Antibacterial activity of the venom of Heterometrus xanthopus.

Heterometrus xanthopus (Scorpion) is one of the most venomous and ancient arthropods. Its venom contains anti-microbial peptides like hadrurin, scorpine, Pandinin 1, and Pandinin 2 that are able to effectively kill multidrug-resistant pathogens. The present study was conducted to evaluate the anti-bacterial activity of H. xanthopus venom. Six Gram-positive and Gram-negative bacterial strains were tested against 1/100, 1/10, and 1/1 fractions of distilled water diluted and crude venom. 1/100 and 1/10 dilutions were not successful in any of the six bacterial strains studied while the 1/1 dilution was effective on Bacillus subtilis ATCC 6633, Salmonella typhimurium ATCC 14028, and Pseudomonas aeruginosa ATCC 27853 with highest zone of inhibition were obtained on B. subtilis. Crude venom was effective against Enterococcus faecalis ATCC 14506, B. subtilis, S. typhimurium, and P. aeruginosa. The most effective results were observed on B. subtilis.

Introduction

Scorpions are venomous arthropod animals belonging to the class Arachnida.[1] There are thirteen families and about 1,400 described species and subspecies of scorpions.[2] All scorpions are venomous but approximately 50 of them have enough poison to kill a person.[34] The oldest known scorpions lived around 430 million years ago in the Silurian period, on the bottom of shallow tropical seas, hence regarded as the oldest terrestrial arthropods.[5]

Venoms from scorpions are complex mixtures of compounds (neurotoxins, enzyme inhibitors, salts, etc.). Scorpions use venoms for immobilization of prey and protection against predators. Scorpion venoms consist of a complex of several toxins that exhibit a wide range of biological properties and actions, as well as chemical compositions, toxicity, pharmacokinetic, and pharmacodynamic characteristics.[6] Small basic proteins present in scorpion venoms are responsible for the neurotoxic activities of the venoms. Keeping in view of all these facts the present study was conducted to evaluate the antibacterial activity of Heterometrus xanthopus by utilizing curde and diluted venom.

Materials and Methods

Materials

All agars, sulphuric acid, barium chloride, sodium chloride, ethyl alcohol, nutrient broth, and other chemicals used in this study were of procured from Oxoid (UK), Fluka Chemika, and Difco Laboratories, USA.

Scorpion Procurement and Extraction Procedure

Scorpion (H. xanthopus) was collected by excavation from burrows in Mehra Alikhan, dry location in Haripur District, Khyber Pakhtunkhwa, Pakistan. The scorpions were fed on spiders, centipedes, grasshoppers, and cockroaches in glass cages (38W×58L×38H cm) with open tops for easy monitoring. Cage floor was covered up till four inches height with soil in order to provide natural habitat. These cages were kept at room temperature.[7]

Electricity control (12 V) Huawei AC/DC Adaptor (Model; UE4112005006) was used for venom extraction [Figure 1]. Venom was obtained from mature H. xanthopus from an opening near the tip of the bulb-shape venomous gland stinger by applying 12 V electrical stimulus. Electrical stimulus was applied by touching electrodes in the joints of last two segments of tail. One drop of sodium chloride solution was applied on each of these two joints to maximize flow of electricity. The venom was collected in 1.5 ml eppendorf tube.

Figure 1

Milking venom from Heterometrus xanthopus scorpion by 12V electric shock

Preparation of Venom Dilutions

Crude venom as well as 1/1, 1/10, and 1/100 dilutions were prepared in sterile distilled water. 1/1 diluted venom was prepared by mixing equal volume of venom and distilled water. 1/10 diluted venom was prepared by mixing 1 part of venom with 9 parts of distilled water (v/v) while 1/100 diluted venom was prepared by mixing 1 part of venom with 99 parts of distilled water with the help of micropipette.

Gram-positive and Gram-negative bacterial strains

Reference bacterial strains, Staphylococcus aureus ATCC 6538, Enterococcus faecalis ATCC 49452, Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 14028, Pseudomonas aeruginosa ATCC 27853, and Bacillus subtilis ATCC 19659 were obtained from American Type Culture Collection (ATCC) and were maintained on Nutrient agar slants (Oxoid, UK) at 4°C

Purity testing of each organism

Each organism is inoculated form working culture of nutrient broth (Merck) on their respective selective media for control as well as for purity testing, i.e., P. aeruginosa on (PCA) Pseudomonas Cetrimide Agar (Oxoid, CM0579), S. typhimurium on (XLD) Xylose Lysine Deoxycholate Agar (Oxoid, CM0469), S. aureus on (MSA) Mannitol Salt Agar (Oxoid, CM0085), E. faecalis on (S and B) Slanetz and Bartley (Oxoid, CM0377), E. coli on (EMB) Eosin Methylene Agar (Oxoid, CM0069), B. subtilis on Mannitol Egg Yolk Polymyxin Agar (MYP; Oxoid, CM0929) and incubated at 37°C for 24 h.

Evaluation of antimicrobial activity

After incubation, one colony of each bacterium from their respective selective agar medium was inoculated into 5 ml nutrient broth and incubated for 4-6 h at 37°C. The inocula were standardized by matching turbidity with a McFarland standard (No. 0.5). The test culture was spread evenly on the surface of pre-sterilized plastic petri dish containing solidified Mueller Hinton Agar (MHA; Oxoid CM 0337) with a sterile cotton swab. A total of 15 μl venom from eppendorf tube was dropped on the surface of each swabbed plate with the help of micropipette. These plates were then incubated at 37°C for 24 h (Culture box, DH-2500). Resulting zones of inhibition were measured in centimeters for all bacterial plates after incubation. Same procedure was repeated for 1/1, 1/10, and 1/100 dilution.

Results

Antimicrobial activity of crude venom

Antimicrobial activity of crude venom was presented in Table 1. H. xanthopus crude venom was active against four bacterial strains. The clear zone of inhibition (30 mm) was seen against B. subtilis followed by S. typhimurium with 20 mm zone of inhibition. E. faecalis and P. aeruginosa showed (12 mm) each zone of inhibition on crude extract. However, the crude venom appeared to lack activity against the E. coli and S. aureus.

Table 1

Venom's activity against bacteria with different concentrations

Inhibitory zone with 1/1, 1/10, and 1/100 diluted venom

1/1 diluted venom formed 30 mm zone of inhibition against B. subtilis showing no change of inhibition in comparison with crude venom [Figure 2]. P. aeruginosa showed zone of inhibition of 20 mm that was much higher than crude venom (12 mm). The possible reason for higher antibacterial activity is due to more activation of antibacterial peptides (AMP) in water in comparison with crude venom as previously described through RNA and DNA sequence by Gao et al.[8] 1/1 diluted venom showed 11 mm zone of inhibition against S. typhimurium. S. typhimurium showed reduced activity on 1/1 diluted venom in comparison with crude venom (20 mm). 1/1 diluted venom was found inactive against E. coli, E. faecalis, and S. aureus. Similar results of inactiveness of E. coli and S. aureus were seen on crude venom but in comparison a higher zone of inhibition of 12 mm against E. faecalis was seen on crude venom.

Figure 2

Venom's activity against bacterial cultures with different concentrations

All bacterial strains showed resistance against 1/10 and 1/100 times diluted venom, with no zone of inhibition on Mueller Hinton Agar.

Discussion

Scorpion species often use to spray venom on their own bodies to disinfect them from possible saprophytic organisms including bacteria and fungi, showing that venom of these scorpions could contain some sort of antibiotic potential.[9] Scorpion venom contains peptides which exhibit anti-microbial properties.[1011] Kievit et al.[12] reported that an ingredient in the venom of the “deathstalker” scorpion could help in gene therapy for effective treatment for brain cancer. The scientists describe a new approach that could solve these problems. Key ingredients of their gene delivery system are chlorotoxin, the substance in deathstalker scorpion venom that can slow the spread of brain cancer, and nanoparticles of iron oxide.[1213]

In this study, crude venom was effective against B. subtilis showed zone of inhibition 30 mm. These results are similar with that of spider venom activity reported by Benli and Yigit.[14] E. coli showed resistance against venom with no zone of inhibition is in relation with least effect of scorpion venom from southern Africa.[15] The results of E. faecalis showed 12 mm inhibition zone, Benli and Yigit[14] reported that E. faecalis was resistant against spider venom, whereas our finding indicates effectiveness of scorpion venom against E. faecalis. The results of P. aeruginosa are in comparison with Benli and Yigit.[14] S. aureus showed no zone of inhibition in our finding, whereas S. aureus showed zone of inhibition on spider venom.[1416]

H. xanthopus venom with 1/1 dilution showed different results. The zone of inhibition of B. subtilis is identical to the results obtained from antibacterial study of two different spider species venom.[1416] E. coli and S. aureus showed resistance against 1/1 diluted venom. These results are in comparison with results of San et al.[17] They obtained similar results with antimicrobial activity of venoms from snakes. 1/1 diluted venom made inhibition zone of 11 mm against S. typhimurium, while 9 mm inhibition zone was reported against S. typhimurium when bee venom was tested against it.[18] All tested bacteria were found resistant to 1/10 and 1/100 diluted H. xanthopus venom. The possible reason behind this might because at some point when venom gets too much diluted the antibacterial activity subside.[1418]

Conclusion

The data presented in the present research provides a strong base that venom peptide/protein is involved in antibacterial responses. This work paves the way for further characterizing immune related components involved in H. xanthopus venom, antibacterial response at the molecular level, which will undoubtedly expand our understandings in innate immunity. Polypeptide component's involvement in antibacterial response of venom, offers clues for the search of new antibacterial template from scorpion resources for drug design and for high potential for clinical use.