Bacteriophage as effective decolonising agent for elimination of MRSA from anterior nares of BALB/c mice

Abstract

Background: Nasal carriers not only pose serious threat to themselves but also to the community by playing an active role in the dissemination of serious and life threatening S. aureus especially MRSA strains. The present study focuses on the use of broad spectrum lytic phage as decolonising agent. In addition, the combined use of lytic phage with mupirocin has also been investigated as an effective decolonising regimen. The effect of phage on the adherence, invasion and cytotoxic effect of MRSA strains on nasal epithelial cells was studied in an ex-vivo model of cultured murine nasal epithelial cells. This was followed by demonstration of therapeutic potential of phage along with mupirocin in decolonising the nares of BALB/c mice using a nasal model of MRSA colonisation.

Results: Phage was able to significantly reduce the in vitro adherence, invasion and cytotoxicity of MRSA 43300 as well as other clinical MRSA strains on murine nasal epithelial cells as compared to untreated control. Also, the frequency of emergence of spontaneous mutants decreased to negligible levels when both the agents (phage and mupirocin) were used together.

Conclusion: Phage MR-10, given along with mupirocin showed an additive effect and the combination was able to effectively eradicate the colonising MRSA population from the nares of mice by day 5.

Figure 1

Schematic representation of the infection and treatment schedule followed for establishing nasal colonization model in BALB/c mice.

Figure 2

Isolated murine nasal epithelial cells as observed under 40X Olympus light microscope on different days post-seeding. A) and B) unstained and stained preparation of isolated single cells seen on the day of isolation C) unstained and D) stained preparation of cultured NEC on day 2 post seeding. Nucleus is clearly evident in all the cells E) and F) cells as seen on day 3 post seeding of different shapes and sizes and G) Polygonal shaped NEC as seen on day 5 with significant increase in size as well. These cells were harvested, counted and used for adherence and invasion studies.

Figure 3

Bacterial burden in terms of A) Mean log CFU/gram of mice tissue ofS. aureus43300 following treatment of colonised nares with different anti-bacterial agents on different days post treatment; Phage counts in terms of B) Mean log PFU/g count in the anterior nares of mice belonging to group 2 and group 4 on various days post phage treatment. Error bars represent the standard deviation.

Figure 4

Mean MPO activity (Units/ml) detected in the homogenates of nares of different groups of mice on different days post treatment. Red dotted line represent the basal MPO activity as seen in healthy BALB/c mice (n = 4). Error bars represent standard deviation.

Figure 5

Histopathological analysis showing. A) Photo micrograph of skin tissue of nasal mucosa of untreated colonised mice on day 2 post colonisation showing mild inflammation with recruitment of few acute inflammatory cells(red arrows) (H and E 100X). B) and C) Photo micrograph of skin tissue of nasal mucosa of untreated colonised mice on day 5 post colonisation showing marked sub epithelial inflammation rich in neutrophils and plasma cells (H and E 100X and 200X). D) and E) Photo micrograph of skin tissue of nasal mucosa of phage treated (group 3) and Mupirocin treated(group 4) mice on day 5 post treatment showing mild infiltration in the sub epithelial lining (H and E 100X). F) Photo micrograph of skin tissue of nasal mucosa of mice receiving combined therapy (group 5) with nearly normal skin (H and E 100X).