Clin Exp Otorhinolaryngol.  2012 Sep;5(3):139-144. 10.3342/ceo.2012.5.3.139.

In Vitro Streptococcus pneumoniae Biofilm Formation and In Vivo Middle Ear Mucosal Biofilm in a Rat Model of Acute Otitis Induced by S. pneumoniae

• Yadav MK
• Chae SW
• Song JJ

• KMID: 2134596
• DOI: http://doi.org/10.3342/ceo.2012.5.3.139

Abstract

OBJECTIVES
Streptococcus pneumoniae is one of the most common pathogens of otitis media (OM) that exists in biofilm, which enhances the resistance of bacteria against antibiotic killing and diagnosis, compared to the free-floating (planktonic) form. This study evaluated biofilm formation by S. pneumoniae on an abiotic surface and in the middle ear cavity in a rat model of OM.
METHODS
In vitro biofilm formation was evaluated by inoculation of a 1:100 diluted S. pneumoniae cell suspension in a 96-well microplate. Adherent cells were quantified spectrophotometrically following staining with crystal violet by measurement of optical density at 570 nm. The ultrastructure of pneumococcal biofilm was assessed by scanning electron microscopy (SEM). For in vitro biofilm study, S. pneumoniae cell suspensions containing 1x10(7) colony forming units were injected through transtympanic membrane into the middle ear cavity of Sprague Dawley rats. The ultrastructure of middle ear mucus was observed by SEM 1 and 2 weeks post-inoculation.
RESULTS
The in vitro study revealed robust biofilm formation by S. pneumoniae after 12-18 hours of incubation in high glucose medium, independent of exogenously supplied competence stimulating peptide and medium replacement. Adherent cells formed three-dimensional structures approximately 20-30 microm thick. The in vivo study revealed that ciliated epithelium was relatively resistant to biofilm formation and that biofilm formation occurred mainly on non-ciliated epithelium of the middle ear cavity. One week after inoculation, biofilm formation was high in 50% of the treated rats and low in 25% of the rats. After 2 weeks, biofilm formation was high and low in 25% and 37.5% of rats, respectively.
CONCLUSION
The results imply that glucose level is important for the S. pneumoniae biofilm formation and S. pneumoniae biofilm formation may play important role in the pathophysiology of OM.

Keyword

Otitis media; Streptococcus pneumoniae; Biofilm

MeSH Terms

Animals
Bacteria
Biofilms
Ear, Middle
Epithelium
Gentian Violet
Glucose
Homicide
Membranes
Mental Competency
Microscopy, Electron, Scanning
Mucus
Otitis
Otitis Media
Pneumonia
Rats
Rats, Sprague-Dawley
Stem Cells
Streptococcus
Streptococcus pneumoniae
Suspensions
Gentian Violet
Glucose
Suspensions

Figure

• 

  Fig. 1 In vitro biofilm formation by Streptococcus pneumoniae R6 strain in trypticase soy broth (TSB) medium supplemented with 0%, 1%, and 2% glucose, incubated at 37℃ for 6, 12, 18, and 24 hours in 5% CO2.

• 

  Fig. 2 Scanning electron microscopic image of Streptococcus pneumoniae R6 strain biofilm formed on a polystyrene microplate. (A) Filament material linked pneumococcal cells to each other and to the intercellular matrix. (B) Apical view of the irregular surface of a pneumococcal biofilm. Micro-colonies of different sizes revealing non-homogeneous microbial populations are evident.

• 

  Fig. 3 Scanning electron microscopic image of middle ear mucosa of a control rat. (A) Non-ciliated area of middle ear mucosa. (B) Ciliated area of middle ear mucosa.

• 

  Fig. 4 Scanning electron microscopic image of middle ear mucosa of treated rat after 1 week of bacterial inoculation. (A) High degree of biofilm formation with rough exopolysaccharide matrix. (B) Conglomerated cilia of ciliated epithelium.

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  Fig. 5 Scanning electron microscopic image of middle ear mucosa of treated rat after 2 weeks of bacterial inoculation. (A) Thick biofilm with irregular surfaced exopolysaccharide matrix of biofilm. (B) Exopolysaccharide matrix connected with thread like structures. (C) Debris of exopolysaccharide matrix attached to ciliated epithelium.

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