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articles Jain et al. Table 3. PAE on Different Bacterial Strains at Inhibitory (10 × MIC) Concentrations of SNP organism Escherichia coli ATCC 117 Pseudomonas aeruginosa ATCC 9027 Salmonella abony NCTC 6017 Salmonella typhimurium ATCC 23564 Klebsiella aerogenes ATCC 1950 Proteus vulgaris NCIB 4157 Bacillus subtilis ATCC 6633 Staphylococcus aureus ATCC 6538 Streptococcus epidermidis ATCC 12228 Pseudomonas sp. # MDR 1 Staphylococcus sp. # MDR 1 a NM ) not measurable. PAE (h) NMa 10.5 4.6 4.9 NM 8.5 3.6 1.3 1.5 9.3 1.8 Figure 4. Kill curve of reference bacteria and MDR cultures at MBC of SNP (error bars not shown as deviations from the mean values are <0.1 × log 10). the higher end of this range. Also, at higher absolute values of K, the bactericidal activity was found to be greater. Among all the strains tested, E. coli and P. aeruginosa were found to be most vulnerable to SNP as they were killed in the least time (3-4 h). In the next experiment, the time required to achieve 3 log10 decrease in viability level of killing was determined from time kill curves at MBC (12.5 μg/mL) of SNP. The results obtained (Figure 4) clearly show that bactericidal action was more rapid as compared to time kill curves seen at MIC. From the analysis of kill curve the time required to achieve 3 log10 decrease in viable cell count (D) was calculated (Table 2). Once again, it is seen that SNP kill Gram-negative bacteria more effectively, achieving a 3 log10 decrease in 5 to 9 h as against 12 h for Gram-positive bacteria such as B. subtilis and S. epidermidis. Three Staphylococcus aureus strains exposed to SNP showed <1 log10 decrease in viability in 8 h and no increase in cell count upon continued incubation for 24 h, suggesting a bacteriostatic effect. Results obtained for the post agent effect studies are presented in Table 3. The PAE was longer for Gram-negative bacteria than Gram-positive bacteria, with maximum effect against P . aeruginosa (P AE 10.5 h) and minimum effect against Staphylococcus aureus (PAE 1.3 h). PAE against E. coli and K. aerogenes could not be measured owing to rapid bactericidal activity while it ranged from 1.3 to 10.5 h for other bacterial strains. As a part of FIC studies, susceptibility of P. aeruginosa to different combinations of SNP and antibiotics was investigated and the results obtained are presented in Table 4. These results clearly show that FICi was synergistic (<0.5) for one combination, i.e., SNP with ceftazidime. Additive effects (FICi >1) were seen for the individual combination of SNP with streptomycin, kanamycin, ampiclox and poly- myxin B. The combination of SNP with chloramphenicol was antagonistic (FICi >2). SNP were found to inhibit the mycelial growth of Aspergillus niger after 72 h of incubation with an IC50 value of 75 μg/mL and a corresponding antifungal index of 55.5%. The MIC50 and MIC90 against C. albicans were 6.25 and 12.5 μg/mL, respectively, whereas the MFC of SNP was 25 μg/mL. The data on the time kill studies are presented in Figure 5. At SNP concentrations equal to MIC, 97% of C. albicans cells were killed after 8 h of incubation. When C. albicans was exposed to higher SNP concentration (MFC), the rate of killing as well as extent of killing increased (99.9%) in 6 h. The PAE of SNP for C. albicans was found tobe1.6h. Studies on inhibition of matrix metalloproteinase activities show that SNP inhibited MMP-2 and MMP-9 enzymes in a concentration-dependent manner (Figure 6). SNP at concen- tration of 50 μg/mL showed ∼75% inhibition of these enzymes while at 100 μg/mL complete inhibition could be seen. The interactions of silver nanoparticles were studied in vitro using an established cell line, viz., Hep G2. Data on cell viability estimated by XTT assay (Figure 7) showed a dose-dependent decrease in viability with IC50 value working out to be 251 μg/mL. To localize the presence of silver nanoparticles, ultrathin sections of Hep G2 cells exposed to ∼(1/2)IC50 SNP for 24 h were visualized under transmission electron microscope. Dark, electron dense, spherical ag- gregates were found inside the mitochondria as compared to unexposed (control) cells (Figure 8, right and left, respectively). When biochemical changes occurring in the SNP treated cells were monitored (with respect to enzyme activity indicative of oxidative stress), interesting data emerged (Figure 9). Treatment with SNP increased GSH levels by ∼1.1-fold (62.09 μmol/mg protein in unexposed cells and 69.12 μmol/mg protein in SNP-exposed cells). There was ∼1.1-fold increase in catalase levels after SNP treatment (124.85 μmol/mg protein as compared to 110.17 μmol/mg 1394 MOLECULAR PHARMACEUTICS VOL. 6, NO. 5PDF Image | Silver Nanoparticles in Therapeutics: Antimicrobial Gel
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