## 4 Discussion  
This study attempted to evaluate the antimicrobial and antib biofilm activities of silver-ion-exchanged zeolite (AgZ) against two marine bacterial pathogens, _V. campbellii and V. parahaemolyticus_. The first stage of this study involved the ICP-OES evaluation of Ag ion incorporation of zeolite synthesized from local bentonite clay (data published elsewhere). The initial ICP-OES analysis of AgZ1, AgZ2, AgZ3 and AgZ4 revealed the successful incorporation of Ag ion into the zeolite samples to be typically in direct correlation with the increase in Ag ion concentration introduced. The highest Ag ion concentration was observed in sample AgZ4 (1.50 M) at 190.511 \(\pm\) 1.989 mg/g. At 0.50 M, the Ag ion concentration of AgZ2 was double that of AgZ1 at 160.041 \(\pm\) 1.328 mg/g compared to 80.949 \(\pm\) 0.389 mg/g. Nonetheless, doubled concentration increases observed for AgZ2 were not indicated in AgZ3 and AgZ4. A possible explanation for this is saturation of available sites in AgZ3 and AgZ4 for the ion exchange to occur. In general, minimum inhibitory concentration (MIC) evaluation against _V. campbellii_ and _V. parahaemolytic_ firstly revealed that the incorporation of Ag ions into the zeolite samples AgZ1, AgZ2, AgZ3 and AgZ4 was successful in inhibiting bacterial growth. Silver (Ag) has long been established in various studies as an effective antibacterial agent. Several studies have also demonstrated the efficiency of Ag-exchanged zeolite against many microbial pathogens [31; 32; 33] and concur with the findings of this study. Additionally, a typically inverse relationship between sample concentration and MIC for AgZ1, AgZ2, AgZ3 and AgZ4 was also observed. As seen in Table 2 a lower MIC, which signifies stronger antibacterial activity, was indicated for each bacterial type as the concentration of metal ion in the zeolite increased. Therefore, the higher the Ag ion loading in the zeolite, the lower the concentration of ion-loaded zeolite A needed to inhibit the growth of the bacteria. There have been many studies that have investigated the mechanism of microbial killing by metal ion. A possible mechanism involves the ability of the metal ion to attach to the bacteria membrane through electrostatic interaction and drastically alter the integrity of the bacterial membrane. Consequently, it promotes the formation of reactive oxygen species, (ROS) which will induce the oxidative stress to the bacteria cell resulting in the oxidation of cellular component, DNA damage, mitochondria damage and disruption of the cell membrane, which lead to the death of the bacteria [36; 37; 38; 39]. MIC studies also revealed that between the two bacterial species, _V. parahaemolyticus_ indicated higher susceptibility against Ag ion compared to _V. campbellii_, as lower MICs were observed across AgZ1-AgZ4. Under antibiotic pressure, bacterial phenotypes such as susceptibility, resistance, tolerance and persistence differ from one bacteria to the other. In a review by [PERSON] et al. [40], the efficacy of antimicrobials are influenced by many factors including bacterial status, host factors and antimicrobial concentrations. A typical feature of bacterial biofilms is the extracellular matrix, which provides protection and structure to the cell population within it. The CV assay is a useful tool for rapid and simple assessment of biofilm formation differences between bacteria, as it stains the extracellular matrix as well as the aggregated bacterial cells [41]. The CV biofilm assays of _V. campbelli_ and _V. parahaemolytic_ isolates grown in AgZ4 overnight showed that biofilm growth was effectively inhibited in the presence of AgZ4. However, _V. parahaemolyticus_ biofilms were indicated to be more susceptible against AgZ4 with a percentage inhibition of 77.3% compared to _V. campbelli_ at 60.2%. This concurred with the MIC assays, which also showed a higher susceptibility of _V. parahaemolyticus_ when compared against _V. campbelli_ and demonstrated significant attenuation of biofilm formation against _V. campbellii_ and _V.