At higher MOI, adherence was reduced to negligible level Similar

At higher MOI, adherence was reduced to negligible level. Similarly, almost minimal invasion and cytotoxic damage to NEC was observed with phage added at MOI-1. At higher phage concentration (MOI-10), the reduction in all the three parameters was highly significant (p < 0.01) and no invasion or cytotoxic damage was seen on NEC. Table 2 depicts the adherence, invasion and cytotoxic damage of five different clinical MRSA strains denoted as CS-1 to CS-5(chosen at random) against which phage (MR-10) showed lytic activity. S. aureus 29213(MSSA) was also studied as

an internal control. All the strains were found to adhere to cultured nasal epithelial cells in significant numbers (>60% adherence). The presence of phage significantly affected the adherence of all the strains (p < 0.01). Maximum CP-690550 chemical structure invasion (33%) and cytotoxicity RG7112 chemical structure (14%) was observed with strain CS-3. The phage at MOI-1 was able to sixgnificantly decrease both the invasion and cytotoxic damage inflicted by all the clinical isolates. At higher MOI-10, no detectable invasion or cytotoxicity was observed Table 2 Effect of phage on adhesion, invasion and cytotoxicity

of NEC by additional clinical strains of S. aureus (MRSA) Strains (Bacteria: NEC- 10:1) Mean percent (%) Adherence Invasion Cytotoxicity (24 h) No phage Phage (MOI-1) Phage (MOI-10) No phage Phage (MOI-1) Phage (MOI-10) No phage Phage (MOI-1) Phage (MOI-10) S. aureus ATCC 43300 (MRSA) 73.7 0.41 0.025 31.9 0.031 No invasion 11.1 0.21 No cytotoxicity S. aureus ATCC 29213 (MSSA) 76.8 0.51 0.034 18.4 0.034 No invasion 10.2 0.23 No cytotoxicity S. aureus CS-1 68.4 0.37 0.066 28.1 0.06 No invasion 11.4 0.41 No cytotoxicity S. aureus CS-2 62.5 0.32 0.074 25.4 0.064 No invasion 10.1 0.43 No cytotoxicity S. aureus CS-3 74.8 0.45 0.084 33.3 0.078 No invasion 14.5 0.64 No cytotoxicity S. aureus CS-4 70.4 0.34 0.081 30.4 0.072 No invasion 14 0.61 No cytotoxicity S. aureus CS-5 72.1 0.33 0.075 32.8 0.066

No invasion 13.3 0.72 No cytotoxicity (CS-1 to CS-5 : these are clinical strains (CS) of MRSA chosen at random to test for their adherence, invasion and cytotoxicity parameters on cultured Mannose-binding protein-associated serine protease murine NEC). . Frequency of resistant mutant development The frequency of emergence of resistant colonies using mupirocin was determined. The mupirocin resistant mutants in vitro appeared at a frequency of (7.1 ± 0.54) × 10−6 and (2.4 ± 0.14) × 10−7 at 2 and 4 μg/ml (2X and 4X MIC) respectively. The calculated bacteriophage insensitive mutant (BIM) frequency at multiplicity of infection (MOI) of 10 was comparatively higher with a value of (7.4 ± 0.21) × 10−7. However, when both the agents were used in combination, mutation rate was below detection limit (<10−9). The results clearly depict the advantage referred by combination treatment in decreasing the frequency of resistant mutant generation.

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