AbstractStaphylococci are normal colonizers of human and animal skin and mucous membranes. However, S. aureus is one of the most pathogenic bacteria isolated from humans. S. aureus produces numerous virulence factors that are responsible for the pathogenicity of this microorganism. Exposure of bacteria to sub-MICs of antibiotics is often encountered clinically, therefore, the effect of sub-MICs on staphylococcal virulence has been well studied on planktonic forms. Nevertheless, few studies have investigated the alteration of biofilm virulence under sub-MIC conditions.
In this study, after several attempts to support the growth of Staphylococcus aureus in chemically defined media containing the basic nutrient requirements, addition of seven amino acids improved the growth yield. However, staphylococcal growth in biofilm required a complex medium, such as brain heart infusion, which was used in the study. The effect of 1/16 MIC of selected antibiotics representing three different groups (cefalexin, a cell wall inhibitor; ciprofloxacin, a DNA replication inhibitor and roxithromycin, a protein synthesis inhibitor), was investigated on the virulence of S. aureus NCTC 11962 biofilms grown in Sorbarod filters. Protease secretion, coagulase production, ability to form biofilm and the superantigen (toxic shock syndrome toxin-1 (TSST-1)) production were evaluated in challenged and control biofilms, eluates and supernatants. Moreover, since sub-MICs of antibiotics have the potential to alter virulence and interfere with metabolic processes of the bacteria, the proliferative effect of TSST-1 was evaluated to determine any modulation in its activity.
SDS-PAGE and 2D-PAGE of biofilm and eluate cells in addition to eluate supernatants exhibited remarkable differences in protein profiles among challenged and control biofilms. Such protein shifts would imply in part, alterations in biofilm virulence.
Proteases were not detected in the eluates of the treated or control biofilms. Coagulase production was reduced in eluates exposed to roxithromycin when compared to control, while TSST-1 production was reduced in biofilms exposed to cefalexin and to a lesser extent to ciprofloxacin. On the other hand, cefalexin has been shown to induce biofilm formation at a wide range of sub-MICs.
TSST-1 was purified from eluate supernatants of treated biofilms and control and the proliferative activity of 10-fold serial dilutions of the toxins on mouse splenocytes were tested using MTS/PMS assay. No significant difference in activity between the treated toxin and the control has been observed. However, the activity of the crude eluate supernatants has been found to be more active when compared to equivalent concentrations of purified TSST-1, an effect ascribed to the presence of enterotoxin A, another superantigen produced by the bacteria. An hypothesis to explain the inhibitory effect of cefalexin on TSST-1 production was also discussed.
The results obtained in this thesis, indicate that antibiotics could potentially interfere with the virulence of S. aureus biofilms, if concentrations of these agents fall below MIC of the infecting strain.
|Date of Award||Mar 2008|
|Supervisor||Phillip Collier (Supervisor)|