## 4 Discussion
In all bacteria, the antibacterial potency of C-CaOH\({}_{2}\) NPs increased with increasing dosage. The highest susceptibilities in all tested strains were at 150 mg/mL concentration, and are considered intermediate susceptibilities as defined in the literature [54, 55]. Additionally, the difference in the ZOIs between the maximum and minimum concentrations was statistically significant in all bacteria except for _S. epidermidis_ and _K. pneumoniae_, for which the results were close. These findings are similar to those reported by [PERSON] et al., which demonstrated a dose-dependent efficacy of CaOH\({}_{2}\) NPs up to 100 mg/mL [28]. Although C-CaOH\({}_{2}\) NPs demonstrated dose-dependent activity, the ZOIs exhibited no statistically significant difference between 75 and 100 mg/mL in all strains except in _E. faecalis_. This should be carefully considered when deciding the most effective dose with least toxic effect for future in vivo and clinical studies. The MIC and MBC tests were used to further confirm the antibacterial potency of C-CaOH\({}_{2}\) NPs. Resazurin-based MIC provides a colorimetric test to determine the lowest concentration where bacteria growth is visibly inhibited [38; 56], whereas the MBC is used to determine the lowest concentration required to kill 99.9% of bacteria [56]. In this study, C-CaOH\({}_{2}\) NPs exhibited high potency against tested strains, as the MIC value effective for aerobes was 3.12 mg/mL, with the exception of _E. faecalis_ and _K. pneumoniae_, which required lower doses (0.78 mg/mL and 1.56 mg/mL, respectively). The MBC value for aerobes was 12.5 mg/mL for most strains, except for _P. aeruginosa_ (3.12 mg/mL) and _K. pneumoniae_ (6.25 mg/mL). Conversely, _P. micra_ required higher effective concentrations, as the MIC was at 15.62 mg/mL and the MBC was at 31.25 mg/mL. These results agree with those reported in the literature about CaOH\({}_{2}\) and carob respectively [19; 26; 57]. C-CaOH\({}_{2}\) NPs can be considered bactericidal as the ratio between MBC and MIC is small [58]. C-CaOH\({}_{2}\) NPs demonstrated biocompatible doses of up to 100 mg/mL in our recent work [30]. In this study, the ZOI activity of C-CaOH\({}_{2}\) NPs was achieved at high doses (100 and 150 mg/mL), which can be argued to compromise biocompatibility. [PERSON] et al. reported a similar dose (100 mg/mL) of CaOH\({}_{2}\) NPs and similar findings [26]. When compared to other biogenic NPs, C-CaOH\({}_{2}\) NPs exhibited a dose-dependent effect similar to titanium oxide NPs. [PERSON] et al. investigated titanium oxide NPs and reported cell viability at 100 mg/mL [59]. They also found that the highest ZOI activity between the different concentrations (50, 100, and 200 mg/mL) was at 200 mg/mL against _S. aureus_ and _E. coli_. Although the C-CaOH\({}_{2}\) NPs' MIC and MBC values were less than the ZOI doses, they were still higher than the 100 mg/mL biocompatible margin. This disparity between the biocompatible and inhibitory doses may influence the effective in vivo dose choice. Conversely, other biogenic NPs, such as zinc oxide and chitosan, require low doses to induce antibacterial effects. [PERSON] et al. assessed chitosan NPs' antibacterial activity at different concentrations and observed superior activity at a 150 mg/mL concentration [60]. Moreover, zinc oxide NPs have been reported to have high antibacterial activity at low doses, ranging from 7.36 to 230 mg/mL [61; 62; 63]. Nevertheless, the current findings warrant future in vitro and in vivo investigations to optimize the results and address limitations. The following possible mechanisms of C-CaOH\({}_{2}\) NP activity are suggested based on the preexisting collective knowledge of NPs, CaOH\({}_{2}\), and carob antibacterial effects, since their exact antibacterial activity has yet to be investigated. NPs are generally known for their small size and high surface area [2], which play roles in their antibacterial efficacy.