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Keywords:

  • L-amino acid oxidase;
  • antibacterial activity;
  • phospholipase A2;
  • toxins;
  • venoms

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Aims:  Venoms of snakes, scorpions, bees and purified venom phospholipase A2 (PLA2) enzymes were examined to evaluate the antibacterial activity of purified venom enzymes as compared with that of the crude venoms.

Methods and Results:  Thirty-four crude venoms, nine purified PLA2s and two l-amino acid oxidases (LAAO) were studied for antibacterial activity by disc-diffusion assay (100 μg ml−1). Several snake venoms (Daboia russelli russelli, Crotalus adamanteus, Naja sumatrana, Pseudechis guttata, Agkistrodon halys, Acanthophis praelongus and Daboia russelli siamensis) showed activity against two to four different pathogenic bacteria. Daboia russelli russelli and Pseudechis australis venoms exhibited the most potent activity against Staphylococcus aureus, while the rest showed only a moderate activity against one or more bacteria. The order of susceptibility of the bacteria against viperidae venoms was –S. aureus > Proteus mirabilis > Proteus vulgaris > Enterobacter aerogenes > Pseudomonas aeruginosa and Escherichia coli. The minimum inhibitory concentrations (MIC) against S. aureus was studied by dilution method (160–1·25 μg ml−1). A stronger effect was noted with the viperidae venoms (20 μg ml−11) as compared with elapidae venoms (40 μg ml−1). The MIC were comparable with those of the standard drugs (chloramphenicol, streptomycin and penicillin).

Conclusion:  The present findings indicate that viperidae (D. russelli russelli) and elapidae (P. australis) venoms have significant antibacterial effects against gram (+) and gram (−) bacteria, which may be the result of the primary antibacterial components of laao, and in particular, the PLA2 enzymes. The results would be useful for further purification and characterization of antibacterial agents from snake venoms.

Significance and Impact of the Study:  The activity of LAAO and PLA2 enzymes may be associated with the antibacterial activity of snake venoms.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Antimicrobial peptides are evolutionarily ancient weapons. Their widespread distribution throughout the animal kingdom suggests that they have served a fundamental role in the successful evolution of complex multicellular organisms (Zasloff 2002). Antimicrobial peptides have been isolated from a variety of natural sources, including antimicrobial secretions and venoms. Snake venoms contain many proteinaceous components, including neurotoxins (pre and postsynaptic), cardiotoxins, myotoxins, cytotoxins, proteases, nucleases (Stiles et al. 1991), l-amino acid oxidase (LAAO) (Iwanaga and Suzuki 1979) and phospholipase enzymes that are biologically active. Previous reports point towards the association of some venom with antibacterial activity (Stocker and Traynor 1986; Theakston et al. 1990; Talan et al. 1991). A direct lytic factor from the venom of cobra Hemachatus haemachatus has been reported to show antibacterial property (Aloof-Hirsch et al. 1968). Various peptides (α-helical, cationic and pore-forming), derived from the venom of scorpions (Opistophtalmus carinatus), also possess broad spectrum of antimicrobial activity against bacteria and fungi (Moerman et al. 2002). The α-helical polycationic peptides (pandinin 1 and 2) from the scorpion venom have been shown to have high antimicrobial activity against a range of gram-positive bacteria (Corzo et al. 2001). Moreover, CsTX peptides from the venom of the scorpion, Cupiennus salei also exhibit antimicrobial properties (Xu et al. 1989). Other than the venoms of snakes and scorpions, the venom of the common honey bee (Apis mellifera) also displays antimicrobial properties (Fennel et al. 1968). Its venom component, mellitin, is more active against gram-positive than gram-negative bacteria. Besides, venoms of the waSPS, honey bees and various snakes also contain antimicrobial peptides, but their functions have not been investigated (Blaylock 2000).

To date, only few studies have been made on the antimicrobial activities of snake venoms (Stiles et al. 1991; Blaylock 2000). Antimicrobial peptides from other nonvenom sources are considered to kill bacteria by permeabilizing and/or disrupting their membranes (Zhao et al. 2002). A possible synergistic action between the antimicrobial peptides and the venom enzymes, like phospholipase A2 (PLA2), has recently been reported (Zhao and Kinnunen 2003), thus suggesting some concerted action between the enzymes and antimicrobial peptides of venoms. This potential synergistic property of venom enzymes may be important, particularly at a time when more antibiotic-resistant bacterial strains are emerging. The enzymatic degradation of phospholipids in the target bacterial membrane may be one of the important factors in the bactericidal property of animal venoms. Hence, antibacterial properties of 34 different venoms from snakes, scorpions, honey bee, and a variety of purified PLA2 enzymes and laao were investigated against some clinical isolates of pathogenic (gram-positive and gram-negative) bacteria to evaluate the antibacterial activity of venom enzymes as compared with that of the crude venoms.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Collection of venoms

Lyophilized venoms were obtained from commercial sources (Venom Supplies Pte Ltd, Tanunda, South Australia). Venoms were collected in a sterile manner under strict laboratory conditions, and were centrifuged at 4°C, frozen and lyophilized within 6 h of extraction. The dried venom was normally packed and stored in dark at −20°C. All the venom enzymes used in the experiment have been purified by successive chromatographic steps with the final purity of at least 95%, as assessed by reversed-phase HPLC. Crotoxin from Crotalus durissus terrificus (South American rattlesnake) has a molecular weight of 24 350 Da and a pI of approximately 4·7. Crotoxin PLA2 enzyme is very stable and supplied at 4°C in phosphate buffered saline (pH 7·4). laao, purified from the venom of Bothrops atrox and Crotalus adamanteus were obtained commercially (Sigma Aldrich, St Louis, MO, USA), reconstituted with 50 mmol l−1 of Tris-HCl buffer (pH 7·4), and stored at −20°C.

Chemicals

The following antimicrobial agents: chloramphenicol (CHL-30) 30 μg/disc, streptomycin (STR-10) 10 μg/disc and penicillin (P-10) 10 μg/disc were obtained from (BBL blank disc, 7-mm diameter) Becton Dickinson Labware, Franklin Lakes, MD 21152, USA, and included in the antibacterial test as drug controls and blank discs. Mueller Hinton (MH) agar medium was obtained from Oxoids Pte Lte, UK. One mole per litre tris-HCl buffer (pH 7·4) was purchased from National University of Medical Institute (NUMI), Singapore. All the buffers were filtered by 0·2 μm Nalgo nung filter (Apogent Technologies, Rochester, NY, USA) before use. Molecular weight precision protein standards and acrylamide were obtained from Bio-Rad Laboratories, Hercules, CA, USA. All other reagents were of analytical grade.

Bacterial strains

Six clinical isolates of bacteria were obtained from the Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore. Bacteria used in the present investigation include Escherichia coli, Enterobacter aerogenes, Proteus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa and Staphylococcus aureus. The bacterial cultures were spread and allowed to grow overnight at 37°C on 20 ml of MH agar (pH 7·4) plates (90-mm diameter), prior to storage at 4°C.

Antibacterial effects of crude venoms

Lyophilized crude venoms (100 μg) dissolved in 1 ml of 50 mmol l−1 of Tris-HCl buffer (pH 7·4), were filtered using 0·22 μm syringe filter (Millipore, NY, USA), and stored at 4°C for assay. Susceptibility test was performed by disc-diffusion method (Bauer et al. 1966) with the following modifications. Bacterial inoculums [200 μl of a 0·1–1·0 A600 culture containing 1·5 × 106 to 3·2 × 108 colony forming units (CFU) ml−1] were spread by using a sterile cotton swab onto 20 ml of sterile MH agar plates (90-mm diameter). The surface of the medium was allowed to dry for about 3 min. Sterile paper discs (7-mm diameter) were then placed onto the MH agar surface and 20 μl each of venom samples (100 μg ml−1) were added per disc in five replicates. The discs containing the following antimicrobial agents: chloramphenicol (CHL, 30 μg/disc), streptomycin (STR, 10 μg/disc) and penicillin (P, 10 μg/disc) were used as drug controls, and the blank disc containing 20 μl of 50-mmol l−1 tris-HCl buffer (pH 7·4), served as a normal control. The plates were incubated at 37°C for 24 h. Zones of inhibition were recorded in millimetre diameters and interpreted as sensitive, intermediate or resistant according to the recommendations of the NCCLS (2002).

Antibacterial effects of purified PLA2

Purified PLA2 enzymes – crotoxin A, crotoxin B, ammodytoxin A, daboiatoxin, mojavetoxin, β-bungarotoxin, taipoxin, mulgatoxin, bee venom PLA2 and two LAAO (B. atrox and C. adamanteus), were obtained commercially. Each enzyme was dissolved in 500 μl of 50 mmol l−1 of Tris-HCl (pH 7·4) buffer and mixed by vortex (Labnet VX100, Labnet International, USA) to a final concentration of 0·5 μmol l−1. In vitro antimicrobial activity was determined by the previously described disc-diffusion method (Bauer et al. 1966) with some modifications.

Minimum inhibitory concentrations

The minimum inhibitory concentrations (MIC) were determined by broth dilution method, proposed by Wu and Hancock (1999), for the testing of antimicrobial peptides. Twenty-four-hour-old bacterial cultures were harvested from fresh MH agar plates. The strains were grown in Mueller Hinton broth (MHB) to a mid-logarithmic phase with absorbance at A600 of 0·1–1·0 (1·5 × 106 to 3·2 × 108 CFU ml−1). Whole venom was reconstituted at the required test concentrations in the range of 160, 80, 40, 20, 10, 5, 2·5 and 1·25 μg ml−1 using 1 mol l−1 of Tris-HCl buffer (pH 7·4). Two hundred microlitre of a mid-logarithmic phase culture of bacteria was added to 20 μl of venom samples in 96-well bottomed plate. Five independent experiments were performed as replicates. One well containing 200 μl of bacterial inoculates served as a bacterial control, while another well containing 200 μl of uninoculated MH broth and 20 μl of 1 mol l−1 of Tris-HCl buffer (pH 7·4) were used as a negative control. As positive controls, 20 μl of antibiotics (80–2·5 μg ml−1) in 1 mol l−1 of Tris-HCl buffer were added to 200 μl of bacterial inoculates for the liquid growth inhibition assays. Culture plates were incubated at 37°C for 24 h. The inhibition of bacterial growth was determined by ELISA reader (Molecular Devices Emax precision microplate reader; Research Instruments, Singapore) measuring the absorbance at 560 nm. Results were expressed as MIC, the lowest concentration of venom that reduces growth by more than 50% of the strains.

Biochemical characterization

PLA2 enzyme activity PLA2 catalyses the hydrolysis of phospholipids at the sn-2 position, yielding a free fatty acid and a lysophospholipid. The Cayman Chemical secretory PLA2 (sPLA2) assay kit was used for the measurement of sPLA2. This assay uses the 1,2-dithio analogue of diheptanoyl phosphatidylcholine, which serves as a substrate for most PLA2 (Reynolds et al. 1992). Enzymatic activity, expressed as an increase in absorbance per minute, was converted to specific activity (i.e. micro moles of fatty acid released per minute per milligram of protein).

Protein assay The protein concentration of the crude venoms was determined using the Bradford (1976) protein assay (Bio-Rad protein assay kit; Hercules, CA, USA). Venom samples were prepared at a concentration of 1 mg ml−1. Bovine serum albumin (1 mg ml−1, A280 = 0·56) was used as the protein standard.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the method of Laemmli (1970). Separating gels containing 10·5% acrylamide and stacking gel of 4·5% acrylamide were used. The protein sample (0·1–1·36 mg ml−1) and Laemmli sample buffer (50 mmol l−1 of Tris-HCl, pH 7·4 containing 2% SDS, 2% 2-mercaptoethanol, 10% glycerol, 0·02% bromophenol blue), were heated for 5 min in a boiling water bath. Twenty microlitre of each protein sample was then loaded onto a gel, and electrophoresis was carried out at a constant voltage (120 V for 2 h). The gel was fixed with 5% acetic acid overnight and stained for 2 h in 0·2% Coomassie blue R-250 in 5% acetic acid solution. Destaining was carried out in a solution containing 35% methanol and 7% acetic acid, until the background became clear. The molecular weights of the protein bands were determined using Precision protein standard markers (10–250 kDa). Gels were imaged using a GS-710 calibrated imaging densitometer scanner (Bio-Rad).

Statistical analysis

The results (mean ± SD, n = 5) were statistically analysed by one way anova with repeated measures used to analyse factors influencing the size of the growth inhibition zone.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Antibacterial effects of crude venoms

Table 1 summarizes the antibacterial activity of 34 different venoms of snake, scorpion and honey bee. The venoms of four viperidaes (Daboia russelli russelli, Echis carinatus, Bitis gabonica rhinoceros and Bitis arietans) and two elapidaes (Pseudechis australis, Naja naja naja) suppress, especially against S. aureus. Among them, P. australis and D. russelli russelli venoms exhibited the maximum inhibitory zones with potency almost equal to that of standard drugs. The standard drugs (chloramphenicol and streptomycin) suppress the S. aureus bacteria, because the activity of chloramphenicol is significantly higher than that of the crude venoms. On the other hand, the venoms of death adder (Acanthophis praelongus), Australian elapid (Pseudonaja textilis) and scorpions (Buthotus hottenota hottenota) showed only moderate antimicrobial effect against S. aureus. Noticeable exceptions were venoms from black scorpion (Acanthophiscrasicuda and B.hottentota), sea snake (Hydrophis cyanocinctus), tiger snake (Notechis ater ater), cobras (Naja kaouthia), Australian elapids (Pseudechis nuchalis and Pseudechis affinis), coastal Taipan (Oxyuranus scutellatus) and tiger keel back (Rhabdophis tigrinus), all of which lacked antimicrobial activity. A broad spectrum of activity was seen with some viperidae venoms, including those of Indian Russell's viper (D. russelli russelli), Pallas (Agkistrodon halys), Northern death adder (A. praelongus), Diamondback rattlesnake (C. adamanteus), Speckled brown snake (Pseudechis guttata), Spitting cobra (Naja sumatrana) and Burmese Russell's viper (Daboia russelli siamensis). Among the gram-positive and gram-negative bacteria tested, S. aureus, P. mirabilis and P. vulgaris were more susceptible to elapid venoms. The order of the susceptibility of the bacteria tested against viperidae venoms is as follows: S. aureus > P. mirabilis > P. vulgaris > E. aerogenes > P. aeruginosa > E. coli.

Table 1.   Antibacterial effect of different animal venoms, tested by disc-diffusion method, against some clinical isolates of gram-positive and negative bacteria
Common nameScientific nameMicro-organism
Sa (+)Ea (−)Pv (−)Pm (−)Pa (−)Ec (−)
  1. The values are presented as mean ± SD (n = 5) and represent a venom inhibition zone in mm, including the 7-mm diameter of the disc, after 24-h incubation. The bacterial inoculum per plate contained 1·5 × 106 to 3·2 × 108 CFU, which were spread onto the agar surface with sterile cotton swap. Sterile paper discs (7-mm diameter) were placed onto the agar surface and 20 μl of venom (100 μg ml−1) added. Micro-organisms: Ec, Escherichia coli; Ea, Enterobacter aerogenes; Pv, Proteus vulgaris; Pm, Proteus mirabilis; Pa, Pseudomonas aeruginosa; and Sa, Staphylococcus aureus. Control (0); no activity (−).

Elapidae
 Death adderAcanthophis augtra20 ± 0·71
 Common death adderAcanthophis antarcticus21·2 ± 1·9314·4 ± 0·84
 Northern death adderAcanthophis praelongus8·4 ± 0·707·7 ± 0·858·1 ± 0·447·2 ± 0·45
 Desert death adderAcanthophis pyrrhus21·4 ± 1·1415·5 ± 0·92
 HectorAndroctonus australis7·2 ± 0·84
 Malayan kraitBungarus candidus25·1 ± 1·23
 Indian cobraNaja naja naja27·8 ± 1·10
 Spitting cobraNaja sumatrana24·4 ± 1·517·9 ± 0·7014·4 ± 0·87
 King brown snakePseudechis australis29·9 ± 0·71
 Speckled brown snakePseudechis guttata15·2 ± 0·8328·8 ± 1·107·7 ± 0·66
 Red-bellied black snakePseudechis porphyriacus22·5 ± 0·50
 Collett's snakePseudechis colletti7·2 ± 0·45
 Peninsula brown snakePseudonaja inframaggula23·3 ± 0·46
 Eastern brown snakePseudonaja textilis15 ± 0·70
Viperidae
 PallasAgkistrodon halys24·1 ± 1·237·4 ± 0·8915·4 ± 0·7417·2 ± 0·837·9 ± 0·74
 Diamondback rattlesnakeCrotalus adamanteus25·4 ± 1·5121·.7 ± 2·2315·6 ± 0·5
 Puff adderBitis arietans26 ± 0·4316 ± 0·83
 West African gaboon viperBitis gabonica rhinoceros27 ± 0·717·7 ± 0·85
 Russell's viperDaboia russelli russelli29·4 ± 0·898 ± 0·7026·4 ± 0·9816·8 ± 0·847·8 ± 0·83
 Burmese viperDaboia russelli siamensis25·2 ± 0·8414·8 ± 0·837·5 ± 0·50
 Saw-scaled viperEchis carinatus28·6 ± 0·81
 Wagler's pit viperTrimeresurus wagleri25·2 ± 1·928·4 ± 0·89
Apiidae
 Honey bee venomApis mellifera23·2 ± 1·09
Scorpionidae
 ScorpionButhotus hottenota hottenota15·4 ± 0·89
 Chinese red scorpionButhus martensii Karsch16·6 ± 0·89
Antibiotics
 Chloramphenicol (CHL)30 μg per disc35·1 ± 1·2627·8 ± 1·0924·6 ± 0·8633 ± 2·3425 ± 0·7031·8 ± 1·64
 Streptomycin (STR)10 μg per disc30·3 ± 0·7715·7 ± 0·4418·2 ± 0·8327·8 ± 1·0916·5 ± 0·5029·4 ± 0·89
 Penicillin (P)10 μg per disc17·4 ± 0·8918·3 ± 0·8516·7 ± 0·9827·8 ± 1·1016·6 ± 0·8915·2 ± 0·84

Antibacterial effects of venom enzymes

The antibacterial activity of the crude venoms was then compared with that of the purified PLA2 enzymes and LAAO. Among the various purified PLA2 enzymes examined for antibacterial effects, crotoxin B, daboiatoxin, mulgatoxin and bee venom PLA2 exhibited significant activity against S. aureus, E. coli, P. aeruginosa and E. aerogenes, with the highest activity noted only for the basic PLA2 crotoxin B (Table 2). LAAO was also found to have strong effect against S. aureus and P. mirabilis. Five enzymes – crotoxin A, ammodytoxin A, mojavetoxin, β-bungarotoxin and taipoxin did not show any effect against all the tested organisms; however, none of the enzymes had any activity against P. vulgaris.

Table 2. In vitro antibacterial activity of purified phospholipase A2 enzymes from snake venoms, tested by disc-diffusion method
Phospholipase A2 enzymesScientific nameMol. wt. (kDa)Conc. μg ml−1Micro-organisms
Sa (+)Ea (−)Pm (−)Pa (−)Ec (−)
  1. The values are presented as mean ± SD (n = 5) and represent a PLA2 inhibition zone in mm, including the 7-mm diameter of the disc, after 24-h incubation. Micro-organisms: Ec, Escherichia coli; Ea, Enterobacter aerogenes; Pv, Proteus vulgaris; Pm, Proteus mirabilis; Pa, Pseudomonas aeruginosa; and Sa, Staphylococcus aureus. Control (0); no activity (−).

l-Amino acid oxidase (LAAO)Bothrops atrox10027·4 ± 0·7326·8 ± 0·60
LAAOCrotalus adamanteus10027·1 ± 0·7224·7 ± 1·19
Crotoxin BCrotalus durissus terrificus24·310027·7 ± 1·1021·2 ± 1·9324·5 ± 0·8625 ± 0·70
MulgatoxinPseudechis australis13·21008·4 ± 0·89
Daboiatoxin (DbTx)Daboia russelli siamensis13·610014·2 ± 0·84
Bee venom PLA2Apis mellifera19·010013·3 ± 0·83

Minimum inhibitory concentrations

The most promising venoms were further studied for MIC by broth dilution method. The inhibitory effect against S. aureus was found to be stronger with the venoms of P. australis, D. russelli russelli and E. carinatus (MIC 20 μg ml−1) than that seen with the venoms of N. sumatrana, C. adamanteus and A. halys (MIC 40 μg ml−1). LAAO from C. adamanteus and B. atrox had the highest inhibition against all the organisms tested. The inhibitory action of the venoms of B. gabonica rhinoceros, C. adamanteus, E. carinatus, D. russelli russelli and LAAO (C. adamanteus) against S. aureus was at least twofold higher than that found against P. vulgaris at all concentrations (MIC 160–1·25 μg ml−1). The inhibitory effect shown by these venoms against S. aureus growth was more or less equal to that of the standard drugs.

PLA2 enzyme activity

The PLA2 activity varied depending on the venom of different species. Maximal PLA2 activity was found for the venoms of D. russelli russelli, A. halys, B. gabonica rhinoceros, E. carinatus, C. adamanteus and P. australis. Viperidae and elapidae venoms exhibited a wide range of PLA2 enzyme activities (Table 3). However, D. russelli russelli (activity 785·2 μmol min−1 mg−1), A. halys (activity 86·5 μmol min−1 mg−1) and P. australis (activity 3949 μmol min−1 mg−1) venoms showed stronger PLA2 activity as compared with that of the other venoms.

Table 3.   Total and specific activity of phospholipase enzyme (PLA2) activity and protein contents of different venom samples
SpeciesPLA2 activity
Total activity (μmol min−1) Specific activity (μmol min−1 mg−1)Yield of protein (mg ml−1)
  1. Total activity of PLA2 enzyme estimated from the whole venoms (μmol min−1).

  2. PLA2 enzymatic activity (μmol min−1 mg−1). Values are presented as mean ± SD (n = 10) of 10 replicates.

Elapidae
Acanthophis augtra3·7474·5 ± 0·50·1
Acanthophis antarcticus190487·5 ± 0·960·78
Acanthophis praelongus241·21416 ± 3·840·34
Acanthophis pyrrhus1381150 ± 0·980·24
Androctonus australis22·285·2 ± 0·270·52
Bungarus candidus34·9166·3 ± 0·560·42
Naja naja naja293·41333 ± 1·70·44
Naja sumatrana406·5903·5 ± 0·60·90
Pseudechis australis434·53949 ± 3·20·22
Pseudechis guttata308·5791 ± 2·00·78
Pseudechis porphyriacus726·73303 ± 1·70·44
Pseudechis colletti15·7111·8 ± 0·890·28
Pseudonaja inframaggula19955945 ± 26·60·66
Pseudonaja textilis416·8832·3 ± 0·61
Viperidae
Agkistrodon halys86·5157·4 ± 0·201·1
Bitis arietans124·1248·2 ± 0·270·54
Bitis gabonica rhinoceros126·5452·4 ± 0·570·56
Bothrops atrox (l-amino acid oxidase)3·86·36 ± 0·061·2
Crotalus adamanteus (l-amino acid oxidase)34·2201·3 ± 0·210·34
Crotalus adamanteus236·4619·4 ± 0·461·56
Echis carinatus53·4106·5 ± 0·491·4
Daboia russelli russelli392·8785·2 ± 0·401·36
Daboia russelli siamensis262·4524·4 ± 0·441·24
Trimeresurus wagleri4·638·2 ± 0·260·24
Apiidae
Apis mellifera3·720·5 ± 0·10·36
 Scorpionidae
Buthotus hottenota hottenota5·138·8 ± 0·10·26
Buthus martensii Karsch4·690·4 ± 0·20·102

Protein determination

The protein content was improved in the antibacterial active crude venoms of C. adamanteus (1·6 mg), D. russelli russelli (1·4 mg), E. carinatus (1·4 mg) when compared with that of nonactive venoms of A. pyrrhus (0·3 mg) and P. colletti (0·28 mg), respectively. The yield of venom protein content generally varies with the snake species (Table 3). The protein profile analysis (SDS-PAGE) also supported the view that the venoms with potent antibacterial activity apparently contain the small molecule 10–15-kDa protein (Fig. 1a–f). Other than small molecule proteins, C. adamanteus venom also expressed the major proteins of 25 and 37 kDa. In D. russelli russelli venom, 50-kDa proteins were more prominently expressed than other molecular weight proteins. Whereas the nonactive venoms (Fig. 1g–h) did not show the small molecules between 10–15 and 25–50 kDa) proteins when compared with the active venoms.

image

Figure 1.  Electrophoretic profile of the crude venoms studied. The venom samples were prepared and run in polyacrylamide gel electrophoresis (SDS-PAGE). Staining was done with coomassie brilliant blue R250. (a) Daboia russelli russelli (Viper), (b) Agkistrodon halys (Pallas), (c) Pseudechis australis (Mulga or Australian king brown snake), (d) Echis carinatus (Saw-scaled viper), (e) Crotalus adamanteus (Diamondback rattlesnake), (f) Bitis guttata (Puff adder), (g) Pseudonaja nuchalis (Western brown), (h) Oxyuranus scutellatus (Coastal taipan), (i) Naja kaouthia (Cobra).

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N-terminal sequencing

A comparison of the N-terminal sequences of crotoxin B with other venom PLA2 sequences shows a moderate degree of similarity (Fig. 2). The hydrophobicity profiles of purified PLA2 from venoms were obtained by placing the hydrophobic indices against the sequence residue numbers (Fig. 3). The sequence of crotoxin B exhibited higher hydrophobicity at the C-terminal region (102–120) than that of the other venom PLA2 enzymes.

image

Figure 2.  Comparison of the amino acid sequences of Crotoxin (CB2) phospholipase A2 enzymes (Crotalus durissus terrificus) with other PLA2 of taipoxin alpha chain (Oxyuranus scutellatus scutellatus), ammodytoxin C [ATXC] (Vipera ammodytes ammodytes), Bothropstoxin [BthTX-I] (Bothrops jararacussu), Mojave toxin basic chain [Mtx-b precursor] (Crotalus scutulatus scutulatus), Ecarpholin S-neutral (Echis carinatus), isozyme PA-12C (Pseudechis australis), AGKHP (Gloydius halys), β-bungarotoxin [A1-chain precursor] (Bungarus multicinctus), CA [Crotapotin] (Crotalus durissus terrificus) and completely conserved residues in all sequences are bolded and marked by asterisks. The gaps are inserted in the sequences in order to attain maximum homology. CLUSTAL W (1·83) multiple sequence alignment.

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image

Figure 3.  Hydropathic profiles of crotoxin b, daboiatoxin, mulgatoxin, taipoxin, ammodytoxin A, bee venom PLA2, β-bungarotoxin and mojavetoxin were calculated by using the kyte-doolittle method.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The present study provides evidence that several venoms of different snake species have antibacterial effects against both gram-positive and gram-negative bacteria. Among the venoms examined, those from four species of viperidae (D. russelli russelli, E. carinatus, B. gabonica rhinoceros and B. arietans) and two species of elapidae (P. australis and N. naja naja) showed strong antimicrobial effects, especially against S. aureus. These venoms exhibited greater zones of inhibition, equivalent to that shown by the standard drugs, chloramphenicol and streptomycin. Compared with the elapidae (P. australis) venom, which was more specific against S. aureus, the venoms of viperidae (D. russelli russelli and A. halys), on the other hand, exhibited a broader spectrum of antibacterial activity. A strong activity was shown against S. aureus by the venoms of viperidae (B. gabonica rhinoceros and B. arietans) and crotalidae (C. adamanteus), while most venoms (A. australis, N. sumatrana, P. guttata, A. halys, B. gabonica rhinoceros, D. russelli russelli) exhibited only a weaker activity against E. coli. In contrast, a stronger activity on E. coli had been reported previously for A. mellifera, Naja sputatrix, V.russellii and C. adamanteus venoms (Stocker and Traynor 1986). Noticeable exceptions were venoms from Buthus hottenlota (black scorpion), Buthus martensii (Chinese red scorpion), H. cyanocinctus (sea snake), R. tigrinus (Tiger keel back), N. ater ater (Krefft's), N. kaouthia (Cobras), P. affinis (Dugite), P. nuchalis (Western brown) and Oxyuranus scutellatus (Coastal taipan), which lacked any antimicrobial activity against all the tested bacteria.

The venoms with potent antibacterial activity were then chosen for further studies on MIC, as determined by broth dilution method. The MIC against S. aureus was found to be higher with the venoms of P. australis, D. russelli russelli and E. carinatus (MIC 20 μg ml−1) at the lowest dilution than that seen with the venoms of N. sumatrana, C. adamanteus and A. halys (MIC 40 μg ml−1). Previously, crotalid (C. adamanteus) venoms were reported to exhibit strong inhibitory effect (MIC 80 μg ml−1) against Staphylococci, P. aeruginosa, Enterobacter, Citrobacter, Proteus and Morganella species (Talan et al. 1991). In the present study, bacterial growth was completely inhibited against P. aeruginosa by C. adamanteus, E. carinatus, P. guttata and D. russelli russelli venoms, but the inhibitory effect was lost with prolonged incubation (after 24 h) for many gram-negative species. Venoms from B. arietans, B. candidus, C. adamanteus and N. naja naja were only moderately effective against P. mirabilis at all dilutions (MIC 160–1·25 μg ml−1). Comparison of the inhibitory effect of the venoms with that of the purified LAAO showed that the inhibition of the C. adamanteus and B. atrox LAAO against P. mirabilis and S. aureus was significantly higher than that of the crude C. adamanteus venom. Association between an antibacterial property of snake venom and LAAO had previously been reported (Stiles et al. 1991). The LAAO from Trimeresurus jerdonii venom inhibited the growth of E. coli, S. aureus, P. aeruginosa and Bacillus megaterium (Lu et al. 2002), while the LAAO from A. halys (Pallas) exhibited inhibitory activity against bacteria (E. coli) and fungi (Yan et al. 2000).

Besides the antibacterial effect exhibited by venom LAAO, the inhibitory activity seen with the venoms may also be to the result of the multiple biological effects of snake venom PLA2 (Chioato and Ward 2003). PLA2 enzymes are present in almost all venoms, but their prevalence is particularly higher in viper venoms. The maximum PLA2 activity was found in the venoms of D. russelli russelli, A. halys, B. gabonica rhinoceros, E. carinatus and C. adamanteus. When the antibacterial activity of purified PLA2 was compared, crotoxin B from the highly toxic South American rattlesnake (C. durissus terrificus) venom exhibited the most potent activity against S. aureus, E. coli, P. aeruginosa and E. aerogenes. Mammalian sPLA2 have been implicated in lipid digestion on host defence mechanisms, including antibacterial defence (Valentin and Lambeau 2000).

A comparison of the N-terminal sequences of crotoxin B with other venom PLA2 shows a moderate degree of similarity. Crotoxin B is a basic neurotoxic PLA2 (C. durissus terrificus) containing three chains which enhance its lethal potency (Bouchier et al. 1991). It exhibits higher hydrophobicity at the C-terminal region (102–120) than those of other PLA2 enzymes. The C-terminal region of basic PLA2Bothrops asper myotoxin-II has been suggested as the segment responsible for its cytotoxic and myotoxic effects (Lomonte et al. 1994, 2003). Antimicrobial peptides inhibit microbes by scrambling of the usual distribution of lipids between the leaflets of the bi-layer of the cell wall, thus resulting in the disturbance of membrane function, and the damaging of critical intercellular targets after internalization of the peptides. It appears that the enzyme hydrophobicity might be playing an important role for the antimicrobial action on bacteria.

In conclusion, the in vitro screening provides convincing evidence that several venoms have most promising antibacterial effects against gram (+) and gram (−) bacteria. The present findings indicate that viperidae (D. russelli russelli, E. carinatus, P. guttata) and elapidae (P. australis) venoms have significant antibacterial effects, which may be the result of the primary antibacterial components of LAAO, and in particular, the PLA2 enzymes. The results will be useful for further purification and characterization of antibacterial agents from snake venoms.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors are thankful to the Defence Science and Technology Agency (DSTA), Singapore, for the financial support (Grant No R-181 000 063 422) to carry out this work. We thank the Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, for their support and permission to use bacterial cultures in this investigation.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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