Investigation of antibacterial and haemolytic activity of Russell’s viper and Echis carinatus venom

AK. Prabhakaran¹*, P. Kumaravel¹, J. Priya¹, G. Melchias², A. Edward³, G. Sridevi⁴

¹Department of Biotechnology, Vysya College, Salem- 636 103, Tamil Nadu, India.

²Department of Botany, St. Joseph's College (Autonomous), Tiruchirappalli- 620 002, Tamil Nadu, India.

³Department of Biotechnology, St. Joseph's College (Autonomous), Tiruchirappalli- 620 002, Tamil Nadu, India.

⁴Department of Antitoxin, King’s Institute of Preventive Medicine and Research, Guindy, Chennai- 600 032, Tamil Nadu, India.

*Corresponding Author E-mail: prabhabiotech@gmail.com

ABSTRACT:

Snake bites represent a serious public health problem around the world. Snake venom known to cause different metabolic disorders by altering the cellular and enzymatic activities in animals. The present study aims to investigate the effect of antibacterial and haemolytic activity of Russell’s viper (RV) and Echis carinatus (EC) venom. Most bacteria’s are highly active against RV and EC venom at this concentration (100µg/ml). The current results showed that Russell’s viper and Echis carinatus venom has a significant antibacterial effect against Escherichia coli, Pseudomonas aeruginosa and Vibrio cholera by comparing with standard antibiotics. The haemolytic activity effect was determined with blood agar plates and finally it confirms the venom having a property of haemolysis.

KEYWORDS: Russell’s viper, Echis carinatus, Antibacterial activity, Haemolytic activity.

1. INTRODUCTION:

Snake is a limbless reptile. Snake produces numerous biological effects through its venom and has therefore proven to be very useful [1,2]. It is acknowledged that snakebite is a serious medical problem in rural India and in April 2009, snakebite was added to world Health Organisation list of neglected tropical diseases [3,4]. A recent study reported that, the worldwide total number of snakebites could be as high as 5.5 million with 94,000 deaths [5,6]. In India, all snakes are protected under the Wildlife Protection Act and as such, snakes cannot be collected or venom extracted without the permission of the state wild life authorities [7]. Bacterial infections are among the ten causes of death worldwide according to the World Health Organization. The presence and emergence of resistant strains make the risk of these infections a universal problem with deleterious effects. Therefore, the discovery of new alternatives is necessary for treatment of infections involving resistant microorganisms [8].

Several antimicrobial studies involving venom of snakes have been described. The majority of bacteria such as Pseudomonas aeruginosa, Klebsiella, Entrobacter, Acinobacter, Salmonella, Staphylococcus, Methicillin Resistant Staphylococcus aureus (MRSA), Enterococcus and Penicillin resistant Streptococcus pneumoniae (PRSP) vancomycin-resistant enterococci have developed several ways to resist antibiotics. Such bacteria are becoming a serious clinical problem throughout the world [9,10].

From earlier days, most Indian patients of snake bites have been the victims of Russell's viper and Echis carinatus [11,12,13]. In India polyvalent Anti snake venom is available which contain antibody against Cobra, Russell’s viper, Common krait and Saw scaled viper [14]. Echis carinatus is one of the most venomous viper snakes in the world, found specifically in India, Pakistan, Afghanistan and Iran [15,16]. Russell’s viper or Daboia (Viper russelli) appears to be the commonest cause of fatal snakebite in Southern India, Pakistan, Bangladesh, Sri Lanka, Burma and Thailand [17]. Viper russelli (Indian subspecies of Russell’s viper) is one of the four-major classes of snakes that cause death in the Indian sub-continent [18].

The aim of the present study was to investigate the antibacterial activity of RV and EC crude venom against Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa) and Vibrio cholera (V. cholera). Also the study extends by investigating the haemolytic activity by blood agar plates by disk diffusion method.

2. EXPERIMENTAL DESIGN:

2.1 Preparation of venom

Lyophilized crude venom of RV and EC venom was obtained from Department of Antitoxin, King’s Institute of Preventive Medicine and Research, Chennai, Tamil Nadu, India. 1g of lyophilized dry form of venom was weighed accurately and dissolved in a 50ml of normal saline (sterile). Centrifuged at 1500 rpm for 15 minutes. After centrifugation, the unwanted mucus and tissues were discarded. The clear supernatant was filtered through membrane filter or cellulose acetate. The volumes of venom were measured and add the equal amount of sterile glycerine. Aliquots were stored in sterile bottles and kept at 4˚C hence the 1% of venom was prepared.

2.2 Antibacterial agents

Different strains like E. coli, P. aeruginosa and V. cholera obtained from the same institution and used for testing antibacterial effect. The slant slope cultures were obtained from the clinical and diagnostic department of King Institute of Preventive Medicine and Research, Chennai. Muller Hinton Agar (MHA) was prepared and poured into the eight sterile petriplates for both RV and EC two for antibiotic plate (control for RV and EC) and six for venom sample plate. Bacterial cultures were streaked using inoculation loop uniformly on MHA plates. Antibiotic discs were used as control, antibiotic discs were soaked into the venom and dried that was used as a test. Dried venom discs were placed with the help of sterile forceps on the MHA plates and incubated for 24 hours.

2.3 Standard antibiotics

Standard antibiotics like Amoxycillin (AM30), Ceftrixone (CTX30), Cefazolin (CZ30) used for RV and variation of Ticarcillin (Ti), Cefixime (CFM5) and Erythromycin (E15) used for testing EC venom. The drug discs were obtained from the clinical and diagnostic department of King Institute of Preventive Medicine and Research, Chennai.

2.4 Haemolytic activity

Blood agar (Horse blood) and plates were obtained from the media section of King Institute of Preventive Medicine and Research, Chennai. Blood agar was prepared by adding 40ml of defibrinated blood with 450ml of nutrient agar. 15ml of blood agar was poured into the three sterile Petri plates and allowed to solidify. Single well (5mm in diameter) was punched on each plate using a gel puncher. 5µl of 1% RV and EC venom was added and then the plates were incubated for 48 hours and zone of lyses were measured.

3. RESULTS AND DISCUSSION:

In our current study the antibacterial effects of RC venom against some pathogenic bacteria such as E. coli, P. aeruginosa and V. cholera were shown in the figure 1 and table 1. Here in, it was clear that RC venom shows broad spectrum antibacterial effect against the above mentioned bacteria, although its shows significant activity against P. aeruginosa in comparison with the standard antibiotics, Amoxycillin (AM30), Ceftrixone (CTX30), Cefazolin (CZ30) has been observed. E. carinatus showed the least antibacterial activity against P. aeruginosa and while other bacterial strains V. cholera and E. coli it was found that the maximum inhibitory concentrations when compared with the standard antibiotics, Ticarcillin (Ti), Erythromycin (E15), Cefixime (CFM5) were shown in the figure 2 and table 2. The modes of antibacterial action of various proteins and enzymes from the venom are by destabilizing the microbial membrane, inhibiting the synthesis of specific membrane proteins or stress proteins, interaction with DNA and arrest of DNA synthesis, may help to inhibit bacterial growth [19,20].

Table 1: Invitro antibacterial activity of RV crude venom tested by disc diffusion and compared to some standard antibiotics.

Antibiotics / Venom	Microorganisms (Zone of inhibition (mm in diameter))
Antibiotics / Venom	*V. cholerae*	*P. aeruginosa*	*E. coli*
RV Venom (100µg/ml)	24	12	28
Amoxycillin (AM30)	30	8	23
Ceftrixone (CTX30)	31	24	34
Cefazolin (CZ30)	13	6	30

Table 2: Invitro antibacterial activity of EC crude venom tested by disc diffusion and compared to some standard antibiotics.

Antibiotics / Venom	Microorganisms (Zone of inhibition (mm in diameter))
Antibiotics / Venom	*V. cholerae*	*P. aeruginosa*	*E. coli*
EC venom (100µg/ml)	22	10	24
Ticarcillin (Ti)	2	6	4
Erythromycin (E15)	30	16	26
Cefixime (CFM5)	28	4	25

Most of the pathogenic bacteria named above are highly active against RV and EC which confirms haemotoxic efficiency for antibacterial activity. However, certain species had become resistant to certain antibiotics and hence slight visible zones were observed. The crude venom was loaded in Blood Agar Plates (BAP). The BAP was observed after 24 hrs of incubation. A zone of lysis of 2cm diameter of RV and 2.1cm diameter of EC was observed were shown in the figure 3. This shows the haemolytic

4. CONCLUSION:

Snake is a limbless reptile. There are about 2,500 species of snake. Some inhabit the sea and others live in freshwater, but the majority live on land. Antisnake venom which is a life saving weapon is the only proven antidote but it’s a double edged sword because of the allergic reactions associated with it. This study indicated that Russell’s viper and Echis carinatus shows maximum effect on antibacterial and haemolytic effects. To determine whether RV and EC venom can influence other pathogens, further studies are needed using a wider spectrum of microbial studies and also other concentrations of this venom.

5. REFERENCES:

[1] C.S. Phua, J. Vejayan, S. Ambu, G. Ponnudurai, A. Gorajana. Purification and antibacterial activities of an L-amino acid oxidase from king cobra (Ophiophagus hannah) venom. J. Venomous Animals and Toxins including Tropical Diseases. 2012;18: 198-207.

[2] A. Gomes, A. Saha, L. Chatterjee, A.K. Chakravarty. Viper and cobra venom neutralization by sitosterol and stigmasterol isolated from the root extract of Pluchea indica Less. (Asteraceae). Phytomedicine. 2007;14: 637-643.

[3] World Health Organization, 2010. WHO Guidelines for the Production Control and Regulation of Snake Antivenom Immunoglobulins. Adopted by the WHO Expert Committee on Biological Standardization at 59th meeting, World Health Organization (WHO), Geneva.

[4] R.A. Harrison, A. Hargreaves, S.C. Wagstaff, B. Faragher, and D.G. Lalloo. Snake envenoming: a disease of poverty. PLoS Negl. Trop. Dis. 2009: 3.

[5] P.S. Gawade. Therapeutic alternatives from venoms and toxins. Indian Journal of Pharmacology. 2007;6: 260-264.

[6] A. Kasturiratne, A.R. Wickremasinghe, N. de Silva, N.K. Gunawardena, and A. Pathmeswaran. The global burden of snake-bite: a literature analysis and modelling based on regional estimates of envenoming and deaths. PLoS Med. 2008; 5.

[7] Indian Wildlife (Protection) Act (1972), Government of India, Amended up to 2008.

[8] A.D. Lopez, C.D. Mathers, M. Ezzati, D.T. Jamison, C.J. Murray. Global Burden of Disease and Risk Factors. Washington, DC, USA: IBRD/ The World Bank and Oxford University Press; 2006.

[9] J.Y. Ang, E. Ezike, and B.I. Asmar. Antibacterial resistance. Symposium series Society for Applied Microbiology Ser Soc Appl Microbiol. 2004;3: 229-239.

[10] A. Shirwaikar, K. Rajendran, R. Bodla, C.D. Kumar. Neutralization potential of Viper russelli russelli (Russell’s viper) venom by ethanol leaf extract of Acalyphaindica. J. Ethnopharmacol. 2004; 94: 267-273.

[11] K.S. Chugh. Snake-bite-induced acute renal failure in India. Kidney International. 1989;35: 891-907.

[12] H.P. Desmond, J.M. Crampton, R.D.G. Theakston. Rapid isolation and partial characterization of two phospholipases from Kenyan Echis carinatus leakeyi (Leakey's saw-scaled viper) venom. Toxicon. 1991;29: 536-539.

[13] Atena Jami al ahmadi, Behrooz Fathi, Abdoula Jamshidi, Hosein Zolfagharian and Abbase Zare Mirakabbadi. Investigation of the Antibacterial Effect of Venom of the Iranian Snake Echis carinatus. Iranian J. of Veterinary Science and Technology. (2010;2: 93-100.

[14] S.P. Dhanya, R. Bindu Lattha, C.G. Hema, T.H. Dhanya. Antisnake venom use: A retrospective analysis in a tertiary care centre. Calicut Medical Journal. 2009; 7(3).

[15] K. Kemparaju, B.N. Prasad, T.V. Gowda. Purification of a basic phospholipase A2 from Indian saw-scaled viper (Echis carinatus) venom: characterization of antigenic, catalytic and pharmacological properties. Toxicon. 1994;32: 1187-1196.

[16] S. Backshall. Steve Backshall's venom: Poisonous creatures in the natural world. New Holland publishers (UK) Ltd, London; 2007.

[17] D.A. Warrel. Clinical toxicology of snakebites in Asia, CRC press, Boca Raton. FL. 1995: 493-594.

[18] A. De Silva, L. Ranasinghe. Epidemiology of snakebite in Sri Lanka: A review. Ceyl. Med. J. 1983;28: 144-154.

[19] R.G. Thomas and F.H. Pough. The effect of rattlesnake venom on digestion of prey. Toxicon. 1979;17: 221-228.

[20] K. De Smet and R. Contreras. Human antimicrobial peptides: defensins, cathelicidins and histatins. Biotechnol. Lett. 2005;27: 1337-1344.

[21] E. Condrea, A. De Vries, J. Mager. Hemolysis and splitting of human erythrocyte phospholipids by snake venoms. Biochimica Biophysica Acta- Specialized Section on Lipids and Related Subjects. 1964;84(1): 60-73.

[22] R. Permual Samy, P. Gopalakrishnakone, M.M. Thwin, T.K.V. Chow, H. Bow, Y.E. Hain, T.W.J. Thong. Antibacterial activity of snake, scorpion and bee venoms: a comparison with purified venom phospholipase A2 enzymes. J. of Applied Microbiology. 2007;102: 650-659.

Received on 31.01.2014 Accepted on 10.02.2014

Asian J. Pharm. Ana. 4(1): Jan.-Mar. 2014; Page 01-04