3.1 Antibiotic Susceptibility of Marine Pathogens
Antibiotic susceptibility of the pathogenic strains V. harveyi, V. alginolyticus, V. parahaemolyticus and S. haemolyticus were tested using antibiotic discs such as Penicillin 10µg, Ampicillin 2µg, Enrofloxacin 5µg, Gentamycin 30µg, Ceftriaxone 30µg, Streptomycin 10µg, and Cefoperazone sulbactam 75µg. From the result, it was identified that MP3, MP4, MP5 tested organisms showed resistance to Penicillin and Ampicillin and were susceptible to all other antibiotics tested (Table 1) wherease V. harveyi (MP1) strain was resisitant to Penicillin 10µg, Ampicillin 2µg, Enrofloxacin 5µg, and Ceftriaxone 30µg.
3.2 Phytochemical analysis of biological extract of Myrobalans
Phytocompounds enriched extracts of Triphala myrobalans was prepared and concentrated. Phytochemical analysis of biological aqueous extract of Triphala myrobalans revealed the presence of carbohydrates, phenols, saponins, terpenoids, flavonoids, alkaloids, terpenoids, anthocyanin, steroids, and carotenoids (Table 2). Medicinal plants produce phenols, flavonoids, terpenoids, and tannins that contribute to their medicinal property. Flavonoids in combination with phenols, polyphenols, saponins, and other tannins exhibit potential antibacterial, anti-tumor, anti-viral properties . Antioxidant and reducing properties of these phytoconstituents help in the reduction of silver nitrate into nanocolloidal silver. The quantitative estimation of carbohydrates confirmed the presence of total carbohydrate (90mg/ml) of concentrated triphala extract. This confirmed the presence of carbohydrates which may include the presence of all polysaccharides. The cellulose is one of the carbohydrate which can be extracted easily through water and accounts for major proportion in the Triphala extract. Previous studies have reported the presence of cellulose as a major biochemical constituent in triphala extract 
3.3 Biogenesis of Myrobalans mediated green nano colloids (MBNc) and physicochemical characterization
Myrobalans mediated green nano colloid (MBNc) was prepared by mixing the concentrated extract with 1mM Silver Nitrate and was kept in dark. Initial colour change was observed from pale brown that gradually changed into brownish green to dark brown nanocolloidal particles (Fig. 1a). This colour change is the preliminary signature which indicates the synthesis of biomolecules mediated nanoparticle synthesis in green colloidal system. At macroscale, the electrons are loosely packed, at nanoscale the electrons are lightly packed with restricted movement, so the intensity of scattering light varies when compared to macroscale and nanoscale. Hence, the nanoscaled particles look dark brown colour and as an indicator for the successful synthesis of nanocolloids . Phytoconstituents such as starch, cellulose, phenols, poly phenols, flavonoids etc. act as reducing and capping agents and aid in the formation of nanoparticles containing colloidal solution. Further, the synthesis of myrobalans mediated green nanocolloids (MBNc) was further confirmed by physicochemical characterization.
UV-Visible spectrometer scanning was performed between 300-800nm. Silver nanoparticles have strong absorption and scattering of light. When the nanoparticles are exposed to particular wavelength, because of conduction of electrons, they undergo oscillation. The oscillation created by nanoparticle will be much stronger than non-plasmonic particles . The oscillation developed by the nanoparticles, will develop strong peak, which is called as SPR peak. The SPR peak of MBNc was observed around 430nm which corresponds to nanoparticles which are spherical in shape (Fig. 1b). Myrobalans extract is reported to have phytocompounds, polymers of carbohydrates, polysaccharides, polyphenols, enzymes, coenzymes and other proteins which act as a bioreductant in reducing the silver ions [24- 27].
The size of MBNc was found as 407.1nm in range, based on Brownian movement of the nanoparticles dispersed in the colloidal solution with the poly dispersive index of 0.299 with intercept of 0.785 (Fig. 1c). The size of MBNc were measured using DLS which was based on random dynamic motion of the particles. PDI value of 0.299 is a highly acceptable value, described as homogenous. The Zeta potential value of MBNc was found as -13.5 with the conductivity of 0.497ms/cm, with the viscosity of 0.8872cP (Fig. 1d). The negative value implies that the negative surface charge of -13.9 will create a repulsive force between the particles and prevent agglomeration in the dispersed medium. The size, charge, and aggregation nature of the MBNc mainly depends on natural polymers and phytocompounds present in myrobalans extract. These phytocompounds and biological macromolecules uniformly laid over the surface of nanoparticles protect as a cap, to prevent the agglomeration. Thus, these biomolecules work synergistically along with silver in imparting effective antibacterial activity [21- 23].
FESEM images revealed that the nanocolloids were embedded with spherical shaped nanoparticles at 1µm scale (Fig. 2a). The mixture of three myrobalans may produce these spherical shaped nanoparticles, which may have its unique role in targeting the bacterial cells through its shapes that helps in penetrating through the bacterial cell wall. EDAX analysis revealed the presence of silver at 3Kev, which confirmed the presence of silver (Fig. 2b). The histogram of EDAX analysis depicts the percentage weight and percentage atom of elements present in MBNc (Fig. 2c). The presence of other element such as cl, which works synergistically with silver in enhancing its antimicrobial activity [36, 37].
HRTEM analysis showed the distribution, average size and shape of the MBNc. HRTEM images of MBNc revealed its polydispersed and spherical (Fig. 3a) nature. The average size of MBNc was found as 23.63nm. SAED patterns are signature for the crystals. Shows the spots which indicate polycrystalline (Fig. 3b) nature of MBNc .
3. 4. Antibacterial activity of MBNc
3.4.1 Detection of antibacterial activity of MBNc through agar well diffusion assay
The preliminary antibacterial effect of MBNc was evaluated against V. harveyi (MP1), V. parahaemolyticus (MP2), V. alginolyticus (MP3) and S. haemolyticus (MP4 and MP5). At 50µg concentration of MBNc maximum zone of inhibition was observed as 26mm, 25mm, 26mm, 20mm for V. harveyi (MP1), V. parahaemolyticus (MP2), V. alginolyticus (MP3) and S. haemolyticus (MP4 and MP5) respectively (Table 3). MBNc when loaded into the wells, diffuses into the maximum area. One of the property of nanoparticles is large surface area where nanoparticles encounter microorganism in maximum area, which inhibits and kills the organism through several mechanisms . The synergistic effect of phytocompounds and silver enhances the antibacterial potential of MBNc.
3.4.2 Detecting the Bacteriostatic and Bactericidal concentration of MBNc
MBNc was validated to detect the concentration at which it completely inhibits the visible growth of organism. The minimum inhibitory concentration for V. harveyi (MP1), V. parahaemolyticus (MP2), V. alginolyticus (MP3) was found as 6.25 µg, 1.56 µg, 3.125 µg respectively, and for S. haemolyticus (MP4 and MP5) bacteriostatic concentration was found as 12.5µg. From this result it was observed that each strain responds differently to MBNc. The cell wall of Gram-negative Vibrio sp requires maximum 6.125µg of MBNc when compared to Gram-positive Staphylococcus haemolyticus, which requires 12.5µg of MBNc (Table 4). Because the cell wall of Gram-positive strains will be thicker than Gram-negative strains. The mode of action of MBNc will require higher concentration to penetrate the cell wall. The bactericidal concentration of MBNc was found as 12.5 µg, 3.125 µg, 6.25 µg for V. harveyi (MP1), V. parahaemolyticus (MP2), V. alginolyticus (MP3) respicteively and 12.5 µg for other 2 strains of Staphylococcus haemolyticus. From the result it was found that MBNc has potent bacteriostatic and bactericidal property against antibiotic-resistant, Gram positive and Gram-negative organisms, even up to the concentration of 12.5µg/ml (Table 4). Because nanocolloids target the bacterial cell through several mechanisms, the first target is the cell membrane. When the nanoparticles interact with the bacterial cell it creates pits on the surface of cell membrane and disrupt membrane permeability. Then the silver ions capped with biomolecules enter the cell through porins, which interfere with the normal functions of the plasma membrane . Previous reports are supporting to the functioning of nanoparticles in such a way that, imbalance of pH gradient will be created which induces the leakage of H+ ions in Vibrio Sp . MBNc may interact with thiol group of many functional proteins and enzymes, which may ultimately inhibit the respiratory chain and block the synthesis of ATP. There is an evidence that silver nanoparticles intercalate with phosphorus and sulphur group of DNA and inhibit the replication of the cell. Further the interaction of Ag ions with signalling pathway may alter the tyrosine phosphorylation, which leads to the adverse effect on mitosis, meiosis and finally leads to cell death. Silver ions also generate more and more ROS inside the cell, which is very toxic and leads to cell death . Our results suggested that the physical property such as size, shape, charge and chemical properties such as interaction within the bacterial cell of MBNc played a crucial role to exert its action as a potent bacteriostatic and bactericidal agent.
3.4.3. MBNc modulates growth kinetics in marine pathogens
The dose dependent activity of MBNc was observed in all the test strains V. harveyi (MP1), V. parahaemolyticus (MP2), V. alginolyticus (MP3) and S. haemolyticus (MP4 and MP5) after 12 hours of treatment (Fig. 4a). To validate the effect of MBNc on growth, different concentrations are used and growth rate was measured at different time interval. And with different concentrations of MBNc (1.5625µg, 3.125µg, 6.25µg and 12.5µg). At the concentration of 1.5625µg the growth rate was decreased by 72%, 81%, 67.5%, 76%, and 61.5% in MP1 to MP5 strains, respectively. At the concentration of 3.125µg the growth rate was decreased by 73.8%, 89.8%, 88.1%, 78.9%, and 63.3% in MP1 to MP5 strains, respectively. At the concentration of 6.25µg the growth rate was decreased by 90.4%, 93.9%, 93.9%, 86.3%, and 80.8% in MP1 to MP5 strains, respectively. At the concentration of 12.5µg the growth rate was decreased by 94.88%, 94.69%, 94.3%, 99.0%, and 93.7% in MP1 to MP5 strains, respectively.
At 24 hours the growth rate was further decreased. At the concentration of 1.5625µg the growth rate was decreased by 80.6%, 86.6%, 40.9%, 40.3%, and 68.5% in MP1 to MP5 strains, respectively. At the concentration of 3.125µg the growth rate was decreased by 82%, 89%, 64.4%, 66.5%, and 83.7% in MP1 to MP5 strains, respectively. At the concentration of 6.25µg the growth rate was decreased by 90%, 90.8%, 91.0%, 82.2%, and 87% in MP1 to MP5 strains, respectively. At the concentration of 12.5µg the growth rate was decreased by 99%, 94%, 93%, 98%, and 98% in MP1 to MP5 strains, respectively. At the concentration of 12.5µg the growth rate of all the marine pathogens V. harveyi (MP1), V. parahaemolyticus (MP2), V. alginolyticus (MP3) and S. haemolyticus (MP4 and MP5) were effectively inhibited even after 24hours of incubation without any further treatment (Fig. 4b). This results suggested that MBNc could be used as potent alternate to control the growth of marine pathogens in marine fishes growing in caged farming.
3.4.4 Effect of MBNc on biofilm formation
Biofilm produced by the bacteria is a virulence mechanism which in turn become more antibiotic resistant strain. The hydrophobic nature of bacterial cell communicates and interact by means of signalling molecules to develop biofilms on the surface. In general, antibiofilm agents will disrupt the communication through cell membrane or will quench the signals from bacterial cells that can inhibit the biofilm formation. To check this hypothesis, MBNc was validated to check the anti-biofilm action on the following marine pathogens V. harveyi (MP1), V. parahaemolyticus (MP2), V. alginolyticus (MP3) and S. haemolyticus (MP4 and MP5). From the antibiofilm assay it was found that MBNc inhibited the biofilm formation in a dose dependent manner of four different concentrations 1.5625µg, 3.125µg, 6.25µg and 12.5µg (Fig. 5a). At the concentration of 1.5625µg the biofilm formation was inhibited up to 82%, 82%, 54%, 77%, and 78.9% for MP1 to MP5, respectively. At the concentration of 3.125µg, the biofilm formation was inhibited up to 90%, 85%, 86.5%, 90.9%, and 83.6% for MP1 to MP5, respectively. At the concentration of 6.25µg the biofilm formation was inhibited up to 92%, 96.8%, 95.6%, 92%, and 88.9% for MP1 to MP5, respectively. At the concentration of 12.5µg the biofilm formation was inhibited maximum up to 97.9%, 99.7%, 97.8%, 94.9%, and 90.8% for MP1 to MP5, respectively.
As per the bacteriostatic and bactericidal assay, the MBNc was effective at the concentration of 12.5µg, supporting to these findings the MBNc also showed the maximum biofilm inhibition potential of 99%. It was reported that smaller the size of nanoparticle greater the antimicrobial effect, according to HRTEM the average size of particles was found around 23.63nm. Hence, MBNc easily penetrate the cell membrane and disrupt the surface of bacterial cell. The quorum sensors are the molecules secreted out for cell to cell communication. The secretion of these signalling compounds could have been inhibited by the nanocolloids, through inhibiting the synthesis of signalling molecules, degrading, or inactivating the signalling enzymes, analogs, disrupting the signalling cascade. MBNc can effectively disrupt the cell membrane, targets the central dogma of cell by disturbing whole mechanism of replication, transcription, and translation, sequentially all the signalling cascade will be disturbed . There were previous reports supporting that nanoparticles controlling the biofilm formation in organism such as S. dysentriae, V. parahaemolyticus and S. infestis through interfering with cell to cell adhesion . Our results suggest that MBNc can be effective anti-biofilm agent, even at lower concentration, which could be used as an effective alternative to antibiotics to control the infection caused by marine pathogen and marine associated food borne pathogens. Thus, this can be effectively used to control the usage of antibiotics in marine cage culturing and in aqua culturing industries.
3.4.5 Effect of MBNc on antibiotic resistant genes
The presence of CTX-M15 and AmpC genes was checked upon treatment with MBNc and Ampicillin. From the result it was observed that AmpC gene was absent in all the tested marine pathogens. However, CTX-M-15 was detected only in V. alginolyticus (MP3). Absence of CTX-M-15 gene in V. harveyi (MP1) and V. parahaemolyticus (MP2) and Staphylococcus haemolyticus (MP4 and MP5) suggested that virulence and antibiotic resistance were governed by some other ESBL genes present in these strains . From the gel documentation, it was found that in control (without treatment), presence of CTX-M-15 gene was observed in V. alginolyticus (MP3). Upon treatment with ampicillin, CTX-M-15 gene was present, but upon treatment with 5µg and 10µg of MBNc, the CTX-M-15 gene was abolished completely. This demonstrates that MBNc may effectively target the CTX-M-15 gene at the transcription and translational level and prevent the expression of CTX-M-15 gene (Fig. 5b). These results suggest that MBNc can effectively target on all strains with and without the expression of CTX-M-15 gene.