Synergism against clinical isolates and positive isolates could also be demonstrated by a broth dilution method. In case of positive controls, MIC for vancomycin with l-arginine plus ceftriaxone (CVA1020) was 0.25 μg/ml for each of S. aureus, S. epidermidis,
E. faecalis and was 0.125 μg/ml for S. pneumoniae, whereas it ranged between 1.0 and 4.0 μg/ml for vancomycin (without l-arginine) and ceftriaxone when tested independently. For clinical isolates, MICs for CVA1020 ranged from 0.125 to 8 μg/ml, whereas 4–5 times higher MICs were observed when vancomycin with and without l-arginine and ceftriaxone were tested independently against the similar clinical isolates ( Table 1). MIC studies were also conducted using other ratios (C:V::1:2or 1:3 or 1:4 or 1:5
and vise versa) of vancomycin with l-arginine Neratinib nmr BMN 673 in vivo and ceftriaxone but significant results were obtained only with 1:1 ratio. l-arginine was not having antibiotic activity. Synergism of CVA1020 against S. aureus, S. epidermidis, S. pneumoniae, E. faecalis, MRSA and hGISA isolates was also demonstrated by a cup-plate agar diffusion method. For all positive controls ( S. aureus, S. epidermidis, S. pneumoniae and E. faecalis) inoculated onto an MHA plate containing CVA1020, an enhanced zone of inhibition (≥5 mm) was seen compared to ceftriaxone and vancomycin alone indicating synergistic activity between the vancomycin and ceftriaxone in presence of non antibiotic adjuvant l-arginine at C:V::1:1 ratio ( Table 1). Similarly for clinical isolates of S. aureus, S. epidermidis, S. pneumoniae, no E. faecalis, MRSA and hGISA, CVA1020 produced a greater zone of inhibition (≥5 mm) when compared with alone ceftriaxone and vancomycin ( Table 1). AST studies conducted using other ratios (C:V::1:2or 1:3 or 1:4 or 1:5 and vise versa) of CVA1020 (vancomycin with l-arginine and ceftriaxone) (results not disclosed) did not show significant synergy. TKC study was performed on all clinical as well as positive controls and results are presented only for one clinical isolates of MRSA and hGISA (Fig. 4). Results of TKC demonstrated an enhancement of killing of selected organisms in the presence of combinations of vancomycin with l-arginine and
ceftriaxone in a ratio of 1:1 (CVA1020), in comparison to vancomycin and ceftriaxone alone. After 12 h of incubation, (CVA1020) exhibited approximately 104–105 log reduction both in MRSA and hGISA whereas when vancomycin was tested alone against these isolates re growth was observed after 6 h, similarly, re growth appeared after 4 h with ceftriaxone alone. TKC studies were also conducted using other ratios (C:V::1:2or 1:3 or 1:4 or 1:5 and vise versa) of vancomycin with l-arginine and ceftriaxone but significant results were obtained only with 1:1 ratio in resistant strains. The decreasing vancomycin susceptibility among clinical isolates of gram positive strains especially staphylococci has imposed great threats for the treatment of infections caused by these isolates.
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