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The potential of the naturally produced inhibitory substance (A345) as a potential therapeutic agent for treating Staphylococcus aureus

Abstract number: P1088

Al-Mahrous M., Burnie J., Tagg J., Upton M.

Objectives: In recent years, there has been much focus on a promising class of bacteriocins known as lantibiotics. The most prominent representative of lantibiotics, nisin, has already a long history of use in the protection of foodstuffs. Lantibiotics have also been considered for application in humans; however they have not yet been used in the setting of chemotherapy on the same scale as traditional antibiotics.

We here investigate the cationic peptide antibiotic (A345) as a future opportunity for treating staphylococcal infections.

Methods: 1. Investigation of S. aureus strain A345 was performed using simultaneous and deferred-antagonism against MRSA.

3. Purification and/or concentration of free inhibitors in broth supernatants was carried out using ammonium-sulphate precipitation, Sep-Pak® cartridge, Speed-Vac®, Cation-exchange; then C18 reverse-phase chromatography.

6. MALDI TOF/TOF was used for mass analysis.

7. Electron microscopy was used for ultra-structure diagnosis.

8. Biological activities were tested using spot-on-loan assay.

Results: The biological activity of A345 is heat-stable and displaying specificity for the closely-related S. aureus.

The high ammonium-sulphate saturation (more than 80%) needed for precipitating A345 suggests its small mass. The cationic-exchange chromatography (pH 5.2) and the late elution from C18 column suggest the cationic nature of A345. MALDI TOF/TOF showed 4 species and sized the mass in a window between 1500.2 and 3200.7 Da. This suggests further purification using high resolution HPLC to eliminate, if any, unrelated species.

The Electron microscopy diagnosis reveals a clear damage in the cell wall (Figure 1).

Figure 1. Thin section of EMRSA-15 strain A208 cell wall after incubation with the inhibitory substance A345 for 24 h at 37°C. An example of a ruptured wall is seen.

Conclusion: 1. The biological activity of the highly-purified extract of the heat-stable small mass inhibitory agent A345, which shows specific inhibitory activity against Epidemic MRSA-15 and strains of MSSA, suggests its nature as a bacteriocin, possibly of Class-I.

3. The cationic-exchange separation and late elution of A345 from C18 reverse-phase column suggest that it is a hydrophobic in nature.

4. Based on the electron microscopy diagnosis, A345 shows obvious damage to the protective cell wall of the sensitive indicators. This postulates the binding of the hydrophobic A345 to negatively-charged lipid-II in the cell-membrane resulting in its lysis.

6. A345 could have potential as topical therapeutic agents for treating highly drug-resistant staphylococcal infections.

Session Details

Date: 16/05/2009
Time: 00:00-00:00
Session name: 19th European Congress of Clinical Microbiology and Infectious Diseases
Subject:
Location: Helsinki, Finland, 16 - 19 May 2009
Presentation type:
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