## 4 Discussion  
The findings of this study show varying activities of the C-CaOH\({}_{2}\) NPs against the different aerobic and anaerobic strains tested. The selected bacterial strains in this study represent common pathogens associated with infections including surgical site, bloodstream, or dental infections [39, 40, 41]. The initial screening test conducted was the agar well diffusion test as the standard in vitro screening method for plant extract materials [42]. We found that _P. aeruginosa_ was the most susceptible of all pathogens, not only among the Gram-negative aerobes. _E. coli_ followed _P. aeruginosa_: they both exhibited the largest inhibition zones at all concentrations. On the other hand, _K. pneumoniae_ was the least susceptible of the Gram-negative aerobes; however, its ZOIs were comparable to those of the Gram-positive aerobes. These findings coincide with the studies of [PERSON] et al. [27] and [PERSON] et al. [28] that showed similar antibacterial efficacy of CaOH\({}_{2}\) NPs against _P. aeruginosa_, _E. coli_, and _K. pneumoniae_. The Gram-positive aerobes _S. aureus_ and _S. epidermidis_ showed very similar results at 75 and 100 mg/mL; however, _S. aureus_ was more susceptible at 150 mg/mL. Similar results have been reported for CaOH\({}_{2}\) NPs, supporting a dose-dependent efficacy against _S. aureus_[28]. Conversely, _E. faecalis_ was the only tested aerobe that exhibited no inhibition zones at lower concentrations. This can be related to _E. Faecalis_'s resistance to CaOH\({}_{2}\) due to the buffering capacity of its cytoplasm that allows it to tolerate high pH levels, as reported [43, 44]. Nevertheless, C-CaOH\({}_{2}\) NPs exhibited efficacy against _E. faecalis_ at 100 and 150 mg/mL concentrations. These results are supported by the study of [PERSON] et al., which reported similar efficacy of CaOH\({}_{2}\) as nanoparticles against _E. faecalis_ at 100 mg/mL concentrations [26]. Generally, we found that Gram-negative aerobes demonstrated greater susceptibility to C-CaOH\({}_{2}\) NPs than the Gram-positive strains. This can be explained by differences in the architecture of cell wall components between Gram-positive and Gram-negative bacteria. It is well known that Gram-positive bacteria cell walls have a thicker peptidoglycan layer than the Gram-negative bacteria, which offers them more protection and hence explains the higher resistance to C-CaOH\({}_{2}\) NPs [45, 46]. Moreover, the thickness variations in the peptidoglycan layer within the Gram-positive strains can further explain the different responses among them [45, 46]. For instance, the cell wall of _S. epidermidis_ is reported to have approximately twice the peptidoglycan thickness of _S. aureus_, which could explain the lower antibacterial activity of C-CaOH\({}_{2}\) NPs against them in this investigation [47, 48]. Another reported variable that may have played a role in the different responses between the Gram-positive and Gram-negative bacteria is their cell wall shape. It has been reported that the rod-shaped _P. aeruginosa_ and _E. coli_ have greater surface area than the spherical-shaped _S. aureus_, which may have allowed higher C-CaOH\({}_{2}\) NP interaction and antibacterial activity [28, 46, 49]. Furthermore, CaOH\({}_{2}\) is more soluble in lipids than in water, which may imply a more favorable interaction with the Gram-negative bacterial outer cell membrane due to its high permeability and selectivity to hydrophilic and lipophilic compounds [43, 50, 51, 52]. For the anaerobic bacteria, we found that C-CaOH\({}_{2}\) NPs had little activity against _P. micra_, even at higher concentrations. Previous studies have reported that Gram-positive facultative anaerobes, such as _P. micra_, are resistant to CaOH\({}_{2}\) as a bulk material [44]. Additionally, a study using agar well diffusion reported that CaOH\({}_{2}\) produced no inhibition zones against _P. micra_[53]. In this study, C-CaOH\({}_{2}\) NPs were proven to have inhibitory activity against _P. micra_ despite the low activity.