram staining with optical microscopy; acridine orange with fluorescent microscopy and with two fluorescence staining (propidium iodide/ fluorescein isothiocyanate-FITC) by CSLM. The ability to form biofilms in vitro of isolated bacteria was measured by adherence to 96-well plates, and subsequent staining with Crystal Violet and extracting with a bleaching solution: ethanol/glacial acetone. The intensity of the coloration was determined by measuring the absorbance at 590 nm. RESULTS: For CSLM, in the tissues of the patients studied, bacterial micro and macro-colonies formed biofilms with some of these being mixed bacterial species, such as Staphylococcus aureus, Streptococcus viridans, Haemophilus influenzae, and Staphylococcus coagulase negative. There are very few precedents for the presence of biofilms in nasal polyps. In vitro studies of the isolated species values from 0.173 to 4.764 (OD 595 nm) were observed when stained with crystal violet. DISCUSSION AND CONCLUSIONS: The full significance of bacterial biofilms in the human chronic rhinosinusitis has yet to be determined, with more work being necessary. However, the corroboration of biofilms in vivo, and the demonstration of the high capacity to form biofilms in vitro for all isolated bacteria, suggests a nexus between chronic rhinosinusitis and chronic inflammation or infection. CSLM has proved a useful tool when analyzing the structure of both the extracellular matrix exopolysaccharide, and the cells that form biofilms immersed in the tissue of the upper airway. However, the possibility of viewing biofilms with an optical technique may also be helpful in ordinary practice within an attempt to global approach of these patients. Further research is required to clarify the impact of these biofilms in the. The pathology and symptoms of patients who develop chronic rhinosinusitis might be directly related to the presence of the form of biofilms. Demonstrating the presence of these biofilms would help to confirm the hypothesis that bacteria are protected from host defenses by biofilms, and continue their metabolism and local production of exotoxins, which would favor the chronicity of the inflammatory response evident by changes in the respiratory mucosa and persistent infections that do not respond to antibiotic treatments. Keywords: biofilms, rhinosinusitis, optical microscopy, confocal scanning laser microscopy.