Bactericidal activity of novel calcium-based core-shell particles and its mechanism of action

Bactericidal activity of novel calcium-based core-shell particles and its mechanism of action | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Bactericidal activity of novel calcium-based core-shell particles and its mechanism of action Mehdi Mohammadi ashani, Noora Naif Darwish, Gopal Ramamourthy, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8378570/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The global rise in antimicrobial resistance (AMR) poses a significant challenge to infection control efforts worldwide. This growing threat highlights the critical need for innovative technologies with advanced antimicrobial properties to improve infection prevention and control strategies. To address this challenge, we developed a novel core–shell \(\:{Ca\left(OH\right)}_{2}\) – \(\:{CaCO}_{3}\) (CSCC) particle exhibiting antibacterial properties against both gram-positive and gram-negative bacteria. Through confocal microscopy imaging and the developed in vitro microbiology protocol, we identified the bacteria-killing mechanism, wherein the bacteria adhere to the \(\:{CaCO}_{3}\) shell and are subsequently killed, by the membrane disruption and aggregation mechanisms of the \(\:{Ca\left(OH\right)}_{2}\) core. The minimum Bactericidal content of CSCC, \(\:{CaCO}_{3}\) and \(\:{Ca\left(OH\right)}_{2}\) was measured using a combination of broth dilution and spot-plating methods. The results indicate that 2.5 mg/mL CSCC kills both gram-positive and gram-negative bacteria, whereas 2.5 mg/mL and 5 mg/mL of \(\:{Ca\left(OH\right)}_{2}\) killed gram-negative and gram-positive bacteria, respectively. No antimicrobial properties were observed for \(\:{CaCO}_{3}\) . We believe the mechanism of binding and killing bacteria may offer a prominent solution to the global challenge of antimicrobial resistance. Accelerated aging tests confirmed that the CSCC particles retained full antibacterial activity against E. coli equivalent to 100 years of natural aging. Biological sciences/Biotechnology Biological sciences/Microbiology antimicrobial antibiotic-resistant bacteria core–shell particle Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. INTRODUCTION The escalating global issue of antimicrobial-resistant bacteria mandates the development of effective antibacterial products and innovative approaches to microbial control [ 1 – 3 ]. There is a critical need for novel long-acting biocidal agents that can be used on surfaces, especially in healthcare, food processing, and other high-contact environments [ 4 – 6 ]. Despite the widespread nature of this challenge, the range of effective antimicrobial tools available for use in these settings remains surprisingly limited [ 7 ]. For instance, traditional chemical sterilizers, such as bleach and hydrogen peroxide, are effective in the short term but pose health risks (e.g., respiratory issues, skin irritation) and environmental harm [ 8 – 10 ]. As a result, there is an unmet need for advanced antimicrobial agents that not only exhibit long-term persistent activity but are also safe for use on a variety of surfaces in critical environments [ 1 ]. To address this challenge, innovative antibacterial agents have emerged for different applications including medical devices [ 11 ], wound dressings [ 12 ], water purification [ 13 ], surface coating [ 14 ], textiles [ 15 ], and food packaging [ 16 ]. In addition, a wide range of materials and particles including metal oxide nanoparticles (NPs) [ 17 ], metal NPs [ 18 ], and carbon nanomaterials [ 19 , 20 ], have been developed with biocidal activity owing to their unique physicochemical properties, including their small size [ 21 , 22 ]. A combination of physical and chemical bactericidal properties has also been explored. For example, \(\:Si\) nano-ripples coated with \(\:Ag\) or \(\:{TiO}_{2}\) NPs damaged bacterial DNA and prevented biofilm formation [ 23 , 24 ]. Biocidal surfaces can kill bacteria and prevent biofilm formation, however the continuous release of antibacterial agents may lead to bacteria resistance and reduce their long-term effectiveness [ 25 ]. To address this, alternative strategies have been proposed, including surfaces that prevent early bacterial adhesion [ 26 , 27 ]. For example, laser-induced periodic surface structures (LIPSS) have been used to create nanostructures on surfaces to inhibit bacteria attachment. Studies show that LIPSS reduced Escherichia coli ( E.coli ) population [ 26 , 28 , 29 ]. Additionally, surface that physically disrupts bacterial membranes have been tested [ 30 , 31 ]. For instance, nanostructured titanium surfaces fabricated through hydrothermal etching were assessed in killing both methicillin- and gentamicin-susceptible and -resistant S. aureus strains [ 31 ]. The nanostructured surfaces effectively killed both strains, hence demonstrating that the physical disruption mechanism is equally effective against susceptible and resistant bacteria [ 31 ]. Nevertheless, antimicrobial nanostructured surfaces are still in their infancy [ 23 ]. Generally, there is still a scarcity of effective, environmentally friendly, and biodegradable antibacterial agents that could be incorporated into a broad range of materials and applications. To address this gap, we present a new core-shell \(\:{Ca\left(OH\right)}_{2}\) – \(\:{CaCO}_{3}\) (CSCC) antibacterial particles and explore their mechanism of action against gram-positive and gram-negative bacteria. \(\:\:{Ca\left(OH\right)}_{2}\) is strongly bactericidal, fungicidal, and virucidal owing to \(\:{Ca\left(OH\right)}_{2}\) ability to maintain high alkalinity (pH 11–13) [ 32 , 33 ]. Chemically, \(\:{OH}^{-}\) damages the microbial membranes, disrupts the metabolic processes, and inhibits DNA replication [ 34 – 36 ]. Physically, \(\:{OH}^{-}\) deprives the microorganisms of essential nutrients and the space needed to multiply [ 34 – 36 ]. In this context, \(\:{Ca\left(OH\right)}_{2}\) acts as a protective barrier that prevents bacterial invasion and further growth by removing available substrates and limiting the area for microbial proliferation [ 34 – 36 ]. In contrast, \(\:{CaCO}_{3}\) is generally chemically inert with no inherent antimicrobial properties [ 37 ]. Nevertheless, \(\:{CaCO}_{3}\) has been used as a carrier for antimicrobial agents, such as \(\:{Ag}^{+}\) or biocidal compounds, improving their delivery to microbial targets [ 38 ]. Different approaches were employed to incorporate materials of interest into \(\:{CaCO}_{3}\) , including adsorption, coprecipitation, and infiltration [ 38 ]. The slow release of these agents from \(\:{CaCO}_{3}\) matrix ensures a prolonged antimicrobial effect [ 39 ]. Moreover, \(\:{CaCO}_{3}\:\) can influence the adhesion of bacterial cells onto surfaces by adsorbing onto bacteria [ 39 ]. 2. MATERIALS AND METHODS 2.1. Materials Technical grade calcium hydroxide, \(\:{Ca\left(OH\right)}_{2}\) (dp = 44 µm, 1.3 wt% moisture, Atlantic Equipment Engineering Inc., USA), was used as a reactant. Liquid carbon dioxide, \(\:{CO}_{2}\) (> 99% pure, Air Liquide, Canada) was supplied from a pressurized cylinder at 5.9 MPa using a siphon. The BD BBL™ Mueller Hinton (MH) II Broth (Cation-Adjusted) was purchased from BD Biosciences (VWR, Canada). The medium was prepared based on the manufacturer guidelines and autoclaved at 121.5 \(\:℃\) for 20 min using a Primus Sterilizer (USA). The MH medium is cooled to room temperature, then stored at 4 \(\:℃\) . Similarly, the MH agar is prepared, autoclaved, poured on plates, and then stored at 4 \(\:℃\) . A crystal violet, 1% (aqueous solution) dye in a solution of ethanol-phenol-methanol-water, was purchased from Harleco-EMD. 2.2. Core-Shell antibacterial particle preparation Detailed CSCC preparation protocol is presented in our previous study [ 40 ]. In brief, \(\:{Ca\left(OH\right)}_{2}\) was mixed with liquid \(\:{CO}_{2}\) at 5.9 MPa, 21 ± 1 \(\:℃\) and 1300 rpm. After 5 min, the pressure was rapidly reduced inducing adiabatic flashing of liquid \(\:{CO}_{2}\) into dry ice and gaseous \(\:{CO}_{2}\) . Dry ice encapsulated the \(\:{Ca\left(OH\right)}_{2}\) particles, which in turn carbonated \(\:{Ca\left(OH\right)}_{2}\) surface and preserved particle aggregation. The dry ice was sublimed to control the coating thickness, producing a 45 wt% − 55 wt% core–shell structure [ 40 ]. This partial carbonation was confirmed using XRD, acid-base titration, and thermogravimetry with calorimetry, while the surface coating was analyzed using FTIR spectroscopy and TEM microtomy [ 40 ]. 2.3. Bacterial sample preparation Strains of Escherichia coli , ATCC 25922, E.coli 25922GFP, Pseudomonas aeruginosa , ATCC 27853, Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300 and Staphylococcus aureus , ATCC 25923 were purchased from Cedarlane lab and stored at \(\:-80\:℃\) . These bacteria were inoculated overnight in 10 mL MH media in a Heracell™ VIOS 250i \(\:{CO}_{2}\) Incubator (Thermo Fisher, Waltham, MA, USA) at 36.5 \(\:℃\) , with an atmosphere of 5% \(\:{CO}_{2}\) and 17.5% \(\:{O}_{2}\) . Thermo NanoDrop™ OneC Spectrophotometer (USA) was used to measure the optical density (OD) of bacterial stock solutions at 600 nm under absorbance mode. These stock solutions were further diluted to achieve an OD600 value of 0.10, followed by two 10-fold dilutions (100x) to obtain a final OD600 value of 0.001. Different masses of CSCC, \(\:{CaCO}_{3}\) , and \(\:{Ca\left(OH\right)}_{2}\) were mixed with MH medium and introduced to the plate to achieve the desired particle content. 2.4. Bactericidal assays A hybrid of broth dilution and spot plating methods was employed to determine the minimum bactericidal concentration (MBC) of the CSCC, \(\:{Ca\left(OH\right)}_{2}\) and \(\:{CaCO}_{3}\) particles. A range of particle content: 0.625, 1.25, 2.5, 5, 10, 25, 50, and 100 mg/mL was used. To ensure a uniform suspension of particles into MH media, a vortex mixer was used for 10 min, followed by vigorous shaking. After preparation, the uniform suspension of particles into MH media was transferred into a 96-well plate. A volume of 20 µL of a bacterial suspension with an optical density (OD) of 0.001 ( \(\:1.45\:\times\:{10}^{5}\:\) and \(\:7.9\:\times\:{10}^{5}\:\) Colony Forming Unit, CFU for S. aureus or P. aeruginosa , respectively) was added to each well. The bacterial strains used in the MBC study included P. aeruginosa (gram-negative) and S. aureus (gram-positive). The 96-well plate was incubated in a Heracell™ VIOS 250i \(\:{CO}_{2}\) incubator (Thermo Fisher, Waltham, MA, USA) at 36.5 \(\:℃\) , with an atmosphere of 5% \(\:{CO}_{2}\) and 17.5% \(\:{O}_{2}\) for 3 h. Following incubation, 10 µL of each sample was transferred to MH agar plates using the spot plating technique [ 41 ]. The agar plates were then cultured for 20 h to allow for bacterial growth and MBC determination. 2.5. Bacteria aggregation assay E.coli was cultured overnight in MH media, then centrifuged at 4300 rpm for 5 min, and resuspended in autoclaved water to achieve an OD of 10. A 100 µL aliquot of particle suspension, prepared by dispersing particles in MH media at a concentration of 50 mg/mL, was added to each well of a 96-well plate. Subsequently, 100 µL of bacterial suspension was introduced into each well. The plate was then incubated for 24 hours at 23°C (room temperature). The supernatant was carefully extracted, and the well was rinsed 3 times with water. A 100 µL of the crystal violet solution, prepared by mixing 3 mL of crystal violet 1% solution with 3 mL of water, was added to the well and incubated at 23 \(\:℃\) for 15 min. The supernatant was discarded, and the well was washed with water 4–5 times, or until no residual crystal violet was observed in the control well. Finally, 100 µL of 95% ethanol was added to each well, and the absorbance was measured at 595 nm. 2.6. Confocal laser-scanning microscopy (CLSM) Optical imaging using confocal laser-scanning microscopy (CLSM, Carl Zeiss, Göttingen, Germany) was collected for three mixtures of CSCC, \(\:{Ca\left(OH\right)}_{2}\) and \(\:{CaCO}_{3}\) with E.coli GFP suspension. A 1 mL of a 20 mg/mL particle suspension in MH medium was combined with 1 mL of E. coli GFP culture, adjusted to an OD of 0.4. The mixture was incubated at 23°C (room temperature) for 30 minutes. Subsequently, 100 µL of the sample was placed on a round glass coverslip for microscopic imaging using an inverted LSM510 CLSM. The images were made in three modes: fluorescence to track GFP-labeled bacteria, reflection to capture the positions of the particles, and overlay to combine both imaging modes. 2.7. Accelerated aging protocol for long-term stability assessment of CSCC particles To assess the long-term physicochemical stability and antimicrobial efficacy of the CSCC particles, an accelerated thermal aging study was performed under tightly controlled conditions. Freshly synthesized CSCC particles, alongside analytical-grade \(\:{Ca\left(OH\right)}_{2}\) as a comparative control, were each evenly distributed in separate inert stainless-steel trays. These trays were placed in a fully enclosed convection dry oven maintained at 150°C for a continuous period of 35 days. The oven chamber (BINDER ED 56) remained sealed throughout the study to ensure thermal homogeneity and prevent environmental fluctuations such as moisture uptake, which could otherwise alter particle morphology or surface chemistry. This accelerated aging approach was modeled using the Arrhenius equation, which describes the temperature dependence of reaction kinetics and degradation processes [ 42 , 43 ]. This thermal stress approach enables a comparative evaluation of the structural and antimicrobial integrity of CSCC versus \(\:{Ca\left(OH\right)}_{2}\) , providing insight into their durability 3. RESULTS Detailed CSCC particle characterization is included in our earlier publication [ 44 ]. In brief, CSCC mean particle size ranged between 20–170 nm with aggregates ranging 0.2–0.6 µm. 3.1. Mechanism of action Optical imaging (CLSM) revealed the morphology of the product of the interaction between the different particles and the bacteria. The positive control (Fig. 1 (a-c)), i.e. in the absence of any treatment, showed visible rod-shaped E.coli GFP exhibiting distinct green fluorescence, suggesting complete viability. Figure 1 (d-f) shows intense GFP fluorescent signals suggesting bacterial viability with \(\:{CaCO}_{3}\) particles and some bacterial binding. Figure 1 (g-i) depicts E.coli GFP onto \(\:{Ca\left(OH\right)}_{2}\) with faded fluorescence, suggesting a loss of viability. Lastly, Fig. 1 (j-l) captures E.coli GFP treated with CSCC particles. Figure 1 (j-l) shows a substantial degree of bacterial binding to CSCC particles, leading to the formation of a chain-like structure. Moreover, E. coli GFP fluorescence was notably reduced, suggesting substantial biocidal activity. To further validate the biocidal attribute of the particles, samples were collected post-experiment and re-cultured on agar plates. The results in Figure S1 confirm that the bacteria remained viable following \(\:{CaCO}_{3}\) treatment, in contrast to CSCC and \(\:{Ca\left(OH\right)}_{2}\) treatments. 3.2. Bacteria aggregation The crystal violet assay was performed to evaluate bacterial behavior under different experimental conditions. As shown in Fig. 2 (a), no bacterial aggregation was observed in the positive control. In contrast, minimal aggregation occurred in the presence of 20 mg/mL \(\:{CaCO}_{3}\) (Fig. 2 (b)), whereas extensive aggregation was evident in the presence of 20 mg/mL \(\:{Ca\left(OH\right)}_{2}\) and CSCC (Fig. 2 (c),(d)), with a substantially higher extent of aggregation for CSCC. To exclude the possibility of artifacts caused by CSCC particles, a control sample in absence of bacteria was included (Fig. 2 (e)). The absence of aggregates in Fig. 2 (e) confirms that the aggregates observed in Fig. 2 (d) originated from interactions between bacteria and CSCC particles. These results suggest that the biocidal activity of CSCC and \(\:{Ca\left(OH\right)}_{2}\) is likely mediated through aggregation-driven mechanisms. It is noteworthy that bacterial aggregation can enhance bacterial survival by forming protective clusters that shield cells from environmental stresses while facilitating nutrient exchange and intercellular communication [ 45 ]. Crystal violet, a cationic dye, binds preferentially to negatively charged bacterial components. In the absence of bacteria, minimal staining is expected for CSCC or \(\:{Ca\left(OH\right)}_{2}\) particles because their positively charged surfaces repel the dye. \(\:{CaCO}_{3}\) may retain limited amounts of dye through physical adsorption associated with its aggregated morphology rather than specific chemical interactions. In the presence of bacteria, the distinct staining patterns observed for \(\:{Ca\left(OH\right)}_{2}\) and CSCC are likely attributed to differences in bacterial adhesion and membrane disruption. To further quantify bacterial aggregation, each well of the 96-well plate was treated with 100 µL of 95% ethanol to solubilize the crystal violet bound to the bacterial cells. The absorbance of the dissolved dye was subsequently measured at 595 nm using a microplate reader. Figure 3 presents the quantitative crystal violet results for E. coli , S. aureus , and MRSA exposed to different particles. Compared with \(\:{\:Ca\left(OH\right)}_{2}\) , CSCC exhibited 25%, 24%, and 18% higher absorbance for E. coli , S. aureus , and MRSA, respectively. When compared to \(\:{CaCO}_{3}\) , CSCC showed 46%, 69%, and 62% higher absorbance for E. coli , S. aureus , and MRSA, respectively. These findings confirm the superior aggregation-inducing capability of CSCC toward both gram-positive and gram-negative bacteria. 3.3. Minimum bactericidal concentration (MBC) The broth dilution method combined with optical density measurement was initially employed. However, the background noise due to optical interference of the particles introduced a significant error in the results (Figure S2). To address this challenge, a combination of broth dilution and spot-plating methods was used to determine the MBC of the different particles. The relevant experimental protocol is given in Figure S3. A range of particle content, 0.625, 1.25, 2.5, 5, 10, 25, 50, and 100 mg/mL, was tested. The results in terms of particle content versus P. aeruginosa and S. aureus growth are presented in Fig. 4 . Figure 4 (a,b) shows the initial time of spot plating following broth dilution and a schematic representation of the spot plating protocol for each particle content and bacteria. Figures 4 (c,d), 4(e,f) and 4(g,h) include the results for, \(\:{CaCO}_{3}\) , \(\:{Ca\left(OH\right)}_{2}\) and CSCC particles, respectively, following 3 h incubation. The results show that 2.5 mg/mL of CSCC was effective in killing both P. aeruginosa and S. aureus . In contrast, no antibacterial effect was observed for \(\:{CaCO}_{3}\) . The MBC for \(\:{Ca\left(OH\right)}_{2}\) was 2.5 mg/mL for P. aeruginosa and 5 mg/mL for S. aureus . These results highlight the role of bacteria adsorption/attachment onto the carbonate shell of CSCC particles as a component of the killing mechanism. To evaluate the impact of accelerated thermal aging on bactericidal efficacy, the minimum bactericidal concentration (MBC) of \(\:{Ca\left(OH\right)}_{2}\) and CSCC particles was determined against E. coli (ATCC 25922). The antimicrobial performance of both fresh and thermally aged CSCC and \(\:{Ca\left(OH\right)}_{2}\) particles was examined across 1.5, 3, 6, 12.5, 25, and 50 mg/mL particle content. All formulations fresh and thermally aged CSCC and \(\:{Ca\left(OH\right)}_{2}\) particles achieved complete bacterial killing at 6 mg mL⁻¹, establishing this concentration as the MBC for all groups (Fig. 5 ). The applied thermal-aging protocol corresponds to more than 100 years of ambient storage based on Arrhenius kinetics, providing a robust model for accelerated stability assessment. Despite similar MBC values, significant differences emerged in bactericidal performance at sub-MBC concentrations. Fresh \(\:{Ca\left(OH\right)}_{2}\) exhibited marked antimicrobial activity at concentrations as low as 1.5 mg/mL, causing a substantial reduction in E. coli colony-forming units (CFUs) compared with untreated controls. In contrast, aged \(\:{Ca\left(OH\right)}_{2}\) displayed markedly reduced efficacy at the same content, with minimal CFU reduction. This decline is likely due to physicochemical transformations during thermal exposure, including particle aggregation. Conversely, both fresh and aged CSCC particles retained strong bactericidal activity even at sub-MBC concentrations. At 1.5 mg/mL, both formulations produced statistically significant E. coli viability reduction, demonstrating that thermal aging did not compromise the antimicrobial performance of the CSCC system. The sustained efficacy of aged CSCC particles supports the stabilizing role of the carbonate shell, which likely protected the particles from major chemical and physical changes while maintaining hydroxide ion availability. Collectively, these findings demonstrate that CSCC particles maintain long-term bactericidal stability. 4. DISCUSSION Antimicrobial materials can kill bacteria through different mechanisms, including protein synthesis, nucleic acid synthesis, membrane permeabilization, and bacterial cell agglutination [ 46 , 47 ]. The confocal microscopy imaging enabled the evaluation of the efficacy of each treatment. As depicted in Fig. 1 , the chain-like structure resulting from the interaction of bacteria adhered to the surface of the CSCC particles suggests that attachment/adsorption plays a major role in the killing mechanism. In contrast, confocal microscopy imaging of \(\:{Ca\left(OH\right)}_{2}\) primarily displayed bacterial aggregation with much less extent of bacterial attachment. Lastly, \(\:{CaCO}_{3}\) displayed limited adhesion likely owing to effective detachment of the viable bacteria. Both CSCC and \(\:{Ca\left(OH\right)}_{2}\) demonstrated antibacterial properties in contrast with \(\:{CaCO}_{3}\) . The antibacterial action of the CSCC particles appears to follow an adsorption-mediated killing mechanism. Bacterial cells first adsorb onto the particle surface—in this case, the \(\:{CaCO}_{3}\) shell—facilitated by electrostatic interactions, hydrogen bonding, and surface roughness. Following adsorption, the \(\:{Ca\left(OH\right)}_{2}\) core releases hydroxyl ions, which locally increase pH and disrupt bacterial membranes, leading to cell death. While the mechanisms of antimicrobial action are not fully understood, the sequential adsorption followed by membrane permeabilization and lysis, has been recently reported for cationic polymers and engineered antimicrobial coatings [ 48 , 49 ]. Notably, Ivanova et al. [ 49 ] demonstrated a purely physical killing mechanism on cicada wings ( Psaltoda claripennis ), where nanopillar structures penetrated and ruptured bacterial cells ( P. aeruginosa ) within minutes of contact, independent of surface chemistry. These cicada wings maintain clean surface not only through self-cleaning hydrophobicity but also via bactericidal action driven by nanoscale surface architecture [ 49 ]. However, while adsorption-killing pathways are well documented for polymers [ 50 – 52 ] and nanostructured surfaces [ 49 , 53 , 54 ], to the best of our knowledge, there is no literature reports pertaining to mineral-based core-shell systems such as CSCC. The Biocidal action of \(\:{Ca\left(OH\right)}_{2}\) is attributed to a damage to the cytoplasmic membranes as well as protein denaturation [ 55 ]. Bacterial aggregation had been previously reported as a response of bacteria to environmental stimuli. This aggregation enhances the survival rate of bacteria under environmental stresses, including pH and osmotic extremes, or exposure to pharmaceutical antimicrobials [ 45 , 56 ]. Figures 2 and 3 demonstrate significant bacterial aggregation in the presence of CSCC compared to \(\:{Ca\left(OH\right)}_{2}\) . CSCC particles aggregate both gram-negative and gram-positive bacterial strains, suggesting that its mechanism for bacterial killing is likely similar for both types of bacteria. CSCC particles disrupt and destabilize the bacterial membrane, leading to aggregation of the bacterial cells. However, due to issues with the particle solubility and its optical properties, we were unable to conduct a membrane permeabilization assay. More discussion on the role of \(\:{Ca}^{2+}\) in bacteria aggregation is given below. The attachment/adsorption of bacteria onto CSCC particles involves several interfacial interactions. Motile bacteria exist in three fluid regions: bulk liquid, where surfaces have no effect; near-surface bulk, influenced by hydrodynamics; and near-surface constrained, affected by both hydrodynamic and physicochemical forces [ 57 ]. Two main theories explain bacterial attachment to solid surfaces [ 58 – 60 ]. The first involves a two-step process, wherein bacteria first approach the surface through van der Waals forces, electrostatic interactions, and hydrophobic effects. At this stage, bacteria still exhibit Brownian motion and can be easily dislodged by fluid shear forces. The second step is irreversible attachment, wherein bacteria anchor themselves using exopolysaccharides or specific ligands such as pili or fimbriae [ 60 – 63 ], by means of short-range covalent and hydrogen bonding, and hydrophobic interactions [ 64 ]. Poortinga et al. [ 65 ] suggested that bacterial attachment also involves electron exchange between the cell surface and the substratum. Busscher and Weerkamp [ 66 ] proposed a three-step model for bacterial attachment to surfaces. In the first step, Lifshitz–van der Waals forces act over several hundred nanometers. In the second step, both Lifshitz–van der Waals forces and electrostatic interactions occur at around 20 nm. In the third step, specific adhesion receptors enable strong attachment at about 5 nm [ 66 ]. Once irreversible attachment occurs, stronger chemical methods are needed to kill the bacteria [ 66 ]. The van der Waals forces, arising from permanent and temporary dipoles in atoms and molecules, are key to initial bacterial contact with the CSCC surface. The interaction is further stabilized by surface charge. The CSCC particles are positively charged, zeta potential = 31.7 mV (Table S1 ), whereas bacterial surfaces are negatively charged owing to components such as teichoic acids or lipopolysaccharides, promoting strong electrostatic attraction [ 67 , 68 ]. Once bacteria adhere to the CSCC surface, the \(\:{OH}^{-}\) ions from the \(\:{Ca\left(OH\right)}_{2}\) core diffuse through the porous calcium carbonate shell to reach the bacteria. This creates a highly alkaline environment near the bacterial cells, which disrupts their cellular processes [ 34 , 36 , 69 ]. The high concentration of \(\:{OH}^{-}\) damages the bacterial cell membrane and wall, causes lipid saponification, protein denaturing, and essential enzymatic activity inhibition, leading to cell structure breakdown [ 34 , 36 , 69 ]. As \(\:{OH}^{-}\) continues to affect the bacteria, the cell membrane loses integrity, resulting in lysis and ultimately bacterial death. This process is especially effective against bacteria that are not adaptive to high alkaline environments, e.g. E. coli, P. aeruginosa , and S. aureus [ 34 , 36 , 69 ]. In addition, \(\:{Ca}^{2+}\) ions and extracellular DNA (eDNA) promote bacterial aggregation and biofilm formation [ 70 ]. \(\:{Ca}^{2+}\) released through the \(\:{CaCO}_{3}\) shell plays an important role in bridging the negatively charged bacterial surfaces, particularly in gram-positive and gram-negative bacteria [ 70 , 71 ]. The negative charges on these surfaces typically arise from components such as lipopolysaccharides (LPS) or teichoic acids in gram-positive bacteria [ 67 , 72 ]. These charged molecules repel each other, but \(\:{Ca}^{2+}\) can bridge these negative charges, leading to surface charge neutralization and promoting bacterial aggregation and biofilm formation [ 70 , 71 ]. This ionic interaction is strongest when bacterial cell wall components, e.g. carboxyl groups, bond with \(\:{Ca}^{2+}\) . In biofilm-forming bacteria, \(\:{Ca}^{2+}\) ions stabilize the biofilm matrix by neutralizing the charge repulsion between bacterial cells. Neutralized bacteria stick together and to surfaces, enhancing the structural integrity of biofilms [ 70 ]. These interactions are thermodynamically favorable, with the binding of \(\:{Ca}^{2+}\) to eDNA strengthening the biofilm structural integrity, making it more resistant to physical stresses and antibiotics [ 70 ]. Nevertheless, once bacteria adhere to the CSCC surface, \(\:{OH}^{-}\) ions from the \(\:{Ca\left(OH\right)}_{2}\) core diffuse through the porous calcium carbonate shell, creating a highly alkaline environment around the bacterial cells. Hydrophobic regions on bacterial surfaces and CSCC particles can also promote hydrophobic interactions, further stabilizing adhesion [ 57 ]. Additionally, physical features of the CSCC surface, such as roughness and porosity, increase the contact points for bacterial cells, trap bacteria in surface irregularities, and make detachment less likely [ 57 ]. Core-shell metal and metal oxide particles, such as \(\:Ag/{Fe}_{3}{O}_{4}\) [ 73 ], \(\:\left(Ni-Cu\right)@Ag\) NPs [ 74 ], \(\:Ag@{Ag}_{2}O\) nanostructures [ 75 ], magnetic \(\:{Fe}_{3}{O}_{4}\) @ \(\:{TiO}_{2}\) [ 76 ], \(\:Au-Ag\) NPs, have been tested as antibacterial particles [ 77 ]. These metal/metal oxide particles are associated with several drawbacks, including high-temperature synthesis [ 78 ], potential heavy metal pollution [ 79 ], and toxicity [ 80 ]. CSCC, on the other hand, is more environmentally friendly and has minimal side effects on human health and the environment. It is noted that both the core and the shell materials are food-grade. We envision the novel core-shell structure of the CSCC particle and its mechanism of hunting and killing bacteria as a promising solution for targeting pathogenic bacteria, particularly resistant strains. The antimicrobial resistance profile is currently being assessed and will be presented in a separate future study; where resistance development will be monitored through serial passage experiments that track changes in MIC or MBC over time [ 81 ]. 5. CONCLUSIONS In this work, we delineated the antimicrobial killing of mechanism of an environmentally friendly core-shell calcium-based biocidal particle. The biocidal efficacy of CSCC particles was assessed against gram-positive and gram-negative bacteria and compared with control samples of \(\:{Ca\left(OH\right)}_{2}\:\) and a \(\:{CaCO}_{3}\) . Using confocal microscopy, we were able to establish the adsorption/killing mechanism of the CSCC particles, wherein the bacteria first adsorb to the carbonate shell and are subsequently killed by the \(\:{Ca\left(OH\right)}_{2}\) core, likely through membrane disruption and aggregation. The minimum bactericidal content of CSCC was 2.5 mg/mL for both gram-positive and gram-negative bacteria, whereas double this dose of \(\:{Ca\left(OH\right)}_{2}\) was required to kill gram-positive bacteria. This observation highlights the importance of the killing mechanism of CSCC particles. We believe that this mechanism of binding and bacterial eradication has the potential to offer a safer and more environmentally friendly alternative. The accelerated aging study demonstrated the role of the carbonate shell in maintaining a biocidal activity of the CSCC particles equivalent to over 100 years at natural conditions. Funding Declaration This research was supported by Mitacs Accelerate (Award No. IT26596), and the Natural Sciences and Engineering Research Council of Canada (NSERC) Alliance Program (Award No. ALLRP 580333–22). Declarations Author Contribution M.M.A. conceived and designed the study and performed the microbiological validation experiments. N.N.D. and M.H. synthesized the particles and formulations, prepared particle samples, and drafted the sections related to particle synthesis and chemistry. G.R. conducted the bacterial aggregation characterization. M.M.A. and N.N.A. drafted the main manuscript. M.M.A. prepared all figures and performed data analysis. M.H. and I.A.L. supervised the experimental work and methodology and critically reviewed and edited the manuscript. All authors reviewed and approved the final manuscript. Acknowledgement The authors would like to thank Biosenta Inc. for sponsoring this project. Also, this work was supported by the Alberta Centre for Advanced Diagnostics (ACAD). Data Availability All data supporting the findings of this study are available within the paper and its Supplementary Information. References Muteeb, G., Rehman, M. T., Shahwan, M. & Aatif, M. Origin of Antibiotics and Antibiotic Resistance, and Their Impacts on Drug Development: A Narrative Review, (in eng). Pharmaceuticals (Basel) . 16 (11), 15 (2023). Podolsky, S. H. The evolving response to antibiotic resistance (1945–2018), Palgrave Communications , vol. 4, no. 1, p. 124, 2018/10/23 2018. Ventola, C. L. The antibiotic resistance crisis: part 1: causes and threats, (in eng), P t , vol. 40, no. 4, pp. 277 – 83, Apr (2015). Kubde, D., Badge, A. K., Ugemuge, S. & Shahu, S. Importance of Hospital Infection Control, (in eng), Cureus , vol. 15, no. 12, p. e50931, Dec (2023). Rutala, W. A. & Weber, D. J. Disinfection and Sterilization in Health Care Facilities: An Overview and Current Issues, (in eng), Infect Dis Clin North Am , vol. 30, no. 3, pp. 609 – 37, Sep 2016. Hua, Z. & Zhu, M. J. Unlocking the Hidden Threat: Impacts of Surface Defects on the Efficacy of Sanitizers Against Listeria monocytogenes Biofilms on Food-contact Surfaces in Tree Fruit Packing Facilities, Journal of Food Protection , vol. 87, no. 2, p. 100213, /02/01/ 2024. (2024). Salam, M. A. et al. Antimicrobial Resistance: A Growing Serious Threat for Global Public Health, (in eng), Healthcare (Basel) , vol. 11, no. 13, Jul 5 (2023). Gorguner, M., Aslan, S., Inandi, T. & Cakir, Z. Reactive airways dysfunction syndrome in housewives due to a bleach-hydrochloric acid mixture, (in eng). Inhal Toxicol , 16 , 2, pp. 87–91, Feb 2004. Sastre, J. et al. Airway response to chlorine inhalation (bleach) among cleaning workers with and without bronchial hyperresponsiveness. American J. Industrial Medicine , 54 , 4, pp. 293–299, 2011/04/01 2011. Clausen, P. A. et al. Chemicals inhaled from spray cleaning and disinfection products and their respiratory effects. A comprehensive review, International Journal of Hygiene and Environmental Health , vol. 229, p. 113592, /08/01/ 2020. (2020). Zander, Z. K. & Becker, M. L. Antimicrobial and Antifouling Strategies for Polymeric Medical Devices. ACS Macro Letters , 7 , 1, pp. 16–25, 2018/01/16 2018. Liang, Y., Liang, Y., Zhang, H. & Guo, B. Antibacterial biomaterials for skin wound dressing, (in eng). Asian J. Pharm. Sci. 17 (3), 353–384 (May 2022). Ciawi, Y. & Khoiruddin, K. Low-Cost Antibacterial Ceramic Water Filters for Decentralized Water Treatment: Advances and Practical Applications. ACS Omega , 9 , 11, pp. 12457–12477, 2024/03/19 2024. Swartjes, J. J. et al. Current Developments in Antimicrobial Surface Coatings for Biomedical Applications, (in eng). Curr. Med. Chem. 22 (18), 2116–2129 (2015). Gulati, R., Sharma, S. & Sharma, R. K. Antimicrobial textile: recent developments and functional perspective, (in eng), Polym Bull (Berl) , vol. 79, no. 8, pp. 5747–5771, (2022). Malhotra, B., Keshwani, A. & Kharkwal, H. Antimicrobial food packaging: potential and pitfalls, (in English). Frontiers Microbiol. Rev. 6 , 2015-June-16 2015. Gold, K., Slay, B., Knackstedt, M. & Gaharwar, A. K. Antimicrobial Activity of Metal and Metal-Oxide Based Nanoparticles. Advanced Therapeutics , 1 , 3, p. 1700033, 2018/07/01 2018. Jiang, H., Li, L., Li, Z. & Chu, X. Metal-based nanoparticles in antibacterial application in biomedical field: Current development and potential mechanisms. Biomedical Microdevices , 26 , 1, p. 12, 2024/01/23 2024. Maleki Dizaj, S., Mennati, A., Jafari, S., Khezri, K. & Adibkia, K. Antimicrobial activity of carbon-based nanoparticles, (in eng). Adv. Pharm. Bull. 5 (1), 19–23 (Mar 2015). Raul, P. K. et al. Carbon Nanostructures As Antibacterials and Active Food-Packaging Materials: A Review. ACS Omega , 7 , 14, pp. 11555–11559, 2022/04/12 2022. Sarojini, S. & Jayaram, S. An Impact of Antibacterial Efficacy of Metal Oxide Nanoparticles: A Promise for Future, in Bio-manufactured Nanomaterials: Perspectives and Promotion, (ed Pal, K.) Cham: Springer International Publishing, 393–406. (2021). Rawat, N. et al. Nanobiomaterials: exploring mechanistic roles in combating microbial infections and cancer. Discover Nano , 18 , 1, p. 158, 2023/12/20 2023. Zheng, S. et al. Implication of Surface Properties, Bacterial Motility, and Hydrodynamic Conditions on Bacterial Surface Sensing and Their Initial Adhesion, (in eng). Front. Bioeng. Biotechnol. 9 , 643722 (2021). Nastulyavichus, A. et al. In Vitro Destruction of Pathogenic Bacterial Biofilms by Bactericidal Metallic Nanoparticles via Laser-Induced Forward Transfer, (in eng), Nanomaterials (Basel) , vol. 10, no. 11, Nov 15 (2020). Niño-Martínez, N. et al. Molecular Mechanisms of Bacterial Resistance to Metal and Metal Oxide Nanoparticles. Int. J. Mol. Sci. 20 (11), 2808 (2019). Zhang, K. et al. Developing a New Generation of Therapeutic Dental Polymers to Inhibit Oral Biofilms and Protect Teeth, (in eng), Materials (Basel) , vol. 11, no. 9, Sep 17 (2018). Chi, M. et al. Novel Bioactive and Therapeutic Dental Polymeric Materials to Inhibit Periodontal Pathogens and Biofilms, (in eng), Int J Mol Sci , vol. 20, no. 2, Jan 11 (2019). Epperlein, N. et al. Influence of femtosecond laser produced nanostructures on biofilm growth on steel, Applied Surface Science , vol. 418, pp. 420–424, /10/01/ 2017. (2017). Luo, X. et al. Biocompatible nano-ripples structured surfaces induced by femtosecond laser to rebel bacterial colonization and biofilm formation, Optics & Laser Technology , vol. 124, p. 105973, /04/01/ 2020. (2020). Reed, J. H. et al. Ultrascalable Multifunctional Nanoengineered Copper and Aluminum for Antiadhesion and Bactericidal Applications, (in eng). ACS Appl. Bio Mater , 2 , 7, pp. 2726–2737, Jul 15 2019. Wandiyanto, J. V. et al. Outsmarting superbugs: bactericidal activity of nanostructured titanium surfaces against methicillin- and gentamicin-resistant Staphylococcus aureus ATCC 33592. J. Mater. Chem. B . 7 (28), 4424–4431. 10.1039/C9TB00102F (2019). Chan, W., Chowdhury, N. R., Sharma, G., Zilm, P. & Rossi-Fedele, G. Comparison of the Biocidal Efficacy of Sodium Dichloroisocyanurate and Calcium Hydroxide as Intracanal Medicaments over a 7-Day Contact Time: An Ex Vivo Study, Journal of Endodontics , vol. 46, no. 9, pp. 1273–1278, /09/01/ 2020. (2020). Rogers M. B. C. G. G. L (Biocidal Coating Composition, 2001). Athanassiadis, B., Abbott, P. V. & Walsh, L. J. The use of calcium hydroxide, antibiotics and biocides as antimicrobial medicaments in endodontics. Australian Dent. Journal , 52 , s1, pp. S64-S82, 2007/03/01 2007. Gomes, I. C., Chevitarese, O., de Almeida, N. S., Salles, M. R. & Gomes, G. C. Diffusion of calcium through dentin, Journal of Endodontics , vol. 22, no. 11, pp. 590–595, /11/01/ 1996. (1996). Kim, D. & Kim, E. Antimicrobial effect of calcium hydroxide as an intracanal medicament in root canal treatment: a literature review - Part I. In vitro studies, (in eng), Restor Dent Endod , vol. 39, no. 4, pp. 241 – 52, Nov (2014). Darwish, N., Ashani, M., Lewis, I. A. & Husein, M. M. Controlled carbonation of Ca(OH)2 surface and its application as an antibacterial particle, Colloids and Surfaces A: Physicochemical and Engineering Aspects , vol. 682, p. 132852, /02/05/ 2024. (2024). Ferreira, A. M., Vikulina, A. S. & Volodkin, D. CaCO3 crystals as versatile carriers for controlled delivery of antimicrobials, Journal of Controlled Release , vol. 328, pp. 470–489, /12/10/ 2020. (2020). Fadia, P. et al. Calcium carbonate nano- and microparticles: synthesis methods and biological applications, (in eng), 3 Biotech , vol. 11, no. 11, p. 457, Nov (2021). Darwish, N., Ashani, M., Mehairi, A. & Lewis, I. A. and M. M. Husein Synthesis of uniform core-shell calcium hydroxide-calcium carbonate biocidal particles via encapsulation into dry ice, The Canadian Journal of Chemical Engineering (submitted Nov responses to reviewers comments has been submitted). (2024). Dodge, R. & Ludington, W. B. Fast Colony Forming Unit Counting in 96-Well Plate Format Applied to the Drosophila Microbiome, (in eng), J Vis Exp , no. 191, Jan 13 (2023). Alsante, K. M. et al. The role of degradant profiling in active pharmaceutical ingredients and drug products, Advanced Drug Delivery Reviews , vol. 59, no. 1, pp. 29–37, /01/10/ 2007. (2007). Blessy, M., Patel, R. D., Prajapati, P. N. & Agrawal, Y. K. Development of forced degradation and stability indicating studies of drugs—A review, Journal of Pharmaceutical Analysis , vol. 4, no. 3, pp. 159–165, /06/01/ 2014. (2014). Darwish, N., Ashani, M. M., Mehairi, A., Lewis, I. A. & Husein, M. M. Synthesis of uniform core-shell calcium hydroxide-calcium carbonate biocidal particles via encapsulation into dry ice, The Canadian Journal of Chemical Engineering , vol. n/a, no. n /a Secor, P. R., Michaels, L. A., Bublitz, D. C., Jennings, L. K. & Singh, P. K. The Depletion Mechanism Actuates Bacterial Aggregation by Exopolysaccharides and Determines Species Distribution & Composition in Bacterial Aggregates, (in English), Frontiers in Cellular and Infection Microbiology , Original Research vol. 12 , 2022-June-16 2022. Kapoor, G., Saigal, S. & Elongavan, A. Action and resistance mechanisms of antibiotics: A guide for clinicians, (in eng). J Anaesthesiol. Clin. Pharmacol , 33 , 3, pp. 300–305, Jul-Sep 2017. Halawa, E. M. et al. Antibiotic action and resistance: updated review of mechanisms, spread, influencing factors, and alternative approaches for combating resistance. (in English) Frontiers Pharmacol. Rev. 14 , 2024-January-12 2024. Benmamoun, Z., Chandar, P., Jankolovits, J. & Ducker, W. A. Time-Resolved Killing of Individual Bacterial Cells by a Polycationic Antimicrobial Polymer. ACS Biomaterials Sci. & Engineering , 10 , 5, pp. 3029–3040, 2024/05/13 2024. Ivanova, E. P. et al. Natural bactericidal surfaces: mechanical rupture of Pseudomonas aeruginosa cells by cicada wings, (in eng), Small , vol. 8, no. 16, pp. 2489-94, Aug 20 (2012). Sochacki, K. A., Barns, K. J., Bucki, R. & Weisshaar, J. C. Real-time attack on single Escherichia coli cells by the human antimicrobial peptide LL-37, (in eng). Proc Natl. Acad. Sci. U S A , 108 , 16, pp. E77-81, Apr 19 2011. Benmamoun, Z., Chandar, P., Jankolovits, J. & Ducker, W. A. Time-Resolved Killing of Individual Bacterial Cells by a Polycationic Antimicrobial Polymer, (in eng). ACS Biomater. Sci. Eng , 10 , 5, pp. 3029–3040, May 13 2024. Barns, K. J. & Weisshaar, J. C. Single-cell, time-resolved study of the effects of the antimicrobial peptide alamethicin on Bacillus subtilis, (in eng). Biochim Biophys. Acta , 1858 , 4, pp. 725 – 32, Apr 2016. Ma, J., Sun, Y., Gleichauf, K., Lou, J. & Li, Q. Nanostructure on taro leaves resists fouling by colloids and bacteria under submerged conditions, (in eng), Langmuir , vol. 27, no. 16, pp. 10035-40, Aug 16 (2011). Truong, V. K. et al. The influence of nano-scale surface roughness on bacterial adhesion to ultrafine-grained titanium, (in eng), Biomaterials , vol. 31, no. 13, pp. 3674-83, May (2010). Mohammadi, Z., Shalavi, S. & Yazdizadeh, M. Antimicrobial activity of calcium hydroxide in endodontics: a review, (in eng), Chonnam Med J , vol. 48, no. 3, pp. 133 – 40, Dec (2012). Stewart, P. S. & William Costerton, J. Antibiotic resistance of bacteria in biofilms, The Lancet , vol. 358, no. 9276, pp. 135–138, /07/14/ 2001. (2001). Tuson, H. H. & Weibel, D. B. Bacteria–surface interactions, Soft Matter , 10.1039/C3SM27705D vol. 9, no. 17, pp. 4368–4380, (2013). Marshall, K. C., Stout, R. & Mitchell, R. Mechanism of the Initial Events in the Sorption of Marine Bacteria to Surfaces, Microbiology , vol. 68, no. 3, pp. 337–348, (1971). Kumar, C. G. & Anand, S. K. Significance of microbial biofilms in food industry: a review, (in eng). Int J. Food Microbiol , 42 , no. 1–2, pp. 9–27, Jun 30 1998. Palmer, J., Flint, S. & Brooks, J. Bacterial cell attachment, the beginning of a biofilm, (in eng), J Ind Microbiol Biotechnol , vol. 34, no. 9, pp. 577 – 88, Sep (2007). Gilbert, P., Evans, D. J., Evans, E., Duguid, I. G. & Brown, M. R. Surface characteristics and adhesion of Escherichia coli and Staphylococcus epidermidis, (in eng), J Appl Bacteriol , vol. 71, no. 1, pp. 72 – 7, Jul (1991). Carpentier, B. & Cerf, O. Biofilms and their consequences, with particular reference to hygiene in the food industry, (in eng). J. Appl. Bacteriol. 75 (6), 499–511 (Dec 1993). van Loosdrecht, M. C., Lyklema, J., Norde, W., Schraa, G. & Zehnder, A. J. Electrophoretic mobility and hydrophobicity as a measured to predict the initial steps of bacterial adhesion, (in eng), Appl Environ Microbiol , vol. 53, no. 8, pp. 1898 – 901, Aug (1987). Jullien, C., Bénézech, T., Carpentier, B., Lebret, V. & Faille, C. Identification of surface characteristics relevant to the hygienic status of stainless steel for the food industry, Journal of Food Engineering , vol. 56, no. 1, pp. 77–87, /01/01/ 2003. (2003). Poortinga, A. T., Bos, R. & Busscher, H. J. Charge transfer during staphylococcal adhesion to TiNOX coatings with different specific resistivity, (in eng), Biophys Chem , vol. 91, no. 3, pp. 273-9, Jul 24 (2001). Busscher, H. J. & Weerkamp, A. H. Specific and non-specific interactions in bacterial adhesion to solid substrata. FEMS Microbiol. Rev. 3 (2), 165–173 (1987). Poxton, I. R. Chapter 5 - Teichoic Acids, Lipoteichoic Acids and Other Secondary Cell Wall and Membrane Polysaccharides of Gram-Positive Bacteria, in Molecular Medical Microbiology (Second Edition) , Y.-W. Tang, M. Sussman, D. Liu, I. Poxton, and J. Schwartzman, Eds. Boston: Academic Press, pp. 91–103. (2015). Poortinga, A. T., Bos, R., Norde, W. & Busscher, H. J. Electric double layer interactions in bacterial adhesion to surfaces, Surface Science Reports , vol. 47, no. 1, pp. 1–32, /06/01/ 2002. (2002). Gomes, I. C., Chevitarese, O., de Almeida, N. S., Salles, M. R. & Gomes, G. C. Diffusion of calcium through dentin, (in eng). J. Endod . 22 (11), 590–595 (Nov 1996). Das, T. et al. Influence of calcium in extracellular DNA mediated bacterial aggregation and biofilm formation, (in eng), PLoS One , vol. 9, no. 3, p. e91935, (2014). Thomas, K. J. & Rice, C. V. 3rd and Revised model of calcium and magnesium binding to the bacterial cell wall, (in eng), Biometals , vol. 27, no. 6, pp. 1361-70, Dec (2014). Wang, X. & Quinn, P. J. Endotoxins: lipopolysaccharides of gram-negative bacteria, (in eng). Subcell. Biochem. 53 , 3–25 (2010). Sharaf, E. M. et al. Synergistic antibacterial activity of compact silver/magnetite core-shell nanoparticles core shell against Gram-negative foodborne pathogens, (in English). Frontiers Microbiol. Original Res. 13 , 2022-September-02 2022. Ahmed, A. A. A., Aldeen, T. S., Al-Aqil, S. A., Alaizeri, Z. M. & Megahed, S. Synthesis of Trimetallic (Ni-Cu)@Ag Core@Shell Nanoparticles without Stabilizing Materials for Antibacterial Applications. ACS Omega , 7 , 42, pp. 37340–37350, 2022/10/25 2022. Elyamny, S., Eltarahony, M., Abu-Serie, M., Nabil, M. M. & Kashyout, A. E. H. B. One-pot fabrication of Ag @Ag2O core–shell nanostructures for biosafe antimicrobial and antibiofilm applications. Scientific Reports , 11 , 1, p. 22543, 2021/11/19 2021. Rani, N. & Dehiya, B. S. Magnetic core-shell Fe3O4@TiO2 nanocomposites for broad spectrum antibacterial applications. IET Nanobiotechnology , 15 , 3, pp. 301–308, 2021/05/01 2021. Fan, X., Yahia, L. H. & Sacher, E. Antimicrobial Properties of the Ag, Cu Nanoparticle System, Biology , vol. 10, no. 2, p. 137, (2021). Huynh, K. H. et al. Synthesis, Properties, and Biological Applications of Metallic Alloy Nanoparticles. Int. J. Mol. Sci. 21 (14), 5174 (2020). Tchounwou, P. B., Yedjou, C. G., Patlolla, A. K. & Sutton, D. J. Heavy metal toxicity and the environment, (in eng), Exp Suppl, vol. 101, pp. 133 – 64, (2012). Hama Aziz, K. H. et al. Heavy metal pollution in the aquatic environment: efficient and low-cost removal approaches to eliminate their toxicity: a review, (in eng), RSC Adv , vol. 13, no. 26, pp. 17595–17610, Jun 9 2023. Belkin, N. L. Bacterial penetration vis-á-vis lint generation, (in eng). J. Hosp. Infect. 52 (4), 315–317 (Dec 2002). Additional Declarations No competing interests reported. Supplementary Files SupplementaryInfo.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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09:21:50","extension":"png","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":279783,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8378570/v1/dbad66c4efea895afc175372.png"},{"id":100664727,"identity":"0d6287b7-41a9-475f-8c71-bb3ec5635cec","added_by":"auto","created_at":"2026-01-20 09:21:38","extension":"png","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":253051,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8378570/v1/53cf530c1fd12f91ba9bb4ab.png"},{"id":100664921,"identity":"2c439f0b-8715-42c7-851f-db69f5f98dbb","added_by":"auto","created_at":"2026-01-20 09:22:47","extension":"xml","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":151980,"visible":true,"origin":"","legend":"","description":"","filename":"d0a7e3d53f2744fcbbe7be2e5092d94f1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8378570/v1/11067d117eda0a8e32ef485d.xml"},{"id":100664841,"identity":"8a55a1c8-79a3-471b-8016-ca5a7d8b47c6","added_by":"auto","created_at":"2026-01-20 09:22:11","extension":"html","order_by":30,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":178116,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8378570/v1/4ba73635eb2ead7abf9cc172.html"},{"id":100665077,"identity":"d3e1fbdb-61cd-4267-9866-ad9a167a52b0","added_by":"auto","created_at":"2026-01-20 09:23:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":589817,"visible":true,"origin":"","legend":"\u003cp\u003eOptical imaging confocal microscope including fluorescence, reflection and overlay for E.coli GFP: (a-c) Positive control (no treatment); (d-f) 20 mg/mL \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;; (g-i) 20 mg/mL \u0026nbsp;\u0026nbsp;; (j-l) 20 mg/mL CSCC.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8378570/v1/58fc79bfaa5116e5e98b4b9b.png"},{"id":100664729,"identity":"4c661d74-4149-46af-bb9f-277fc66a48c7","added_by":"auto","created_at":"2026-01-20 09:21:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":643919,"visible":true,"origin":"","legend":"\u003cp\u003eE.coli bacteria aggregation in the presence of particles 20 mg/ml (a) Positive Control (no particles), (b) \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;, \u0026nbsp;\u0026nbsp;,. (d) CSCC, (e) \u0026nbsp;CSCC particles with only media.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8378570/v1/838df21b80bd4d47f8d9bac1.png"},{"id":100664731,"identity":"73e85e18-5fe9-42d2-b667-cb83c38622fb","added_by":"auto","created_at":"2026-01-20 09:21:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":59670,"visible":true,"origin":"","legend":"\u003cp\u003eAbsorbance of dissolved crystal violet at 595 nm used to quantify the aggregation of E. coli, S. aureus, and MRSA in the presence of 20 mg/mL of various treatments, including the positive control (no treatment), \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;, \u0026nbsp;\u0026nbsp;, and CSCC.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8378570/v1/9c7a4d3be2475bf21bc14e91.png"},{"id":100664874,"identity":"cc61205b-f684-4ff9-ab46-367ce9e8271f","added_by":"auto","created_at":"2026-01-20 09:22:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":204636,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8378570/v1/6ab7e13d2a60e209deba38c6.png"},{"id":100664837,"identity":"6448b3e4-042f-40cd-8c28-5db0b73c32e2","added_by":"auto","created_at":"2026-01-20 09:22:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":643298,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8378570/v1/05e0410ab18323aaf7cb7aea.png"},{"id":101943039,"identity":"18614f31-7553-44ec-8ed1-c846d7e137d1","added_by":"auto","created_at":"2026-02-05 09:39:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3149686,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8378570/v1/af38adb9-176e-4ccd-adbc-f08902188fa8.pdf"},{"id":100664807,"identity":"1b83053e-ed03-489f-b329-ab123cc39d68","added_by":"auto","created_at":"2026-01-20 09:22:03","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":3744921,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInfo.docx","url":"https://assets-eu.researchsquare.com/files/rs-8378570/v1/00dc37f0499d88a53fed637b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bactericidal activity of novel calcium-based core-shell particles and its mechanism of action ","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eThe escalating global issue of antimicrobial-resistant bacteria mandates the development of effective antibacterial products and innovative approaches to microbial control [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. There is a critical need for novel long-acting biocidal agents that can be used on surfaces, especially in healthcare, food processing, and other high-contact environments [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Despite the widespread nature of this challenge, the range of effective antimicrobial tools available for use in these settings remains surprisingly limited [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. For instance, traditional chemical sterilizers, such as bleach and hydrogen peroxide, are effective in the short term but pose health risks (e.g., respiratory issues, skin irritation) and environmental harm [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. As a result, there is an unmet need for advanced antimicrobial agents that not only exhibit long-term persistent activity but are also safe for use on a variety of surfaces in critical environments [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo address this challenge, innovative antibacterial agents have emerged for different applications including medical devices [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], wound dressings [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], water purification [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], surface coating [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], textiles [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and food packaging [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In addition, a wide range of materials and particles including metal oxide nanoparticles (NPs) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], metal NPs [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], and carbon nanomaterials [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], have been developed with biocidal activity owing to their unique physicochemical properties, including their small size [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. A combination of physical and chemical bactericidal properties has also been explored. For example, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:Si\\)\u003c/span\u003e\u003c/span\u003e nano-ripples coated with \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:Ag\\)\u003c/span\u003e\u003c/span\u003e or \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{TiO}_{2}\\)\u003c/span\u003e\u003c/span\u003e NPs damaged bacterial DNA and prevented biofilm formation [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Biocidal surfaces can kill bacteria and prevent biofilm formation, however the continuous release of antibacterial agents may lead to bacteria resistance and reduce their long-term effectiveness [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. To address this, alternative strategies have been proposed, including surfaces that prevent early bacterial adhesion [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. For example, laser-induced periodic surface structures (LIPSS) have been used to create nanostructures on surfaces to inhibit bacteria attachment. Studies show that LIPSS reduced \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE.coli\u003c/em\u003e) population [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Additionally, surface that physically disrupts bacterial membranes have been tested [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. For instance, nanostructured titanium surfaces fabricated through hydrothermal etching were assessed in killing both methicillin- and gentamicin-susceptible and -resistant \u003cem\u003eS. aureus\u003c/em\u003e strains [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The nanostructured surfaces effectively killed both strains, hence demonstrating that the physical disruption mechanism is equally effective against susceptible and resistant bacteria [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Nevertheless, antimicrobial nanostructured surfaces are still in their infancy [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Generally, there is still a scarcity of effective, environmentally friendly, and biodegradable antibacterial agents that could be incorporated into a broad range of materials and applications.\u003c/p\u003e \u003cp\u003eTo address this gap, we present a new core-shell \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e\u0026ndash;\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{CaCO}_{3}\\)\u003c/span\u003e\u003c/span\u003e (CSCC) antibacterial particles and explore their mechanism of action against gram-positive and gram-negative bacteria. \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003eis strongly bactericidal, fungicidal, and virucidal owing to \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e ability to maintain high alkalinity (pH 11\u0026ndash;13) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Chemically, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{OH}^{-}\\)\u003c/span\u003e\u003c/span\u003e damages the microbial membranes, disrupts the metabolic processes, and inhibits DNA replication [\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Physically, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{OH}^{-}\\)\u003c/span\u003e\u003c/span\u003e deprives the microorganisms of essential nutrients and the space needed to multiply [\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In this context, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e acts as a protective barrier that prevents bacterial invasion and further growth by removing available substrates and limiting the area for microbial proliferation [\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In contrast, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{CaCO}_{3}\\)\u003c/span\u003e\u003c/span\u003e is generally chemically inert with no inherent antimicrobial properties [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Nevertheless, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{CaCO}_{3}\\)\u003c/span\u003e\u003c/span\u003e has been used as a carrier for antimicrobial agents, such as \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ag}^{+}\\)\u003c/span\u003e\u003c/span\u003e or biocidal compounds, improving their delivery to microbial targets [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Different approaches were employed to incorporate materials of interest into \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{CaCO}_{3}\\)\u003c/span\u003e\u003c/span\u003e, including adsorption, coprecipitation, and infiltration [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The slow release of these agents from \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{CaCO}_{3}\\)\u003c/span\u003e\u003c/span\u003e matrix ensures a prolonged antimicrobial effect [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Moreover, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{CaCO}_{3}\\:\\)\u003c/span\u003e\u003c/span\u003ecan influence the adhesion of bacterial cells onto surfaces by adsorbing onto bacteria [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eTechnical grade calcium hydroxide, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e (dp\u0026thinsp;=\u0026thinsp;44 \u0026micro;m, 1.3 wt% moisture, Atlantic Equipment Engineering Inc., USA), was used as a reactant. Liquid carbon dioxide, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{CO}_{2}\\)\u003c/span\u003e\u003c/span\u003e (\u0026gt;\u0026thinsp;99% pure, Air Liquide, Canada) was supplied from a pressurized cylinder at 5.9 MPa using a siphon. The BD BBL\u0026trade; Mueller Hinton (MH) II Broth (Cation-Adjusted) was purchased from BD Biosciences (VWR, Canada). The medium was prepared based on the manufacturer guidelines and autoclaved at 121.5 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:℃\\)\u003c/span\u003e\u003c/span\u003e for 20 min using a Primus Sterilizer (USA). The MH medium is cooled to room temperature, then stored at 4 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:℃\\)\u003c/span\u003e\u003c/span\u003e. Similarly, the MH agar is prepared, autoclaved, poured on plates, and then stored at 4 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:℃\\)\u003c/span\u003e\u003c/span\u003e. A crystal violet, 1% (aqueous solution) dye in a solution of ethanol-phenol-methanol-water, was purchased from Harleco-EMD.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Core-Shell antibacterial particle preparation\u003c/h2\u003e \u003cp\u003eDetailed CSCC preparation protocol is presented in our previous study [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In brief, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e was mixed with liquid \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{CO}_{2}\\)\u003c/span\u003e\u003c/span\u003e at 5.9 MPa, 21\u0026thinsp;\u0026plusmn;\u0026thinsp;1 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:℃\\)\u003c/span\u003e\u003c/span\u003e and 1300 rpm. After 5 min, the pressure was rapidly reduced inducing adiabatic flashing of liquid \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{CO}_{2}\\)\u003c/span\u003e\u003c/span\u003e into dry ice and gaseous \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{CO}_{2}\\)\u003c/span\u003e\u003c/span\u003e. Dry ice encapsulated the\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e particles, which in turn carbonated \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e surface and preserved particle aggregation. The dry ice was sublimed to control the coating thickness, producing a 45 wt% \u0026minus;\u0026thinsp;55 wt% core\u0026ndash;shell structure [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. This partial carbonation was confirmed using XRD, acid-base titration, and thermogravimetry with calorimetry, while the surface coating was analyzed using FTIR spectroscopy and TEM microtomy [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Bacterial sample preparation\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eStrains of \u003cem\u003eEscherichia coli\u003c/em\u003e, ATCC 25922, \u003cem\u003eE.coli\u003c/em\u003e 25922GFP, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, ATCC 27853, Methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA) ATCC 43300 and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, ATCC 25923 were purchased from Cedarlane lab and stored at \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:-80\\:℃\\)\u003c/span\u003e\u003c/span\u003e. These bacteria were inoculated overnight in 10 mL MH media in a Heracell\u0026trade; VIOS 250i \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{CO}_{2}\\)\u003c/span\u003e\u003c/span\u003e Incubator (Thermo Fisher, Waltham, MA, USA) at 36.5 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:℃\\)\u003c/span\u003e\u003c/span\u003e, with an atmosphere of 5% \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{CO}_{2}\\)\u003c/span\u003e\u003c/span\u003e and 17.5% \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{O}_{2}\\)\u003c/span\u003e\u003c/span\u003e. Thermo NanoDrop\u0026trade; OneC Spectrophotometer (USA) was used to measure the optical density (OD) of bacterial stock solutions at 600 nm under absorbance mode. These stock solutions were further diluted to achieve an OD600 value of 0.10, followed by two 10-fold dilutions (100x) to obtain a final OD600 value of 0.001. Different masses of CSCC, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{CaCO}_{3}\\)\u003c/span\u003e\u003c/span\u003e, and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e were mixed with MH medium and introduced to the plate to achieve the desired particle content.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Bactericidal assays\u003c/h2\u003e \u003cp\u003eA hybrid of broth dilution and spot plating methods was employed to determine the minimum bactericidal concentration (MBC) of the CSCC, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{CaCO}_{3}\\)\u003c/span\u003e\u003c/span\u003e particles. A range of particle content: 0.625, 1.25, 2.5, 5, 10, 25, 50, and 100 mg/mL was used. To ensure a uniform suspension of particles into MH media, a vortex mixer was used for 10 min, followed by vigorous shaking. After preparation, the uniform suspension of particles into MH media was transferred into a 96-well plate. A volume of 20 \u0026micro;L of a bacterial suspension with an optical density (OD) of 0.001 (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:1.45\\:\\times\\:{10}^{5}\\:\\)\u003c/span\u003e\u003c/span\u003eand \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:7.9\\:\\times\\:{10}^{5}\\:\\)\u003c/span\u003e\u003c/span\u003e Colony Forming Unit, CFU for \u003cem\u003eS. aureus\u003c/em\u003e or \u003cem\u003eP. aeruginosa\u003c/em\u003e, respectively) was added to each well. The bacterial strains used in the MBC study included \u003cem\u003eP. aeruginosa\u003c/em\u003e (gram-negative) and \u003cem\u003eS. aureus\u003c/em\u003e (gram-positive). The 96-well plate was incubated in a Heracell\u0026trade; VIOS 250i \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{CO}_{2}\\)\u003c/span\u003e\u003c/span\u003e incubator (Thermo Fisher, Waltham, MA, USA) at 36.5 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:℃\\)\u003c/span\u003e\u003c/span\u003e, with an atmosphere of 5% \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{CO}_{2}\\)\u003c/span\u003e\u003c/span\u003e and 17.5% \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{O}_{2}\\)\u003c/span\u003e\u003c/span\u003e for 3 h. Following incubation, 10 \u0026micro;L of each sample was transferred to MH agar plates using the spot plating technique [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The agar plates were then cultured for 20 h to allow for bacterial growth and MBC determination.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Bacteria aggregation assay\u003c/h2\u003e \u003cp\u003e \u003cem\u003eE.coli\u003c/em\u003e was cultured overnight in MH media, then centrifuged at 4300 rpm for 5 min, and resuspended in autoclaved water to achieve an OD of 10. A 100 \u0026micro;L aliquot of particle suspension, prepared by dispersing particles in MH media at a concentration of 50 mg/mL, was added to each well of a 96-well plate. Subsequently, 100 \u0026micro;L of bacterial suspension was introduced into each well. The plate was then incubated for 24 hours at 23\u0026deg;C (room temperature). The supernatant was carefully extracted, and the well was rinsed 3 times with water. A 100 \u0026micro;L of the crystal violet solution, prepared by mixing 3 mL of crystal violet 1% solution with 3 mL of water, was added to the well and incubated at 23 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:℃\\)\u003c/span\u003e\u003c/span\u003e for 15 min. The supernatant was discarded, and the well was washed with water 4\u0026ndash;5 times, or until no residual crystal violet was observed in the control well. Finally, 100 \u0026micro;L of 95% ethanol was added to each well, and the absorbance was measured at 595 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Confocal laser-scanning microscopy (CLSM)\u003c/h2\u003e \u003cp\u003eOptical imaging using confocal laser-scanning microscopy (CLSM, Carl Zeiss, G\u0026ouml;ttingen, Germany) was collected for three mixtures of CSCC, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{CaCO}_{3}\\)\u003c/span\u003e\u003c/span\u003e with \u003cem\u003eE.coli\u003c/em\u003e GFP suspension. A 1 mL of a 20 mg/mL particle suspension in MH medium was combined with 1 mL of \u003cem\u003eE. coli\u003c/em\u003e GFP culture, adjusted to an OD of 0.4. The mixture was incubated at 23\u0026deg;C (room temperature) for 30 minutes. Subsequently, 100 \u0026micro;L of the sample was placed on a round glass coverslip for microscopic imaging using an inverted LSM510 CLSM. The images were made in three modes: fluorescence to track GFP-labeled bacteria, reflection to capture the positions of the particles, and overlay to combine both imaging modes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Accelerated aging protocol for long-term stability assessment of CSCC particles\u003c/h2\u003e \u003cp\u003eTo assess the long-term physicochemical stability and antimicrobial efficacy of the CSCC particles, an accelerated thermal aging study was performed under tightly controlled conditions. Freshly synthesized CSCC particles, alongside analytical-grade \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e as a comparative control, were each evenly distributed in separate inert stainless-steel trays. These trays were placed in a fully enclosed convection dry oven maintained at 150\u0026deg;C for a continuous period of 35 days. The oven chamber (BINDER ED 56) remained sealed throughout the study to ensure thermal homogeneity and prevent environmental fluctuations such as moisture uptake, which could otherwise alter particle morphology or surface chemistry. This accelerated aging approach was modeled using the Arrhenius equation, which describes the temperature dependence of reaction kinetics and degradation processes [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. This thermal stress approach enables a comparative evaluation of the structural and antimicrobial integrity of CSCC versus \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e, providing insight into their durability\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cp\u003eDetailed CSCC particle characterization is included in our earlier publication [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In brief, CSCC mean particle size ranged between 20\u0026ndash;170 nm with aggregates ranging 0.2\u0026ndash;0.6 \u0026micro;m.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Mechanism of action\u003c/h2\u003e \u003cp\u003eOptical imaging (CLSM) revealed the morphology of the product of the interaction between the different particles and the bacteria. The positive control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(a-c)), i.e. in the absence of any treatment, showed visible rod-shaped \u003cem\u003eE.coli\u003c/em\u003e GFP exhibiting distinct green fluorescence, suggesting complete viability. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(d-f) shows intense GFP fluorescent signals suggesting bacterial viability with \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{CaCO}_{3}\\)\u003c/span\u003e\u003c/span\u003e particles and some bacterial binding. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(g-i) depicts \u003cem\u003eE.coli\u003c/em\u003e GFP onto \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e with faded fluorescence, suggesting a loss of viability. Lastly, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (j-l) captures \u003cem\u003eE.coli\u003c/em\u003e GFP treated with CSCC particles. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (j-l) shows a substantial degree of bacterial binding to CSCC particles, leading to the formation of a chain-like structure. Moreover, \u003cem\u003eE. coli\u003c/em\u003e GFP fluorescence was notably reduced, suggesting substantial biocidal activity.\u003c/p\u003e \u003cp\u003eTo further validate the biocidal attribute of the particles, samples were collected post-experiment and re-cultured on agar plates. The results in Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e confirm that the bacteria remained viable following \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{CaCO}_{3}\\)\u003c/span\u003e\u003c/span\u003e treatment, in contrast to CSCC and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e treatments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Bacteria aggregation\u003c/h2\u003e \u003cp\u003eThe crystal violet assay was performed to evaluate bacterial behavior under different experimental conditions. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(a), no bacterial aggregation was observed in the positive control. In contrast, minimal aggregation occurred in the presence of 20 mg/mL \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{CaCO}_{3}\\)\u003c/span\u003e\u003c/span\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(b)), whereas extensive aggregation was evident in the presence of 20 mg/mL \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e and CSCC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(c),(d)), with a substantially higher extent of aggregation for CSCC. To exclude the possibility of artifacts caused by CSCC particles, a control sample in absence of bacteria was included (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(e)). The absence of aggregates in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(e) confirms that the aggregates observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(d) originated from interactions between bacteria and CSCC particles. These results suggest that the biocidal activity of CSCC and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e is likely mediated through aggregation-driven mechanisms. It is noteworthy that bacterial aggregation can enhance bacterial survival by forming protective clusters that shield cells from environmental stresses while facilitating nutrient exchange and intercellular communication [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCrystal violet, a cationic dye, binds preferentially to negatively charged bacterial components. In the absence of bacteria, minimal staining is expected for CSCC or \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e particles because their positively charged surfaces repel the dye. \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{CaCO}_{3}\\)\u003c/span\u003e\u003c/span\u003e may retain limited amounts of dye through physical adsorption associated with its aggregated morphology rather than specific chemical interactions. In the presence of bacteria, the distinct staining patterns observed for \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e and CSCC are likely attributed to differences in bacterial adhesion and membrane disruption.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further quantify bacterial aggregation, each well of the 96-well plate was treated with 100 \u0026micro;L of 95% ethanol to solubilize the crystal violet bound to the bacterial cells. The absorbance of the dissolved dye was subsequently measured at 595 nm using a microplate reader. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e presents the quantitative crystal violet results for \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e, and MRSA exposed to different particles. Compared with \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\:Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e, CSCC exhibited 25%, 24%, and 18% higher absorbance for \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e, and MRSA, respectively. When compared to \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{CaCO}_{3}\\)\u003c/span\u003e\u003c/span\u003e, CSCC showed 46%, 69%, and 62% higher absorbance for \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e, and MRSA, respectively. These findings confirm the superior aggregation-inducing capability of CSCC toward both gram-positive and gram-negative bacteria.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Minimum bactericidal concentration (MBC)\u003c/h2\u003e \u003cp\u003eThe broth dilution method combined with optical density measurement was initially employed. However, the background noise due to optical interference of the particles introduced a significant error in the results (Figure S2). To address this challenge, a combination of broth dilution and spot-plating methods was used to determine the MBC of the different particles. The relevant experimental protocol is given in Figure S3.\u003c/p\u003e \u003cp\u003eA range of particle content, 0.625, 1.25, 2.5, 5, 10, 25, 50, and 100 mg/mL, was tested. The results in terms of particle content versus \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e growth are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(a,b) shows the initial time of spot plating following broth dilution and a schematic representation of the spot plating protocol for each particle content and bacteria. Figures\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(c,d), 4(e,f) and 4(g,h) include the results for, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{CaCO}_{3}\\)\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e and CSCC particles, respectively, following 3 h incubation. The results show that 2.5 mg/mL of CSCC was effective in killing both \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e. In contrast, no antibacterial effect was observed for \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{CaCO}_{3}\\)\u003c/span\u003e\u003c/span\u003e. The MBC for \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e was 2.5 mg/mL for \u003cem\u003eP. aeruginosa\u003c/em\u003e and 5 mg/mL for \u003cem\u003eS. aureus\u003c/em\u003e. These results highlight the role of bacteria adsorption/attachment onto the carbonate shell of CSCC particles as a component of the killing mechanism.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the impact of accelerated thermal aging on bactericidal efficacy, the minimum bactericidal concentration (MBC) of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e and CSCC particles was determined against \u003cem\u003eE. coli\u003c/em\u003e (ATCC 25922). The antimicrobial performance of both fresh and thermally aged CSCC and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e particles was examined across 1.5, 3, 6, 12.5, 25, and 50 mg/mL particle content. All formulations fresh and thermally aged CSCC and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e particles achieved complete bacterial killing at 6 mg mL⁻\u0026sup1;, establishing this concentration as the MBC for all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The applied thermal-aging protocol corresponds to more than 100 years of ambient storage based on Arrhenius kinetics, providing a robust model for accelerated stability assessment. Despite similar MBC values, significant differences emerged in bactericidal performance at sub-MBC concentrations. Fresh \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e exhibited marked antimicrobial activity at concentrations as low as 1.5 mg/mL, causing a substantial reduction in \u003cem\u003eE. coli\u003c/em\u003e colony-forming units (CFUs) compared with untreated controls. In contrast, aged \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e displayed markedly reduced efficacy at the same content, with minimal CFU reduction. This decline is likely due to physicochemical transformations during thermal exposure, including particle aggregation. Conversely, both fresh and aged CSCC particles retained strong bactericidal activity even at sub-MBC concentrations. At 1.5 mg/mL, both formulations produced statistically significant \u003cem\u003eE. coli\u003c/em\u003e viability reduction, demonstrating that thermal aging did not compromise the antimicrobial performance of the CSCC system. The sustained efficacy of aged CSCC particles supports the stabilizing role of the carbonate shell, which likely protected the particles from major chemical and physical changes while maintaining hydroxide ion availability. Collectively, these findings demonstrate that CSCC particles maintain long-term bactericidal stability.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eAntimicrobial materials can kill bacteria through different mechanisms, including protein synthesis, nucleic acid synthesis, membrane permeabilization, and bacterial cell agglutination [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The confocal microscopy imaging enabled the evaluation of the efficacy of each treatment. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the chain-like structure resulting from the interaction of bacteria adhered to the surface of the CSCC particles suggests that attachment/adsorption plays a major role in the killing mechanism. In contrast, confocal microscopy imaging of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e primarily displayed bacterial aggregation with much less extent of bacterial attachment. Lastly, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{CaCO}_{3}\\)\u003c/span\u003e\u003c/span\u003e displayed limited adhesion likely owing to effective detachment of the viable bacteria. Both CSCC and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e demonstrated antibacterial properties in contrast with \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{CaCO}_{3}\\)\u003c/span\u003e\u003c/span\u003e. The antibacterial action of the CSCC particles appears to follow an adsorption-mediated killing mechanism. Bacterial cells first adsorb onto the particle surface\u0026mdash;in this case, the \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{CaCO}_{3}\\)\u003c/span\u003e\u003c/span\u003e shell\u0026mdash;facilitated by electrostatic interactions, hydrogen bonding, and surface roughness. Following adsorption, the \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e core releases hydroxyl ions, which locally increase pH and disrupt bacterial membranes, leading to cell death. While the mechanisms of antimicrobial action are not fully understood, the sequential adsorption followed by membrane permeabilization and lysis, has been recently reported for cationic polymers and engineered antimicrobial coatings [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Notably, Ivanova et al. [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] demonstrated a purely physical killing mechanism on cicada wings (\u003cem\u003ePsaltoda claripennis\u003c/em\u003e), where nanopillar structures penetrated and ruptured bacterial cells (\u003cem\u003eP. aeruginosa\u003c/em\u003e) within minutes of contact, independent of surface chemistry. These cicada wings maintain clean surface not only through self-cleaning hydrophobicity but also via bactericidal action driven by nanoscale surface architecture [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. However, while adsorption-killing pathways are well documented for polymers [\u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] and nanostructured surfaces [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], to the best of our knowledge, there is no literature reports pertaining to mineral-based core-shell systems such as CSCC. The Biocidal action of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e is attributed to a damage to the cytoplasmic membranes as well as protein denaturation [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Bacterial aggregation had been previously reported as a response of bacteria to environmental stimuli. This aggregation enhances the survival rate of bacteria under environmental stresses, including pH and osmotic extremes, or exposure to pharmaceutical antimicrobials [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Figures\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e demonstrate significant bacterial aggregation in the presence of CSCC compared to \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e. CSCC particles aggregate both gram-negative and gram-positive bacterial strains, suggesting that its mechanism for bacterial killing is likely similar for both types of bacteria. CSCC particles disrupt and destabilize the bacterial membrane, leading to aggregation of the bacterial cells. However, due to issues with the particle solubility and its optical properties, we were unable to conduct a membrane permeabilization assay. More discussion on the role of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca}^{2+}\\)\u003c/span\u003e\u003c/span\u003e in bacteria aggregation is given below.\u003c/p\u003e \u003cp\u003eThe attachment/adsorption of bacteria onto CSCC particles involves several interfacial interactions. Motile bacteria exist in three fluid regions: bulk liquid, where surfaces have no effect; near-surface bulk, influenced by hydrodynamics; and near-surface constrained, affected by both hydrodynamic and physicochemical forces [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Two main theories explain bacterial attachment to solid surfaces [\u003cspan additionalcitationids=\"CR59\" citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. The first involves a two-step process, wherein bacteria first approach the surface through van der Waals forces, electrostatic interactions, and hydrophobic effects. At this stage, bacteria still exhibit Brownian motion and can be easily dislodged by fluid shear forces. The second step is irreversible attachment, wherein bacteria anchor themselves using exopolysaccharides or specific ligands such as pili or fimbriae [\u003cspan additionalcitationids=\"CR61 CR62\" citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e], by means of short-range covalent and hydrogen bonding, and hydrophobic interactions [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Poortinga et al. [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e] suggested that bacterial attachment also involves electron exchange between the cell surface and the substratum. Busscher and Weerkamp [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e] proposed a three-step model for bacterial attachment to surfaces. In the first step, Lifshitz\u0026ndash;van der Waals forces act over several hundred nanometers. In the second step, both Lifshitz\u0026ndash;van der Waals forces and electrostatic interactions occur at around 20 nm. In the third step, specific adhesion receptors enable strong attachment at about 5 nm [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Once irreversible attachment occurs, stronger chemical methods are needed to kill the bacteria [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. The van der Waals forces, arising from permanent and temporary dipoles in atoms and molecules, are key to initial bacterial contact with the CSCC surface. The interaction is further stabilized by surface charge. The CSCC particles are positively charged, zeta potential\u0026thinsp;=\u0026thinsp;31.7 mV (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), whereas bacterial surfaces are negatively charged owing to components such as teichoic acids or lipopolysaccharides, promoting strong electrostatic attraction [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Once bacteria adhere to the CSCC surface, the \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{OH}^{-}\\)\u003c/span\u003e\u003c/span\u003e ions from the \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e core diffuse through the porous calcium carbonate shell to reach the bacteria. This creates a highly alkaline environment near the bacterial cells, which disrupts their cellular processes [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. The high concentration of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{OH}^{-}\\)\u003c/span\u003e\u003c/span\u003e damages the bacterial cell membrane and wall, causes lipid saponification, protein denaturing, and essential enzymatic activity inhibition, leading to cell structure breakdown [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. As \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{OH}^{-}\\)\u003c/span\u003e\u003c/span\u003e continues to affect the bacteria, the cell membrane loses integrity, resulting in lysis and ultimately bacterial death. This process is especially effective against bacteria that are not adaptive to high alkaline environments, e.g. \u003cem\u003eE. coli, P. aeruginosa\u003c/em\u003e, and \u003cem\u003eS. aureus\u003c/em\u003e [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. In addition, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca}^{2+}\\)\u003c/span\u003e\u003c/span\u003e ions and extracellular DNA (eDNA) promote bacterial aggregation and biofilm formation [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca}^{2+}\\)\u003c/span\u003e\u003c/span\u003e released through the \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{CaCO}_{3}\\)\u003c/span\u003e\u003c/span\u003e shell plays an important role in bridging the negatively charged bacterial surfaces, particularly in gram-positive and gram-negative bacteria [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. The negative charges on these surfaces typically arise from components such as lipopolysaccharides (LPS) or teichoic acids in gram-positive bacteria [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. These charged molecules repel each other, but \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca}^{2+}\\)\u003c/span\u003e\u003c/span\u003e can bridge these negative charges, leading to surface charge neutralization and promoting bacterial aggregation and biofilm formation [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. This ionic interaction is strongest when bacterial cell wall components, e.g. carboxyl groups, bond with \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca}^{2+}\\)\u003c/span\u003e\u003c/span\u003e. In biofilm-forming bacteria, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca}^{2+}\\)\u003c/span\u003e\u003c/span\u003e ions stabilize the biofilm matrix by neutralizing the charge repulsion between bacterial cells. Neutralized bacteria stick together and to surfaces, enhancing the structural integrity of biofilms [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. These interactions are thermodynamically favorable, with the binding of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca}^{2+}\\)\u003c/span\u003e\u003c/span\u003e to eDNA strengthening the biofilm structural integrity, making it more resistant to physical stresses and antibiotics [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. Nevertheless, once bacteria adhere to the CSCC surface, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{OH}^{-}\\)\u003c/span\u003e\u003c/span\u003e ions from the \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e core diffuse through the porous calcium carbonate shell, creating a highly alkaline environment around the bacterial cells. Hydrophobic regions on bacterial surfaces and CSCC particles can also promote hydrophobic interactions, further stabilizing adhesion [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Additionally, physical features of the CSCC surface, such as roughness and porosity, increase the contact points for bacterial cells, trap bacteria in surface irregularities, and make detachment less likely [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCore-shell metal and metal oxide particles, such as \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:Ag/{Fe}_{3}{O}_{4}\\)\u003c/span\u003e\u003c/span\u003e [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e], \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\left(Ni-Cu\\right)@Ag\\)\u003c/span\u003e\u003c/span\u003e NPs [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e], \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:Ag@{Ag}_{2}O\\)\u003c/span\u003e\u003c/span\u003e nanostructures [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e], magnetic \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Fe}_{3}{O}_{4}\\)\u003c/span\u003e\u003c/span\u003e@\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{TiO}_{2}\\)\u003c/span\u003e\u003c/span\u003e [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e], \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:Au-Ag\\)\u003c/span\u003e\u003c/span\u003e NPs, have been tested as antibacterial particles [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. These metal/metal oxide particles are associated with several drawbacks, including high-temperature synthesis [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e], potential heavy metal pollution [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e], and toxicity [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. CSCC, on the other hand, is more environmentally friendly and has minimal side effects on human health and the environment. It is noted that both the core and the shell materials are food-grade. We envision the novel core-shell structure of the CSCC particle and its mechanism of hunting and killing bacteria as a promising solution for targeting pathogenic bacteria, particularly resistant strains. The antimicrobial resistance profile is currently being assessed and will be presented in a separate future study; where resistance development will be monitored through serial passage experiments that track changes in MIC or MBC over time [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e].\u003c/p\u003e"},{"header":"5. CONCLUSIONS","content":"\u003cp\u003eIn this work, we delineated the antimicrobial killing of mechanism of an environmentally friendly core-shell calcium-based biocidal particle. The biocidal efficacy of CSCC particles was assessed against gram-positive and gram-negative bacteria and compared with control samples of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\:\\)\u003c/span\u003e\u003c/span\u003eand a \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{CaCO}_{3}\\)\u003c/span\u003e\u003c/span\u003e. Using confocal microscopy, we were able to establish the adsorption/killing mechanism of the CSCC particles, wherein the bacteria first adsorb to the carbonate shell and are subsequently killed by the \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e core, likely through membrane disruption and aggregation. The minimum bactericidal content of CSCC was 2.5 mg/mL for both gram-positive and gram-negative bacteria, whereas double this dose of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e was required to kill gram-positive bacteria. This observation highlights the importance of the killing mechanism of CSCC particles. We believe that this mechanism of binding and bacterial eradication has the potential to offer a safer and more environmentally friendly alternative. The accelerated aging study demonstrated the role of the carbonate shell in maintaining a biocidal activity of the CSCC particles equivalent to over 100 years at natural conditions.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFunding Declaration\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThis research was supported by Mitacs Accelerate (Award No. IT26596), and the Natural Sciences and Engineering Research Council of Canada (NSERC) Alliance Program (Award No. ALLRP 580333\u0026ndash;22).\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM.M.A. conceived and designed the study and performed the microbiological validation experiments. N.N.D. and M.H. synthesized the particles and formulations, prepared particle samples, and drafted the sections related to particle synthesis and chemistry. G.R. conducted the bacterial aggregation characterization. M.M.A. and N.N.A. drafted the main manuscript. M.M.A. prepared all figures and performed data analysis. M.H. and I.A.L. supervised the experimental work and methodology and critically reviewed and edited the manuscript. All authors reviewed and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors would like to thank Biosenta Inc. for sponsoring this project. Also, this work was supported by the Alberta Centre for Advanced Diagnostics (ACAD).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data supporting the findings of this study are available within the paper and its Supplementary Information.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMuteeb, G., Rehman, M. T., Shahwan, M. \u0026amp; Aatif, M. Origin of Antibiotics and Antibiotic Resistance, and Their Impacts on Drug Development: A Narrative Review, (in eng). \u003cem\u003ePharmaceuticals (Basel)\u003c/em\u003e. \u003cb\u003e16\u003c/b\u003e (11), 15 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePodolsky, S. H. The evolving response to antibiotic resistance (1945\u0026ndash;2018), \u003cem\u003ePalgrave Communications\u003c/em\u003e, vol. 4, no. 1, p. 124, 2018/10/23 2018.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVentola, C. L. The antibiotic resistance crisis: part 1: causes and threats, (in eng), \u003cem\u003eP t\u003c/em\u003e, vol. 40, no. 4, pp. 277\u0026thinsp;\u0026ndash;\u0026thinsp;83, Apr (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKubde, D., Badge, A. K., Ugemuge, S. \u0026amp; Shahu, S. Importance of Hospital Infection Control, (in eng), \u003cem\u003eCureus\u003c/em\u003e, vol. 15, no. 12, p. e50931, Dec (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRutala, W. A. \u0026amp; Weber, D. J. Disinfection and Sterilization in Health Care Facilities: An Overview and Current Issues, (in eng), \u003cem\u003eInfect Dis Clin North Am\u003c/em\u003e, vol. 30, no. 3, pp. 609\u0026thinsp;\u0026ndash;\u0026thinsp;37, Sep 2016.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHua, Z. \u0026amp; Zhu, M. J. Unlocking the Hidden Threat: Impacts of Surface Defects on the Efficacy of Sanitizers Against Listeria monocytogenes Biofilms on Food-contact Surfaces in Tree Fruit Packing Facilities, \u003cem\u003eJournal of Food Protection\u003c/em\u003e, vol. 87, no. 2, p. 100213, /02/01/ 2024. (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalam, M. A. et al. Antimicrobial Resistance: A Growing Serious Threat for Global Public Health, (in eng), \u003cem\u003eHealthcare (Basel)\u003c/em\u003e, vol. 11, no. 13, Jul 5 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGorguner, M., Aslan, S., Inandi, T. \u0026amp; Cakir, Z. Reactive airways dysfunction syndrome in housewives due to a bleach-hydrochloric acid mixture, (in eng). \u003cem\u003eInhal Toxicol\u003c/em\u003e, \u003cb\u003e16\u003c/b\u003e, 2, pp. 87\u0026ndash;91, Feb 2004.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSastre, J. et al. Airway response to chlorine inhalation (bleach) among cleaning workers with and without bronchial hyperresponsiveness. \u003cem\u003eAmerican J. Industrial Medicine\u003c/em\u003e, \u003cb\u003e54\u003c/b\u003e, 4, pp. 293\u0026ndash;299, 2011/04/01 2011.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClausen, P. A. et al. Chemicals inhaled from spray cleaning and disinfection products and their respiratory effects. A comprehensive review, \u003cem\u003eInternational Journal of Hygiene and Environmental Health\u003c/em\u003e, vol. 229, p. 113592, /08/01/ 2020. (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZander, Z. K. \u0026amp; Becker, M. L. Antimicrobial and Antifouling Strategies for Polymeric Medical Devices. \u003cem\u003eACS Macro Letters\u003c/em\u003e, \u003cb\u003e7\u003c/b\u003e, 1, pp. 16\u0026ndash;25, 2018/01/16 2018.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiang, Y., Liang, Y., Zhang, H. \u0026amp; Guo, B. Antibacterial biomaterials for skin wound dressing, (in eng). \u003cem\u003eAsian J. Pharm. Sci.\u003c/em\u003e \u003cb\u003e17\u003c/b\u003e (3), 353\u0026ndash;384 (May 2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCiawi, Y. \u0026amp; Khoiruddin, K. Low-Cost Antibacterial Ceramic Water Filters for Decentralized Water Treatment: Advances and Practical Applications. \u003cem\u003eACS Omega\u003c/em\u003e, \u003cb\u003e9\u003c/b\u003e, 11, pp. 12457\u0026ndash;12477, 2024/03/19 2024.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSwartjes, J. J. et al. Current Developments in Antimicrobial Surface Coatings for Biomedical Applications, (in eng). \u003cem\u003eCurr. Med. Chem.\u003c/em\u003e \u003cb\u003e22\u003c/b\u003e (18), 2116\u0026ndash;2129 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGulati, R., Sharma, S. \u0026amp; Sharma, R. K. Antimicrobial textile: recent developments and functional perspective, (in eng), \u003cem\u003ePolym Bull (Berl)\u003c/em\u003e, vol. 79, no. 8, pp. 5747\u0026ndash;5771, (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMalhotra, B., Keshwani, A. \u0026amp; Kharkwal, H. Antimicrobial food packaging: potential and pitfalls, (in English). \u003cem\u003eFrontiers Microbiol. Rev.\u003c/em\u003e \u003cb\u003e6\u003c/b\u003e, 2015-June-16 2015.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGold, K., Slay, B., Knackstedt, M. \u0026amp; Gaharwar, A. K. Antimicrobial Activity of Metal and Metal-Oxide Based Nanoparticles. \u003cem\u003eAdvanced Therapeutics\u003c/em\u003e, \u003cb\u003e1\u003c/b\u003e, 3, p. 1700033, 2018/07/01 2018.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang, H., Li, L., Li, Z. \u0026amp; Chu, X. Metal-based nanoparticles in antibacterial application in biomedical field: Current development and potential mechanisms. \u003cem\u003eBiomedical Microdevices\u003c/em\u003e, \u003cb\u003e26\u003c/b\u003e, 1, p. 12, 2024/01/23 2024.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaleki Dizaj, S., Mennati, A., Jafari, S., Khezri, K. \u0026amp; Adibkia, K. Antimicrobial activity of carbon-based nanoparticles, (in eng). \u003cem\u003eAdv. Pharm. Bull.\u003c/em\u003e \u003cb\u003e5\u003c/b\u003e (1), 19\u0026ndash;23 (Mar 2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRaul, P. K. et al. Carbon Nanostructures As Antibacterials and Active Food-Packaging Materials: A Review. \u003cem\u003eACS Omega\u003c/em\u003e, \u003cb\u003e7\u003c/b\u003e, 14, pp. 11555\u0026ndash;11559, 2022/04/12 2022.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSarojini, S. \u0026amp; Jayaram, S. An Impact of Antibacterial Efficacy of Metal Oxide Nanoparticles: A Promise for Future, in Bio-manufactured Nanomaterials: Perspectives and Promotion, (ed Pal, K.) Cham: Springer International Publishing, 393\u0026ndash;406. (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRawat, N. et al. Nanobiomaterials: exploring mechanistic roles in combating microbial infections and cancer. \u003cem\u003eDiscover Nano\u003c/em\u003e, \u003cb\u003e18\u003c/b\u003e, 1, p. 158, 2023/12/20 2023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng, S. et al. Implication of Surface Properties, Bacterial Motility, and Hydrodynamic Conditions on Bacterial Surface Sensing and Their Initial Adhesion, (in eng). \u003cem\u003eFront. Bioeng. Biotechnol.\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, 643722 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNastulyavichus, A. et al. In Vitro Destruction of Pathogenic Bacterial Biofilms by Bactericidal Metallic Nanoparticles via Laser-Induced Forward Transfer, (in eng), \u003cem\u003eNanomaterials (Basel)\u003c/em\u003e, vol. 10, no. 11, Nov 15 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNi\u0026ntilde;o-Mart\u0026iacute;nez, N. et al. Molecular Mechanisms of Bacterial Resistance to Metal and Metal Oxide Nanoparticles. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cb\u003e20\u003c/b\u003e (11), 2808 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, K. et al. Developing a New Generation of Therapeutic Dental Polymers to Inhibit Oral Biofilms and Protect Teeth, (in eng), \u003cem\u003eMaterials (Basel)\u003c/em\u003e, vol. 11, no. 9, Sep 17 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChi, M. et al. Novel Bioactive and Therapeutic Dental Polymeric Materials to Inhibit Periodontal Pathogens and Biofilms, (in eng), \u003cem\u003eInt J Mol Sci\u003c/em\u003e, vol. 20, no. 2, Jan 11 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEpperlein, N. et al. Influence of femtosecond laser produced nanostructures on biofilm growth on steel, \u003cem\u003eApplied Surface Science\u003c/em\u003e, vol. 418, pp. 420\u0026ndash;424, /10/01/ 2017. (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuo, X. et al. Biocompatible nano-ripples structured surfaces induced by femtosecond laser to rebel bacterial colonization and biofilm formation, \u003cem\u003eOptics \u0026amp; Laser Technology\u003c/em\u003e, vol. 124, p. 105973, /04/01/ 2020. (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReed, J. H. et al. Ultrascalable Multifunctional Nanoengineered Copper and Aluminum for Antiadhesion and Bactericidal Applications, (in eng). \u003cem\u003eACS Appl. Bio Mater\u003c/em\u003e, \u003cb\u003e2\u003c/b\u003e, 7, pp. 2726\u0026ndash;2737, Jul 15 2019.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWandiyanto, J. V. et al. Outsmarting superbugs: bactericidal activity of nanostructured titanium surfaces against methicillin- and gentamicin-resistant Staphylococcus aureus ATCC 33592. \u003cem\u003eJ. Mater. Chem. B\u003c/em\u003e. \u003cb\u003e7\u003c/b\u003e (28), 4424\u0026ndash;4431. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1039/C9TB00102F\u003c/span\u003e\u003cspan address=\"10.1039/C9TB00102F\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChan, W., Chowdhury, N. R., Sharma, G., Zilm, P. \u0026amp; Rossi-Fedele, G. Comparison of the Biocidal Efficacy of Sodium Dichloroisocyanurate and Calcium Hydroxide as Intracanal Medicaments over a 7-Day Contact Time: An Ex Vivo Study, \u003cem\u003eJournal of Endodontics\u003c/em\u003e, vol. 46, no. 9, pp. 1273\u0026ndash;1278, /09/01/ 2020. (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRogers \u003cem\u003eM. B. C. G. G. L\u003c/em\u003e (Biocidal Coating Composition, 2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAthanassiadis, B., Abbott, P. V. \u0026amp; Walsh, L. J. The use of calcium hydroxide, antibiotics and biocides as antimicrobial medicaments in endodontics. \u003cem\u003eAustralian Dent. Journal\u003c/em\u003e, \u003cb\u003e52\u003c/b\u003e, s1, pp. S64-S82, 2007/03/01 2007.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGomes, I. C., Chevitarese, O., de Almeida, N. S., Salles, M. R. \u0026amp; Gomes, G. C. Diffusion of calcium through dentin, \u003cem\u003eJournal of Endodontics\u003c/em\u003e, vol. 22, no. 11, pp. 590\u0026ndash;595, /11/01/ 1996. (1996).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim, D. \u0026amp; Kim, E. Antimicrobial effect of calcium hydroxide as an intracanal medicament in root canal treatment: a literature review - Part I. In vitro studies, (in eng), \u003cem\u003eRestor Dent Endod\u003c/em\u003e, vol. 39, no. 4, pp. 241\u0026thinsp;\u0026ndash;\u0026thinsp;52, Nov (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDarwish, N., Ashani, M., Lewis, I. A. \u0026amp; Husein, M. M. Controlled carbonation of Ca(OH)2 surface and its application as an antibacterial particle, \u003cem\u003eColloids and Surfaces A: Physicochemical and Engineering Aspects\u003c/em\u003e, vol. 682, p. 132852, /02/05/ 2024. (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerreira, A. M., Vikulina, A. S. \u0026amp; Volodkin, D. CaCO3 crystals as versatile carriers for controlled delivery of antimicrobials, \u003cem\u003eJournal of Controlled Release\u003c/em\u003e, vol. 328, pp. 470\u0026ndash;489, /12/10/ 2020. (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFadia, P. et al. Calcium carbonate nano- and microparticles: synthesis methods and biological applications, (in eng), \u003cem\u003e3 Biotech\u003c/em\u003e, vol. 11, no. 11, p. 457, Nov (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDarwish, N., Ashani, M., Mehairi, A. \u0026amp; Lewis, I. A. and M. M. Husein Synthesis of uniform core-shell calcium hydroxide-calcium carbonate biocidal particles via encapsulation into dry ice, \u003cem\u003eThe Canadian Journal of Chemical Engineering (submitted Nov\u003c/em\u003e responses to reviewers comments has been submitted). (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDodge, R. \u0026amp; Ludington, W. B. Fast Colony Forming Unit Counting in 96-Well Plate Format Applied to the Drosophila Microbiome, (in eng), \u003cem\u003eJ Vis Exp\u003c/em\u003e, no. 191, Jan 13 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlsante, K. M. et al. The role of degradant profiling in active pharmaceutical ingredients and drug products, \u003cem\u003eAdvanced Drug Delivery Reviews\u003c/em\u003e, vol. 59, no. 1, pp. 29\u0026ndash;37, /01/10/ 2007. (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlessy, M., Patel, R. D., Prajapati, P. N. \u0026amp; Agrawal, Y. K. Development of forced degradation and stability indicating studies of drugs\u0026mdash;A review, \u003cem\u003eJournal of Pharmaceutical Analysis\u003c/em\u003e, vol. 4, no. 3, pp. 159\u0026ndash;165, /06/01/ 2014. (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDarwish, N., Ashani, M. M., Mehairi, A., Lewis, I. A. \u0026amp; Husein, M. M. Synthesis of uniform core-shell calcium hydroxide-calcium carbonate biocidal particles via encapsulation into dry ice, \u003cem\u003eThe Canadian Journal of Chemical Engineering\u003c/em\u003e, vol. n/a, no. n\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e/a\u003c/span\u003e\u003cspan address=\"http:///a\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSecor, P. R., Michaels, L. A., Bublitz, D. C., Jennings, L. K. \u0026amp; Singh, P. K. The Depletion Mechanism Actuates Bacterial Aggregation by Exopolysaccharides and Determines Species Distribution \u0026amp; Composition in Bacterial Aggregates, (in English), \u003cem\u003eFrontiers in Cellular and Infection Microbiology\u003c/em\u003e, Original Research vol. \u003cb\u003e12\u003c/b\u003e, 2022-June-16 2022.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKapoor, G., Saigal, S. \u0026amp; Elongavan, A. Action and resistance mechanisms of antibiotics: A guide for clinicians, (in eng). \u003cem\u003eJ Anaesthesiol. Clin. Pharmacol\u003c/em\u003e, \u003cb\u003e33\u003c/b\u003e, 3, pp. 300\u0026ndash;305, Jul-Sep 2017.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHalawa, E. M. et al. Antibiotic action and resistance: updated review of mechanisms, spread, influencing factors, and alternative approaches for combating resistance. \u003cem\u003e(in English) Frontiers Pharmacol. Rev.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, 2024-January-12 2024.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBenmamoun, Z., Chandar, P., Jankolovits, J. \u0026amp; Ducker, W. A. Time-Resolved Killing of Individual Bacterial Cells by a Polycationic Antimicrobial Polymer. \u003cem\u003eACS Biomaterials Sci. \u0026amp; Engineering\u003c/em\u003e, \u003cb\u003e10\u003c/b\u003e, 5, pp. 3029\u0026ndash;3040, 2024/05/13 2024.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIvanova, E. P. et al. Natural bactericidal surfaces: mechanical rupture of Pseudomonas aeruginosa cells by cicada wings, (in eng), \u003cem\u003eSmall\u003c/em\u003e, vol. 8, no. 16, pp. 2489-94, Aug 20 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSochacki, K. A., Barns, K. J., Bucki, R. \u0026amp; Weisshaar, J. C. Real-time attack on single Escherichia coli cells by the human antimicrobial peptide LL-37, (in eng). \u003cem\u003eProc Natl. Acad. Sci. U S A\u003c/em\u003e, \u003cb\u003e108\u003c/b\u003e, 16, pp. E77-81, Apr 19 2011.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBenmamoun, Z., Chandar, P., Jankolovits, J. \u0026amp; Ducker, W. A. Time-Resolved Killing of Individual Bacterial Cells by a Polycationic Antimicrobial Polymer, (in eng). \u003cem\u003eACS Biomater. Sci. Eng\u003c/em\u003e, \u003cb\u003e10\u003c/b\u003e, 5, pp. 3029\u0026ndash;3040, May 13 2024.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarns, K. J. \u0026amp; Weisshaar, J. C. Single-cell, time-resolved study of the effects of the antimicrobial peptide alamethicin on Bacillus subtilis, (in eng). \u003cem\u003eBiochim Biophys. Acta\u003c/em\u003e, \u003cb\u003e1858\u003c/b\u003e, 4, pp. 725\u0026thinsp;\u0026ndash;\u0026thinsp;32, Apr 2016.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa, J., Sun, Y., Gleichauf, K., Lou, J. \u0026amp; Li, Q. Nanostructure on taro leaves resists fouling by colloids and bacteria under submerged conditions, (in eng), \u003cem\u003eLangmuir\u003c/em\u003e, vol. 27, no. 16, pp. 10035-40, Aug 16 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTruong, V. K. et al. The influence of nano-scale surface roughness on bacterial adhesion to ultrafine-grained titanium, (in eng), \u003cem\u003eBiomaterials\u003c/em\u003e, vol. 31, no. 13, pp. 3674-83, May (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMohammadi, Z., Shalavi, S. \u0026amp; Yazdizadeh, M. Antimicrobial activity of calcium hydroxide in endodontics: a review, (in eng), \u003cem\u003eChonnam Med J\u003c/em\u003e, vol. 48, no. 3, pp. 133\u0026thinsp;\u0026ndash;\u0026thinsp;40, Dec (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStewart, P. S. \u0026amp; William Costerton, J. Antibiotic resistance of bacteria in biofilms, \u003cem\u003eThe Lancet\u003c/em\u003e, vol. 358, no. 9276, pp. 135\u0026ndash;138, /07/14/ 2001. (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTuson, H. H. \u0026amp; Weibel, D. B. Bacteria\u0026ndash;surface interactions, \u003cem\u003eSoft Matter\u003c/em\u003e, 10.1039/C3SM27705D vol. 9, no. 17, pp. 4368\u0026ndash;4380, (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarshall, K. C., Stout, R. \u0026amp; Mitchell, R. Mechanism of the Initial Events in the Sorption of Marine Bacteria to Surfaces, \u003cem\u003eMicrobiology\u003c/em\u003e, vol. 68, no. 3, pp. 337\u0026ndash;348, (1971).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar, C. G. \u0026amp; Anand, S. K. Significance of microbial biofilms in food industry: a review, (in eng). \u003cem\u003eInt J. Food Microbiol\u003c/em\u003e, \u003cb\u003e42\u003c/b\u003e, no. 1\u0026ndash;2, pp. 9\u0026ndash;27, Jun 30 1998.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePalmer, J., Flint, S. \u0026amp; Brooks, J. Bacterial cell attachment, the beginning of a biofilm, (in eng), \u003cem\u003eJ Ind Microbiol Biotechnol\u003c/em\u003e, vol. 34, no. 9, pp. 577\u0026thinsp;\u0026ndash;\u0026thinsp;88, Sep (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGilbert, P., Evans, D. J., Evans, E., Duguid, I. G. \u0026amp; Brown, M. R. Surface characteristics and adhesion of Escherichia coli and Staphylococcus epidermidis, (in eng), \u003cem\u003eJ Appl Bacteriol\u003c/em\u003e, vol. 71, no. 1, pp. 72\u0026thinsp;\u0026ndash;\u0026thinsp;7, Jul (1991).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarpentier, B. \u0026amp; Cerf, O. Biofilms and their consequences, with particular reference to hygiene in the food industry, (in eng). \u003cem\u003eJ. Appl. Bacteriol.\u003c/em\u003e \u003cb\u003e75\u003c/b\u003e (6), 499\u0026ndash;511 (Dec 1993).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan Loosdrecht, M. C., Lyklema, J., Norde, W., Schraa, G. \u0026amp; Zehnder, A. J. Electrophoretic mobility and hydrophobicity as a measured to predict the initial steps of bacterial adhesion, (in eng), \u003cem\u003eAppl Environ Microbiol\u003c/em\u003e, vol. 53, no. 8, pp. 1898\u0026thinsp;\u0026ndash;\u0026thinsp;901, Aug (1987).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJullien, C., B\u0026eacute;n\u0026eacute;zech, T., Carpentier, B., Lebret, V. \u0026amp; Faille, C. Identification of surface characteristics relevant to the hygienic status of stainless steel for the food industry, \u003cem\u003eJournal of Food Engineering\u003c/em\u003e, vol. 56, no. 1, pp. 77\u0026ndash;87, /01/01/ 2003. (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePoortinga, A. T., Bos, R. \u0026amp; Busscher, H. J. Charge transfer during staphylococcal adhesion to TiNOX coatings with different specific resistivity, (in eng), \u003cem\u003eBiophys Chem\u003c/em\u003e, vol. 91, no. 3, pp. 273-9, Jul 24 (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBusscher, H. J. \u0026amp; Weerkamp, A. H. Specific and non-specific interactions in bacterial adhesion to solid substrata. \u003cem\u003eFEMS Microbiol. Rev.\u003c/em\u003e \u003cb\u003e3\u003c/b\u003e (2), 165\u0026ndash;173 (1987).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePoxton, I. R. Chapter 5 - Teichoic Acids, Lipoteichoic Acids and Other Secondary Cell Wall and Membrane Polysaccharides of Gram-Positive Bacteria, in \u003cem\u003eMolecular Medical Microbiology (Second Edition)\u003c/em\u003e, Y.-W. Tang, M. Sussman, D. Liu, I. Poxton, and J. Schwartzman, Eds. Boston: Academic Press, pp. 91\u0026ndash;103. (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePoortinga, A. T., Bos, R., Norde, W. \u0026amp; Busscher, H. J. Electric double layer interactions in bacterial adhesion to surfaces, \u003cem\u003eSurface Science Reports\u003c/em\u003e, vol. 47, no. 1, pp. 1\u0026ndash;32, /06/01/ 2002. (2002).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGomes, I. C., Chevitarese, O., de Almeida, N. S., Salles, M. R. \u0026amp; Gomes, G. C. Diffusion of calcium through dentin, (in eng). \u003cem\u003eJ. Endod\u003c/em\u003e. \u003cb\u003e22\u003c/b\u003e (11), 590\u0026ndash;595 (Nov 1996).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDas, T. et al. Influence of calcium in extracellular DNA mediated bacterial aggregation and biofilm formation, (in eng), \u003cem\u003ePLoS One\u003c/em\u003e, vol. 9, no. 3, p. e91935, (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThomas, K. J. \u0026amp; Rice, C. V. 3rd and Revised model of calcium and magnesium binding to the bacterial cell wall, (in eng), \u003cem\u003eBiometals\u003c/em\u003e, vol. 27, no. 6, pp. 1361-70, Dec (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, X. \u0026amp; Quinn, P. J. Endotoxins: lipopolysaccharides of gram-negative bacteria, (in eng). \u003cem\u003eSubcell. Biochem.\u003c/em\u003e \u003cb\u003e53\u003c/b\u003e, 3\u0026ndash;25 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharaf, E. M. et al. Synergistic antibacterial activity of compact silver/magnetite core-shell nanoparticles core shell against Gram-negative foodborne pathogens, (in English). \u003cem\u003eFrontiers Microbiol. Original Res.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 2022-September-02 2022.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhmed, A. A. A., Aldeen, T. S., Al-Aqil, S. A., Alaizeri, Z. M. \u0026amp; Megahed, S. Synthesis of Trimetallic (Ni-Cu)@Ag Core@Shell Nanoparticles without Stabilizing Materials for Antibacterial Applications. \u003cem\u003eACS Omega\u003c/em\u003e, \u003cb\u003e7\u003c/b\u003e, 42, pp. 37340\u0026ndash;37350, 2022/10/25 2022.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElyamny, S., Eltarahony, M., Abu-Serie, M., Nabil, M. M. \u0026amp; Kashyout, A. E. H. B. One-pot fabrication of Ag @Ag2O core\u0026ndash;shell nanostructures for biosafe antimicrobial and antibiofilm applications. \u003cem\u003eScientific Reports\u003c/em\u003e, \u003cb\u003e11\u003c/b\u003e, 1, p. 22543, 2021/11/19 2021.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRani, N. \u0026amp; Dehiya, B. S. Magnetic core-shell Fe3O4@TiO2 nanocomposites for broad spectrum antibacterial applications. \u003cem\u003eIET Nanobiotechnology\u003c/em\u003e, \u003cb\u003e15\u003c/b\u003e, 3, pp. 301\u0026ndash;308, 2021/05/01 2021.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFan, X., Yahia, L. H. \u0026amp; Sacher, E. Antimicrobial Properties of the Ag, Cu Nanoparticle System, \u003cem\u003eBiology\u003c/em\u003e, vol. 10, no. 2, p. 137, (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuynh, K. H. et al. Synthesis, Properties, and Biological Applications of Metallic Alloy Nanoparticles. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cb\u003e21\u003c/b\u003e (14), 5174 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTchounwou, P. B., Yedjou, C. G., Patlolla, A. K. \u0026amp; Sutton, D. J. Heavy metal toxicity and the environment, (in eng), Exp Suppl, vol. 101, pp. 133\u0026thinsp;\u0026ndash;\u0026thinsp;64, (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHama Aziz, K. H. et al. Heavy metal pollution in the aquatic environment: efficient and low-cost removal approaches to eliminate their toxicity: a review, (in eng), \u003cem\u003eRSC Adv\u003c/em\u003e, vol. 13, no. 26, pp. 17595\u0026ndash;17610, Jun 9 2023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBelkin, N. L. Bacterial penetration vis-\u0026aacute;-vis lint generation, (in eng). \u003cem\u003eJ. Hosp. Infect.\u003c/em\u003e \u003cb\u003e52\u003c/b\u003e (4), 315\u0026ndash;317 (Dec 2002).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"antimicrobial, antibiotic-resistant bacteria, core–shell, particle","lastPublishedDoi":"10.21203/rs.3.rs-8378570/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8378570/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe global rise in antimicrobial resistance (AMR) poses a significant challenge to infection control efforts worldwide. This growing threat highlights the critical need for innovative technologies with advanced antimicrobial properties to improve infection prevention and control strategies. To address this challenge, we developed a novel core\u0026ndash;shell \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e\u0026ndash;\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{CaCO}_{3}\\)\u003c/span\u003e\u003c/span\u003e (CSCC) particle exhibiting antibacterial properties against both gram-positive and gram-negative bacteria. Through confocal microscopy imaging and the developed in vitro microbiology protocol, we identified the bacteria-killing mechanism, wherein the bacteria adhere to the \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{CaCO}_{3}\\)\u003c/span\u003e\u003c/span\u003e shell and are subsequently killed, by the membrane disruption and aggregation mechanisms of the \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e core. The minimum Bactericidal content of CSCC, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{CaCO}_{3}\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e was measured using a combination of broth dilution and spot-plating methods. The results indicate that 2.5 mg/mL CSCC kills both gram-positive and gram-negative bacteria, whereas 2.5 mg/mL and 5 mg/mL of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Ca\\left(OH\\right)}_{2}\\)\u003c/span\u003e\u003c/span\u003e killed gram-negative and gram-positive bacteria, respectively. No antimicrobial properties were observed for \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{CaCO}_{3}\\)\u003c/span\u003e\u003c/span\u003e. We believe the mechanism of binding and killing bacteria may offer a prominent solution to the global challenge of antimicrobial resistance. Accelerated aging tests confirmed that the CSCC particles retained full antibacterial activity against \u003cem\u003eE. coli\u003c/em\u003e equivalent to 100 years of natural aging.\u003c/p\u003e","manuscriptTitle":"Bactericidal activity of novel calcium-based core-shell particles and its mechanism of action","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-20 08:27:16","doi":"10.21203/rs.3.rs-8378570/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d2695f97-9e99-40d3-a898-7b728a1132cf","owner":[],"postedDate":"January 20th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":61282009,"name":"Biological sciences/Biotechnology"},{"id":61282010,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-01-22T19:53:55+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-20 08:27:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8378570","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8378570","identity":"rs-8378570","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}