Antibacterial Activity of Venom from the Puff Adder (Bitis arietans), Egyptian Cobra (Naja haje), and Red Spitting Cobra (Naja pallida)

Bitis arietans (Puff adder), Naja haje (Egyptian cobra), and Naja pallida (Red spitting cobra) venoms were tested for antimicrobial activity. This evaluation employed disc diffusion and microbroth dilution techniques. Gram-positive bacteria (Bacillus cereus and Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli, Klebsiella pneumonia, and Salmonella typhi) were used. Aztreonam (30 µg), cefpodoxime (10 µg), cefoxitine (30 µg), streptomycin (25 µg), ceftriaxone (30 µg), nalidixic acid (30 µg), tetracycline (30 µg), and sulfamethoxazole (25 µg) were used as controls. All tests were conducted in triplicate (n = 3). Results. The activity of B. arietans venom against Gram-negative bacteria was significantly lower (p < 0.001) than that of controls. The efficacy of B. arietans venom and sulfamethoxazole against both Gram-positive and Gram-negative bacteria was not significantly different (p > 0.9999). The efficacy of B. arietans venom against Gram-positive bacteria was significantly lower (p < 0.001) than cefoxitin, streptomycin, and tetracycline. The efficacy of N. haje venom against Gram-negative bacteria was significantly lower (p < 0.001) than that of controls. There was no significant difference in the antimicrobial efficacy of N. haje venom and controls against Gram-positive bacteria (p=0.3927 to p=0.9998). There was no significant difference in the efficacy of N. pallida venom and controls against Gram-negative bacteria (p=0.3061 to p=0.9981). There was no significant difference in the efficacy of N. pallida venom and controls against Gram-positive bacteria (p=0.2368 to p > 0.9999). Conclusions. Of all the tested venoms, only Naja pallida venom showed good efficacy against both Gram-positive and Gram-negative bacteria.


Background
Bacillus cereus, Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae are some of the pathogens of medical importance in the developing World [1][2][3][4]. Tese pathogens have been implicated in food poisoning [5,6], sepsis [7], and neonatal infections [8,9]. Reports on drug resistance for clinical isolates of these pathogens are rife in the scientifc literature [10][11][12]. Terefore, new and innovative therapies are urgently required to mitigate the unfolding AMR crisis. Venom from animals comprises a complex cocktail of pharmacological molecules that could help bolster the ranks of the current antimicrobial agents. Bitis arietans (puf adder, a viper), Naja haje (Egyptian cobra, a nonspitting cobra), and Naja pallida (Red spitting cobra, a spitting cobra) are snakes of medical importance in Subsaharan Africa [13]. Venom from these snakes could be useful in combating medically important pathogens in the region including Bacillus cereus, Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae. However, data to support their efcacy against common bacterial pathogens are not available. Te aim of the present study was therefore to determine the antibacterial activities of venoms from Bitis arietans, Naja haje, and Naja pallida against Bacillus cereus, Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae.

Materials and Methods
2.1. Venom. Venoms from Bitis arietans (Puf adder), Naja haje (Egyptian Cobra), and Naja pallida (Red spitting Cobra) were collected from wild-caught snakes at the East African Venom Supplies (Kenya). Tey were lyophilized and stored at −4°C at the Pharmacology and Toxicology Lab, Faculty of Veterinary Medicine, University of Nairobi. Each of the lyophilized venoms were accurately weighed (0.25 g) in an analytical balance and triturated using a pestle and mortar. Te triturated venoms were transferred to 10 mL volumetric fasks and made up to the mark with phosphate bufered saline to make a 25 mg/mL concentration.

Antimicrobial Discs.
Antimicrobial discs were prepared by punching Whatman Filter paper number 1 using an ofce paper punch. Te prepared discs were sterilized by autoclaving at 121°C for 15 minutes. Te discs were then soaked for 5 minutes in the prepared venom concentrations (25 mg/ mL) using Petri dishes. Te venom-soaked discs were then gently picked using forceps and dried in an oven at 37°C for 30 minutes.

Growth Media.
Mueller Hinton Agar (MHA) was prepared by dissolving 19 g of the media in distilled water, heating in an oven to boil and dissolve for 5 minutes. Tis was followed by sterilization via an autoclave at 121°C for 15 minutes. Te medium was allowed to cool in a 45°C water bath, poured into plates under the sterile laminar fow hood, and left to solidify for 10 minutes. Te prepared Mueller Hinton Agar (MHA) plates were transferred to an oven and allowed to dry at an angle of 45°C for 10 minutes.

Microbial
Inoculum. 0.85% normal saline was placed in the same tubes as used in the McFarland standard and sterilized by autoclaving at 121°C for 15 minutes. Te inoculum of the microorganisms was prepared by suspending isolated bacterial colonies from the pure microbial subcultures in 0.85% normal saline, and the turbidities were standardized to 0.5 McFarland units.
2.6. Antimicrobial Activity Assay. Te disk difusion test and minimum inhibitory concentration (MIC) determination were used to screen for antibacterial activity according to standard techniques described in previous publications [14][15][16]. In brief, the bacterial strains were grown in trypticase soy agar at 37°C for 16-18 hours before being adjusted to 0.5 McFarland standards (1.5 × 10 8 colony forming unit/ mL) with sterile normal saline [14][15][16]. A sterile swab was dipped into the inoculum, and excess inoculums were removed by pressing frmly against the side of the tube above the liquid level. Te swab was streaked three times across the surface of Mueller−Hinton agar plates [14][15][16]. Each snake venom solution was prepared at a concentration of 25 mg/ mL by dissolving it in sterile deionized water. 1 µL solutions of the prepared venom concentration were applied to Whatman paper discs (6 mm diameter) and placed on bacterial culture in the triplicate assay, which was then incubated for 16-24 hours at 35 ± 2°C [14][15][16]. Te zones of inhibition around the discs were measured using a digital Vernier caliper. Distilled water discs were used as the negative control. Positive controls included aztreonam (30 µg), cefpodoxime (10 µg), cefoxitin (30 µg), streptomycin (25 µg), ceftriaxone (30 µg), nalidixic acid (30 µg), tetracycline (30 µg), and sulfamethoxazole (25 µg). Te snake venoms with the largest inhibition zone diameters were chosen for minimum inhibitory concentration (MIC) determination [14][15][16]. Te Clinical Laboratory Standards Institute (CLSI 2014) broth microdilution method was used for the MIC test [14][15][16]. Te culture was diluted to 10 6 CFU/ mL after 3 hours of the bacterial growth. Te snake venom was diluted twice with Mueller−Hinton broth, and then the diluted bacterial culture was added to achieve a fnal concentration ranging from 0.39 mg/mL to 25.0 mg/mL. After 16-18 hours of incubation at 35 ± 2°C, the MIC was defned as the lowest concentration of venom or antibiotic preventing the visible bacterial growth when compared to the positive growth control (medium plus bacteria without venom or antibiotic) with high turbidity and to the negative growth control (medium plus bacteria without venom or antibiotic) [14][15][16].  Figure 1 illustrates the efect of conventional antibiotics and venoms on selected bacteria. Table 1 shows the mean size of the clearing zones or zones of inhibition values of venoms and conventional antibiotics against Gram-positive and Gram-negative bacteria.

Results
Te efect of the conventional antibiotics and venoms on E. coli was in the order ATM > CRO > TCY > CPD > CXT > NAL > SMZ > STM > NHV > NPV > BAV as shown in Table 1. Te efect of the conventional antibiotics and venoms on K. pneumoniae was in the order TCY International Journal of Microbiology shown in Table 1. Te efect of the conventional antibiotics and venoms on S. aureus was in the order STM > TCY > CRO > CXT > NHV > NPV > ATM∼CPD∼NAL∼SMZ∼BAV as shown in Table 1. Te efect of the conventional antibiotics and venoms on B. cereus was in the order STM > TCY > CXT > CRO > NPV > NHV > ATM∼CPD∼NAL∼BAV as shown in Table 1. Te efect of the conventional antibiotics and venoms on S. typhi was in the order CRO > CXT > STM > TCY > NHV > NPV > ATM∼NAL∼SMZ∼BAV as shown in Table 1. Figure 2 is a comparison of the antibacterial efect of venom from Bitis arietans venom and conventional antibacterial agents against Gram-negative bacteria. Te efect of B. arietans venom on E. coli was signifcantly lower (p < 0.001) than the efect of cefpodoxime, cefoxitin, streptomycin, ceftriaxone, nalidixic acid, tetracycline, and sulfamethoxazole as shown in Figure 2. Te efect of B. arietans venom on K. pneumoniae was signifcantly lower (p < 0.001)than the efect of cefpodoxime, cefoxitin, streptomycin, ceftriaxone, nalidixic acid, tetracycline, and sulfamethoxazole as shown in Figure 2. Tere was no signifcant diference (p > 0.9999) in the efect of Bitis arietans venom and Sulfamethoxazole on K. pneumoniae as shown in Figure 2.    Figure 3 shows a comparison of the antibacterial efect of Bitis arietans venom and conventional antimicrobial agents against Gram-positive bacteria. Te efect of B. arietans venom on S. aureus was signifcantly lower (p < 0.001) than the efect of cefoxitin, streptomycin, ceftriaxone, and tetracycline as shown in Figure 3. Tere was no signifcant diference (p > 0.9999) in the efect of Bitis arietans venom, aztreonam, cefpodoxime, nalidixic acid, and sulfamethoxazole on S. aureus as shown in Figure 3. Te efect of B. arietans venom on S. typhi was signifcantly lower (p < 0.001) than the efect of aztreonam, cefpodoxime, cefoxitin, streptomycin, ceftriaxone, and tetracycline as shown in Figure 3. Tere was no signifcant diference (p > 0.9999) in the efect of Bitis arietans venom, nalidixic acid, and sulfamethoxazole on S. typhi as shown in Figure 3. Figure 4 shows a comparison of the antibacterial efect of Naja haje venom and conventional antimicrobial agents against Gram-negative bacteria. Te efect of N. haje venom on E. coli and K. pneumoniae was signifcantly lower (p < 0.001) than the efect of aztreonam, cefpodoxime, cefoxitin, ceftriaxone, nalidixic acid, tetracycline, and sulfamethoxazole as shown in Figure 4.  to S. typhi, there was no signifcant diference between the efect of Naja haje venom and aztreonam (p � 0.6349), Naja haje venom and cefpodoxime (p � 0.8358), Naja haje venom and cefoxitin (p � 0.9829), Naja haje venom and ceftriaxone (p � 0.9999), Naja haje venom and nalidixic acid (p � 0.3946), Naja haje venom and tetracycline (p � 0.8931), and Naja haje venom and sulfamethoxazole (p > 0.9999) as shown in Figure 5. Figure 6 is a comparison of the antibacterial efects of Naja pallida venom and conventional antimicrobial agents against Gram-negative bacteria. With regard to E. coli, there was no signifcant diference between the efect of Naja pallida venom and aztreonam (p � 0.3439), Naja pallida venom and cefpodoxime (p � 0.3061), Naja pallida venom and cefoxitin (p � 0.8041), Naja pallida venom and ceftriaxone (p � 0.3725), Naja pallida venom and nalidixic acid (p � 0.8608), Naja pallida venom and tetracycline (p � 0.5628), and Naja pallida venom and sulfamethoxazole (p � 0.9971) as shown in Figure 6. With regard to K. pneumoniae, there was no signifcant diference between the efect of Naja pallida venom and aztreonam (p � 0.9993), Naja pallida venom and Cefpodoxime (p � 0.9981), Naja pallida venom and Cefoxitin (p � 0.9804), Naja pallida venom and ceftriaxone (p � 0.9967), Naja pallida venom and nalidixic acid (p � 0.9144), Naja pallida venom and tetracycline (p � 0.9955), and Naja pallida venom and sulfamethoxazole (p � 0.8690) as shown in Figure 6. Figure 7 is a comparison of the antibacterial efects of Naja pallida and conventional antimicrobial agents against Gram-positive bacteria. With regard to B. cereus, there was no signifcant diference between the efect of Naja pallida venom and aztreonam (p � 0.5583), Naja pallida venom and cefpodoxime (p � 0.6217), Naja pallida venom and cefoxitin (p � 0.9741), Naja pallida venom and streptomycin (p � 0.5142), Naja pallida venom and ceftriaxone (p > 0.9999), Naja pallida venom and nalidixic acid (p � 0.5583), Naja pallida venom and tetracycline (p � 0.5310), and Naja pallida venom and sulfamethoxazole (p � 0.9786) as shown in Figure 7.

Discussion
In 2017, the World Health Organization published a list of antibiotic resistant bacteria. Tis list was dubbed as the "WHO priority pathogen list." It is divided into three key priorities based on the urgency and need for new antibiotics, i.e., priority 1: critical, priority 2: high, and priority 3: medium.
In the present study, we evaluated the antimicrobial activities of Bitis arietans, Naja haje, and Naja pallida against Gram-positive (B. cereus, S. aureus, and S. typhi) and Gramnegative (E. coli and K. pneumoniae). Organisms such as E. coli and K. pneumoniae are WHO priority 1 (critical) pathogens while organisms such as S. typhi and S. aureus are WHO priority 2 (high) pathogens.
Te clinical and laboratory standard institute (CLSI) has developed zone diameter and minimum inhibitory concentration breakpoints of various antibiotics when tested against various pathogens [17]. Based on these breakpoints, it is possible to determine whether the pathogens are sensitive, intermediate, or resistant to a test substance/compound [17]. When these criteria are considered, E. coli was found to be sensitive to aztreonam, cefpodoxime, cefoxitin, ceftriaxone, nalidixic acid, and tetracycline. K. pneumoniae was sensitive to streptomycin and tetracycline but resistant to sulfamethoxazole, aztreonam, and cefpodoxime. S. aureus was sensitive to cefoxitin, streptomycin, ceftriaxone, and tetracycline but resistant to cefpodoxime, nalidixic acid, and sulfamethoxazole. S. typhi was sensitive to cefoxitin and ceftriaxone but resistant to aztreonam, sulfamethoxazole, nalidixic acid, streptomycin, and tetracycline.    [18][19][20][21]. It is expected that snake mouth bacteria may be inoculated during a dry bite or envenomation resulting in infection. However, it is fascinating that snakebite victims rarely sufer from complications arising from bacterial infections [21]. One of the frst reports to evaluate the antimicrobial properties of snake venom was by Glaser in 1948 [22]. Since then, the feld has grown exponentially to the point that individual snake venom proteins are being explored for antimicrobial activity [23][24][25][26][27].
Te results of the present study suggest that both Grampositive and Gram-negative bacteria were resistant to B. arietans venom as no inhibition was observed in the antibacterial assay. Tese results are contrary to those of Al-Asmari and colleagues who reported that Bitis arietans venom from captive bred snakes in Saudi Arabia was effective against S. aureus, E. fecalis, and P. aeruginosa [28].
Elapid venoms (e.g., Naja haje and Naja pallida venoms) have been reported to have signifcantly higher percentages of three fnger toxins (3FTx's) than viperid venoms (Bitis arietans venom) [29,30]. Te three fnger toxins (3FTx's) have been reported to have higher specifc activity towards the lipids contained within the Gram-positive plasma membranes than those found in Gram-negative bacterial membranes [31]. Not unexpectedly, the elapid venoms studied were more efective than at inhibiting Gram-positive bacteria than Gram-negative bacteria. Similar observations were made by Charvat and colleagues [32].
Te exact mechanism of antimicrobial activity of 3FTxs is not known. However, it is postulated that 3FTxs cause membrane destabilization and release of cytoplasmic materials in bacteria [32][33][34]. L-amino acid oxidases (LAAOs) in venoms have also been implicated in morphological alterations in bacteria including disruption of the mitochondrial membranes leading to total destruction and/or loss of organelles [35,36].

Conclusions
In conclusion, these fndings suggest that Naja haje and Naja pallida venoms have better antibacterial activity than some of the antibiotics which are currently in use for microbial infections. However, the venom of Bitis arietans appears to be inefective against common bacterial pathogens.

Limitations.
A limited number of venoms (from one viper, one spitting cobra, and one nonspitting cobra) was used in this study. Future studies should employ a broader range of snake venoms including those from the Mambas and colubrids found in Subsaharan Africa.

Data Availability
Te datasets used and/or analyzed during this study are available from the corresponding author on reasonable request.

Ethical Approval
Tis research was approved by the University of Nairobi, Institutional Ethics and Review Committee. BAUEC/2019/220.

Conflicts of Interest
Te authors declare that they have no conficts of interest.

Authors' Contributions
MOO is responsible for conceptualization, data curation, formal analysis, investigation, methodology, project administration, resources, visualization, writing original draft,   writing review, and editing. KLE is responsible for investigation, methodology, validation, resources, writing review, and editing. LKB is responsible for conceptualization, investigation, methodology, supervision, resources, validation, writing review, and editing. NG is responsible for investigation, methodology, validation, supervision, resources, writing review, and editing.