ABSTRACT
Microbial diseases have always been proved destructive to human health and economy. Many drugs are available against harmful bacteria, but they are developing resistance against these drugs day by day so, we need more antibiotics with environment friendly nature. Natural resources for these new drugs are considered to be more suitable than synthetic ones. Spiders of genus Argiope have been proved in this work to produce silk that has antimicrobial nature. Web silk and egg silk extracted from these spiders were dissolved in six different solvents (methanol, ethanol, acetone, distilled water, 5% NaOH and urea solution) and checked for the antibiotic potential against four different bacterial species, Pseudomonas aeruginosa (gram negative), Klebsiella pneumoniae (gram negative), Escherichia coli (gram negative) and Staphylococcus aureus (gram positive). Results of present study revealed that web silk dissolved in all solvents except distilled water inhibit the growth of tested bacteria. The observed inhibition zones didn’t vary from zones of negative control group. However, observed diameter of inhibition zones for positive control group were significantly higher than negative control and treatments. It is concluded from the present study that web and egg silks of genus Argiope don’t have antibacterial activity. The inhibition zones observed in case of silk dissolved in different solvents were due to antibacterial activity of these solvent.
INTRODUCTION
1.1 Background:
Bacterial diseases and infections are among the most common fatal conditions of human populations. According to WHO (2017) 56.4 million deaths occurred worldwide in 2015. Among these, half were due to infectious diseases. Microbes are not only responsible for health problems. These are also the major causes of food spoilage (Gram et al., 2002) and about 25% food throughout the world is lost annually due to microbial attacks (Bondi et al., 2014). Number of efforts were made to cure bacterial diseases and prevention of bacterial destructions. Such as the discovery and production of chemicals against these pathogens, but there exists a rapid evolution in this organism and it developed rapid tolerance against the applied chemical (Okeke et al., 2005). So, pathogens became multi-drug resistant and the main cause for resistance is misuse and over use of broad-spectrum antibiotics (Zuridah et al., 2008).
1.2 Statement of the Problem:
World health organization had described 12 different families of bacteria which became multidrug resistant and against them new antibiotics are required urgently (WHO, 2017). So, there is a dire need to discover new antibiotics. Many antibiotics are reported to be present in animal and their products (Hoskin & Ibrahim, 2012; Hoskin & Ramamoorthy, 2008). One of the major biological sources for these antibiotics is “Spider silk” (Roozbahani et al., 2014).
1.3 Objectives of the Study:
Objectives of the study are as follow.
- Collection of spiders of genus Argiope.
- Recovery of silk.
- Checking for the better solvent to dissolve silk.
- Evaluating the Argiope silk for antibacterial potential by disc diffusion method.
1.4 Scope and Significance of the Study:
This work may prove beneficial to improve the economy of Pakistan by controlling epidemics, food poisoning and other destructions made by bacteria in future.
1.5 Outline of the Study/ Organization of Thesis:
Spider silk consists of fibrous protein and is acidic in nature (Altman et al., 2003). The first report for the antibiotic activity of spider silk due to its acidic nature was given by Heimer (1988). Silk is also important for its maximum mechanical strength (Bourzac, 2015), biodegradability (Rising, 2014) and biocompatibility (Gellynck et al., 2008; Vollrath et al., 2002). So, the silk producing spiders are important for further researches on antibiotics.
Spider’s evolutionary history is about 374-380 million years old and the known oldest spider is Attercopus fimbriamguis (Selden et al., 1991). According to World Spider Catalog (Version 20.0, 2019), now we have about 48,053 species of spiders with 3935 genera and 117 families throughout the world. Spiders are cosmopolitan in distribution. These have a large range of habitat (Foelix, 1996) and the global distribution ranking status of spiders is at 7th position (Coddington & Levi, 1991). Among such a large diversity of spiders about 41,000 species was reported in 2010 which can produce silk (Agnarsson et al., 2010) but up till now this figure is further increased.
Spiders produce different kinds of web according to specific role of the web such as for the attraction of prey, capturing of food, prey enclosing for storage, the sensation of vibrations in silk and egg capsule formation (Hoefler, 2007; Winkler & Kaplan, 2000). Different spider species produce non-identical web in distinct manners that depends on their habitat, prey and lifestyle (Foelix, 1996). This web also protects the food of spider from external influences including microbial attacks against decomposition (Mishra et al., 2012).
The Araneidae (Clerck, 1757) spiders can produce seven different kinds of web (Andersen, 1970). These webs have various proteins with different properties, functions and uses. The organization of fibers to construct the silk structure is highly specific for their particular function. Such as “aciniform silks” are considered to be the hardest form of silk which is used to immobilize the prey and construct the egg sacs (Hayashi et al., 2004; Liivak et al., 1997). The maximum mechanical strength containing silks are produced by orb weavers and are known as “flagelliform silks” (Lewis, 2006). The function of this silk along with the “spiral capture silk” is to absorb the excess amount of mechanical energy produced by the prey when it collides the silk and become entangled in it (Lin et al., 1995).
Major ampullate gland produce the silk which has extreme elasticity and toughness and is known as “dragline silk” it is produced to form the radii and main frame of the web. “Glue like silk” is produced by Aggregate gland and cause the prey to stick with the web. “Cocoon silk” is produced by the cylindrical glands and is used to construct the egg casing. Aciniform glands produce “wrapping silk” which is used to wrap the captured prey (Lewis, 2006). These different kinds of spider silk have some proteins which are antibiotic in nature (Tahir et al., 2017). Spider store its food in silk cocoons in which the food remains preserved against the attack of fungus and bacteria due to the presence of antimicrobial substances in silk (Eberhard et al., 2006). Spider silk of some species have some proteins which are antibiotic in nature (Amaley et al., 2014; Chakraborty & Das, 2009; Mirghani et al., 2012; Roozbahani et al., 2014; Saravanan, 2006; Saleem et al., 2010; Simon & Goodacre, 2012; Sharma, 2014; Tahir et al., 2017; Wright & Goodacre, 2012).
Spider silk of family Araneidae has antimicrobial potential (Mirghani et al., 2012; Tahir et al., 2015; Tahir et al., 2017). It inhibits the growth of bacteria because these proteins are acidic in nature, so, they may show bacteriostatic effect (Saleem et al., 2010). The venom of some spiders and scorpions contain different proteins such as neurotoxins (Corzo et al., 2000), defensins, antimicrobial peptides, proteinases and phospholipases that have medicinal importance (Cordeiro et al., 2015). The use of spider silk by humans is historical for wound healing purposes because of its antiseptic nature, its ability to stimulate immune system and the presence of a large amount of vitamin K in it (Heimer, 1988). Phospholipids hydrate and potassium nitrate present in the spider silk prevent the growth of bacteria and fungi (Chakraborty & Das, 2009; Gomes et al., 2010). There are number of components in the spider silk that have antimicrobial potential such as soluble or non-soluble growth resisting factors and surface or structural proteins (Bhushan, 2011).
The mechanism through which potassium hydrogen phosphate resist bacterial growth was reported in detail, this substance is present in spider silk and release protons in the aqueous solution creating an acidic environment with pH of about 4 which is not suitable for many pathogens and fungi to grow (Heimer, 1988). Spider silk’s applications in medical field is increasing day by day (Bourzac, 2015). It is not only a natural source for the restriction of bacterial growth but is also bio-friendly with less chances for pathogens to develop resistance against it (Craig et al., 1997). This research was designed to check the antibacterial properties of spider silk belonging to genus Argiope.
REVIEW OF LITERATURE
The evolutionary origins of spiders are not completely known but we fossil record suggests that the Araneae was originated in the late Silurian (420 Ma). In the late Paleozoic (250 Ma) or in early Mesozoic there happened adaptive radiation of Anareomorphae (Coddington & Levi, 1991). The true spiders originated about 380-374 Ma (Selden, 1990) and Attercopus fimbriamguis is the oldest known species of true spiders. Order Araneae has three sub-orders named Mesothelae (350 species), Mygalomorphae (1,500 species) and Anareomorphae. First two contains almost 10% spider species while remaining 90% species belong to suborder Anareomorphae (Foelix, 1996).
There are some specific anatomical features that are common among all spiders and are considered as distinguishing characters for spiders. These are, body is divided into two segments the abdomen and the cephalothorax which are joined together through a stalk known as pedicel, eight legs, either six or eight eyes and spinnerets to release silk. Silk glands and spinnerets are among the most important part of spider’s body for research. But the body parts of spider that are of interest for this research are silk glands and the spinnerets. The silk is produced in silk glands which are located inside the abdomen, after its formation, silk is released out of the body through spigots which are basically the opening present on spinnerets. The spinnerets are located on ventral side of the posterior abdomen in the form of three pairs, the posterior, the median and the anterior. The head and thorax region of spider are not separate entities and are collectively known as cephalothorax on which all the body parts such as legs, palps, mouth parts, eyes are present while the spinnerets are present on the abdomen. Central nervous system, poison glands and stomach are located in cephalothorax while silk glands are located in abdomen (Roberts, 1995).
Chelicerae are located in front of the mouth and are used to bite, cut and inject the venom through the fangs present on chelicerae. One pair of palps is present behind chelicerae which are more developed in sexually mature males where they are functional in sperm transfer. Sperms on the male palps are deposited in a sperm web made up of silk. Four pairs of legs are present behind the palps and each leg is divided into seven segments which includes coxa, trochanter, femur, patella, libra, metatarsus and tarsus. Tarsus is the last segment on which claws are present which are used in locomotion on web, spinning of web and prey capturing. Web spinning spiders has three claws on each tarsus (Roberts, 1995).
The Araneae is a carnivorous group which mainly feeds on insects, sometimes on other arthropods and even can feed on other spiders (Wise, 1993). There exist variations in mechanical properties of silk produced by the same individual under different environmental conditions (Madsen et al., 1999; Vollrath, 1999). Intraspecific variability was larger than interspecific variability in amino acid composition of dragline silk of Araneidae (Work & Young, 1987). Orb-web spiders eat their web daily; they construct circular “spiral” web and can digest it even at the same day (Roberts, 1995). Silk is formed in a liquid state within the silk glands and it becomes solid when exposed to air, the weight of liquid silk is ten times lighter than its solid form and it is water soluble when it is in liquid form but becomes water insoluble when it solidifies (Foelix, 1996). Liquid silk produced in vitro lack the property of solidification, it only solidifies when it is produced naturally, with water as solvent at ambient temperatures (Vollrath & Knight, 2001).
Dragline silk is produced in major ampullate gland which is a complex system which consists up of a long tail and a sac like structure which leads to a funnel shaped tube which takes the shape of three loops in which fibers are formed and the whole system is covered by a sheath. These fibers are further processed within fine tubular system to recover extra water rapidly. Then it is released outside the body through spigots and hydrophilic silk then crystallize to attain hydrophobic property, due to this change in property silk release its water contents and becomes solidify. The speed and method of silk movement along the system is also important for the composition of silk (Vollrath & Knight, 2001). But still our understanding about the silk is not complete.
Ehrlich discovered the first antibiotic in 1909 which was named as “606 compound” so called because he was succeed to discover it after his 606th experiment on animals but it was not so much effective (Jones & Ricke, 2003). Penicillin was the first true antibiotic discovered in 1928 and introduced to the world in 1940s by Alexander Fleming, Professor of bacteriology at St. Mary’s Hospital in London, began the era of antibiotics (National Historic Chemical Landmarks, 1999). Now antimicrobials are searched against mechanisms of microbes that do not occur in humans (Norrby et al., 2005).
First report for the antibiotic potential of spider silk was given by Heimer (1988), it was stated that some antimicrobial peptides are present in spider silk which create an acidic environment in aqueous medium by releasing protons which make the pH of the medium round about 4 which is unsuitable for the microbes to grow. Potassium nitrate is present in spider silk which is also responsible to inhibit microbial growth and silk degradation (Heimer, 1988). So, it was an important discovery to cure diseases, transport of food, medicines, vaccines and many other products (Lammel et al., 2010).
Since 1988 until now the silk of spider remain the center of interest due to its antibiotic potential. It was observed that spider silk (Cyclosa confraga, Araneae: Araneidae) has anti-bacterial properties against both gram positive and gram-negative bacteria but the zones of inhibition of growth for Acinetobactor sp. (gram negative bacteria) are larger than those for Streptococcus sp. (gram positive bacteria) (Tahir et al., 2017).
Silk recovered from common house spider Tegenaria domestica (Araneae: Agelenidae) has anti-bacterial potential against gram-positive bacteria, Bacillus subtilis but no significant inhibitory potential against gram-negative bacteria, Escherichia coli. Further, the anti-bacteria potential against B. subtilis was observed to be short lived thus the inhibitory effect must be bacteriostatic in nature rather than bactericidal and because this silk shown no inhibitory effect against mammalian cells so it appeared to be just anti-bacterial (Wright & Goodacre, 2012), so, it is supposed to be a good therapeutic material for humans. Spider silk also has anti-fungal effect, for example silk recovered from Neoscona theisi inhibit fungus to grow on bread (Tahir et al., 2015).
In order to observe the anti-bacterial activity of spider silk, it must be dissolved in some suitable solvent such as ethanol, methanol, acetone and distilled water in different concentrations and the dilution time for each solvent must be specific. The effect of these dilutions was observed by disc diffusion method and acetone was reported to be the best solvent in which the silk inhibited the growth of both gram-positive bacteria, Bacillus subtilis and gram-negative bacteria Escherichia coli quite within similar magnitudes (Mirghani et al., 2012).
Similarly, during a study in 2017 spider silk was dissolved in ethanol, distilled water and acetone to check its antibacterial potential. Eight bacterial species, Enterobacter cloacae, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Proteus mirabilis, Bacillus subtilis, Staphylococcus aureus, Streptococcus spp, was used to test the effect of silk on their growth patterns and acetone was reported to be the best solvent for silk dilution to inhibit bacterial growth while the silk diluted in distilled water and ethanol showed no effect on bacterial growth. Even that, different concentrations of silk in acetone gave different diameters of inhibition zones and 40 mg/ml was observed to be the best concentration against gram-negative bacteria while 10 mg/ml concentration gave the lowest effect (Al-Kalifawi & Kadem, 2017).
Spiders, Pholcus phalangioides, grown in sterile conditions produce silk which has more anti-microbial effect for gram-positive bacteria, Listeria monocytogenes, than gram-negative bacteria, E. coli, and this effect was recorded through well diffusion method and macro broth dilution method (Roozbahani et al., 2014).
About 30 different spider webs was used to illustrate the antibacterial effect on gram-negative bacteria, Pseudomonas fluorescens, colonies of these bacteria shown no-growth regions or inhibition zones (Border et al., 2001). Anti-bacterial potential of web recovered from Crossopriza lyoni, daddy longlegs spider, for both gram-positive and gram-negative bacteria was recorded in 2009 (Chakraborty, 2009). Anti-bacterial activities are either due to the presence of soluble or non-soluble inhibitory substances (Suto et al., 1992) or owing to structural or surface properties of a material (Bhushan, 2011).
A very important study was made in 2014 to check whether spider silk actively inhibits and resist the growth of bacteria or is naturally resistant to microbes. Two experiments were made, first was for the presence of inhibitory agent if any and second was scanning electron microscope analysis to check whether microbes adhere to spider silk or not. Dragline silk recovered from Argiope aurantia was used against Escherichia coli gram-negative bacteria, Bacillus subtilis gram-positive bacteria and Pseudomonas aeruginosa, gram negative bacteria. Results of organic extraction from silk and bacterial grown curves showed the absence of any inhibitory agent in the dragline silk and the results of SEM showed low adherence of bacteria on the surface of dragline silk. Therefore, results shown that the silk extracted from Argiope aurantia did not actively inhibit the growth of bacteria but resist the adherence of bacteria (especially gram-negative) due to its unique surface properties (Sharma, 2014).
Another important study was made in 2011 according to which not all silk producing spiders can produce silk having antimicrobial potential, even among those spiders who produce antimicrobial silk, the inhibitory potential against microbes varies and different types of silk recovered from same spider have different antimicrobial potential. Many spiders were used in that research but only the egg silk of Pityohyphantes phrygianus and web silk recovered from Tegenaria domestica had significant antimicrobial property. The antimicrobial effect of T. domestica silk was reported to be short lived because it inhibited only 24 hours of growth and could not inhibited the growth at 48 hours. Whereas, the egg silk of P. phrygianus can inhibit the growth of microbes at any time stage. T. domestica could not inhibit the growth of funji. Spiders belonging to genera Zilla can inhibit the growth of bacteria but it is not significant. Genera Araneus and Lasiodora was also tested and recorded not to produce antibacterial silk. In the same research different treatments on the silk was also made which shown that if UV light was subjected to silk or it was treated with Proteinase K or heated at 80oC then it was no more potent with antimicrobial activity which shown that the antimicrobial agent of silk may be protein in nature. Tegenaria silk was tested against mammalian cell where it could not inhibit the growth of cells, and it did not cause autoimmune response in human cells, so, it can be used as medicine (Wright, 2011).
Spider web is traditionally used for wound healing purposes because it has a very good potential to heal wounds, re-epithelization and neovascularization (Kumari et al., 2012). Human immune system does not respond to proteins of spider silk because spider silk is a good biocompatible material. Recombinant spider silk (fusion proteins) is more biocompatible and has more potential for biomedical applications (Rising et al., 2011). Spider silk is an ideal biomaterial but its biomedical applications are not so much studied due to difficulties in its production on a large scale but the production of recombinant spider silk on required scale is possible (Widhe et al., 2011).
Three different and new fusion proteins (biomaterials) were designed through recombinant DNA technology by combining the nucleotide sequences for dragline spider silk with the nucleotide sequences for three different anti-microbial peptides. Nucleotide sequences for these three anti-microbial peptides were taken from human, i.e., the nucleotide sequences for human neutrophil defensin 2 (HNP-2), hepcidin and human neutrophil defensins 4 (HNP-4). Designed new fusion proteins were cloned, expressed and assessed for function and demonstrated as more reliable antibacterial chemicals against gram-negative E. coli and gram-positive Staphylococcus aureus (Gomes et al., 2011).
A similar effort was again made in 2016, AMPs Magainin I, Lactoferricin and a synthetic AMP (WGR) were fused with recombinant spider silk 4RepCT. The designed fusion protein was demonstrated to be more effective against Staphylococcus aureus but not significantly more effective against E. coli (Floderus, 2016).
The goal of all these researches is to discover new antibiotics in spider silk and then design the discovered drug artificially through genetic engineering and amplify it on a large scale to get the desired amount of antibiotic but there are many problems to amplify the discovered drug. The major problem is the limitation of knowledge about the true physical form of silk proteins stored in the silk gland at a high concentration before transform into silk fibers (Parent et al., 2018). In the same research it was demonstrated that in Latrodectus hesperus, or black widow spiders the dragline silk is stored in the major ampullate gland in the predominating form of two proteins (spidroins) and the concentration of these proteins is very high (26–28) (25–50 wt %).
All the researches mentioned above and many others had been made to discover more and more antibiotics because bacteria have very high and rapid rate of evolution against applied drug. So, each discovered new antibiotic is temporary due to the emergence of new strains of bacteria which resist this antibiotic thus we need to discover and establish new antibiotics continuously. Even that many bacteria become multi drug resistance due to the use and misuse of antibiotics (Levy & Marshall, 2004).
Bacterial rate of evolution is further accelerated due to selective pressure exerted by the use of synthetic antibiotics (Witte et al., 2000). On the other hand, natural antibiotics are superior to synthetic antibiotics because biologically relevant chemical space is largely occupied by natural antibiotics (Rosen et al., 2009). Different strains of bacterial community form a biofilm which plays an important role against drug resistance, in such biofilms the community is embedded in self-secreted substances (Mohammed et al., 2013). So, the purpose of present work was to check for the presence antibacterial chemicals in spider silk Argiope.
RESEARCH METHODOLOGY
3.1 Principle:
This method is based on the fact that “if the solution of silk is antibiotic in nature then it must inhibit the growth of bacteria (bacteriostatic) on culture plate in the form of inhibition zone”. Bacteria was spread on agar plates then 6 mm diameter disks of filter paper placed upon the culture and the solution of silk was added on these disks, solvent of the respective solution was poured on another disk on the same plate as negative control. Prepared tetracycline disks of 6 mm diameter were used as positive control. Each culture plate contained an egg silk solution dose, a web silk solution dose, a negative control and a positive control. Applied agent would be proved bacteriostatic if it caused a zone of inhibition and the diameter of this zone show the intensity of effectiveness.
3.2 Sampling of Argiope:
For the study of antibiotic potential of silk recovered from spiders of family Araneidae, genus Argiope, spiders were collected from described locations by following certain key characters of these spiders such as the web of Argiope has a characteristic large and vertical orb or circular shape with very precisely arranged radial spokes, a characteristic zig zag fashioned silk pattern in this orb web can be seen, as in picture. 1, due to which these are commonly known as “Signature spiders”. Further, Argiope spiders align one pair of its legs with each of the four arms of a “X” when these are in resting position and waiting for the prey. Argiope spiders were found extensively at “Jallo Park, Lahore” and “Changa Manga Forest” south-west of Lahore, Punjab, Pakistan. It was only found there in October, November and December and sampling was done at dawn and dusk.
Figure1.1: Me and my tools with Argiope at Jallo Park, Lahore.
3.3 Habitat:
It constructs the web in the exposed air in/between the medium sized green bushes of undisturbed areas of the field.
3.4 Sampling Methods:
There are different methods for capturing spider as follow.
- Jerk method
- Pitfall trap method
- Sweep net method
- Leaf litter exploration method
- Hand picking method, and many others.
We collected all the specimens simply by “Hand picking method”. In this method a plastic box was used. The mouth of the box was placed over the spider, resting on his web, and slightly disturb the spider from the other side of the web or from the ventral side of the spider without damaging the web, when spider went into the box then the mouth of the box was closed with net cloth for aeration. The web was also collected in a clean Eppendorf tube if it was clean and has no remains of prey. Only adult specimens were collected and brought to the laboratory for silk harvest.
3.5 Spider keeping:
Adult specimens were kept in large sterile plastic boxes (1feet. 1feet. 1feet) the lids of which had fine pores for aeration. Sterile glass sticks were also placed in the boxes for radial web’s support as the spider anchor its radial web around these spicks. Each box was loaded with a single specimen because there exists cannibalistic behavior among these spiders. The containers were placed at a least disturbed area, so spiders could make silk to their maximum. But these spiders gave a very small amount of silk, they did not survive more than two weeks in laboratory. Spider hatchling were also reared in small containers where they gave a good quantity of web silk due to their large number but they also died within two months of hatching and their size did not grow up to a considerable extent. The “Web silk” used in this study was collected from the field as mentioned above, from the hatchlings and small amount from the adults. The “Egg silk” used in this study was only collected from the adult female spiders that we kept in sterile conditions of laboratory.
As the project deals with the bacterial studies, I used sterile containers and sticks to get as less contamination as possible.
3.5.1 Feed:
House flies were provided to the spiders after every 3 days, number of flies per feed was according to the size and age of the spider. We provided 2 to 3 alive flies to the active specimens which had increased rate of metabolism, and only 1 or 2 alive flies was provided to the spider which had laid down the eggs, because these was relatively inactive and rarely feed and mostly died before the hatching of young ones. Spider hatchlings were provided with a solution of honey and glucose D in milk which was absorbed on a foam. Drosophila was provided to the specimens which just had left the artificial synthetic feed.
Since, house flies are very active, so we used to keep them at about 0 oC for 1 to 1.5 minutes to sedate them so they could be easily transferred to the spider container with the help of forceps.
3.5.2 Humidity:
The containers were made slightly humid with the help of atomizer before every feed. But the humidity for adults was provided within limits because it causes the formation of molts which contaminate the silk.
3.5.3 Temperature:
Since, spiders are ectotherms so they were kept at room temperature. Spiders can tolerate a wide range of temperature but low or too much high temperature can affect their feeding behavior and stops the formation of silk.
3.5.4 Cleaning:
Specimens were observed daily without disturbing them and containers were cleaned by removing the remaining of the prey when the spiders had finished it.
3.6 Silk Extraction:
After 1 to 1.5 weeks of the feed the silk was collected from the container with the help of sterile glass rod, kept it in a sterile labeled Eppendorf and stored at room temperature. Just after silk extraction the next feed was provided so the spiders construct new web to capture the prey. Next day slight humidity was provided by spraying water with the help of atomizer. During all this process the spiders were least disturbed.
Those containers in which the spiders laid down eggs and formed cocoons were not disturbed and eggs were allowed to hatch.
3.7 Rearing of Argiope Hatchlings:
Hatchlings were maintained in small cylindrical containers. Sand was laid down in the bottom of the container over which circular filter paper was placed to absorb extra moisture. Mixture of honey, milk and glucose D was made as food which was absorbed in small pieces of foam, then these pieces were places in the containers and about 50 hatchlings were kept in one container in which these suck the juice from the foam pieces. Silk from these containers was collected after 2 days and with the silk collection the hatchlings were transferred to the freshly prepared containers because fungal attack happened in such conditions of containers within 4 to 5 days.
3.8 Testing of Antimicrobial Potential of Silk:
The antimicrobial action of silk recovered from Argiope was checked by “disk diffusion method”, by dissolving silk in suitable solvents as described below.
3.8.1 Solvents for Silk Dilution:
Six different solvents, 5% NaOH, methanol, ethanol, distilled water, acetone and U-Solution (U = urea), were used to dissolve the recovered silk and to test their antibacterial potential. Composition of urea solution is as follow.
3.8.2 Composition of Urea Solution (U-Solution):
Actually, it was a mixture of two solutions as follow.
Sol. 1 = 12.25 gm CaCl2 + 10.66 ml Dis.H2O + 9.75 ml ethanol
Sol. 2 = 4 gm urea + 8.33 ml ethanol
These two solutions were mixed in 1:1 proportion and resulting solution was boiled for 2 mints for sterilization providing the mouth of the beaker was sealed with aluminium foil.
3.8.3 Silk Dilution:
Two types of silk of Argiope were used separately, as egg silk and web silk. 0.035 gm of silk was measured and it was treated to get its fluffy form to increase the surface area, then this weight of silk was dissolved in 1 ml of solvent in an Eppendorf and then label it. Silk solutions for each of the 6 solvents described above were prepared just before dosing. These solutions were heated at 470C for 1 hour just before centrifugation at 7000 rpm for 30 mints. So, the supernatant of these solutions after centrifugation was used for dosing on bacteria.
3.9 Bacterial Species:
Four strains of bacteria Escherichia coli (gram negative), Pseudomonas aeruginosa (gram negative), Klebsiella pneumoniae (gram negative), Staphylococcus aureus (gram positive) were used to check the antibiotic effect of silk recovered from Argiope.
3.10 Bacterial Growth:
Bacteria was grown in broth media from where bacterial inoculums were taken to infect the uncontaminated agar plates to grow pure cultures of bacterial colonies as follow.
3.10.1 Preparation of Nutrient Broth Media:
To prepare Nutrient broth media 0.3 gm of yeast extract and 0.5 gm of peptone were added in 100ml of distilled water (Pelczar et al., 5th edition) in a 500 ml flask. The resulting solution was gently shaken for about 3 to 4 minutes. Then broth media was transferred to the eight universal 20 ml test tubes, 10 ml in each test tube, two tubes for each bacterial strain. These test tubes were made air tight by placing cotton plugs in their mouths and then covered them by aluminum foil and further sealed with scotch tape. Then these test tubes were autoclaved along with petri dishes at 121.75oC and 15 psi pressure for 15 minutes. After removing them from autoclave these were cooled down up to 37oC. Now the nutrient broth media is ready for bacteria of a specific strain to grow.
3.10.2 Bacteria Revival Technique:
These eight test tubes were labeled, two test tubes for each strain. 50ul inoculum was transferred to the respective labeled test tubes with the help of micropipette from the tubes in which the specific strains were maintained. All this work was done in the “laminar air flow” to avoid contamination. These test tubes were placed in incubator at 37oC for 24 hours after which fresh bacterial strains was ready to use. Since, 2 sets of four strains were made, labeled as set “A” and set “B”. The set “A” was used for making colonies of pure culture on agar plates and the set “B” remained undisturbed to avoid any type of contamination. The set “B” was used in next bacterial revival. Strains was revived after every 3 days.
3.11 Preparation of Agar Plates:
Colonies of bacteria were formed on agar plats for each of the four stains by making “nutrient growth media” as follow.
3.11.1 Preparation of Nutrient Agar Media:
In order to make agar plates for bacterial colonies nutrient agar media was prepared. A sterile 500ml flask was taken in which 0.3 gm of yeast extract, 0.5 gm of peptone and 1.5-2 gm of nutrient agar were added along with 100ml of distilled water and gently shake it. The flask was made air tight by placing cotton plugs in its mouths and then covered by aluminium foil and further sealed with scotch tape. Then the flask was autoclave at 121.75oC and 15 psi pressure for 15 mints along with clean, fully dry and properly sealed petri plates, Gilson tips along with their rack and filter paper disks of 6mm diameter. All the material that I placed in autoclave is properly air tight and the disks was kept in a petri plate. After removing them from autoclave directly place all the apparatus in laminar air flow to avoid contamination and let them to cool down up to 45-50oC. Now the nutrient growth media is ready for pouring.
3.11.2 Pouring:
The growth media was poured in each petri plate up to the uniform depth of 6mm. Then all the petri dishes were piled up and let the media to solidify for about 10 minutes and then place them into the incubator in an inverted style at a temperature of 37oC for 24 hours.
Next day before spreading the uncontaminated plates were selected for inoculum of bacterial strain by observing the small local colonies of bacteria in contaminated petri dishes, such contaminated petri dishes were rejected for further use.
3.12 Inoculation and Spreading:
Before applying the inoculum of a particular bacterial strain each uncontaminated petri plate was labeled for positive control, negative control (these were belonged to the control group) egg silk dose and web silk dose (these two were belonged to the experimental group).
Then an inoculum of 50ul of a particular bacterial strain from its test tube (test tube of set “A”) by using micropipette and autoclaved Gilson tip was poured to the center of the respective labeled agar plate and spread it uniformly with the help of sterile glass spreader. In the same way spreading of all the petri dishes was done in laminar air flow.
3.13 Dosing:
Silk solutions were made, heated at 47oC for 1 hour and centrifuged at 7000 rpm for 30 mints just before use. Within 15 mints of spreading an autoclaved filter paper disk of 6mm was placed in the centers of negative control region, eggs silk dose region and web silk dose region. Then 10ul liquid of negative control, egg silk solution and web silk solution were poured on the centers of their respective disks. A prepared 30 mg tetracycline disk of 6mm diameter was placed in the center of positive control region which was the positive control.
3.13.1 Bacteriostatic Effect:
To study the bacteriostatic effect of the silk the dosage was applied just after the spreading.
3.14 Result Recording:
After 24 hours of dosing the cultured plates were observed for results. Transparent radial zones (zone of inhibition) around the filter paper disks observed against rest of the area of petri dish as opaque due to the presence of uniform bacterial colony. The diameters of the zones of inhibition were measured in centimeters with the help of Vernier Caliper.
The diameter was considered directly proportional to the antibiotic potential of the agent used.
3.14.1 Comparison of Experimental Group with Control Group:
The diameter of the zone of inhibition of sample was divided by that of the negative control to eliminate the effect of the solvent used for silk dilution then the resulting value was compared with the diameter of the zone of inhibition of positive control to illustrate the antibiotic potential of the silk with respect to that of the standard antibiotic used in the positive control.
RESULTS
This study was designed to check the antibacterial potential of silk recovered from spiders belonging to genus Argiope. Two types of pure silk were extracted from these orb-web spiders that were egg silk and web silk which were dissolve in six different solvents (ethanol, methanol, acetone, 5% NaOH, distilled water and urea-solution). These dilutions were applied against four different species of bacteria i.e., Escherichia coli (gram negative), Pseudomonas aeruginosa (gram negative), Klebsiella pneumoniae (gram negative) and Staphylococcus aureus (gram positive). 6mm diameter disks of tetracycline 30μg were used as positive control while the respective solvent of the applied silk solution was used as negative control. Egg silk solution and web silk solution were treated groups. Results were demonstrated in “cm” of diameters of inhibition zones on the petri dishes while these were converted to “mm” before statistical analysis. Antibacterial activity of the mentioned treatments and control groups are as follow.
4.1 Antibacterial activity of silk dissolved in 5% NaOH:
After 24 hours of incubation clear zones of inhibition were observed on petri plates. Positive control always gave largest zone of inhibition while rest of the three zones were different in diameters as shown in figures 4.1 to 4.4 of four selected bacterial species. Statistical analysis (P-Value = 0) showed that clear variations existed among inhibition zones of positive control, negative control, egg silk solution and web silk solution against all assessed bacterial species. The antibacterial action of egg silk and web silk against Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Staphylococcus aureus was due to 5% NaOH in which silk was dissolved as depicted in table 4.1.
Table 4.1: Statistical analysis showing variation between control and treated groups when silk was dissolved in 5% NaOH.
| Bacterial Species | Treatments | Mean ± S. E | d/f | F | P |
| Escherichia coli | Control (+ve) | 25.0000b ± 1.15470 | 3,8 | 149.141 | 0 |
| Control (-ve) | 9.0000a ± 0.57735 | ||||
| Egg Silk | 8.5000a ± 0.28868 | ||||
| Web Silk | 8.6667a ± 0.16667 | ||||
| Pseudomonas aeruginosa | Control (+ve) | 24.6667b ± 1.76383 | 3,8 | 51.720 | 0 |
| Control (-ve) | 9.0000a ± 0.57735 | ||||
| Egg Silk | 8.3333a ± .33333 | ||||
| Web Silk | 9.0000a ± 1.15470 | ||||
| Klebsiella pneumoniae | Control (+ve) | 22.6667b ± 1.85592 | 3,8 | 39.840 | 0 |
| Control (-ve) | 9.3333a ± 0.88192 | ||||
| Egg Silk | 9.5000a ± 0.28868 | ||||
| Web Silk | 9.5000a ± 0.28868 | ||||
| Staphylococcus aureus | Control (+ve) | 23.6667b ± 1.76383 | 3,8 | 52.510 | 0 |
| Control (-ve) | 8.6667a ± 0.33333 | ||||
| Egg Silk | 8.6667a ± 0.88192 | ||||
| Web Silk | 9.1667a ± 0.44096 |
4.2 Antibacterial activity of silk dissolved in Acetone:
24 hours of incubation gave clear zones of inhibition on petri plates. Positive control gave largest zone of inhibition while rest of the zones were different in diameters as showed in figures 4.5 to 4.8. Statistical analysis (P-Value = 0 for E. coli & S. aureus, P-value = 0.002 for P. aeruginosa & P-value = 0.008 for K. pneumoniae) showed that significant variation present among positive control and other treatments against a specific bacterial species. When dissolved in acetone, both egg silk and web silk were proved effective statistically against all tested strains. Antibiotic activity of silk was due to solvent in which these were dissolved (Table 4.2).
Table 4.2: Statistical analysis showing variation between control group and treated group when silk was dissolved in Acetone.
| Bacterial Species | Treatments | Mean ± S. E | d/f | F | P |
| Escherichia coli | Control (+ve) | 25.6667b ± 1.20185 | 3,8 | 41.451 | 0.000 |
| Control (-ve) | 6.0000a ± 0.00000 | ||||
| Egg Silk | 9.6667a ± 2.18581 | ||||
| Web Silk | 11.0000a ± 1.00000 | ||||
| Pseudomonas aeruginosa | Control (+ve) | 23.6667b ± 3.17980 | 3,8 | 12.316 | 0.002 |
| Control (-ve) | 10.6667a ± 1.45297 | ||||
| Egg Silk | 9.0000a ± 1.73205 | ||||
| Web Silk | 10.3333a ± 0.33333 | ||||
| Klebsiella pneumoniae | Control (+ve) | 23.3333b ± 2.60342 | 3,8 | 8.175 | 0.008 |
| Control (-ve) | 10.6667a ± 2.33333 | ||||
| Egg Silk | 9.6667a ± 2.02759 | ||||
| Web Silk | 10.3333a ± 2.18581 | ||||
| Staphylococcus aureus | Control (+ve) | 23.0000b ± 1.73205 | 3,8 | 25.187 | 0.000 |
| Control (-ve) | 6.1667a ± 0.16667 | ||||
| Egg Silk | 7.3333a ± 1.33333 | ||||
| Web Silk | 8.5000a ± 2.25462 |
4.3 Antibacterial activity of silk dissolved in ethanol:
Clear zones of inhibition were appeared on petri plates after incubation of 24 hours. Positive control showed biggest zones of inhibition while rest of the zones were smaller in diameters as showed in figures 4.9 to 4.12. Statistical analysis (P-Value = 0 for E. coli, P-Value = 0.049 for S. aureus, P-value = 0.010 for P. aeruginosa & P-value = 0.022 for K. pneumoniae) showed that variation exist among positive control and treatments against all the specific bacterial species. No significant difference was recorded between control, egg silk and web silk as depicted in table 4.3.
Table 4.3: Statistical analysis showing variation between control group and treated group when silk was dissolved in ethanol.
| Bacterial Species | Treatments | Mean ± S. E | d/f | F | P |
| Escherichia coli | Control (+ve) | 24.3333b ± 1.20185 | 3,8 | 26.705 | 0.000 |
| Control (-ve) | 7.3333a ± 1.33333 | ||||
| Egg Silk | 12.6667a ± 1.20185 | ||||
| Web Silk | 13.0000a ± 1.73205 | ||||
| Pseudomonas aeruginosa | Control (+ve) | 25.0000b ± 2.64575 | 3,8 | 7.651 | 0.010 |
| Control (-ve) | 13.0000a ± 1.52753 | ||||
| Egg Silk | 11.6667a ± 2.96273 | ||||
| Web Silk | 15.0000a ± 1.00000 | ||||
| Klebsiella pneumoniae | Control (+ve) | 23.3333b ± 1.76383 | 3,8 | 5.690 | 0.022 |
| Control (-ve) | 8.6667a ± 2.66667 | ||||
| Egg Silk | 9.0000a ± 3.00000 | ||||
| Web Silk | 10.0000ab ± 4.00000 | ||||
| Staphylococcus aureus | Control (+ve) | 23.6667a ± 1.76383 | 3,8 | 4.090 | 0.049 |
| Control (-ve) | 10.3333a ± 3.38296 | ||||
| Egg Silk | 11.3333a ± 3.52767 | ||||
| Web Silk | 11.3333a ± 3.52767 |
4.4 Antibacterial activity of silk dissolved in methanol:
Figures 4.13 to 4.16 show different zones of inhibitions for control group and treated groups where positive control gave largest diameters of zones. Statistical analysis (P-Value = 0) for E. coli and S. aureus, P-value = 0.002 for P. aeruginosa and K. pneumoniae showed that significant variations existed among all treatments against a specific bacterial species. When dissolved in methanol only web silk solution was discovered to have antibacterial potential against E. coli, P. aeruginosa, K. pneumoniae & S. aureus providing elimination of the effect of solvent as illustrated by Mean ± S. E values in table 4.4.
Table 4.4: Statistical analysis showing variation between control group and treated
group when silk was dissolved in methanol.
| Bacterial Species | Treatments | Mean ± S. E | d/f | F | P |
| Escherichia coli | Control (+ve) | 26.0000c ± 1.52753 | 3,8 | 97.198 | 0.000 |
| Control (-ve) | 6.0000a ± 0.00000 | ||||
| Egg Silk | 6.6667a ± 0.66667 | ||||
| Web Silk | 11.6667b ± 0.88192 | ||||
| Pseudomonas aeruginosa | Control (+ve) | 24.0000b ± 2.00000 | 3,8 | 12.525 | 0.002 |
| Control (-ve) | 9.6667a ± 1.85592 | ||||
| Egg Silk | 9.3333a ± 1.76383 | ||||
| Web Silk | 10.6667a ± 2.33333 | ||||
| Klebsiella pneumoniae | Control (+ve) | 22.6667b ± 2.66667 | 3,8 | 12.531 | 0.002 |
| Control (-ve) | 9.6667a ± 2.02759 | ||||
| Egg Silk | 8.0000a ± 1.52753 | ||||
| Web Silk | 12.0000a ± 0.57735 | ||||
| Staphylococcus aureus | Control (+ve) | 23.0000b ± 2.51661 | 3,8 | 31.257 | 0.000 |
| Control (-ve) | 7.0000a ± 0.00000 | ||||
| Egg Silk | 6.0000a ± 0.00000 | ||||
| Web Silk | 11.3333a ± 1.20185 |
4.5 Antibacterial activity of silk dissolved in urea solution:
Figures 4.17 to 4.20 showed different zones of inhibitions due to control group and treated group where positive control gave biggest zones of inhibition. Statistical analysis (P-Value = 0 for E. coli, S. aureus and P. aeruginosa while P-value = 0.001 for K. pneumoniae) showed that significant variation existed among positive control, negative control, egg silk solution and web silk solution against the specific bacterial species. Silk dissolved in urea solution showed more antibacterial potential than negative control against E. coli & P. aeruginosa, further, the web silk was observed more effect than the egg silk. In case of K. pneumoniae & S. aureus non-of the silk showed any effect by themselves but small effect due to the solvent in which they were dissolved as showed in table 4.5.
Table 4.5: Statistical analysis showing variation between control group and treated group when silk was dissolved in urea solution.
| Bacterial Species | Treatments | Mean ± S. E | d/f | F | P |
| Escherichia coli | Control (+ve) | 25.6667c ± 1.20185 | 3,8 | 120.500 | 0.000 |
| Control (-ve) | 6.6667a ± 0.66667 | ||||
| Egg Silk | 7.0000ab ± 0.57735 | ||||
| Web Silk | 10.6667b ± 0.66667 | ||||
| Pseudomonas aeruginosa | Control (+ve) | 24.0000b ± 1.52753 | 3,8 | 25.964 | 0.000 |
| Control (-ve) | 9.3333a ± 1.76383 | ||||
| Egg Silk | 8.3333a ± 0.66667 | ||||
| Web Silk | 11.0000a ± 1.52753 | ||||
| Klebsiella pneumoniae | Control (+ve) | 23.3333b ± 2.60342 | 3,8 | 17.439 | 0.001 |
| Control (-ve) | 9.6667a ± 2.02759 | ||||
| Egg Silk | 7.8333a ± 0.92796 | ||||
| Web Silk | 9.6667a ± 0.33333 | ||||
| Staphylococcus aureus | Control (+ve) | 23.6667b ± 1.76383 | 3,8 | 28.632 | 0.000 |
| Control (-ve) | 9.6667a ± 1.45297 | ||||
| Egg Silk | 8.3333a ± 0.88192 | ||||
| Web Silk | 9.3333a ± 1.20185 |
4.6 Antibacterial activity of silk dissolved in distilled water:
Figures 4.21 to 4.24 depict that only positive control gave zones of inhibition. So, silk dissolved in dist. water is not effective against any of the four selected bacterial species.
4.7 Comparison of Antibiotic Effect of Silk Dissolved in Different Solvents:
The six solvents were used to dissolve the silk, the resulting solutions showed different antimicrobial potential against the same as well as different species of bacteria.
4.7.1 Silk Solutions Against Escherichia Coli:
P-value = 0, showed that significant variation existed among 12 solutions of silk against E. coli in six solvents as mentioned in table 4.7. Maximum antibiotic effect of silk was revealed in ethanol, then it was considerably reduced in acetone. Web silk was always remained more potent than the egg silk. Methanol and urea solution were almost equally potent with slight variations. Silk in distilled water did not show antibiotic activity because silk did not get dissolve in it and 5 % NaOH was on second last position as indicated in table 4.7 in the form of Mean ± S. E.
Table 4.7: Statistical analysis of six solvents to dissolve silk to show its antibiotic properties against Escherichia coli.
| Solvents | Treatments | Mean ± S. E | d/f | F | P |
| 5% NaOH | Egg silk | 8.5000abc ± 0.28868 | 11,24 | 6.241 | 0.000 |
| Web silk | 8.6667abc ± 0.16667 | ||||
| U-Solution | Egg silk | 7.0000ab ± 0.57735 | |||
| Web silk | 10.6667abc ± 0.66667 | ||||
| Acetone | Egg silk | 9.6667abc ± 2.18581 | |||
| Web silk | 11.0000abc ± 1.00000 | ||||
| Methanol | Egg silk | 6.6667ab ± 0.66667 | |||
| Web silk | 11.6667bc ± 0.88192 | ||||
| Ethanol | Egg silk | 12.6667c ± 1.20185 | |||
| Web silk | 13.0000c ± 1.73205 | ||||
| Dist. Water | Egg silk | 6.0000a ± 0.00000 | |||
| Web silk | 6.0000a ± 0.00000 |
4.7.2 Silk solution against Pseudomonas aeruginosa:
P-value = 0.016, showed that significant variation existed among 12 solutions of silk against P. aeruginosa in six solvents as mentioned in table 4.8. Maximum antibiotic effect of silk was observed in ethanol, then acetone and methanol were equally but slightly less potent than web silk of urea solution. Web silk was always remained more potent than the egg silk. Silk in distilled water showed no antibiotic activity because silk failed to dissolve in it and 5 % NaOH was on second last position as indicated in table 4.8 in the form of Mean ± S. E.
Table 4.8: Statistical analysis of six solvents to dissolve silk to show its antibiotic properties against Pseudomonas aeruginosa.
| Solvents | Treatments | Mean ± S. E | d/f | F | P |
| 5% NaOH | Egg silk | 8.3333ab ± 0.33333 | 11,24 | 2.846 | 0.016 |
| Web silk | 9.0000ab ± 1.15470 | ||||
| U-Solution | Egg silk | 8.3333ab ± 0.66667 | |||
| Web silk | 11.0000ab ± 1.52753 | ||||
| Acetone | Egg silk | 9.0000ab ± 1.73205 | |||
| Web silk | 10.3333ab ± 0.33333 | ||||
| Methanol | Egg silk | 9.3333ab ± 1.76383 | |||
| Web silk | 10.6667ab ± 2.33333 | ||||
| Ethanol | Egg silk | 11.6667ab ± 2.96273 | |||
| Web silk | 15.0000b ± 1.00000 | ||||
| Dist. Water | Egg silk | 6.0000a ± 0.00000 | |||
| Web silk | 6.0000a ± 0.00000 |
4.7.3 Silk solution against Klebsiella pneumoniae:
P-value = 0.498, showed that variations did not exist among 12 solutions of silk against K. pneumoniae in six solvents as mentioned in table 4.9. Comparison of individual solvents with each other showed that maximum antibiotic effect was showed by web silk in methanol, rest of the web silk solutions were almost equally potent. All the egg silk solutions showed almost equal effect except urea solution and dist. water. Silk in distilled water proved too has no antibiotic activity because silk did not dissolve by dist. water and urea solution was on second last position as indicated in table 4.9 in the form of Mean ± S.E.
Table 4.9: Statistical analysis of six solvents to dissolve silk to show its antibiotic properties against Klebsiella pneumoniae
| Solvents | Treatments | Mean ± S. E | d/f | F | P |
| 5% NaOH | Egg silk | 9.5000a ± 0.28868 | 11,24 | 0.970 | 0.498 |
| Web silk | 9.5000a ± 0.28868 | ||||
| U-Solution | Egg silk | 7.8333a ± 0.92796 | |||
| Web silk | 9.6667a ± 0.33333 | ||||
| Acetone | Egg silk | 9.6667a ± 2.02759 | |||
| Web silk | 10.3333a ± 2.18581 | ||||
| Methanol | Egg silk | 8.0000a ± 1.52753 | |||
| Web silk | 12.0000a ± 0.57735 | ||||
| Ethanol | Egg silk | 9.0000a ± 3.00000 | |||
| Web silk | 10.0000a ± 4.00000 | ||||
| Dist. Water | Egg silk | 6.0000a ± 0.00000 | |||
| Web silk | 6.0000a ± 0.00000 |
4.7.4 Silk solution against Staphylococcus aureus:
P-value = 0.256, showed that statistically variations did not exist among 12 solutions of silk against S. aureus in six solvents as mentioned in table 4.10. Comparison of individual solvents with each other showed that maximum antibiotic effect was showed by web silk in methanol which was equal to the effect of both types of silk in ethanol. Rest of the web silk solutions were almost equally potent. All the egg silk solutions showed almost equal effect except dist. water in which silk sis not dissolved as indicated in table 4.10 in the form of Mean ± S. E.
Table 4.10: Statistical analysis of six solvents to dissolve silk to show its antibiotic properties against Staphylococcus aureus.
| Solvents | Treatments | Mean ± S. E | d/f | F | P |
| 5% NaOH | Egg silk | 8.6667a ± 0.88192 | 11,24 | 1.356 | 0.256 |
| Web silk | 9.1667a ± 0.44096 | ||||
| U-Solution | Egg silk | 8.3333a ± 0.88192 | |||
| Web silk | 9.3333a ± 1.20185 | ||||
| Acetone | Egg silk | 7.3333a ± 1.33333 | |||
| Web silk | 8.5000a ± 2.25462 | ||||
| Methanol | Egg silk | 6.0000a ± 0.00000 | |||
| Web silk | 11.3333a ± 1.20185 | ||||
| Ethanol | Egg silk | 11.3333a ± 3.52767 | |||
| Web silk | 11.3333a ± 3.52767 | ||||
| Dist. Water | Egg silk | 6.0000a ± 0.00000 | |||
| Web silk | 6.0000a ± 0.00000 |
Discussion
Egg silk and web silk extracted from spiders of genus Argiope and dissolved in six different solvents (ethanol, methanol, acetone, 5% NaOH, distilled water and urea solution) were checked for their antibacterial potential against four different bacterial species, Escherichia coli (gram negative), Pseudomonas aeruginosa (gram negative), Klebsiella pneumoniae (gram negative) and Staphylococcus aureus (gram positive). In present study although web silk and egg silk inhibit growth of tested bacteria but it was due to activity of solvents in which these were dissolved.
These results are contrary with the results of Roozbahani et al. (2014). They used spider silk extracted from Pholcus phalangioides and state that antibacterial compounds are present in the solution spider silk. Our results are also different from the results of Tahir et al. (2017). They used spider silk of Cyclosa confraga and evaluated its anti-bacterial potential against both gram-positive and gram-negative bacteria. Wright and Goodacre (2012) evaluate the antimicrobial effect of Tegenaria domestica against gram-positive bacteria which is opposite to our results. The first report about antibiotic effect of spider silk (Heimer, 1988) also differs from our results. Heimer (1988) discovered that it was potassium hydrogen phosphate in the spider silk which create an acidic environment which in terns was not good for bacterial growth and potassium nitrate in spider silk also inhibit the growth of bacteria. Chakraborty and Das (2009) also evaluated similar antibacterial results of spider silk.
Wright Simon (2011) evaluated that spider silk was effective against gram-positive bacteria B. subtilis, but this effect was not proved common to all the spiders examined and only the web silk of Tegenaria domestica and egg silk of Pityohyphantes phrygianus were proved antibiotic in nature. In the same way spider silk was proved antibiotic in nature against gram-positive bacteria only in Iraq for the 1st time in 2017. Many gram-positive bacteria were killed by acetone solution of spider silk but it was observed that gram-negative bacteria were not killed by spider silk (Al-Kalifawi & Kadem, 2017). So, present results match with study of Iraq in the case of gram-negative bacteria. In the same way our results are resembles to the results of Wright and Goodacre (2012) in the case of gram-negative bacteria as they failed to kill them by using spider silk.
According to Sharma (2014) there was no inhibitory agent found in the silk of Argiope aurantia applied against E. coli (gram-negative), Bacillus subtilis (gram-positive) and Pseudomonas aeruginosa (gram-negative) due to which silk failed to kill any of these bacterial species. Our results are in accordance to these results. In the same study Sharma concluded through Scanning Electron Microscope analysis that these were the physical features of the silk due to which very low adherence of bacteria happened on the surface of silk.
From the above discussion it becomes clear that silk of certain spider species is antibiotic in nature. Silk of different spiders is affective against different species of bacteria, silk of one spider species may prove antibiotic only against gram-positive bacteria or gram-negative bacteria or against both. These antimicrobial properties are may be due to some antibacterial peptides in the silk, due to its acidic nature or due to just the physical repulsion between the molecules of silk and that of bacterial cells. It needs a lot of research work to discover the true mechanism through which spider silk inhibit the growth of microbes. There also exist many spider species which produce silk with no antibacterial properties as observed in present study.
CONCLUSION AND RECOMMENDATIONS
It is concluded from the present work that Argiope’s silk is not effective against Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia and Pseudomonas aeruginosa statistically. Spider silk is a cocktail of marvelous and potent components so, future studies are recommended to characterize the silk to isolate durable and potent components to explore their antibacterial effects against different bacteria.