INTRODUCTION:

Nanoparticle technology has been a very important field to study for its importance in different branches of science, such as medical diagnosis and therapy (1). Silver nanoparticles (AgNPs) are among the well-known nanoparticles with properties of antibacterial activity. The common methods of (AgNp) production are mainly physical and chemical methods which both have many disadvantages (2). On the other hand, biosynthesis of AgNps come with many advantages such as its simple process in living organisms that mimic bio machines, they possess enzymes which they use in the process of producing the nanoparticles. This can be seen in some fungus and bacterial species (3). Biomolecules or substances extracted from plants and algae are also used in the process of nanoparticle production (4).

In the case of golden apple snail egg extract, it is considered as an important reducing and stabilizing material, due to its low cost, widely availability and its high content of proteins (5). The golden apple snail (Pomaceacanliculata) naturally spread on the edges of water in rivers, their eggs are highly filled of polysaccharides, proteins, and carotenoids (6), these snails lay their eggs outside the water where their shells are cemented and presented with a bright pink color (7).

The good source of reducing and stabilizing agent in these eggs make it an interesting material for biosynthesis of AgNps. In this study, we investigate the ability of egg extract to produce AgNps and examine it as an antibacterial and antibiofilm agent.

MATERIAL AND METHODS:

Collection of golden apple snails:

Fresh apple snail eggs were collected from water rims in summer from Diayla providence/Iraq, transferred to the laboratory and washed with sterilized distilled water to reduce contamination then brought all together and weighed in a sterile container and prepared for further experiments, (Fig .1).

Figure 1: Pink clusters of golden snail eggs in its normal habitat.

Egg extract preparation:

Eggs (5gm) were crushed and homogenized, then suspended in 10ml distilled water. The egg solution was then centrifuged at 3000 rpm for 10 min., the supernatant was then transferred to a sterile clean plane tube which represent the egg extract of the golden apple snail (Fig. 2).

Figure 2: Pink homogenized egg extract.

Detection of antibacterial activity of egg extract:

The egg extract was diluted to the concentrations (2, 4, 8mg/ml) and these dilutions were used to detect the antibacterial activity of the egg extract. We used well diffusion method for detection of antibacterial activities of egg extract. Pathogenic bacteria (Staphylococcus aureus, Staphylococcus epidemidis, Salmonella, Escherichia coli, Klebsiella, Pseudomonas aeruginosa, Streptococcus pyogens and Enterobacter) cultures were prepared and inoculated in broth before (18 h), the concentration of each bacteria was subjected to 1.5*10⁸(CFU/ml) that confront McFarland tube (0.5 turbidity).

Sterilized Nutrient agar was poured in disposable Petri dishes and then seeded with previously cultured pathogenic bacteria, by using cotton swap, streaked on nutrient agar, after 5-10 minutes wells were punched by using the end of pasture pipette which was inserted in the agar to produce the well. The egg extract solution (200 µL) for each concentration was added. The solution spread through the agar, and then incubated in 37C̊ for 24 hr. Inhibition zone was measured in millimeters (mm)(8).

Production of AgNps by egg extract of golden apple snail:

To study whether the egg extract contain a reducing agent for further nanoparticle synthesis, the extract of apple snail eggs at different final concentration (2, 4 , and 8 mg/ml) was mixed with 1M AgNO3(0.25ml), in presence of 2 M glucose (0.5ml)glucose, then adjusting the reaction volume to 5.5 ml with deionized water. The solutions were continuously stirred at 60 C̊ for 48 hr. in the dark and formation of AgNPs was monitored at the wavelength of 300-700 nm (9) (Fig. 3).

Figure 3: Synthesized AgNPs characterized as deep brown color after 48 hr.

Characterization of synthesized AgNPs:

The morphology and size of the AgNPs synthesized by egg extract of the golden snail was analyzed by pictures taken by Atomic force Microscope (AFM) which permits resultant microscopic information on to plot topographies showing the surface alleviation and the structure of surface (10). The sample was imaged over a wide space detecting dimensions at higher resolution. Pictures were taken with the Scanning Probe Microscopy (SPM).

Antibacterial activity of AgNps by well diffusion method:

Dilutions of AgNPs were prepared from the stock concentration (200mg/ml). Four double dilutions were prepared (100, 50, 25, 12.5 mg/ml). We used well diffusion method for detection of antibacterial activities of AgNPs which include prepared bacterial culture (mentioned previously).

Sterilized Nutrient agar was prepared (mentioned previously). The AgNPs solution (200µL) for each concentration was added. Waited until the solution spread through the agar, and then incubated in 37C̊ for 18 hr. Inhibition zones were measured in (mm).

Results were subjected to the student's t-test, P< 0.001 and P<0.05 was considered as significant.

Antibacterial activity of AgNps by broth dilution method:

Antibacterial activity of the AgNPs was examined by broth dilution method against some Gram negative and positive bacteria (mentioned previously) each bacterium was first inoculated in a 10ml nutrient broth tube and incubated at 37 C̊ for 18hr.Same dilutions of AgNPs previously prepared were used, 100µl of each AgNp concentration was mixed with the same volume of bacterial culture after adjusting its concentration to 1*10⁶CFU/ml in a 96 well plate and cultured at 37C̊ for 24hr. the lowest concentration of AgNPs that showed no bacterial growth was detected as the lowest inhibitory concentration (MIC) (9).

Volumes of 100 µl was poled from the bacterial cultures at MIC value as well as two higher concentrations and were spread on nutrient agar plates, which were incubated at 37C̊ for 24hr. The lowest concentration of AgNPs that killed 100% of the initial bacterial growth was determined as the minimal bactericidal concentration (MBC).

Detection of antibiofilm activity of AgNps:

The ability of AgNPs to inhibit biofilm production were evaluated by using crystal violet staining technique in polystyrene microtiter plats and then Optical density (O.D) was determined at 490 nm (11).

Overnight cultures of (pathogenic bacteria mentioned previously) in trypticase soy broth (TSB) supplemented with (1% glucose) were diluted to 10⁸ CFU/ml. Individual wells offlat-bottomed 96 well polystyrene plates were filled with 100 µl aliquots of the cultures then 100µl of each AgNPs concentration was added which were further incubated for 24 hr. at 37C°, next the wells were washed 3 times with 200 µl of sterile phosphate buffer saline (pH: 7.2). Biofilms were fixed with heating at 60 C° during 15 min. 200 µl of crystal violet solution (0.1% wt. /vol.) was added to all wells and left for (15 min). Excess crystal violet was rinsed with distilled water and air dried overnight. Bounded crystal violet was released by adding 200 µl of 96% ethanol. Absorbance was measured spectrophotometrically at 490 nm (A₄₉₀). The test was performed in duplicates, negative control wells contained TBS only. The results were calculated according to (12), where absorbance was proportional to biofilm biomass.

RESULTS AND DISCUSSION:

Antibacterial avtivity of egg extract:

Results showed no effect of egg extract on the growth of pathogenic bacteria, this result was also reported by other studies (13) which indicated the toxic effect of the egg extract only on vertebrate organisms especially rats and mice (14) due to its protienetious neurotoxin it contains beside a high percentage of polysaccharides (15).

Characteristics of synthesized AgNPs:

Atomic force microscopy technique refers to digital images that allow quantitative measurements of surface features, such as root mean square roughness (Rq), or average roughness (Ra) and the analysis of images from different perspectives including 3D simulation (16). (Fig. 4a,b) illustrates the two and three dimensional AFM images of the AgNPs synthesized by egg extract of golden snail. Average grain sizes in diameter of AgNPs was 58.98 nm which was calculated by granularity cumulating distribution chart (Fig.5). It is important to note that the mean values were obtained. This size was larger than AgNPs synthesized from algae or plant extracts (1-30 nm) but less than those synthesized by bacterial sources which are 100nm and more (18).

Figure (4): AFM images of AgNPs. Two dimensional (a) and three dimensional (b).

Figure 5: The granularity cumulating distribution chart of AgNPs synthesized by egg extract of golden snail

Antibacterial activity of AgNPs:

Results illustrate a variation impact of AgNPs synthesized by egg extract on both Gram negative and positive microbes. This could be due to the structure composition of the cell wall between Gram positive and negative bacteria particularly peptidoglycan. The highest inhibition zone was detected against Staphylococcus aureus (22.5mm) in the Gram positive group of pathogenic bacteria, Fig (6a) whereas the highest inhibition zone against Gram negative bacteria was against Klebsiella (25mm) using the well diffusion method, (Fig.7 a), (Table 2).

Table 2: Inhibition zones of AgNPs against pathogenic bacteria.

Name of bacteria	Inhibition zone (mm)					t-test	P value
	mg/ml
	12.5	12.5	12.5	12.5	12.5
Escherichia coli	12.0	12.5	13.5	15.0	16.5	9.23	0.002**
Klebsiella pneumonae	14.0	15.5	16.0	19.5	25.0	6.8	0.04*
Enterobacter	9.0	10.5	10.5	12.0	14.5	12.5	0.007**
Pseudomonas aeruginosa	11.5	12.0	12.5	14.5	17.0	10.9	0.002**
Streptococcus pyogens	15.0	21.0	21.0	22.0	22.0	12.2	0.003**
Staphylococcus epidermidis	21.0	22.0	23.0	23.0	23.5	11.9	0.003**
Staphylococcus aureus	19.0	16.0	17.0	17.0	22.5	4.2	0.03*
Salmonella	13.0	16.5	18.0	19.0	21.0	5.7	0.05*
LSD	55.6	110	85.9	66.8	76.5		0.000**

** P<0.01 ; *P<0.05

These results were similar to other studies that used other sources to synthesize AgNPs such as plant extracts (18). Nanoparticles are very important sources of antibacterial activity and has been used in different medical applications as an effective material, economic and do not cause bacterial resistance, but its safety has been a controversial issue between scientists, because we actually do not know what is the fate of the material inside the body, some papers referred to its risk in triggering cancer or causing organ failure which is also an unclear subject and need further studies.

MIC and MBC estimations of produced AgNPs against bacteria were assessed. The MIC of AgNPs against S.pyogens was 6.25 mg/ml and MBC was 12.5 mg/ml , while the MIC of AgNPs against both S.epidermis and S.aureus was 12.5 mg/ml and the MBC was 25 mg/ml, whereas the MIC of AgNPs against all Gram negative bacteria used in this study was 25 mg/ml and the MBC was 50 mg/ml. these results show the effect of AgNPs produced by egg extract was more effective on Gram positive bacteria at higher dilutions then its effect on Gram negative bacteria. There are different reasons behind this effect; due to structural characteristics of nanoparticles they can inter the cell wall of bacteria and shut down enzyme and protein functions that cause oxidative damage, DNA degradation and finally cell death (19). The antibacterial activity of nanoparticleswas also related to its capacity to increase membrane permeability of the bacteria with an induction of free radicals (20).

One of the important advantages of utilizing AgNPs is its quick and simple production, it very well may be created from various common sources with very good quality, it additionally does not need special trained team or expensive material therefore it is a good and safe choice to be used in epidemic zones (21). AgNPs can be the future of alternative use to antibiotics that show high resistance from pathogenic bacteria since the prolonged exposure to AgNPs has not shown resistance against pathogenic bacteria moreover it has a broad spectrum activity as it targets many locations on or in the bacterial cell, Although long term studies are as yet required for the toxicity, carcinogenicity and different impacts of AgNPs which are necessary to clear up and support the safe utilization of such material in treatment of infectious diseases (22, 23).

Antibiofilm activity of AgNPs:

Antibiofilm activity of AgNPs synthesized by egg extract of golden snail was detected in this study by using the crystal violet microtiter plate method, the results showed a decrease ability of biofilm production as the concentration of synthesized AgNPs is increased, the O.D value decreased significantly to zero when AgNPs reaches 100mg/ml and more in the Gram positive bacteria group (P≤0.01),( Fig.8,a) whereas in the Gram negative bacteria it reached zero when the concentration of AgNPs reaches 50mg/ml and more (P≤0.01) (Fig.8,b).

Figure 8: Mean OD absorbance values of Crystal Violet in Gram positive bacteria (a); and Gram negative bacteria (b) grown in Tryptic soy broth at different concentrations of AgNPs.

Biofilms are extracellular polymeric matrix which is produced by bacterial communities; these biofilms can be found on different kind of surfaces such as living tissues, medical devices, water pipes and others (24). Biofilms are known to increase pathogenicity and antibiotic resistance in a wide range of pathogenic bacteria such as P.aeruginosa, E.coli, Klebsiella, And others. Immune response can be activated by biofilm components of such as cyclic dinucleotide which are recognized by the pattern recognition receptors of the immune system, this sort of immune response trigger the activation of IFN-1 by the host (25).

A few methodologies have been introduced to clarify the antibiofilm activity of bacterial isolates; some incorporate the interference with cell to cell communication which is known as quorum sensing inhibitors (26). There are additionally compounds, for example, antibiofilm polysaccharides produced by bacteria that demonstrated great promise as inhibitors of biofilm production (27). Another strategy used to control biofilm synthesis was the utilization of bacteriophage treatment by penetrating biofilm and directly kill host bacteria or produce enzymes that degrade the exopolysacharide matrix resulting in losing the intact structure of the biofilm (28).

Nanotechnology has achieved to prevent biofilm production through the use of nanoparticles which alter the function of surface biomaterials either by coating (29) or by impregnation (30), also by embedding nanoparticles (31). The increased efficacy of these nanoparticles is due to its permeability through the cell envelope. AgNPs are among the most nanoparticles that show antimicrobial impact which is known to be less dangerous than silver ions. AgNPs have been utilized as a major aspect of sensors for determination of chronic diseases and in delivery of therapeutic agents (32). The shape and size of the AgNPs may change its bactericidal effect, as long as the particle size is smaller the surface area of the AgNPs become larger, this will prompt to increased material interaction with the surroundings (33).Zeta potential affect the biological activity of AgNPs by assuming an important role in the capacity of such particles to infiltrateinside the cell (34).Zeta potential has been demonstrated an effect on biological activity of AgNPs by assuming an important role in the capacity of such particles to infiltrate inside the cell (35).

CONCLUSION:

The egg extract of the apple snail was used as an alternative of plant extracts and chemicals for the production of nanoparticles, the egg extract contained different components that have a reducing activity to reduce Ag+to Ag0. The egg extract alone was not effective against pathogenic bacteria, while when used to transfer AgNO₃to AgNPs the nanoparticles showed antibacterial and antibiofilm activity against pathogenic bacteria. The AgNPs synthesized by egg extract of the golden snail may be considered as a promising method to produce antibacterial and antibiofilm substances which may solve many problems in the field of infectious diseases, treatments, disinfectants or water purification.

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Received on 25.02.2019 Modified on 21.03.2019

Research J. Pharm. and Tech. 2019; 12(7):3444-3450.

DOI: 10.5958/0974-360X.2019.00583.3