Antibacterial Activity of Silk of Cyrtophora citricola (Araneae: Araneidae)

ABSTRACT

Bacteria are responsible for causing serious threat to human health and are becoming resistant day by day. To combat these challenges, it is urgent need to discover new antibacterial compound with better efficacy. Many researchers reported that antibacterial compound obtained from natural sources are more effective than synthetic compound. There are less chances to develop resistant by bacteria against antibacterial substances obtained from natural sources. In the present work antibacterial activity of Cyrtophora citricola silk was checked against four pathogenic bacteria i.e., Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae.

Silk extracted from spiders were dissolved in five different solvents i.e., Methanol, Ethanol, Acetone, 10% NaOH and distilled water but silk was entirely dissolved in 10% NaOH and partially dissolved in other four solvents. Silk dissolved in 10% NaOH solution inhibited the growth of all selected bacterial species both (gram positive and gram negative) but inhibition zones against gram positive bacteria were greater than gram negative bacteria. Silk dissolved in methanol, ethanol, acetone and distilled water did not efficiently inhibit bacterial growth. Moreover, inhibition zone of silk dissolved in methanol, ethanol, acetone and distilled water were not clear (fuzzy) and such zones were not considered in the study. It is concluded that silk of C. citricola inhibited the growth of gram positive and gram negative bacteria.

 INTRODUCTION

Bacterial resistance against antibiotics is a common problem all over the world (Okeke et al., 2005). To combat these challenges, it is an urgent need to discover the new antibacterial compounds with better efficacy. Many researchers reported that antibacterial compounds can be obtained from natural resources such as plants and animals (Gyawali & Ibrahim, 2012; Zuridah., Fairuz., Zakri & Rahim, 2008). These compounds are important to control bacterial infections (Hoskin & Ramamoorthy, 2008; Saleem et al., 2010).

Heimer (1988) reported that spider silk possesses antiseptic properties. It is acidic in nature and contains potassium nitrate that inhibit bacterial growth (Chakraborty & Das, 2009; Gomes, Leonor, Mano, Reis & Kaplan, 2010). Spiders wrap extra food with silk to protects it from fungus and bacteria for a month and sometime even years (Eberhard, Barrantes & Weng, 2006). Number of studies have shown antibacterial potential of silk obtained from some spider species (Amaley, Gawali & Akarte, 2014; Mirghani, Kabbashi, Elfaki & Zulkifi, 2012; Saravanan, 2006; Sharma, 2014; Simon & Goodacre, 2012).

Statement of the problem

Microbial diseases are main threat to human health all over the world. Multidrug resistant bacteria are emerging due to mis use and overuse of antibiotics (Zuridah et al., 2008). In this way bacteria are becoming resistant day by day (Alanis, 2005; Nordmann, Naas, Fortineau & Poirel, 2007). These bacteria transfer drug resistance genes to other hosts and make treatment difficult (Benoit, Ellingson, Waterman & Pearson, 2014; Rossolini, Arena, Pecile & Pollini, 2014; Xu, Fan, Feng, Mi & Weng, 2014).

Significance of the study:

Antibacterials obtained from natural sources proved more effective and biofriendly. So, such natural antibacterial agents are considered the best than synthetic (Rosen, Gottfries, Muresan, Backlund & Oprea, 2009; Vandal, Abou-Zaid, Ferroni & Leduc, 2015). Natural antibiotics have lesser side effects and toxicity as compared to synthetic antibiotics. There is also less chance to develop resistance against antibiotics obtained from the living sources (Marasini et al., 2015; Vandal et al., 2015; Yu et al., 2005). The present research will prove an important contribution for the discovery of new antibiotics with better efficacy.

Objectives

No one can deny the dire need of the discovery of new antibacterial drugs with better efficacy. I set following objectives to achieve this goal

• Collection of Cyrtophora citricola.
• Recovery of spider silk.
• Check efficacy of recovered silk to inhibit bacterial growth.

Outline of the study

According to the previous study there are various proteins present in spider silk. Seven different types of glands are present in spider abdomen. Each type of gland produces a specific silk and the morphological structure of each silk are different because in each silk unique proteins are present. The configuration of these unique proteins is different in all type of silk and each silk engineered for a specific function (Saravanan, 2006).

The composition and properties of silk are also different in all spider species (Garrido, 2002; Vollrath & Knight, 2001). In the species of spider silk composition of amino acid is also different in each type of silk (Craig, 1997; Craig & Riekel, 2000; Work & Young, 1987; Wilson, Valluzzi & Kaplan, 2000). Large proteins are present in silk with molecular weight >200 KDa (Altaman et al., 2003). Non-polar and hydrophobic amino acids such as glycine, alanine and proline are also present in silk (Lombardi & Kaplan, 1990; Peakall, 1969; Romer & Scheibel, 2008). The structure of spider silk is visualized by using scanning electron microscope. The magnification of the visualization of silk is up to 35,000X (Saravanan, 2006).

Spider silk is a hardest material and extend tremendously 140% of their original length without breaking. According to Vollrath and Knight (2001) spider silk is five time stronger than steel. Stretching capacity of spider silk in dry form is 30% and in wet form stretching capacity of spider silk is 300%. The weight of silk in solid form is ten times greater than liquid form. The silk is initially soluble in its liquid form but insoluble in its solid form (Foelix, 1996). The spider silk can also be used to improve the brightness and smoothness of products in the cosmetics (Vendrely & Scherzer, 2007).

Spider silk is used as wound healer in traditional system of different countries of the world. Atypus silk is used as a bandage for wound healing. Atypus silk is a good water absorber and making the condition less ideal for microbial growth (Heimer, 1988). The silk of Nephila clavipes is used for neuronal regeneration in mammals (Allmeling, 2008; Jokuszies, Allmeling, Reimers, Kall & Vogt, 2006). Spider silk is also used in surgical thread (use to stitch injured site), bandages, biodegradable bottles and rust-free panels on boats (Singha, Maity & Singha, 2012). Birds use the spider silk for making nests which silk provides smooth texture to the nest and also act as antibacterial agents (Vankhede, 2013).

High amount of vitamin K is present in spider silk which can promote blood clotting. Several hundred years ago silk was used as gauze pads to stop bleeding (Heimer, 1988). Spider silk can prevent the growth of fungi and bacteria because phospholipids hydrate and potassium nitrate are present in the spider silk (Chakraborty & Das, 2009; Gomes et al., 2010). Spider silk has great strength and flexibility and the most versatile material in nature. It is used in various fields like material sciences as well as life sciences for various purposes and uses in agriculture. The spider silk also reduces plants damage by insect pest in agriculture (Edwards, Butler & Lofty, 1976; Jeyaparvathi, Baskaran & Bakavathiappan, 2013; Sunderland, Fraser & Dixon, 1986).

Spiders produce silk for their biological activity. Spiders use silk for different functions like swathing prey, arresting a fall, making egg cases and building a web (Breslauer & Karplan, 2012; Hoefler, 2007; Romer & Scheibel, 2008; Winkler & Kaplan, 2000). There are many traditional uses of spider silk in medicines. In 18^th century peasants of Carpathian mountain used silk in the field of oncology (Heimer, 1988). The natural history of spider silk was discussed in 17^th century. Spider silk was used in dermatological applications like antihemorrhagic, antipyretic and advancement of cell regeneration (Newman, 1995).

More than 4600 spider species construct orb webs. Web ranged in size from 1cm- 2cm in diameter (Blackedge et al., 2009; Gregoric, Agnarsson, Blackledge & Kuntner, 2011). Orb weavers’-built orb web to increase prey capture rate in the environmental conditions. The expedience of the web depends on its diameter location and orientation (Blackedge & Eliason, 2007; Opell, Bond & Warner 2006). In same species of spider variations are present in web structure due to internal and external conditions of individuals.

Internal factors are body weight, leg length, development stages, carapace width and experience of individual (Eberhard, 1988; Heiling & Herberstein, 1998; Herberstein & Heiling, 1999; Murakami, 1983; Olive, 1980; Vollrath, 1987; Waldorf, 1976; Zschokke & Vollrath, 2000). Web structure is also affected due to environmental factors (temperature, humidity, wind, gravity), quantity and quality of prey, habitat structure and presence of predators and parasites (Eberhard, 1989; Eberhard, 2000; Higgins, 1992; Heiling & Herberstein, 1999; Sandoval, 1994; Vollrath, 1986, 1988; Wu, Blamires, Wu & Tso,  2013). In the web architecture plasticity and flexibility may be response of abundance and size of prey (Blackedge & Zevenbergen, 2006). Web characteristics also depends on weight of spider, body length of spider, leg length of spider and cephalothorax width. CTL (capture thread length) increases in carapace width. CTL increases in heavy weight spider than low weight spider (Eberhard, 1988).

Seven different types of silk identified are major ampullate, minor ampullate, capture spiral, tubiliform, aciniform, flagelliform uniform (Andersen, 1970; Denny 1976; Jones, 2003). Capture spiral silk is very sticky. It is very flexible, strong and spider use this type of silk for capturing prey (Guerette, Ginzinger, Weber & Gosline 1996; Lin, Edmonds & Vollrath, 1995). Stretching capacity of capture spiral silk is 500%-1000% and tensile strength estimated at 1 Gpa (Kohler & Vollrath, 1995; Opell & Band, 2000; Vollrath & Edmonds, 1989). Tubiliform silk is highly elastic and is not secreted on daily basis and it is used to protect egg sacks of spider (Zhao et al., 2005). Aciniform silk is two to three time stronger than other types of silk (Zortea & Fischer, 2009).

Aciniform silk 50% stronger than major ampullate (Hayashi, Blackledge & Lewis, 2004). During the construction of webs minor ampullate silk used for temporary scaffolding and also different amino acid composition in minor ampullate silk than other type of silk (Liivak, Flores, Lewis & Jelinski, 1997). Denny (1976) reported that major ampullate silk also called dragline silk. Tensile strength of dragline silk is 200,000 Pressure per square inch (psi) and elastic capacity is 35 %. Orb weaving spiders produce major and minor ampullate silk for the formation of webs (Benjamin & Zschokke, 2004). The webs of the Orb weaving spiders are two dimensional having major strands from a common origin and minor strands circling and connecting the major strands (Scheibel, 2004).

LITERATURE REVIEW

 About 374-380 million year ago spiders were evolved, and the oldest species of spider is Attercopus fimbriamguis (Selden, 1990, 1991). Coddington and Levi (1991) reported that Araneae originated in the late silurian (430Mya) period. Foelix (1996) reported that Araneae order is divided into three sub-orders that are Mesothelae, Mygalomorphae and Anareomorphae. Mesothelae contains almost 350 spider species, Mygalomorphae has approximately 1500 species and remaining 90% species of spiders belong to Anareomorphae.

Spiders belonging to Araneidae family are also called orb weavers Worldwide occurrence of spiders at present time is 114 families, 3,935 genera and 48334 species (Platnick, 2014; World Spider Catalog Version 20.5). The maximum diversity of spiders are present in the tropical rain forest (Lamoral, 1968). Spiders are widely dispersed in hot and dry environment such as deserts (Foelix, 1996). Almost 41,000 species of spiders produce silk (Agnarsson, Kuntner & Blackledge, 2010).

Family Araneidae has 168 genera and 3029 species and subspecies of spiders are currently included in this family (Platnick, 2012). In Turkey 53 species and one subspecies of Araneidae are known (Bayram, Kunt & Danisman, 2012). Simon (1895) first time established subfamily Crytophorinae in the genus Cyrtophora. Spiders of subfamily Cyrtophorinae and genus Cyrtophora are different from other Araneid spiders due to tent-web (Scharff & Coddington, 1997; Scharff & Schmidt, 2008). Legs of Cyrtophora are relatively heavy than the other Araneid spiders. Proportions of the legs of Cyrtophora also differ from other Araneids having the femur slightly larger than the combined patella and tibia of the same leg and combined patella and tibia also shorter than the combined metatarsus and tarsus of the same leg (Levi, 1997).

Arrangement of the posterior eye row of Cyrtophora differ from Argiope, Gea, Kapogea and Mecynogea and lateral eyes of Cyrtophora are slightly separated so that the Cyrtophora are also different from other Araneids (Levi, 1997). In tropical and subtropical regions of the world 43 species and 9 subspecies of Cyrtophora are distributed (Platnick, 2012). In biological control web of C. citricola is considered as both useful and harmful agent. Web of C. citricola are bulky and permanent web (Edwards, 2006).

C. citricola produce a non-sticky silk for the composition of specialized orb web which consists of horizontal sheet in which irregular barrier web above and below is present (Lubin, 1973). In the upper barrier web of C. citricola, there is a retreat of dead leaves above the hub (Kullmann, 1958; Blanket, 1972). C. citricola web are very strong, durable and sometime renewed (Lubin, 1973). According to Levi and Levi (1968) the maximum body length of C. citricola is 15mm and these spiders adopted open habitat such as roadside and wooded Savanna. C. citricola lives in colonies and the habitation of these spider cacti, ornamental shrubs and fences (Levi & Levi, 1968; Lubin, 1973). Orb weavers feed on insects, other arthropods and other spiders. These spiders are categories in carnivorous group (Wise, 1993).

The First documented study by Heimer stated that spider silk is acidic and may be bacteriostatic in nature (Heimer, 1998). Gas chromatography and Mass spectroscopy analysis showed the presence of 12-mehtyltetradecanoic and 14-methylhexadecanoic acid in the silk of Linyphiatri angularis. These acids are present in the lipid of the silk of Linyphiatri angularis. These acids also show antibacterial activity (Saravanan, 2006). According to the Pohl, Kock and Thibane (2011) 12-methyltetradecanoic acid inhibit the growth of Magnaporthe oryzae (Rice pathogen).

Aciniform and tubuliform glands of spider produce silk. In this silk small coating peptides are present. Mass spectroscopy of Aciniform and tubuliform glands silk suggested the presence of two small coating peptides, named SCP-1 and SCP-2. These peptides (SCP-1 and SCP-2) help to protect silk from microorganisms (Hsia, Gnesa, Jeffery, Tang & Vierra, 1996; Hu et al. 2005). In cell wall of gram-negative bacteria lipopolysaccharides are present. These lipopolysaccharides play a fundamental role in their attachment to any biotic or abiotic surface (Donlan, 2002; Harmsen, Yang, Pamp & Tolker-Nielsen, 2010). Components of spider silk are a source of hope to overcome current barriers (infectious disease) in the development of curative agents. Some important properties of spider silk includes thermal stability, controllable degradation and biocompatibility (Chau, 2008; Vepari & Kaplan, 2007).

Spider silk is composed of many amino acids like Glycine, Alanine and large amount of Pyrrolidine. Due to these amino acids moisture is maintain in spider silk and these amino acids also prevent the silk from drying. Due to acidic property silk prevent the formation of biofilm and bacterial growth (Cotter & Hill, 2003; Dagorn et al., 2013). Formation of biofilm in gram negative bacteria is prevented due to spider silk (Sharma, 2014).

Gamma amino butyric acid (GABA) present in spider silk prevents the formation of biofilm in gram negative bacteria (Sharma, 2014; Stromstedt, Felth & Bohlin, 2014). GABA is a non-protein amino acid commonly present in the environment. It is a four-carbon amino acid (Bouche, Lacombe & Fromm, 2003). Mohammed, Nerland, Al-Haroni & Bakken (2013) reported that biofilm formation plays important role in bacterial resistance. Biofilm are well organized communities of bacteria. Nevin et al. (2009) reported that biofilms help bacteria to remove wastes by forming heterogeneous structure and nutrient uptake. Francolini, Norris, Piozzi, Donelli & Stoodley (2004) reported that natural products in the silk prevent the formation of biofilm.

Bacteria are more resistant due to excessive and improper use of antibiotic for non-bacterial diseases (Levy & Marshall, 2004; Witte, 2000). An antibacterial property of spider silk is beneficial for spider because they store food for months and even years. Spiders rolling silk on food prevents food from bacterial and fungal attack (Eberhard et al., 2006). Antimicrobial compounds are also present in spider silk (Roozbahani, Asmar, Ghaemi & Issazadeh, 2014). Glycoproteins are coated on the surface of spider silk. These are about 150-250nm thick and show antibacterial property (Augsten, Muehlig & Herrmann, 2000; Wright & Goodacre, 2012).

In spider silk antimicrobial compounds are present to induce the inhibition zone against the growth of both gram-negative bacteria Escherichia coli and positive bacteria Listeria monocytogenes (Roozbahani et al., 2014). In 2001 the first documented study of spider silk showed antimicrobial property to inhibit the growth of Pseudomonas fluorescens (Gram negative bacteria) (Border et al., 2001). Another study reported in 2009 that Crossopriza lyoni also known as daddy longless spider produce sheet web silk which show antimicrobial effect against Gram-negative and Gram-positive bacteria (Chakraborty & Das, 2009). Tengenaria domestica silk also shows antimicrobial properties.

The silk of T. domestica showed antimicrobial effects against B. subtilis (Gram positive bacteria) and E. coli (Gram negative bacteria). Spider silk has strong bacteriostatic effect against B. subtilis but no significant effect against E. coli (Gram negative bacteria) (Mondal, 2007; Kaur, Rajkhowa, Afrin, Tsuzuki & Wang, 2013; Wright & Goodcare, 2012).

Spider silk show higher effect on the growth of gram-positive bacteria Listeria monocytogenes than gram negative bacteria Escherichia coli (Mirghani et al., 2012). Some spiders such as Zilladiodia, Linyphiidae and Lasiodora parahybana also show the antibacterial activity. Silk of Zilladiodia and Linyphiidae spiders inhibit the growth of gram-positive bacteria named Bacillus subtilis (Wright, 2011). Silk of Nephila pilipes spider have an inhibitory effect on the growth of gram-negative bacteria Escherichia coli and gram-positive bacteria Staphylococcus atreus (Amaley et al., 2014). Pholcus phalangioides silk show antimicrobial activity againsts gram positive bacteria Listeria monocytogenes (Roozbahani et al., 2014). Walckenaer (1837) reported the plasticity and flexibility in web of Neoscona theisi. Web of Neoscona theisi plays important role to control insect pests in agriculture fields (Tahir & Butt, 2008)

Extraction of spider silk is done by using different solvents like acetone, ethanol, methanol and water. Some gram negative and gram-positive bacteria were used in antibacterial assay such as Bacillus subtilis (gram positive) and Escherichia coli (gram negative). Acetone extract showed 10mm of diameter of inhibition zone for Bacillus subtilis and 9mm of diameter of Escherichia coli. The maximum inhibition zone of Bacillus subtilis was 15mm and of Escherichia coli was 12mm after 48 hours with concentration of 0.035g/ ml.  The best antibacterial activity was shown by acetone extract as compare to the other solvents (Tahir, Qamar, Sattar, Shaheen & Samiullah, 2017).

            Spider silk show antibacterial activity due to the presence of some bisphosphonate (phosphonates) peptides. Spider silk is a great therapeutic compound and also shows low immunogenicity (Gellynck et al., 2006, 2008). Major ampullate spider silk seeded with human Schwann cell to demonstrate biocompatibility for the future treatment of nerve injuries (Allmeling, Jokuszies, Reimers, Kall & Vogt, 2006). Earlier studies showed that for the better nerve conduction spider silk fibers promote myelin formation, Schwann cell migration and axonal regrowth (Radtke et al., 2011; Tokareva, Jacobsen, Buehler, Wong & Kaplan, 2014). Spider silk promote the regeneration of silk cell and nerve cell (Wendt et al., 2011).

Silk of Cyclosa confraga showed antibacterial potential (Thorell, 1892). C. confraga is an orb web spider that is common in citrus orchards of the Punjab province, Pakistan. Its silk inhibits the growth of wide range of bacteria. C. confraga silk extract inhibit the growth of both gram positive and gram-negative bacteria. Inhibition zone was observed against both bacteria gram positive and gram-negative bacteria. Inhibition zone for Acinebactor sp (gram negative bacteria) was larger than streptococcus sp gram positive bacteria (Tahir et al., 2017).

Wright (2011) reported that silk of certain spider species has ability to inhibit the growth of both gram negative (Acinebactor sp) and gram positive (B. subtilis) but this ability does not appear in all common spiders. Pityohyphantes phrygianus egg silk significantly inhibited the growth of bacteria. Bacterial inhibition zone was observed after 24 to 72 hours. Tengenaria domestica web silk also showed temporary antimicrobial effect. Genera Zilla and a Linyphiid spiders silk also showed antimicrobial.

Amaley et al. (2014) reported that silk of Nephila pilipes showed antibacterial activity against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. Inhibitory effect was more against gram negative bacteria (E. coli and P. aeruginosa) than gram positive bacteria (S. aureus).

The first study was conducted in Iraq in which Al-Ankabut’s home extract Spider web was used. Different solvents is used to dissolve spider web (Distilled water, Ethanol and Acetone). Acetone extract show antibacterial activity against pathogen bacteria but distilled water and ethanol extract of Al-Ankabut’s home has no effect against bacteria. Acetone extract showed maximum zone of inhibition against bacteria. Zone of inhibition was higher against gram positive bacteria as compare to gram negative bacteria.

The inhibition zone diameter against gram positive bacteria are, 12mm for Streptococcus sp, 14mm for Bacillus subtilis and 16mm for Staphylococcus aureus and the diameter against gram negative bacteria are 8mm for Escherichia coli and Pseudomonas aeruginosa, 10mm for Klebsiella pneumonia and Proteus mirabilis and 12mm for Enterobacter cloacae. Different concertation of Acetone extract of Al-Ankabut’s home were used against both gram-negative and gram-positive bacteria. The best concentration was 40mg/ml which gives the highest effect. The zone of inhibition in this concentration was 12mm for against all negative bacteria. The inhibition zone in this concentration against gram positive bacteria are 12mm for B. subtilis and Streptococcus sp and 17mm for S. aureus. Whereas the lowest effect was at 10mg/ml concentration of Acetone extract of Al-Ankabut’s home.

The zone of inhibition was 8mm for K. pneumonia, E. cloacae, B. subtilis and Streptococcus sp and 10mm for S. aureus at 10mg/ml. This study showed that the Acetone extract of Al-Ankabut’s home could be used as a therapeutic agent for the treatment of microbial diseases in humans (Al-Kalifawi & Kadem 2017).

Various studies have investigated antimicrobial potential of spider silk. Sharma (2014) performed two different experiments to check antibacterial activity of spider silk. Spot assay (to check bactericidal or bacteriostatic action and presence of non-soluble inhibitory agents). Scanning Electron Microscopy analysis (SEM) is used to examine the bacterial adherence onto the surface of spider silk.

Dragline silk of orb-weaving spider Argiope aurantia was used in both experiments. In spot analysis organic extracts of spider silk did not show any bactericidal or bacteriostatic action against both gram negative (Escherichia coli and Pseudomonas aeruginosa) and gram-positive bacteria (Bacillus subtilis) but scanning electron microscopy analysis showed low bacterial attachment onto the surface of spider silk. Dragline spider silk did not show resistance against gram positive bacteria (B. subtilis) attachment onto the surface of silk but showed resistance to adherence by gram negative bacteria (E. coli and P. aeruginosa). Unique surface properties are present in dragline spider silk of Argiope that prevents attachment by gram negative bacteria (Sharma, 2014). Suto, Domagala, Roland, Mailloux & Cohen (1992) reported that in spider silk soluble or non-soluble inhibitory agents are present. These inhibitory agents show antibacterial potential. Bhushan (2011) also reported that silk show antibacterial activity due to surface or structural property of silk.

Eriovixia excelsa is an orb-web spider. Silk of this spider show antibacterial activity against both gram-negative and gram-positive bacteria. When silk concentration was increased inhibitory potential was also increased (Tahir et al., 2019).

In the present study four bacterial strains were used which are Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa. Pseudomonas aeruginosa is a gram-positive bacteria and Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae are gram negative bacteria.

Escherichia coli is a gram-negative, nonsporing, motile, rod shape bacteria and both sex pili and adhesive fimbriae (Mahon & Manuselis, 1995). E. coli belongs to family Enterobacteriaceae. All members of this family are facultative (Sherris, 1984). E. coli was initially considered a non-harmful member of the colon flora reside in the gut, but various infections and diseases are associated with E. coli such as meningeal infection, gastrointestinal infection, urinary tract infection, wound infection and bacteremia infection in all age groups (Mahon & Manuselis, 1995). Some other infections such as peritonitis, cholecystitis, septic wounds, bedsores, lower respiratory passages and endotoxic shock in debilitated patients are caused by E. coli (Mackie & Mccartney,1989).

Domain: Bacteria

                                    Phylum: Proteobacteria

                                                Class: Gammaproteobacteria

                                                            Order: Enterobacteriales

                                                                        Family: Enterobacteriaceae

                                                                                    Genus: Escherichia

                                                                                                Specie: E. coli

Staphylococcus aureus is a gram-positive round shape, non-motile and non-spore forming bacteria and are facultative anaerobe that grows at an optimum temperature of 37˚C, (Brooks, Carroll, Butel, Morse & Mietzner, 2007). These belong to genus Staphylococcus and are arranged in the form of grape like clusters (Ryan & Ray, 2004). Various infections are caused by S. aureus such as, skin infection, soft tissues infection, bone and lung infections, heart, brain and blood infections (Irving, Ala’Aldeen & Boswell, 2005; Jouda, 2013). Other infections caused by S. aureus include pneumonia, endocarditis, septic arthritis (Sherris, 1984).

Domain: Bacteria

                                    Phylum: Firmicutes

                                                Class: Bacilli

                                                            Order: Bacillales

                                                                        Family: Staphylococcaceae

                                                                                    Genus: Staphylococcus

                                                                                                Specie: S. aureus

Pseudomonas aeruginosa is a gram negative, motile and rod shape bacteria and belong to genus Pseudomonas (Mackie & McCarteny, 1989). P. aeruginosa grows well at 37 to 42˚C and occurs as single bacteria, in pairs, in short chains and sometimes producing a grape like structure (Baron, 1996; Brooks et al., 2007).  Various infections in humans are associated with P. aeruginosa such as Urinary tract infections, Pneumonia, Surgical site infection, Respiratory infections, Ocular infections, Ear infections, Skin and soft tissue infections and Burn sepsis (Trautmann, Halder, Hoegel, Royer & Haller, 2008).

 

 

 

Domain: Bacteria

                                    Phylum: Proteobacteria

                                                Class: Gammaproteobacteria

                                                            Order: Pseudomonadales

                                                                        Family: Pseudomonadaceae

                                                                                    Genus: Pseudomonas

                                                                                                Specie: P. aeruginosa

Klebsiella pneumoniae is a rod shape gram negative bacterium and belong to family Enterobacteriaceae. K. pneumoniae is a pathogen that is found in mouth, skin, intestine, urinary tract, respiratory tract and blood (Podschun & Ullmann, 1998; Paczosa & Mecsas, 2016). Various infections are associated with K. pneumoniae such as pyogenic liver abscesses, meningitis, necrotizing fasciitis, endophthalmitis and severe pneumonia (Li, Zhao, Liu, Chen & Zhou, 2014).

Domain: Bacteria

                                    Phylum: Proteobacteria

                                                Class: Gammaproteobacteria

                                                            Order: Enterobacteriales

                                                                        Family: Enterobacteriaceae

                                                                                    Genus: Klebsiella

                                                                                                Specie: K. pneumonia

METHODOLOGY

 

• Spiders Identification:

In this study silk extracted from Cyrtophora citricola was used to check antibacterial potential. Body colour of the spider is highly variable. Cephalothorax and legs are brownish with yellowish patches. Abdomen is greyish with yellow and brown patches. Legs are short, strong and stout. Hairs and spines are present on legs (Tikder, 1982).

Sampling:

Spiders were collected from the botanical garden of University of Punjab, botanical garden of Jallo park Lahore, Change Manga forest park and roadside bushes in Lahore.

• Sampling Methods:

Jerking method and Hand picking was used for sampling. In this method clean plastic jars were used. Only large spiders were collected and brought to laboratory, Department of Zoology, University of Education Lower Mall Campus Lahore.

• Spider keeping

Specially designed Wooden boxes were used to keep spiders in laboratory. Size of these boxes was 1×1 feet. Spiders were transferred from plastic jar to these boxes carefully without damaging body part of the spiders. As any disturbance may result in the decrease in the quantity of silk, boxes were placed at the least disturbance area of laboratory. Spiders can survive for months if they were not disturbed.

• Humidity:

Water soaked cotton was placed in boxes and were also sprayed with water to maintain humidity. Spider were observed on daily basis and the boxes were cleaned without disturbing the spiders.

• Temperature:

Spiders can tolerate a wide range of temperature. They were kept at room temperature in the laboratory.

• Feed:

Spider were fed on house flies. House flies were provided according to the size of spider after one week.

• Silk Extraction:

            Silk was collected by sterile glass rod before feeding without disturbing the spiders. Silk was kept in sterile eppendorf tubes.

Figure 3.4.      Silk extraction from wooden boxes in laboratory of Department of Zoology University of Education Lower Mall Campus, Lahore.

• Evaluation of Antibacterial Activity:

            The antibacterial activity of silk extracted from C. citricola was checked via agar disc diffusion method (Tendencia, 2004).

• Solvents for silk Dilution:

Silk was dissolved in five different solvents that are Acetone, Ethanol, Methanol, distilled water and 10% Sodium Hydroxide solution to make stock solution. Further dilutions were made by using the stock solution.

• Composition of 10% NaOH:

10g sodium hydroxide was dissolved in 100ml distilled water to form 10% sodium hydroxide solution.

• Preparation of silk solution:

Silk (0.03g) was measured with the help of weight balance and dissolved in 2ml solvent (Acetone, Ethanol, Methanol, Distilled water and 10% sodium hydroxide) in a test tube. Test tube was covered with cotton and labeled. After that the tubes were heated for 10 to 15 minutes on sprit lamp. Silk solution was cooled at room temperature and transferred the solution in sterile label eppendorf tube and centrifuged at 8000 rpm for 30 minutes. Mixture of each solvent was considered as stock solution and the percentage of each mixture was 1.5%. Two different dilutions (1% and 0.5%) of silk dissolved in 10% NaOH.

• Microbial strains:

Antibacterial activity of silk was checked against pathogenic bacteria (gram positive & gram negative). Four bacterial strains were used named as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae. S. aureus is gram positive bacteria and other three E. coli, P. aeruginosa and K. pneumoniae are Gram negative. 

• Preparation of Nutrient Broth Medium:

            Nutrient broth medium was prepared by mixing 0.3 gm yeast extract and 0.5 gm peptone in 100 ml distilled water. This media was prepared in 250ml flask and solution was mixed gently, the broth media was transfered in eight sterile test tubes, 10ml media poured in each test tube with the help of pipette. The cotton plug was placed in the mouth of these test tubes and then covered by Aluminum foil.  Later on, it was autoclaved at 15 atm pressure and 121.5^ᵒC temperature for 15 to 20 minutes.

• Revival of bacteria:

The broth media and test tubes were cooled at room temperature. 100ul bacteria was taken from bacterial culture and transferred to the labelled test tubes with the help of micropipette.  Whole procedure was conducted in laminar air flow. Then the test tubes were kept in incubator at 37^ᵒC for 24 hours. After 24 hours these test tubes were kept in fridge at 4^ᵒC. The bacterial strains were on the agar plates and other set was used for next revival of bacteria. Whole procedure was repeated after one week.

• Preparation of Nutrient Agar Medium:

            Nutrient Agar medium was prepared by mixing 0.3 gm yeast extract, 0.5 gm Peptone and 1.5 gm Agar and the mixture was dissolved in 100ml distilled water. This mixture was prepared in 500ml flask. Flask was covered by cotton plug and aluminum foil properly. The medium, petri plates, filter paper discs and Gilson tips were autoclaved at 15 atm pressure and 121. 5^ᵒC temperature for 15 to 20 minutes.

• Pouring:

After autoclaving nutrient agar media and petri plates were cooled at room temperature. These petri plates and agar medium was kept in the laminar air flow. Agar media was poured in petri plates and these poured petri plates allowed to solidify for 25 to 30 minutes the petri plates were stored in incubator at 37^ᵒC for 24 hours.

 

• Inoculation of plates:

             After 24 hours contamination free agar plates were divided into three regions by using marker. These agar plates were inoculated with the bacterial strains (100ul) obtained from freshly prepared nutrient broth medium with the help of micropipette and poured in the center of the petri plates. Sterile glass spreader was used to make sure the proper spreading of the bacteria on the agar plates.

• Dosing:

Silk solution was centrifuged at 8000 rpm for 30 minutes before use. After spreading bacteria, sterile filter paper discs of the size 6mm diameter were placed on each segment of agar plate. 10ul freshly prepared 1.5% standard antibiotic Amoxicillin solution was poured at center of filter paper disc with the help of micropipette for positive control. For negative control 10ul of respective solvent and 10ul spider silk solution was used. Later on, the petri dishes were incubated at 37^ᵒC for 24 hours. This whole procedure was performed in Laminar air flow (Bauer, Kirby, Sherris & Turck, 1996).

• Result:

            After 24 hours of dosing measure the inhibition zone was measured around the filter paper disc. Vernier Caliper was used for the measurement. Diameter of inhibition zone was measured in centimeters, which was converted into (mm) for further analysis.

• Statistical Analysis:

One-way ANOVA was used to analyze the result via SPSS (Version 16.0).

 

RESULTS

 

Antibacterial potential of silk extracted from Cyrtophora citricola was assessed in the present study. Silk was dissolved in five different solvents (10% NaOH, Distilled Water, Acetone, Ethanol and Methanol). After heating silk solution silk was entirely dissolved in 10% NaOH and partially dissolved in other four solvents. These prepared samples of silk were applied against four bacterial species (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus). Amoxicillin was used as positive control while, respective solvents were used as negative control and silk solutions were used as treatment. The diameter of inhibition zone was measured in centimeter (cm) with the help of Vernier Caliper and was converted to “mm’’ for statistical analysis.

• Antibacterial Potential of 1.5% Silk Solution in 10% NaOH:

            After 24 hours of inoculation of bacteria and dosing on petri plates clear inhibition zones were observed. Inhibition zones of positive control, negative control and 1.5% silk solution (10% NaOH) against all selected bacterial species are represented in figures 4.1 to 4.4. Statistical analysis showed the presence of clear difference in inhibition zones of control and treated groups against specific bacterial species (P-Value = 0 for E. coli, P.  aeruginosa & S. aureus, P-Value = 0.009 for K. pneumoniae). Clear difference in Mean of inhibition zones of control and treated groups were observed in case of E. coli. Diameter of inhibition zone for positive control was larger than treated group and negative control. However, diameter of inhibition zone for treatment was statically larger than negative control. Statistical analysis didn’t show any effect of silk solution against P. aeruginosa, S. aureus and K. pneumonia (Table 4.1).

 

Table 4.1

Statistical analysis showing variation in inhibition zones of control (+ve & -ve) and treated groups (1.5% silk solution in 10% NaOH) against selected bacterial species.

Bacterial Species       Treatments          Inhibition zone              d/f         F           P

                                                                  (Mean ± S.E)

       Control (+ve)            19.6667^c ± 0.88192

11. coli Control (-ve)             11.8000^a ± 0.20000        2,6     56.745   0.000

                               Treatment (Silk)        14.7333^b ± 0.13333

                               Control (+ve)            20.0000^b  ± 1.00000 

12. aeruginosa Control (-ve)             12.6000^a  ± 0.60000       2,6    11.555    0.009

                               Treatment (Silk)       14.5667^a  ± 1.56667                                              

       Control (+ve)            21.5333^b ± 0.46667

12. aureus Control (-ve)      12.9333^a ± 0.63596      2,6    43.809    0.000  

                               Treatment (Silk)       15.0667^a ± 0.86667                                   

                               Control (+ve)            21.0000^b ± 0.57735 

13. pneumoniae Control (-ve)      13.0000^a ± 0.57735      2,6    39.529    0.000

                               Treatment (Silk)       14.6000^a ± 0.83267                                   

 

• Antibacterial Potential of 1% Silk Solution in 10% NaOH:

After 24 hours of inoculation of bacteria and dosing. Inhibition zones were clearly observed on petri plates. Diameter of Inhibition zone recorded for Positive control was always largest as compared to negative control and treatment zones against all selected bacterial species as shown in figure 4.5 to 4.8. Statistical analysis (P-Value = 0.002 for E. coli & P. aeruginosa, P-Value = 0.001 for S. aureus & K. pneumoniae) revealed significance difference among positive control and other treatments against specific bacterial species. Clear difference in Mean of inhibition zones of control and treated groups were observed in case of S. aureus. Diameter of inhibition zone for positive control was larger than treated group and negative control. However, diameter of inhibition zone for treatment was larger than negative control. Statistical analysis didn’t show any effect of silk solution against P. aeruginosa, E. coli and K. pneumonia (Table 4.2)

Table 4.2

Statistical analysis showing variation in inhibition zones of control (+ve & -ve) and treated groups (1% silk solution in 10% NaOH) against selected bacterial species.

Bacterial Species       Treatments          Inhibition zone              d/f         F           P

                                                                  (Mean ± S.E)

       Control (+ve)            23.0000^b ± 0.57735

13. coli Control (-ve)             13.2333^a ± 1.50702       2,6     22.079   0.002

                               Treatment (Silk)       15.4000^a ± 0.98489

                               Control (+ve)            20.3367^b ± 0.44845 

12. aeruginosa Control (-ve)             12.7667^a ± 0.39299      2,6    20.489    0.002

                               Treatment (Silk)       15.4667^a ± 1.34825                                               

       Control (+ve)            21.6333^c ± 0.63333

13. aureus Control (-ve)      13.4333^a ± 0.79655      2,6    33.610    0.001  

                               Treatment (Silk)       16.9667^b ± 0.68880

                               Control (+ve)            21.0000^b ± 0.51316

14. pneumoniae Control (-ve)      14.2333^a ± 0.90615      2,6    33.610    0.001

                               Treatment (Silk)       15.3667^a ± 0.68880                                   

 

• Antibacterial activity of 0.5% Silk Solution in 10% NaOH:

Clear inhibition zones were observed on petri plates after 24 hours of inoculation and dosing for all selected bacterial species. Diameter of positive control and treatment zones were slightly different, but diameter of inhibition zone for negative control was much smaller than positive, and treatment as depicted in figures 4.9 to 4.12. Statistical analysis (P-Value = 0.000 for E. coli & S. aureus, P-Value = 0.003 for P. aeruginosa, P-Value = 0.034 for K. pneumoniae) showed that 0.5% Silk solution effective against E. coli and S. aureus but Silk solution highly effective against P. aeruginosa and K. pneumoniae. Clear difference in inhibition zones of control and treated groups were observed in case of P. aeruginosa, K. pneumonia, E. coli and S. aureus. Diameter of inhibition zone for positive control was larger than treated group and negative control. However, diameter of inhibition zone for treatment was statically larger than negative control (Table 4.3).

 

Table 4.3

Statistical analysis showing variation in inhibition zones of control (+ve & -ve) and treated groups (0.5% silk solution in 10% NaOH) against selected bacterial species.

Bacterial Species       Treatments          Inhibition zone              d/f         F           P

                                                                  (Mean ± S.E)

       Control (+ve)            21.2333^c ± 0.66916

11. coli Control (-ve)             11.8667^a ± 0.41767        2,6     91.429   0.000

                               Treatment (Silk)       17.6667^b ± 0.33333

                               Control (+ve)            20.6000^b ± 0.30551

15. aeruginosa Control (-ve)             15.2333^a ± 0.39299       2,6    18.677    0.003

                               Treatment (Silk)       18.9667^b ± 0.98376                                               

       Control (+ve)            22.1667^c ± 0.44096

15. aureus Control (-ve)      15.1667^a ± 0.76884      2,6    41.705    0.000  

                               Treatment (Silk)       18.1000^b ± 0.32146                                   

                               Control (+ve)            19.6667^b ± 0.33333 

12. pneumoniae Control (-ve)      12.8333^a ± 1.16667      2,6    6.263      0.034

                               Treatment (Silk)       18.3667^a,b± 2.19874                                 

 

• Antibacterial Potential of Silk dissolved in Methanol:

After 24 hours of dosing, inhibition zones were observed on petri plates. Positive control always biggest and clear inhibition zone but negative control and treatment were not clear inhibition zones against all selected bacterial species as represented in figures 4.13 to 4.16. According to Disc diffusion method ignore those zones in which present individual colonies within the fuzzy zone so these were neglected in the study (Tendencia, 2004).

 

• Antibacterial Potential of Silk dissolved in Ethanol:

After 24 hours of dosing, inhibition zones were observed on petri plates. Positive control always biggest and clear inhibition zone but negative control and treatment were not clear inhibition zones against all selected bacterial species as shown in figures 4.17 to 4.20. According to Disc diffusion method ignore those zones in which present individual colonies within the fuzzy zone so these zones were not recorded for further analysis  (Tendencia, 2004).

Petri plates were observed for inhibition zones after 24 hours of inoculation and dosing. Inhibition zones for positive control were always bigger and clear. While no clear zone observed for negative control for all selected bacterial species. However, treatment zones were fuzzy against all bacterial strains as showed in figures 4.25 to 4.28. According to Disc diffusion method fuzzy zones are not considered, so we did not consider these zones.

• Comparison among control groups (+ve, -ve) and different treatments (1.5%, 1% & 0.5% solution in 10% NaOH) apply against all selected bacterial strains:

Different dilutions of silk (1.5%. 1% and 0.5%) in same solvent were used to check antibacterial potential of silk against four bacterial strains (E. coli, S. aureus, P. aeruginosa and K. pneumoniae).

• Comparison among control groups (+ve, -ve) and different treatments (1.5%, 1% & 0.5% solution in 10% NaOH) applied against E. coli:

Statistical analysis (P-Value = 0) showed significant variations in inhibition zones of treatment against E. coli. 0.5% silk dilution showed maximum antibacterial potential against E. coli. Diameter of inhibition zone for positive control is larger than treatment group and diameter of inhibition zone for treatment is larger than negative control in 0.5% silk solution, but 1.5% and 1% dilution of silk showed slight effect against E. coli as showed in table 4.4.

 

Table 4.4

Comparison of inhibition zones of control groups and treatment of 1.5%, 1% and 0.5% silk solution (10%NaOH as solvevent) used against E. coli.

Bacterial Species       Treatments          Inhibition zone              d/f         F           P

                                                                  (Mean ± S.E)

Control (Group 1)  Positive                         21.3000^c ± 0.96193

Control (Group 2) Negative                         12.2999^a ± 0.46695        

1. coli            Treatment (1.5% Silk)    14.7333^a,b ± 0.13333     4,10     25.717   0.000
2. coli                Treatment (1% Silk)       15.4000^a,b ± 0.98489
3. coli Treatment (0.5% Silk)     17.6667^b ± 0.33333                                           

 

• Comparison among control groups (+ve, -ve) and different treatments (1.5%, 1% & 0.5% solution in 10% NaOH) applied against S. aureus:

Statistical analysis (P-Value = 0) showed significant variations in inhibition zones against S. aureus. Clear difference in inhibition zones of control and treated groups was observed in case of S. aureus. Diameter of inhibition zone for positive control is larger than treatment group and diameter of inhibition zone for treatment is larger than negative control in case of 1% and 0.5% silk solution. Silk solution of 1% and 0.5% show maximum antibacterial potential against S. aureus as compare to 1.5% solution. 1.5% dilution showed minor antibacterial effect against S. aureus as depicted in table 4.5.

Table 4.5

Comparison of inhibition zones of control groups and treatment of 1.5%, 1% and 0.5% silk solution used against S. aureus when silk was dissolved in 10% NaOH.

Bacterial Species       Treatments          Inhibition zone              d/f         F           P

                                                                  (Mean ± S.E)

Control (Group 1)  Positive                             21.7776^d ± 0.19670

Control (Group 2)  Negative                            13.8444^a ± 0.67671    4,10    25.758    0.000    

1. aureus        Treatment (1.5% Silk)      15.0667^a,b ±  0.86667      
2. aureus Treatment (1% Silk)      16.9667^b,c ±  0.68880    
3. aureus Treatment (0.5% Silk)   18.1000^c ± 0.32146                                                                

 

 

• Comparison among control groups (+ve, -ve) and different treatments (1.5%, 1% & 0.5% solution in 10% NaOH) applied against P. aeruginosa:

Statistical analysis (P-Value = 0.006) revealed that significant variation is present among inhibition zones of treatmeants against P. aeruginosa. 0.5% and 1% silk dilution showed maximum antibacterial potential against P. aeruginosa in comparison to 1.5% dilution of silk dissolved in 10% NaOH but 1.5% dilution showed trivial antibacterial effect against S. aureus as represented in table 4.6.

Table 4.6

Comparison of inhibition zones of control groups and treatment of 1.5%, 1% and 0.5% silk solution used against P. aeruginosa when silk was dissolved in 10% NaOH.

Bacterial Species       Treatments          Inhibition zone              d/f         F           P

                                                                  (Mean ± S.E)

Control (Group 1) Positive                            20.3122^c ± 0.17364 

Control (Group 2) Negative                          13.5333^a ± 0.85134     4,10     7.131    0.006   

1. aeruginosa Treatment (1.5% Silk)   14.5667^a,b ± 1.56667       
2. aeruginosa Treatment (1% Silk)    15.4667^a,b,c ± 1.34825    
3. aeruginosa Treatment (0.5% Silk) 18.9667^b,c ± 0.98376                                                  

• Comparison among control groups (+ve, -ve) and different treatments (1.5%, 1% & 0.5% solution in 10% NaOH) applied against K. pneumoniae:

Statistical analysis (P-Value = <0.01) revealed difference in treatments inhibition zones against K. pneumoniae. 0.5% and 1% silk dilutions slightly inhibit growth of K. pneumoniae in comparison to 1.5% dilution that did not show any inhibitory effect (Table 4.7).

Table 4.7

Comparison of inhibition zones of control groups and treatment of 1.5%, 1% and 0.5% silk solution used against K. pneumoniae when silk was dissolved in 10% NaOH.

Bacterial Species       Treatments          Inhibition zone              d/f         F           P

                                                                  (Mean ± S.E)

Control (Group 1)    Positive                         20.5567^b ± 0.44333 

Control (Group 2)    Negative                        13.3553^a  ± 0.44147    4,10     6.122    <0.01  

1. pneumoniae Treatment (1.5% Silk) 14.6000^a  ± 0.83267      
2. pneumoniae Treatment (1% Silk)    15.3667^a,b  ± 1.08064   
3. pneumoniae Treatment (0.5% Silk) 18.3667^a,b  ± 2.19874                                                 

DISCUSSION

In the present study silk obtained from C. citricola was dissolved in five different solvents (Ethanol, Methanol, Acetone, Distilled Water and 10% NaOH) and checked for antibacterial activity against four selected bacterial strains (E. coli, P. aeruginosa, K. pneumonia and S. aureus). Silk was entirely dissolved in 10% NaOH and partially dissolved in other four solvents (Methanol, Ethanol, Acetone and Distilled water). 1.5% Silk (10% NaOH) showed inhibitory effect against bacteria. So, further dilution (1% & 0.5%) were made. Results showed that 0.5% silk solution more effective against all selected bacterial strains (E. coli, P. aeruginosa, K. pneumonia and S. aureus) as compared to other silk solution (1% & 1.5%).  1.5% silk solution showed significant effect against E. coli and silk show negligible effect against P. aeruginosa, K. pneumonia and S. aureus. 1% silk solution more effective against S. aureus and negligible effect against other selected bacteria. 0.5% silk solution inhibit the growth of all selected bacteria. Silk was dissolved in other four solvents (Methanol, Ethanol, Acetone and Distilled water) they did not show inhibition zones against selected bacteria.

            Tahir et al. (2019) reported that antibacterial agents present in the silk of Eriovixia excelsa. After 24 hours of incubation silk of E. excelsa show inhibitory effect against Acinetobactor sp. and Streptococcus sp. Spider silk show more effect against Acinetobactor sp. (gram negative) bacteria as compared to Streptococcus sp. (gram positive) bacteria. Tahir et al. (2018) reported that spider silk recovered from Pholcus phalangiodes show antibacterial potential against both gram-negative and gram-positive bacteria. Four bacterial species were used in this study two gram positive (Streptococcus pneumoniae and Staphylococcus aureus) and two gram negative (Acinetobacter baumannii and Pasteurella multocida) but spider silk showed great antibacterial potential against A. baumannii and S. aureus. Inhibitory effect was slight against P. multocida and S. pneumoniae. Tahir et al. (2017) reported that silk of Cyclosa confraga show inhibitory effect against the growth of both gram-negative and gram-positive bacteria. These results are quite similar to Amaley et al. (2014). They used spider silk recovered from Nephila pilipes. Its silk showed inhibitory effect against E. coli, S. aureus and P. aeruginosa.  It was more effective against gram negative bacteria as compared to gram positive bacteria. Wright and Goodacre (2012) reported that bacteriostatic activity of silk is different in different species of spider. Silk recovered from Tengenaria domestica showed antibacterial effect against Bacillus subtilis (gram positive bacteria) but there was no effect against E. coli (gram negative). Roozbahani et al. (2014) reported that silk of Pholcus phalangioides showed significant effect against Listeria monocytogenes (gram positive bacteria) and negligible effect against gram negative bacteria E. coli. Tahir et al. (2015) also reported antifungal potential of web silk recovered from Neoscona theisi.

Heimer (1988) reported that spider silk contains antibacterial compounds and is also acidic in nature. Chakraborty and Das (2009) reported that spider silk has phospholipids hydrate and potassium nitrate which make it acidic in nature and show inhibitory effect against both gram negative and gram-positive bacteria. Silk extracted from Crossopriza lyoni also known as daddy longless spider show inhibitory effect against both gram negative and gram-positive bacteria. According to wright (2011) Zilladiodia and Linyphiidae spiders silk show inhibitory effect against B. subtilis (gram positive bacteria). Mirghani et al. (2012) also reported that spider silk shows higher inhibitory effect against Listeria monocytogenes (gram positive) than E. coli (gram negative) bacteria.

 According to Phartale, Kadam, Bhosale, Karale & Garimella (2019) silk recovered from Pardosa brevivulva was show antibacterial effective against microbes. By the analysis of FT-IR, ¹³C & ¹H NMR and C₁₈ column RP- HPLC silk of Pardosa brevivulva was characterized into antimicrobial compounds. These antimicrobial compounds inhibit the growth of Bacillus megaterium, Salmonella typhi, K. Pneumonia, Aspergillus flavus, Candida albicans, Ustilago maydis and A. solani.

Al-Kalifawi & Kadem (2017) reported that spider silk is dissolved in different solvents i.e., Acetone, Ethanol and Distilled water. However, Acetone extract is more effective to inhibit bacterial growth as compare to other solvents extract. It showed inhibition zone against gram-negative (Escherichia coli and Pseudomonas aeruginosa) and gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis). Inhibition zones was higher against gram-positive bacteria as compare to gram-negative bacteria.

From the above discussion it is clear that silk of certain spider species shows antibacterial potential. Silk of different spiders effect the growth of different species of bacteria. Some spider silk shows antibacterial potential against gram-positive bacteria, some show against gram-negative bacteria and some show effect against both. These antibacterial properties of silk may be due to acidic nature of silk.

 

CONCLUSION AND RECOMMENDATIONS

 It is concluded from present study that silk of Cyrtophora citricola (dissolved in 10% NaOH) has inhibitory effect against all assessed bacterial species, but inhibitory effect against gram positive bacteria (Staphylococcus aureus) is higher than gram negative bacteria (Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae). Further studies are recommended to characterize spider silk for antibacterial peptides.