Study On The Adsorptive And Antibacterial Activity Of Cu-loaded Silicate Nanocomposite On Aquacultural Pathogenic Bacteria, Applied Effects In Nile Tilapia And Approach To The Mechanism

Bacterial disease is a major problem encountered in the rapidly developing fish farming industry. Although vaccines are being developed and marketed, they cannot be used alone as a universal disease control measure in aquaculture. Treatment with antibiotics continues to be important disease control measures in the aquaculture industry. However, abuse or overuse of antibiotics causes various side-effects and also results in the emergence and increase of bacteria resistant to antibiotics. Therefore, it is necessary to develop new types of antibacterial agents that can replace antibiotics.The metal-carrying inorganic antibacterial materials, being carried by nano materials, have attracted attention by durable antibacterial properties, non-resistance, and safety. The Layer silicate material, MMT, is one of the natural minerals that have the highly commercial value to be able to make nanocomposite. The nature nano-property of MMT from samdwich, layer charge and exchangeable cation etc is the base for its nanometer processing and the processing. In the present study, Layer silicate nanoparticle (LSN) and Cu-loaded silicate Nanocomposite (CSN) were prepared taking MMT as the raw material. The adsorptive and antibacterial activity of CSN on aquacultural pathogenic bacteria was studied. Skin, gill and intestinal epithelial cells were used to study the adhesion of Aeromonas hydrophila to fish epithelial cells and the effects of CSN on the adhesion of bacteria and on cell membrane injury due to A. hydrophila. Feed experiments were conducted to study the applied effects of CSN in Nile tilapia. Effects of CSN on water quality and bacteria flora in water were also determined.The results were as follows:1. The average size of CSN was 41 ran. Most of the loaded Cu²⁺ entered into the interlayer position of silicate as hydrated or composite cation by ion exchange, and a small fraction penetrated into Si-0 tetrahedral and Al-O octahedral micro-pore by chemical adsorption, suggesting that the loaded Cu²⁺ reduced the negative charge of silicate, which could enhance electrostatic interaction between CSN and bacteria. Ascompared to the LSN, the external surface area, pore volume and pore diameter of CSN were relatively higher, while total surface area, micro-pore surface area and micro-pore volume were lower implying that treatment with Cu2+ could change the surface characteristic of silicate nanoparticles.2. The Zeta potentials of of CSN was increased with increasing pH and transformed from the negative to positive value when pH=4.8. The Zeta potentials of LSN, A. hydrophila, P. fluorescens, V. parahaemolyticus were all decreased with increasing pH in the range of 2—10, The Zeta potentials of bacteria, LSN and CSN were all increased with increasing ionic strengt in the range of 0.0001⁰.1 mol/L NaNO3 at constant pH 6.0.3. The results of in vitro bacterial-mineral adsorptive trial showed that the adsorption of bacteria onto LSN and CSN reached equilibrium in less than 60 min and 120 min, respectively. The percent of bacteria adsorbed was 37.4-46.5% and 94.4-99.4%, respectively. The extent of bacterial adsorption onto LSN was pH dependent, decreasing with increasing pH in the range of 2—10. However, the bacterial adsorption onto CSN was decreased with increasing pH in the range of 2 ^- 4.8 and increased with increasing pH when pH>4.8. The bacterial adsorption onto LSN and CSN was both decreased with increasing ionic strength in the range of 0.0001 ⁰.1 mol/L NaNO3.4. The results obtained from antibacterial trial in vitro indicated that LSN had no antibacterial activity. The minimum inhibitory concentration and bactericidal concentration of CSN on A. hydrophila, P. fluorescens, V. parahaemolyticus were 150, 150, 75 mg/L and 600, 600, 300 mg/L, respectively. The result of the Cu release from CSN into TSB showed that in the first 2 hours, Cu concentration in the supernatant reaches saturated value, about 1.22-2.27 % of the overall Cu in CSN suspended in the medium. Enzymatic activity of bacteria was examined and the results showed treatment with CSN could lead to significant release of intracellular enzymes from the tested bacteria. The rate of oxygen consumption was measured using a SP-II-type oxygen electrode analyzer and data from oxygen consumption of bacteria showed that CSN inhibited the TCA pathway of the bacterial respiration metabolism. The mechanism for the antibacterial activity of CSN may involve the enhanced affinity of CSN for bacteriaand the antibacterial activity of Cu2+. Moreover, CSN could rupture bacterial wall, and then the contents in cell leaked and bacterium died.5. Skin, gill and intestinal epithelial cells were developed using primary cultures of mucosal cells (isolated from healthy Nile tilapia) to study the adhesion of A. hydrophila to fish epithelial cells and the effects of CSN on the adhesion of bacteria and on cell membrane injury due to A. hydrophila. CSN significantly inhibited adhesion of bacteria to skin, gill and intestinal epithelial cells. The cell membrane was injured due to A. hydrophila. The activity of cytosolic free calcium concentration and membranous phospholipase A2, extracellular LDH and cell damage percent (indicating cell membrane permeability), cell membrane fluorescence polarization and microviscosity (indicating cell membrane fluidity) were all increased significantly. CSN can significantly reduced cell membrane injury caused by the adhesion of A. hydrophila.6. A total of 450 Nile tilapia at an average initial body weight of 3.9 g were used to investigate the effects on growth, digestion, microbial ecology and immunity. Fish were allocated to five dietary treatments (three replicates of 30 fish/tank) for 56 days. The dietary treatments were: 1) control, 2) control+30 mg/kg copper as CUSO4; 2) control +0.15% LSN; 2) control +0.15% CSN; 2) control+4 mg/kg Flavomycin. The results were as follows: (1) As compared with the control, supplementation with CSN significantly improved growth performance and survival rate, reduced the total intestinal aerobic bacterial counts, affected the composition of intestinal microflora with a tendency of Aeromonas, Pseudomonas, Flavobacterium, Acinetobacter, Alcaligence, Enterobacteriaceae, Vibrio decreasing, improved the apparent nutrient digestion and the activities of the intestinal digestive enzymes, increased villus and microvillus heights at different sites of the intestine, increased the phagocytic activity of leukocytes, lysozyme activity and percentage of erythrocyte C3b receptor, increased Na, K-ATPase activity in gill. (2) Supplementation with LSN has a tendency to improve growth, digestion, microbial ecology and immunity. The apparent digestibility of dry matter and crude protein, intestinal protease, and villus and microvillus heights at hindgut and midgut were significantly improved. (3) Supplementation with Flavomycin significantlyimproved average daily weight gain and specific growth rate, reduced the total intestinal aerobic bacterial counts, improved the apparent digestibility of dry matter and crude protein and intestinal protease, increased phagocytic percent of leukocytes. (4) Supplementation with CUSO4 had no significant effect on on growth, digestion, microbial ecology and immunity.7. A total of 180 Nile tilapia at an average initial body weight of 43.5 g were used to investigate the effects of CSN on water quality and bacteria flora in water. Fish were allocated to two dietary treatments (three replicates of 30 fish/tank) for 15 days without replacing water. The dietary treatments were: 1) basal diet, 2) basal diet +0.15% CSN. The results showed that supplementation with CSN significantly improved growth performance and survival rate. The ammonia (NH4+-N), nitrates (NO3^-N), nitrites(NO2 -N), CODmii and the total aerobic bacterial counts in the cultured water werereduced. The composition of microflora in the cultures water was affected by CSN addition, with a tendency of some gram-negative bacteria decreasing and some gram-positive bacteria increasing.The results above suggest that CSN exhibits antibacterial activity. It can inhibit effectively the growth and proliferation of pathogenic bacteria in the intestinal tract. At the same time, CSN has the ability to prevent bacteria from adhesion to intestinal epithelial cells, which can protect intestinal mucosa from invasion of pathogenic bacterium and toxins. These actions of CSN result in a positive effect on the health and function of intestinal mucosa, and then promote the digestion and absorption of dietary nutrition, also improve immunity function. The improvement of feed efficency results in lower feed excretion to the water. CSN excreted from feed and dejecta to the water has antibacterial activity too.