The bactericidal effect of lysostaphin coupled with liposomal vancomycin as a dual combating system applied directly on methicillin-resistant Staphylococcus aureus infected skin wounds in mice 

Abstract

Background and aim

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common causes of surgical infection, and its resistance to numerous conventional antibiotics makes treatment difficult. Although vancomycin is often an effective agent for the initial therapy of MRSA, clinical failure sometimes occurs. Therefore, there is an urgent need to develop better therapies. Here, we prepared some vancomycin-loaded nanoliposomes coupled with anti-staphylococcal protein (lysostaphin) and evaluated their in vitro and in vivo efficacy as a topical MRSA therapy.

Methods

Vancomycin was encapsulated in liposomes, and the coupling of lysostaphin with the surface of liposomes was carried out through cyanuric functional groups. The bactericidal efficacies and a full characterization were evaluated. To define different nanoliposomal–bacterium interactions and their bactericidal effect, flow cytometry was employed. Finally, in vivo, the topical antibacterial activity of each formulation was measured against surgical wound MRSA infection in a mouse model.

Results

High encapsulation and conjugation efficiency were achieved for all formulations. All the formulations showed a significant reduction in bacterial counts (p<0.05). The targeted liposomes more effectively suppress bacterial infection in vitro and in vivo relative to equivalent doses of untargeted vancomycin liposome. The flow cytometry results confirmed liposome–bacterium interactions, which increased during the incubation time. The maximum binding rate and the bactericidal effect were significantly higher in targeted liposomes (p<0.05) compared with control liposomes.

Conclusion

Our data suggest a novel nano-vehicle (lysostaphin-conjugated coupled liposomal vancomycin) which could be used as a great topical antimicrobial construct for treatment of MRSA skin infections.

Keywords:

• MRSA
• antibacterial activity
• encapsulation efficiency
• conjugation efficiency
• lysostaphin

Acknowledgments

The authors gratefully thank the vice-chancellor of Hamadan University of Medical Sciences and Iran University of Medical Sciences for funding support. The authors would like to thank Dr. Shapiro for reading, editing and giving some helpful comments to improve the manuscript. This work was supported by research grants from the Hamadan University of Medical Sciences (grant/award number: 94-10-29-6134) and the Iran University of Medical Sciences (grant/award number: 94-05-31-26873).

Ethical approval

This study was approved by the Ethics Review Body of Animal Experimentation of Iran University (IR.UMSHA.REC.1394.134).

Supplementary materials

Figure S1 Agarose Gel Electrophoresis for the detection of the femA gene (132 bp) (A) and mecA gene (297 bp) (B) hla gene (209 bp) (C) in MRSA clinical isolate by PCR. Lanes: L, molecular marker (100 bp DNA ladder); 1, Staphylococcus aureus ATCC 3591 as positive control; 2, MRSA clinical isolate; 3, E. coli ATCC 25922 as negative control. The Real Time PCR amplification curve (D) and gene expression for hla gene (E) in which gmK gene was used as internal control

Figure S2 Mortality in mice caused by MRSA wound infection. A region of the skin was excised by biopsy punch and the wound infected through different CFU counts of MRSA including 10⁷, 10⁸ and 10⁹ then survival of mice on a daily basis was observed detected. Mice, receiving 10⁹ 10⁸ and 10⁷ CFU, died after 2, 3 and 4 days respectively

Disclosure

The authors declare no conflicts of interest in this study.