INTRODUCTION:

Snake bite is a common occupational hazard amongst farmers, people working outdoors in rural areas resulting in high morbidity and mortality¹. Every year worldwide approximately 5.4 million snake bites occur, out of which 80,000-1,40,000 are fatal². Among the reported cases of snake bites, 4,00,000 results in severe disabilities leading to loss of livelihood². The reports suggest that in India 35,000-50,000 deaths occur annually due to snakebites^3–6 and actual number may be estimated to be even more as they may go unreported. When snake bites, venom is injected into the bite site and the toxic effect of the venom is due to enzymatic and non-enzymatic components in it. Toxicity can be aggravated by the immune response of the victim's body.

Anti-snake venom (ASV) is the only effective antidote for snake bites which are produced by hyper immunizing the horses with snake venom, thereafter, extorting and purification the serum collected from horses. Since venoms are of different composition in different snakes, ASVs can be produced as species specific (monovalent) or against several species (polyvalent).

Monovalent ASV produced against venom of a particular snake species may have cross-neutralizing reaction against other types of venom. Usually, this reaction occurs from a closely related snake species. This type of reaction is called para-specific activity. As per the recommendations of the World Health Organization, administration of monovalent ASV is the most effective treatment for snakebite⁷.

When the snake bites, apart from injecting the venom, it also deposits the organisms present in its mouth at the bite site. Most snakes harbor different types of bacteria like aerobic and anaerobic bacteria in their buccal cavity. The disability and death caused due to snakebites are not only due to the action of the snake venom but also due to the virulence factors of bacteria resulting in secondary infection. The buccal flora of snakes have many types of microorganisms, comprehending members of Enterobacteriaceae family like Escherichia coli, Morganella species, others including Streptococcus species, Aeromonas species, Staphylococcus aureus and anaerobes such as Clostridium species are reported globally⁸.

However, environmental conditions and geographical location predisposes to the presence of a particular dominant microorganism in the snake of that region. Hence, microflora of snakes are different from region to region. These numerous microbes within a snake’s mouth cavity can be nonpathogenic avirulent bacterial environs and pathogenic opportunistic bacterial species of humans⁹. Following a snakebite, the venom causes inflammation or contusion resulting in local manifestations around the site of bite. This creates a congenial environment for microorganisms to establish from the snake’s mouth and salivary secretion¹⁰. Many bacterial species, including aerobic and anaerobic microbes could give rise to secondary infections. Infection due to snake bite can result in gas gangrene by anaerobic bacteria. Bacteria which are gram positive, can cause infections, like boils or infection of the subcutaneous tissue or sometimes they can enter the blood and multiply or cause inflammation of the meninges, and infection of the urinary tract. Gram-negative bacterial infection can cause gastroenteritis or diarrhea, fever, sepsis, or infection of the respiratory tract, soft tissue inflammation, and inflammation of the meninges^11–14.

Though, ASV can neutralize the toxic effects of the snake venom in humans, the study regarding its use as antibiotic is not well studied. Our study was to find out whether the ASV could act also as an antibiotic against the standard strains of bacteria.

MATERIALS AND METHODS:

Polyvalent ASV was obtained from Bharath Serums and Vaccines Private Limited, Ambernath, Maharastra, India. The contents of the entire vial were dissolved using distilled water provided by the manufacturer.

Antimicrobial activity:

Agar well diffusion method was employed to study antibacterial susceptibility ^15,16. Antimicrobial susceptibility was determined against the following strains. The American Type Culture Collection (ATCC) strains of bacteria Staphylococcus aureus (ATCC 25923), Klebsiella pneumoniae (ATCC 700603), Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853) were incorporated in our research work.

Muller-Hinton agar (MHA) was used, which was obtained from Hi-media, Mumbai. The above-mentioned bacterial strains were restored by inoculation on the simple media, nutrient agar plate respectively. These agar culture plates were incubated overnight at 37°C to pick the isolated mono-bacterial strains. Bacteria was identified by using standard procedure. Segregated colonies of bacteria were then transferred to sterile nutrient broth for overnight incubation. Bacterial growth concentration in the nutrient broth was adjusted to 105 CFU/ml by comparing it with 0.5 McFarland’s turbidity standard. For the controls, Ampicillin antibiotic disc (10μg) and antibiotic disc containing (1μg) of Oxacillin , secured from Hi-Media, Mumbai were employed.

Determination of antibacterial activity:

Sterile petri plates were filled with 20ml of MHA. With the help of a sterile cotton swab and by using lawn culture technique, bacteria present in the culture was smeared all over the surface of the agar plate. A diameter of 6mm wells were punched into the agar^17–22for the application of reconstituted ASV. For each bacterial strain, four different concentrations (10, 50, 75 and 100μg/ml) of ASV used to determine the antibacterial activity. The inoculated plates were maintained at 37°C for 18 hours to determine the antibacterial activity of the ASV. Antibacterial activity was measured by zone of inhibition^23,24. Tests were performed in triplicates and the average of the three tests were calculated and considered for this research study.

RESULTS:

Diameter produced in the inhibition zone was measured for the antibacterial action

Table 1: Zone of inhibition by ASV against various standard bacterial strains

	100 μg/ml	50 μg/ml	25 μg/ml	10 μg/ml
*Staphylococcus aureus* (ATCC 25923)	21mm	18mm	15mm	12mm
*Escherichia coli* (ATCC 25922)	-	-	-	-
*Pseudomonas aeruginosa* (ATCC 27853)	-	-	-	-
*Klebsiella pneumoniae* (ATCC 700603)	-	-	-	-

(-): Indicates no zone of inhibition

DISCUSSION AND CONCLUSION:

Snake bite is a frequent, harmful occupational hazard. This has a great influence on the socioeconomic burden among the agricultural dependent population of India. It is a serious cause of the human illness and death considering years. Venomous snakebite causes systemic and local reactions. Snakebite wound leading to infection by microbes occurred in one out of three cases²⁵. Study conducted by R D Theakston et al in Thailand showed Malayan pit vipers (Calloselasma rhodostoma) snake’s mouth contained many types of microorganisms, especially gut related bacilli like Enterobacter species and Pseudomonas species and cocci like Staphylococcus species and Clostridium species²⁶. Study conducted by Lin CC et al showed the dominant microbial species in the snake’s mouth was Enterococcus faecalis and Morganella morganii, followed by less frequent microbes like Staphylococcus, Corynebacterium and Enterobacter species²⁷.

Study conducted by Shaikh IK et al had a total number of 205 strains of bacteria obtained from the throat region of snakes, which constituted the habitual pathogens, especially gram-negative bacteria like Aeromonas hydrophila, Escherichia coli, Morganella morganii, Pseudomonas aeruginosa, or gram-positive cocci like Staphylococcus species, Micrococcus species, bacilli like Bacillus species and anaerobic Clostridium perfringens¹¹. Microbial flora in snakes is variable.

Study conducted by Al-Asmari et al showed snake venom having antibacterial activity against gram negative bacteria²⁸. This study showed neutralizing ASV had antibacterial effect against Staphylococcus aureus, at maximum zone of inhibition at concentration of 100 μg/ml, but for other gram negative bacteria, there was no zone of inhibition (Table 1).

In India herbal drugs derived from plants are also used in the treatment of snakebites^29–31. Glycosides, terpenoids, flavonoids and saponins of these herbal drugs are responsible for the antibacterial efficacy ³²but the exact mode of antibacterial action of these drugs has to be studied.

At present, the only standard treatment regimen for snakebite toxin diffusion in body is treatment with standard ASV comprising of immunoglobulins or its segments obtained from the blood plasma of bigger animals (commonly horses, and other animals like goats, rabbits, or sheep). These animals must have received earlier nontoxic venomous doses and immunized^33,34. Commercial anti-venoms comprised of the polyvalent and monovalent ASV antibodies are administered for snake bite from snake species of the group or single species, respectively³⁵, these ASV apart from neutralizing the venom, has antibacterial effect against gram positive coccus like Staphylococcus species which is reflected in the study. Thus, in cases of the snake bite inoculating Staphylococcus species along with the venom, ASV by itself can act as antibacterial agent in controlling the infection. Staphylococcus species is also able to form biofilms which makes antimicrobial treatment difficult^.36. Further work can be carried out to find out the antigenic structure of the Staphylococcus species present in the snake’s buccal cavity for neutralization reaction with the ASV.

CONFLICT OF INTEREST:

Nil.

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Received on 19.04.2024 Revised on 15.07.2024

Accepted on 19.09.2024 Published on 28.01.2025

Available online from February 27, 2025

Research J. Pharmacy and Technology. 2025;18(2):819-822.

DOI: 10.52711/0974-360X.2025.00121

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