Fish diseases are a major obstacle to the sustainable development of the fisheries sector. Aeromonas hydrophila is a major infectious aquatic pathogen, believed to be responsible for ulcerative disease, fin rot, tail rot, and fish septicemia and has developed resistance to many available antibiotics. To manage the health of the fish suggests more effective prevention than cure. Disinfection technique does not remove all potential pathogens in the ecoregion of fish. Stressful conditions such as suboptimal water quality, poor nutrition and immunosuppression create an environment conducive to opportunistic bacteria such as A. hydrophila [ 6 ], và các chất chống vi trùng như kháng sinh thường được sử dụng để ngăn ngừa dịch bệnh bùng phát. Tuy nhiên, báo cáo gần đây về A. hydrophila từ các mô cá khác nhau cho thấy mầm bệnh đã phát triển khả năng kháng với nhiều loại kháng sinh như amoxicillin, ampicillin, lincomycin, novobiocin, oxacillin, penicillin, rifampicin và tetracycline [ 7], and therefore, research efforts to find antibiotic alternatives are gaining momentum [ 8 ].Nanosilver (AgNPs) are medically and bioavailable nanoparticles. They are widely used in commercial products for wound dressings, diagnostics, treatments, catalysts, biosensors, air and water purifiers, paints, food packaging. [ 9 – 14 ] v.v … Nanomaterial synthesis is the main and most important concern in nanotechnology research and application.
Among a large number of aquatic pathogens, gram-negative Aeromonas hydrophila has been identified from a variety of freshwater fish, and is capable of growing in both aerobic and anaerobic conditions . Accelerates outbreaks of various fish diseases such as ulcers, fin rot, tail rot, septicemia, etc.. [ 3 , 4 ]. In humans, it is reported to cause various water-mediated gastrointestinal infections in children and immunocompromised people . Its infectivity increases with environmental pollution, high water temperature and the addition of aquatic stressors.
Nanosilver (AgNP) are medically and bioavailable nanoparticles. They are widely used in commercial products for wound dressings, diagnostics, treatments, catalysts, biosensors, air and water purifiers, paints, food packaging. [ 9 – 14 ] v.v … Nanomaterial synthesis is the main and most important concern in nanotechnology research and application.
Many conventional chemical processes are available to synthesize AgNP, but many additional chemicals and substances that play a key role in the synthesis and enhancement of stability have been found to be environmental pollutants. and toxic substances, thus detrimental to the flora. The biosynthesis of AgNPs involving environmentally benign biological substances is a relatively new approach [ 15– 17 ],
Nanosilver materials and synthesis methods
Synthesis of nanosilver
The fresh leaves of mango, banana, papaya and eucalyptus are locally collected and washed thoroughly in tap water followed by distilled water twice. Leaf broth was prepared by taking 2 g of leaves in 50 ml of deionized water and heating for 45 minutes at 55 ° C. Leaf extracts were collected by decantation and filtration of the broth. 2 ml of each was added separately to 20 ml of 1 mM aqueous solution of silver nitrate, and the reaction mixture was maintained at ambient temperature until a change in color of the mixture was observed. yellow-brown color as a confirmation of nanoparticle formation.
Properties of AgNPs by spectroscopy analysis UV – Vis
The reduction and conversion of Ag + ions to AgNPs were evaluated by recording the absorbance of the reaction mixture at 380–520 nm using UV-Vis spectrophotometer (UV-1800, Shimadzu).
Features of HR-TEM and EDX
The size of the Nanosilver were determined by HR-TEM (JEM 2100, JEOL) active at 300 kV and their elemental components were determined by EDX spectroscopy (INCAx-Sight, Oxford Instruments) connected to HR. -STAMP.
Antimicrobial test of AgNPs
Silver nanoparticles have been tested for their antibacterial properties by a good diffusion method. A. hydrophila culture was obtained from the Fish Health Management Division of the Central Freshwater Aquaculture Institute (CIFA), Kausalyaganga, Bhubaneswar. Bacteria were regenerated and cultured in LB medium. Five wells with a diameter of 6 mm are made up of two copies per dedicated LB agar plate. Four wells were loaded with 50 μl AgNP, with a concentration of 153.6, 76.8, 30.7 and 15.3 μg / ml of the original nanoparticle solution, and a fifth well with equivalent volumes of leaf extract pure (as control). The cultures were incubated at 37 ° C in an incubator for 48 h, and the diameters (in mm) of clear areas were measured to show qualitative as well as quantitative performance.
Results and discussions on the efficacy of nanosilver
The experiment describes that all formulations exhibited color development with different incubation times. The time period for the color to turn brown or golden brown, showing nano-silver formation in aqueous solution varies from 3 to 4 hours in eucalyptus and papaya; 4–5 hours for the mango and over 6 hours for the banana. This color change pattern marks the conversion of bulk silver to AgNPs that occurs due to surface plasmon resonance . The difference in the times these reaction mixtures are observed for color development may be due to differences in the reduction capacity of the leaf extracts due to the different composition of the leaf extracts. them.
The extracts of the mango, eucalyptus, banana and papaya leaves synthesized the nanoparticles with the maximum absorption at 442, 465, 454 and 442 nm, respectively. Except for nano-silver synthetic papaya leaf extract, synthesized after 72–96 hours of synthesis, the other substances were relatively stable. All the AgNPs synthesized were stable for 24 hours in addition to color change as shown by a graphical representation of absorbance and wave length at defined time intervals (Fig. 2). The wavelength relative to the absorption curve shows that over time the absorption maximum shifts to the 420–450 nm range, due to the progressive formation of silver nanoparticles. The expansion of the peaks confirmed the multi-dispersion nature of AgNPs.
Figure 2. UV-Vis spectroscopy of AgNPs in synthesis using an Eucalyptus, b mango, papaya and banana leaf extract
Elemental size and composition analysis
Part 3 data, presenting HR-TEM, SAED, EDX spectra of AgNPs synthesized from leaf extracts of mango, eucalyptus, papaya and banana respectively. HR-TEM microscopy images of AgNPs extracted from mango, eucalyptus, papaya and banana show their sizes and shapes as 50–65 and ovoid, 60–150 and oval, respectively 25–40 and round, and 10–50 nm and irregular. Since the size of the ‘banana’ silver nanoparticles is the smallest (10–50 nm) and that of the ‘eucalyptus’ is the largest (60–150 nm), their variable size is due to differences in chemical composition. by phytoextracts. Banana peel extracts synthesized gold nanoparticles (AuNPs) have been reported in the 300 nm range [ 19 ]. An encouraging trend in the current study is the smaller synthesized ‘banana’ AgNPs. An earlier study [ 20] the size and shape of AgNPs synthesized Eucalyptus hybrida leaf extract were 50–150 nm and shape, while similar data in the present study were 60–150 nm and oval. Energy scatter X-ray spectroscopy for determining and confirming the elemental composition of the elementary element in nano silver showed a sharp peak at 2.9 keV in all four EDX spectrophotometers, thus confirming the Nanoparticles are of silver. Another vertex describes the presence of copper, a basic material in the grid. It is clear that phyto-extracts contain various compounds that, in addition to being reducing agents, also act as capping agents. The collective effect of the constituent proteins, enzymes, carbohydrates and vitamins can reduce silver ions [ 15 – 17]. FTIR analyzes have shown that polyols such as hydroxyl flavones and catechins, etc. The availability in the leaves reduces silver ions during nanoparticle formation [ 21 ].
Figure 3.a Size, b size distribution diagram, c HEED and d EDX of AgNPs synthesized from Eucalyptus leaf extract
Figure 4.a Size, b size distribution chart, c HEED and d EDX of AgNPs synthesized from mango leaf extract
Figure 5.a Size, b size distribution chart, c HEED and d EDX of AgNPs synthesized from papaya leaf extract
Figure 6.a Size, b size distribution diagram, c HEED and d EDX of AgNPs synthesized from banana leaf extract
Thử nghiệm kháng khuẩn
The leaf extract alone (without Ag + treatment) did not show any significant antimicrobial activity against A. hydrophila as the Nanosilver showed the same way (Fig. 7). AgNPs of ‘papaya’ showed the highest activity and from the eucalyptus tree showed the least. The regions for inhibiting AgNPs are shown in Table 1. It is thus clear that the nanoparticles were significantly active against A. hydrophila, possibly due to a variety of mechanisms. Ag + reduces the uptake of phosphates in Escherichia coli and promotes the outflow of cumulative phosphates as well as that of mannitol, succinate, glutamine and proline, and disrupts the proton dynamics thus causing microbial cell death. [ 22 ]. Studies also suggest that AgNPs can interact with sulfur-containing proteins and inactivate disulfide-linked amino acids. In addition, these particles can form holes in the cell wall that make it easy to seep through and thus lead to the destruction of bacteria. [ 23]. Smaller nanoparticles with a larger ratio of surface area to volume are more harmful because of the large surface area of interaction [24, 25]. Therefore, the antibacterial activity of Nanosilver is dependent on shape and size. The bonding of AgNPs is also dependent on its available surface area. Triangular and truncated silver nanoparticles were the most powerful biocide , similar to the findings in the current study (Table 1) at a concentration of 153.7 μg / ml. Consequently, the change in antimicrobial activity is due to the smaller size of the ‘papaya’ AgNPs and the larger size of the eucalyptus-derived plant, consistent with previous reports. [ 27 ].
- The adjustment for virulence factors is very important for the infectious and transmissive processes of the pathogen. Bacterial virulence factor is reported to be under the control of signaling molecules such as acylated homoserine lactone (AHL) produced during cell density-dependent phenomenon (QS). [ 28]. Aeromonas hydrophila has also been discovered to produce such molecules for growth in hosts [ 29 ]. Furthermore, it has become increasingly resistant to almost all currently recommended antibiotics due to the development of defense mechanisms against antibiotics by exploiting their large genetically modified source. [ 27]. Therefore, it is important to target these virulence elements with molecular weapons produced by other microorganisms to prevent infection by this bacterium and subsequent diseases. [ 30 , 31 ].
Fig. 7 Antibacterial activity of nanosilver synthesized using an Eucalyptus, mango, papaya and banana leaf extract, against Aeromonas hydrophila. Wells ii, iii, iv and v were loaded with 153,6, 76,8, 30,7 and 15,3 μg / ml AgNP, while well i (control) was in each case loaded with whole leaf extract alone. corresponding substance.
Nanosilver have been reported to inhibit biofilm formation by disrupting the QS signaling . It also acts intracellularly across multiple sites to inactivate important physiological functions such as cell membrane transport, nucleic acid synthesis and transcription, protein function and folding, and electron transport [22, 23]. Therefore, since it has to undergo simultaneous mutations at every important function in a single generation to get rid of the influence of the antimicrobial agent, it is difficult for any microorganism to develop resistance. nanosilver again [ 33 ]. Several phytochemicals known as de-quorumants (QQ) have been reported to inhibit the QS signaling. [ 34]. Therefore, phyto-synthesized AgNPs may have natural and synthetic QQ accumulation effects for application in aquaculture as a potential candidate as a drug and / or antiseptic for infection treatment. A. hydrophila and related diseases.
Research on biodegradable polymers for the controlled release of nanoparticles in field conditions is gaining momentum. Integrated processes for the production of bioenergy and biofilmers are also becoming increasingly important. Fictional works on PHB production by Bacillus not photosynthesizing under different culture conditions [ 35 ], and generation of nanoparticles by B. thuringiensis under modified physiological conditions  has been recently reported. Another recent study compared antibacterial treatments of packaging films through gamma irradiation and Nanosilver impregnation. [ 36 ] Reported that the total number of aerobic aerobic bacteria, Enterobacteriaceae, E. coli and Clostridium perfringens showed a greater inhibitory zone when irradiated at 4 kGy without AgNPs and at 2 kGy with nano silver impregnation, this is more reinforces the antimicrobial view of AgNPs.
Arabinda Mahanty, Snehasish Mishra, Ranadhir Bosu, UK Maurya, Surya Prakash Netam, and Biplab Sarkar