Minimum inhibitory concentration (MIC) assay

The turbidity of the inoculated solutions after 24 h incubation was measured at 595 nm.

Figure 2.2 shows the turbidty of the nanomaterial and their bulk in physiological saline.

The reference microplates (the test solutions with no added bacteria) were used to correct the data by subtracting the turbidity caused by the particles alone from the inoculated solution (Figure 2.2). This resulted in negative values for most of the solutions at the high concentrations of these materials, but did adequately correct for particle turbidity at lower concentrations in each dilution series. According to the turbidity measurements, most of the test substances had a concentration-dependent effect on the growth of S. sanguinius. All the dilutions of AgNO3 are significantly lower than the control physiological saline, as the turbidity of inoculated physiological saline (mean ± S.E.M) was 0.401 ± 0.012, and the highest concentration of AgNO3 (400 mg/l) was showed a negative value (- 0.168 ± 0.004), whereas the other dilutions of AgNO3 were showed some growth but significantly lower than the control (physiological saline, Figure 2.2). AgNPs also showed a siginficant difference with physiological saline, as 400 mg/l showed a negative values. Whereas 200 and 100 mg/l of AgNPs showed 33.6 % and 37 % growth inhibition compared to control. Other dilutions of the AgNPs showed higher absorbance values compared to the control.

The high concentrations of TiO2 nanoparticles and bulk also showed negative values and the more diluted solutions showed a concentration-effect on bacterial growth.

Likewise, mHA and nHA also had a concentration-effect on the bacterial growth.

Chlorhexidine caused full growth inhibition at concentrations of 1, 0.5, and 0.25 %.

While the lower concentrations of chlorhexidine (0.125, 0.06, and 0.03%) showed no significant difference with the control. The turbidity of milliQ water (without added

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physiological saline) was significantly lower than control (physiological saline), as the bacterial grwoth in milliQ water was lower by 78 % compared to physiological saline.

Figure 2.3 shows the results of turbidity of the nanomaterials and their bulk after 24 h exposure in 0.85 % NaCl. The high concentrations of AgNO3 (400, 200, and 100 mg/l) showed high absorbance values compared to the control (0.85 % NaCl alone), even after subtracting the natural turbidity of the particles (Figure 2.3), whereas the turbidity of the more diluted solutions was siginificantly lower than the control. The AgNPs series of dilutions showed growth inhibition down to a concentration of 25 mg/l, whereas the more diluted AgNPs showed no growth inhibition. P25 and TiO2 showed negative values at high concentrations, whereas the low concetrations of both materials were not significantly different from control.

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Figure 2.2 the effect of nanomaterials on the growth of S. sanguinuis in physiological saline over 24 h in the minimum inhibitory concentration test (MIC). The absorbance values were read at 595 nm. B) Absorbance values after subtracting the natural turbidity of the particles. (+) shows a significant difference from the physiological saline control (Kruskal-Wallis, p<0.05). Within each test solution, different letters indicate a statistically significant dilution effect (Kruskal-Wallis, p<0.001). The absence of letters indicates no statistical differences between any of the dilutions within the series.

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Figure 2.3 the effect of nanomaterials on S. sanguinuis in 0.85% NaCl over 24 h in the minimum inhibitory concentration test (MIC). The absorbance values read at 595 nm. B) Absorbance values after subtracting the natural turbidity of the particles. (+) shows the significant difference from the control (Kruskal- Wallis, p< 0.001). Within each test solution, different letters indicate a statistically dilution effect (Kruskal-Wallis, p< 0.001). The absence of letters indicates no statistical differences between any of the dilutions within the series.

35 2.3.3 Lactate production

The lactate produced by S. sanguinis was measured after 24 h exposure to different nanomaterials and their bulk/metal salts in both 0.85% NaCl and physiological saline (Figure 2.4). In physiological saline (Figure 2.4 A), there was no lactate production in any of the dilutions of AgNO3. Whereas all dilutions of AgNPs showed full lactate production like the control, except 400 mg/l of AgNPs which showed a 99.5 % decrease in lactate production compared to the control. The remaining materials (P25, TiO2, nHA, and mHA) showed no significant difference with the control; high level of lactate was measured in all dilutions. Chlorhexidine showed a concentration-effect on the lactate production. No lactate was detected in 1 % (v/v) chlorhixidine, whereas the lactate levels in 0.5 % and 0.25 % (v/v) chlorhixidine were lower than the control by 99.5 % and 91.1 % respectively. MilliQ water had a concentration-effect on lactate production; lactate production in 100% milliQ water was 80.3 % lower than the control, whereas further diluted samples showed a negligible decrease in lactate production compared to physiological saline.

Overall, S. sanguinis produced lower concentration of lactate in 0.85 % NaCl compared to physiological saline (Figure 2.4 B); the maximum lactate production was 4.355 mM in 0.85 % NaCl whereas in physiological saline was 20.363 mM. Figure 3 B shows that the lactate levels in all AgNO3 dilutions were lower than the control by approximately 90 %. Among the nanoparticles, AgNPs showed antibacterial activity against S. sanguinis in the 400, 200, 100, and 50 mg/l dilutions; the lactate production was 91 % lower than the control and lactate levels remained statistically lower than the control in the further dilutions of AgNPs. The lactate production in the P25 and bulk TiO2 dilutions were statistically lower than the 0.85 % NaCl control, but still were at millimolar levels (> 3.5 mM).

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Intrestingly, the high concentrations of mHA (400, 200, 100, and 50 mg/l) showed higher lactate levels compared to the control (0.85 % NaCl); the lactate level was (in mM) 7.052 ± 0.17, 6.180 ± 0.068, 5.17 ± 0.11, 4.936 ± 0.31 for 400, 200, 100, and 50 mg/l of mHA, respectively. While lower concentrations of mHA (25, 12.5, 6.25, and 3.125 mg/l) showed no significant difference with the control. nHA showed no significant difference with the control in all dilutions. Chlorhexidine showed a concentration-dependent antibacterial effect against S. sanguinis; the lactate levels in 1%, 0.5 %, and 0.25 % (v/v) dilutions were upto 90 % lower than the control. The lactate production in 0.125 % and 0.06 % (v/v) chlorhixidine was lower than the control by 65 % and 24 % respectively. While the 0.03 % dilution showed no significant difference compared to the control. MilliQ water did not show a negative effect on the lactate production compared to the control. In contrast, 100 % milliQ water (with no added 0.85 % NaCl) showed higher lactate production compared to control.

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B A

P25

Figure 2.4 the concentration of lactate (mean ± S.E.M in mM) produced by S. sanguinuis exposed to different nanomaterials and their bulk after 24 h. A) Nanomaterials and their bulk in physiological saline. B) Nanomaterials and their bulk in 0.85% NaCl. “+” shows significant difference from the control group (physiological saline and 0.85% NaCl) (Kruskal-Wallis, p<0.001), within the test solution, different letters indicate statistically significant dilution effect (Kruskal-Wallis, p<0.001) between the dilution series.

B

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