ASSESSMENT OF ANTIMICROBIAL ACTIVITY OF ECHIS OCELLATUS (CARPET VIPER) VENOM AGAINST BACTERIA AND FUNGI ISOLATED FROM CLINICAL SAMPLES IN NORTHEASTERN NIGERIA

334 Nigerian Journal of Pharmaceutical and Biomedical Research Vol. 8 Issue.3 December, 2024. p-ISSN: 2579-1419 e-ISSN: 2814-1423 ASSESSMENT OF ANTIMICROBIAL ACTIVITY OF ECHIS OCELLATUS (CARPET VIPER) VENOM AGAINST BACTERIA AND FUNGI ISOLATED FROM CLINICAL SAMPLES IN NORTHEASTERN NIGERIA Yahaya Tijani1, Askira, U.M2., Abubakar Settima3, Dige Mohammed Abba4, Mustapa Babagana5, Salisu Muhmmed1, Adamu Akawo1, Abbas Garba1, Maryam Umar Bukar1, Muhammad Musa Jaji1, Pam Haruna Samuel1, Lassafi Commander Bulus1, Hassan Zanna1 1Department of Biochemistry, Faculty of Life Sciences, University of Maiduguri, Borno State, Nigeria. 2Department of Medical Laboratory Science, University of Maiduguri. Borno State, Nigeria. 3Department of Microbiology, Faculty of Life Sciences, University of Maiduguri, Borno State, Nigeria. 4Department of Biotechnoloy, Faculty of Life Sciences, University of Maiduguri, Borno State, Nigeria. 5Department of Laboratory Science Technology, Ramat Polytechnic, Maiduguri, Borno State. Nigeria. Corresponding author: e-mail:yahay[email protected] http://doi.org/10.55639/607.phar.101201.0012 ABSTRACT Bacterial infections involving multidrug-resistant strains are among the top leading causes of death worldwide. Healthcare systems across the globe have been suffering from an extraordinary burden in looking for new and more potent antimicrobial compounds. The study was conducted in Maiduguri northeastern Nigeria on 5 different bacteria and 2 fungi isolated from clinical samples, to determine the antimicrobial activity of venomous snake’s carpet viper using a prepared venom sensitivity disc of different concentrations 25µg/ml, 50µg/ml and 100µg/ml respectively and a control antibiotics ciprofloxacin 30µg/ml and Nystatin 10000IU. Seven microorganisms demonstrate different variations in the zones of inhibition. the antibacterial activity of Carpet Viper venom against gram-positive bacteria at different venom concentrations (25 µg/mL, 50 µg/mL, and 100 µg/mL) alongside Ciprofloxacin (30 µg) as the control, the result have shown that the venom at different concentration have shown significant activity against staphylococcus aureus and streptococcus agalaqctae in all the concentration with highest in 100µg/ml (0.00mm±1.0mm) and least in 25µg/ml (0.00±0.1mm) while the control ciprofloxacin30µg/ml(0.00mm±0.8mm). The results of gram-negative bacteria have shown that no activity was seen against Salmonella typhimurium and proteus mirabilis at all the concentrations only for the control with 25.00 + 0.034mm and 11.2+0.15mm in diameter respectively. With respect to Haemophilus influenza, there is a remarkable activity in all the concentrations with the highest in 100µg/ml (10.3 ± 0.14) followed by 50µg/ml (10.2 ± 0.19) and the least 25µg/ml (09.1 ± 0.11) mm respectively. the antifungal activity of Carpet Viper venom against fungi at different venom concentrations (25 335 µg/mL, 50 µg/mL, and 100 µg/mL) alongside nystatin (30 µg) as the control the result has shown that at different concentrations there is significant activity of the venom on fungal isolate, candida albicans and Trichophytum mentagrophytes with highest activity in Candida at 50µg/ml (14.0 ± 10.36) and T. mentagrophyte at 100µg/ml (12.00±15.00mm). The inhibition zone diameter was measured in millimeters to assess the effectiveness of each concentration. In conclusion, the Carpet Viper’s venom demonstrates notable antimicrobial activity against bacteria and fungi through multiple mechanisms including membrane disruption and protein synthesis inhibition. Keywords: Snake, Venom, Venom sensitivity Disc, Antifungal, Antibacterial, Maiduguri. Introduction Bacterial infections have become increasingly difficult to treat as microorganisms have been developing resistance to a variety of antimicrobial agents, for example, Pseudomonas, Enterobacter, Klebsiella, Acinetobacter, Mycobacterium, Salmonella, Staphylococcus, Streptococcus and Enterococcus spp. Antibiotic-resistance is a serious problem and it is a major challenge in medicine since not many new antibiotics are being produced. Moreover, bacteria resistant to currently available drugs are increasing. To overcome the multidrug-resistant bacteria (MDR), new antimicrobial agents that are broadly effective are strategic. It has been reported that natural products are an important source of medicinal compounds and they are able to kill bacteria. Currently, it is known that venoms can be useful and valuable as pharmacological substances in drug research Snake venoms contain many proteinaceous and nonproteinaceous components. Their venoms are a mixture of proteins and peptides (90-95%), including nucleotide, amino acids, lipids, carbohydrates, and metallic elements bound to proteins (5%), for example; neurotoxin, cytotoxins, myotoxins, proteases, nuclease, and cytotoxins, myotoxins, proteases, nuclease, and peptides (Fry et al,.2004). Most venomous snakes belong to two families, the Elapidae (cobras, mambas, kraits, coral snake, and sea snake) and Viperidae (rattlesnakes, copperheads, cottonmouths, European vipers, etc.). Snake venoms vary in proportions and characteristics of the specific biochemical activities among different species. They contain numerous components of bioactive compounds. The broad spectrum of snake venom activities results from the actions of their constituents. It has been reported that snake venoms and other animal venoms are a rich source of protein and non-protein of pharmacological interest. Antibacterial components in snake venoms may protect the host after eating prey contaminated with pathogens (Abdulrahman et al., 2015). Several antimicrobial of snake venoms have been described in the literature, the inhibitory effects of Naja naja sputatrix, Vipera russelli, and Crotalus adamanteus in E. coli (Skarnes et al., 2013), the antibacterial activities of 30 different snake venoms had been studied by Sitles et al and found that the Asian and African snakes (Naja sp.), Australian elapids (Notechis scutatus and Pseudechis australis) and North American snakes (Crotalus spp.) presents the highest 336 antibacterial activities. Antibacterial effects of viperid venoms have been described (Skarnes et al., 2013). Bactericidal effects of rattlesnake venoms were observed on gram-positive Sarcina species, but there was little effect against Bacillus subtilis, E. coli, or S. aureus. More recently reported, snake venoms have shown promising activities against common infectious bacteria, such as S. aureus, E. coli, Pseudomonas aeruginosa, B. subtilis, Proteus mirabilis, Proteus vulgaris, and Enterobacter aerogenes. Gomes et al. (2005) reported that crude venom and isolated peptides from the Bothrops jararaca showed activity against S. aureus and different fungi. Despite several works reported on this field, the present study was conducted to evaluate the antibacterial activity of snake venoms against different strains of gram-positive and gram-negative bacteria (Izidoro, 2014) Materials and methods Venom Lyophilized venom of Echis ocellatus was obtained from the Department of Biochemistry University of Maiduguri. Preparation of Venom Exactly, 1mg of the venom sample was transferred into a test tube and dissolved in 4 ml of phosphate buffer solution, resulting in venom concentrations of 0.25mg/ml. This made an aqueous solution of 250 µg/ml of stock solution. The solutionwas then kept in the refrigerator under optimum temperature. Preparation of venom disc Whatman filter paper number 1 was cut to disk size. Three (3) different concentrations (25µg, 50µg, and 100µg) of the venom were prepared in separate test tubes. The filter paper was labeled with the concentrations and socked to impregnate the venom appropriately and was dried in an oven. Preparation of Culture Media Muller Hinton agar was prepared by weighing 38 g of the powder and dissolved in 1litre of distilled water and sterilized at 121o C for 15 min after that the media was poured onto a sterile petri dish and was allowed to solidify. Antimicrobial sensitivity using impregnated disc Pure isolates of organisms were inoculated into Nutrient broth and incubated at 37oC for up to 5 hours until turbidity equals 0.5 Mcfarland turbidity scale. This turbidity scale was prepared by adding 0.6mL of 1% aqueous solution of barium chloride in 99.4mL of 1% sulphuric acid giving an approximate bacterial density of 1.2x 109 cfu/mL (Cheesbrough, 2006). The disc was placed alongside antibiotic and antifungal disc which serve as control. Zones of inhibition were determined according to the National Committee for Clinical Laboratory Standard (CLSI, 2009). Results Table 1 presents the antimicrobial activity of Echis ocellatus venom at different venom concentrations (25µg/mL, 50µg/mL, and 100µg/mL) alongside Ciprofloxacin (30µg) and Nystatin 10000IU as the control. The inhibition zone diameter was measured in 337 millimeters to assess the effectiveness of each concentration Table 1. Antimicrobial Susceptibility Activity of Echis ocellatus Venom Organisms Concentration of Venom (µg/mL)/Zones of Inhibition (mm) 25 50 100 Control (ciprofloxacin 30µg) S.. aureus 0.00±0.2 0.00mm±0.4 0.00±1.0 0.00±0.7 S. agalactae 0.00±0.2 0.0±0.4 0.00±1.0 0.00±0.7 Salmonella spp 0.00±0.00 0.00±0.00 0.00±0.00 25.0±0.034 T. mentagrophyte 0.00±2.05 0.00±6.00 12.00±15.00 19.00±21.00 Proteus spp 0.00±0.00 0.00±0.00 0.00±0.00 11.2±0.15 C. albicans 0.00±0.43 14.0±10.36 0.00±2.88 15.0±12.54 H. influenza 09.1±0.11 10.2±0.19 10.3±0.14 30.3±0.16 Mean value frequency 0.00±0.00 Table 2 presents the antibacterial activity of Carpet Viper venom against gram-positive bacteria at different venom concentrations (25 µg/mL, 50 µg/mL, and 100 µg/mL) alongside Ciprofloxacin (30 µg) as the control, the result have shown that the venom at different concentration showed a significant activity on Staphylococcus aureus and Streptococcus agalactae in all the concentration with highest in 100µg/mL (0.00±1.0mm) and least in 25µg/ml (0.00±0.1mm) while the control ciprofloxacin 30µg/mL(0.00±0.8mm). The inhibition zone diameter was measured in millimeters to assess the effectiveness of each concentration Table 2. Antibacterial Activity of Carpet Viper Venom against Gram-Positive Bacteria Organisms Concentration of venom (µg/mL)/ zones of inhibition (mm) 25 50 100 30(Control (ciprofloxacin) S. aureus 0.00±0.1 0.00±0.3 0.00±1.0 0.00±0.7 S. agalactae 0.00±0.2 0.00±0.2 0.00±1.0 0.00±0.8 Mean value frequency 0.00±0.00 Table 3 presents the antibacterial activity of Carpet Viper venom against gram-negative bacteria at different venom concentrations (25 µg/mL, 50 µg/mL, and 100 µg/mL) alongside Ciprofloxacin (30 µg) as the control. The results have shown that no activity was seen with Salmonella typhimurium and proteus mirabilis at all the concentrations only for the control with 25.00 + 0.034 and 11.2+0.15 respectively. With 338 respect to Haemophilus influenza, there is remarkable activity in all the concentrations with the highest in 100µg/mL (10.3±0.14) followed by 50µg/mL (10.2±0.19) and the least 25µg/ml (09.1 ± 0.11) mm respectively. The inhibition zone diameter was measured in millimeters to assess the effectiveness of each concentration Table 3. Antibacterial Activity of Carpet Viper Venom against Gram-Negative Bacteria Organisms Concentration of venom (µg/mL)/ zones of inhibition (mm) 25 50 100 30 (Control, Ciprofloxacin) Salmonella spp 0.00±0.00 0.00±0.00 0.00±0.00 25.0±0.034 Proteus spp 0.00±0.00 0.00±0.00 0.00±0.00 11.2±0.15 H. influenzae 09.1±0.11 10.2±0.19 10.3±0.14 30.3±0.16 Table 4 presents the antifungal activity of Carpet Viper venom against fungi at different venom concentrations (25 µg/mL, 50 µg/mL, and 100 µg/mL) alongside nystatin 10000IU as the control the result has shown that at different concentration there is a significant activity of the venom on fungal isolate, C. albicans and Trichophytum mentagrophytes with highest activity in C. albicans at 50µg/mL (14.0±10.36) and T. mentagrophyte at 100µg/mL (12.00±15.00mm). The inhibition zone diameter was measured in millimeters to assess the effectiveness of each concentration Table 4. Antifungal activity of Carpet Viper venom against fungal Strain Organism Organisms Concentration of venom (µg/ml)/ zones of inhibition (mm) 25 50 100 Control T. mentagrophyte 0.00±2.05 0.00±6.00 12.00±15.00 19.00±21.00 C. albicans 0.00 ± 0.43 14.0 ± 10.36 0.00 ± 2.88 15.0 ± 12.54 Discussion Snake venoms are complex mixtures of proteins, peptides, and enzymes that have evolved primarily for predation and defense. Recent studies have highlighted the potential of snake venom components as antimicrobial agents. The Carpet Viper (Echis ocellatus), known for its potent venom, has been the subject of research to evaluate its antibacterial properties against various pathogens, including Streptococcus agalactiae. Venoms of snakes are a mixture of proteins and peptides including nucleotides, free lipids, and carbohydrates, which are bound to 339 proteins. They have consistently shown high levels of heterogeneity and intra and interspecies variation and this could be due to local adaptation for feeding on different prey. The venom of C. viper had long been recognized for it complexity of molecular composition. Several studies have described the antimicrobial effect of snake venoms, which enlightened the emergence of bioactive peptides as therapeutic alternatives to combat the antibiotic-resistant microorganisms by Priya et al. (2022). Previous studies have reported comparable results by Dean et al. (2011) and Zhang et al. (2010). The antibacterial activity of Carpet Viper venom against gram-positive bacteria at different venom concentrations (25 µg/mL, 50 µg/mL, and 100 µg/mL) alongside Ciprofloxacin (30 µg) as the control, result has shown that the venom at different concentrations have shown significant activity on staphylococcus aureus and streptococcus agalaqctae in all the concentration with highest in 100µg/mL (0.00±1.0)mm and least in 25µg/mL (0.00±0.1)mm while the control ciprofloxacine30µg/mL (0.00±0.8mm) This is in agreement with the study conducted by Nair et al. (2007). The result of gram-negative bacteria have shown that no activity was seen with Salmonella typhimurium and proteus mirabilis at all the concentrations only for the control with 25.00 + 0.034 and 11.2+0.15 respectively. With respect to Haemophilus influenza, there is remarkable activity in all the concentrations with the highest in 100µg/mL (10.3±0.14)mm followed by 50µg/ml (10.2±0.19) and the least 25µg/mL (09.1±0.11) mm respectively. This agrees with studies conducted by Devine, 2002 the antifungal activity of Carpet Viper venom against fungi at different venom concentrations (25 µg/mL, 50 µg/mL, and 100 µg/mL) alongside nystatin (30 µg) as the control the result has shown that at different concentrations there is significant activity of the venom on fungal isolate, C. albicans and T. mentagrophytes with highest activity in candida at 50µg/ml (14.0 ± 10.36) and T. mentagrophyte at 100µg/mL (12.00±15.00) mm. The inhibition zone diameter was measured in millimeters to assess the effectiveness of each concentration. This study disagrees with the result obtained by Asoda (2016). In this study, a considerably high median of 100µg (4mg/kg) was obtained from the venom milked from Carpet viper. This implies that the venom recorded in this concentration of this study is highly toxic to the T. Mentagrophyte growth at the zone of inhibition 12.00±15.00mm, coinciding with the 20.00±27.00mm zone of inhibition for the control agent. This finding is similar to the findings of Yunusa et al. (2017) that reported 80µg carpet viper venom with 10.00±7.00mm inhibition zone and 15.00±10.00mm inhibition zone (1.24 mg/kg) coupled with Ernst and Zug, (1996) that reported 90µg carpet viper Venom of 12.00±14 inhibition zone (0.23 mg/kg) the same snake venom against T. mentagrophyte in their respective studies. The differences in the inhibition zone could be due to differences in the geographical locations of the snakes, sex, diet, and 340 seasonal variation. Also, it could be due to differences in the compositions, method of concentration, and relative abundance of venom toxins (Izidoro et al, 2014). The ability of snake venoms (SVs) to prevent mortality induced by the T. Mentagrophyte. The efficacy of venoms to neutralize the toxicity of medically relevant T. Mentagrophyte has been demonstrated through meticulous preclinical studies. The 25µg concentration of Venom demonstrated a small zone of inhibition and was a slight zone of inhibition on the second plate with 6.00mm and 25.00mm zones of inhibitions of the control agent. This could be due to the low concentration of organisms used respectively. There this study found that the snake venom of carpet viper should be used to treat T. Mentagrophyte infection in high concentration and Nystatin should also be used as a substitute. However, a crucial observation emerges when comparing the venom's efficacy to a standard antibiotic and antifungal agent. The maximum inhibition zone achieved with the venom is significantly lower than that of the antibiotic and antifungal. This indicates that while the venom possesses antibacterial properties, its potency is limited compared to established antimicrobial agents. This finding underscores the need for further investigation. It's essential to isolate and characterize the specific antimicrobial compounds within the venom. By understanding the molecular mechanisms behind the venom's antibacterial activity researchers can potentially optimize its potency and develop novel therapeutic strategies. Moreover, safety considerations are paramount. Before any therapeutic application, rigorous studies on the venom's toxicity and potential side effects are crucial. It is imperative to balance the potential benefits with the risks associated with using a complex biological substance like venom further research is needed to isolate and characterize the specific antimicrobial compounds within the venom. Additionally, studies on the venom's toxicity and potential side effects are crucial before considering its therapeutic applications. The Carpet Viper’s venom demonstrates notable antimicrobial activity against bacteria and fungi through multiple mechanisms including membrane disruption and protein synthesis inhibition. While these findings are promising for future therapeutic applications, extensive research is necessary to fully understand the implications for human health and safety. Further research should focus on isolating specific active compounds within the venom responsible for its antibacterial properties. This could lead to the development of novel antibiotics derived from snake venom components. While in vitro results are promising, it is crucial to conduct in vivo studies to evaluate the safety and efficacy of these compounds in living organisms. 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