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 of Applied Sciences 10 (1 9): 2328-2332, 2010
ISSN 1812-565-4
0 2010 Asian Network for Scientific Informat.ion

Screening Antimicrobial Activity of Venoms from

Snakes Commonlv Found in Malaysia
]T.M, San,
Vejayan, 1K. Shanmugan and 21-1. IbrahiTl
I Jeffrev Cheah School of Medicine and Health Sciences, N,'lonash University Sunway Campus,
Jalan Lagoon Selatan, Bandar Sunway, 461 50, Selangor Darul Ehsan, Vlalaysia
21nstitute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia

Abstract: The study was carried out to screen antimicrobial activity in venoms obtained from 11 species of
snakes that are all common in lvfalaysia. Snake venom, which constitutes a diverse range of proteins, has
been identified as a potential source of
long therapeutics. Therefore, in times when antimicrobial
becoming an Increasingly severe Issue,
resistance is it. is unsurprising t.hat snake venoms are being
Investigated for antimicrobial cornponents- Antibacterial activity was assessed using the hole-plate
method. Venorns of Calloselasma rhodostoma and Ophiophagus hannah were capable of producing the
most prominent bacterial inhibition zones with maxi-mum values as high as 12 mm, while the other venoms
screened only produced bacterial inhibition zones that were not more than
mm. These bvo venoms were selected for further
deterrnination of Minirnurn Inhibitory Concentration (NflC). The WIIC values were tested with Staphylococcus
aureus ATCC25923, ATCC29213 and ATCC43300. The
values obtained for C.alloselasma rhodostoma

tested against S. aureus ATCC29213. NflC values obtained for Ophiophagus hannah were 250 gg mL-l when
tested against all three strains. Since the potential of snake venorns for ant-irnicrobial activity has been
established, further study is in the progress to purify the active antimicrobial component and to screen a
wider range of bacterial strains.

Key words: Venorn, therapeutic use, bact.eria, Staphylococcus aureus, viper

INTRODUCTION Given its vast potential as a source of' therapeutics,

it is, therefore,
Resistance of bacteria against antimicrobial agents has that snake venoms have also been
always been an investigated for antimicrobial components. Despite
I-mmense burden to healthcare
worldwide. High resist.arlce of Staphylococcus aureus against heavy colonization of pathogenic bacteria in the oral cavit.y
of the snake, srlake bite vict.ims are not. frequerltly
ciprofloxacin, amoxicillin and chlorarnphenicol has
observed to
problem has led to wider search for antimicrobial agents suffer from wound infection. This
been documented in a study (Nafeesa et al., 2001 The observation led to the theory that snake venom may have
bacterial sh•ains. Among these new sources are plant antimicrobial activity ('l'alan et al., 1991). While, the
from other sources against the ant.ibiot.ics resistance significa.nce of antimicrobial cornrx)ne.nt in sna.ke venoms is
methicillin resistant S. aureus (MRSA), vancomycin yet to be fully elicited, it has been hypothesized that these
components in the oral secretions of the snake have been
extracts, which have shown promising activities against developed under evolutionary pressure as a defense
S. aureus (VISA) (Dadg•ar et al., 2006, ZakaHa
mechanism of the snake against. the microorganisms on its
resistant S. aureus (VRSA) a.nd vancomycln intermediat.e prey (Shivik, 2006).
Snake venom is another new source of antimicrobialam2CD7).
Among some of the
common antimicrobial
agents that we will describe in this study. components that have been isolated from snake venoms
Snake venoms const.itute a. diverse range of proteins a.re t.he
enzymes L-amino oxidase (LAAO) and
that include
neurotoxins, and peptides. LAAO
phospholipase A, (PLA,) The purified from
Consequently, enzy•rnes
the idea of harvesting proteins from snake Pseudechis australis
venoms to activity against
showed significant antimicrobial
developed as commercial, therapeutic
products has not been new. different strains of Aeromonas (Stiles et al.- 1991 ).
PLAZ purified from Crotalus durissus

Corresponding Author: Jaya Vejayan, Jeffrey Cheah School of Medicine and Health Sciences,
Monash University Sunway Campus, Jalan Lagoon Selatan, Bandar Sunway 461
50, Selangor, Darul Ehsan, Ndalaysia
 J. Apphed sci., 10 (19).• 2328-2332, 2010

terrificus and Daboia pusselli siamens•is have shown

Antibacterial
antibact.erial activity against the Gram-negative bacteria (1 mg mL¯l)
Bu.rkholderia pseudomallei (Sarny et al., 2006). PLA3
isolated from Agkistrodon halys also showed significant
inhibition against Staphylococcus
screening assays: Lyophilized venoms
were prevnred in deionized water. Three
milliliter of bacterial inæulum (cfu mL¯l = 105. = 0.1
was spread onto an agar plate and left standing for 3 min.
aureus, Proteus

E.xcessive inoculum '.vas poured away and the agar plate

vulgaris and Proteus mirabilis (Samy et al., 2008). was left. standing for 3 min again. IJsir1g a modified
Apart frorn
enzyrnatic proteins, antllnicrobial peptides hole-plate met.hod, 4 mm diamet.er wells were made onto
have also been
purified in recent studies. Cathelicidins
the agar usmg an immunodiffusion cutter (Nair et al.,
isolated from
Bungarus fasciatus and Ophiophagus
2007). Thirty microliter of each dissolved venom sample
hannah have reported potent antimicrobial activity
was loaded
into
each
well.
Vancornycin and
against many strams of Grarn
Escherichia coli. negative bacteria
methicillin (30 Mg ml.-l,) were used as drug controls. The
(Wang et al., 2W8•, Chen et al., 2009). A small peptide
bacterial plates were incubated at 37 oc for 18 h and the
vgf-l, isolated from Naja atra, has shown to have
inhibition zones were measured in
antimycobacterial activity (Xie et al.. 2003). Omwaprl-n, diameters.
ptilffied from Oxyuranus microlepidotu.s, is report.ed t.o Minimum inhibitory concentration: Minimum Inhibitory
antimicrobial activity against Gram positive
Concentration (NflC) was determined using a modified
bacteria such
as
Staphylococcus wagneri
and
Bacillus megaterium (Nan- et al., 2007). method described by Nair et al. (2007). •I'he two venoms
which showed the highest diameter of inhibition zones
Tn t.his study, sve describe the prelimina.1Y screening

were chosen for NflC determination. Crude venom of

of I I snake venoms for antibacterial activity against three

concentration 1 mg mL¯l '.•vas serially diluted in the range
different strains of Staphylococcus aureus and m,vo Gram
negative bacteria,
of 0.5, 0.25, 0.125. 0.1625 and 0.03125 mg mL-l using
Pseudomonas aeruginosa and
cation-adjusted DvLH broth. Three milliliter of bacterial
Similar screenmg focusing on
inx•ulum was spread onto 30 mL, of NIH agar as described
antimicrobial property has not been attempted previously above för the ant.ibact.erial screening assays. Six wells of
among indigenous Malaysian snakes. diamet.er 4 mm were made ont.o t.he agar plate. Each •well
was loaded with 30 PL of one concentration of the diluted
MATERIALS METHODS venom. The plates '.vere incubated at 37 oc for 18 h and
MIC was defined as the lowest concentration of venom
All venoms which gave a detectable inhibition zone.
Venoms: used were of common venomous
snakes in Malaysia, obtained from the snake farm in
RESULTS
Sungai Batu PahaL Perlis, Malaysia in the year of
2CÅ)9- The venorns were all, freeze-dried and stored
Table I shows the antibacterial activitv of II crude
in -20ac. venoms tested against five different strains of bacteria,

Bacterial strains: The
strams used in the
antibacterial screelling assays were Staphylococcus
aureus ATCC25923, Staphylococcus aureus ATCC29213,
Methicillin-resistant Staphylococcus aureus (lvfRSA)
ATCC43300, Pseudomonas aeruginosa API'CC25873 and
F.scherichia coli ATCC25922. The bact.eria were streaked against. thc Gram posit.ive bact.eria vvrere föund to be more
onto Mueller-Hinton (NIH) agar plates, incubated
tfree different strains of S. aureus and two different
strains of Gram negative bacteria. From this preliminary
screening, the Gram negative were found to be
resistant
against all the crude venoms as no Inhibition
zone was
exhibited in the antibacterial assay.
'I'he antibacterial activ ities of the crude venom
significant a.mong the Viperidae species. Crude venoms
overnight for growth and stored at 40C..
from all four Viperidae species demonstrat.ed significant.

that. would be
Bacterial inoculum: Bact.erial inoculum

rhodosloma showed the largest inhibition zones between

used for antibacterial screening assays 'vvas prepared
of 10.2-12.5 rnm.
using the standard method of log-phase growth. A
Among the
crude venoms from the six Elapidae
single-unit colony was picked from the bacterial culture
species, only
plates a.nd added to 1 0 mL of Nfueller-Hint.on (IVTH) brot.h. exhibited antibacterial activity.
The inhibition zones of Bungarus candid.us and

The inoculated I'vff-l broth was incubated at 37 oc until a Bungarus fasciatus demonstrated against the tfree
colony-forming (CFU) ml,-l value of 103. This was
strains of S. aureus were only between 6.3-8.0 mm.
confirrned by measuring the absorbance value of the
Ophiophagus hannah was the only exception among the
inoculum with the spectrophotometer (Shimadzu) to give Elapidae species, exhibiting large inhibition zones
befiveen 9.5-11.5 mm.
 J. Apphed sci., 10 (19).• 2328-2332, 2010

Table I : Ant.lbact«ial activity of I I crude venoms against. five st.rains of bacteria tested using hole-plat.e method

Bacterial inhibition zone (mm)

Scientific name Common nmne SA ATC.C2.S923 SA ATCC2921.3 SA ATCC4.3.3(X) PA .ATCC2.f87.3 EC A T C.C,2.S922

Viperidae
Calloselasma r'UÖstoma Malayan Pit Vipa 10.2±0.5 12.0±2.8 12.5±1.0
Trimere.surus Many-ove Pit Vipa 10.4±0.7 10.0±0.0
purpureom«.ulatur
Trimoesurus "'Cleri Waglcr's Pit Viper 10.9±28
Trimoesurus swnavan.if Sumatran's Pit Viper 7.2±0.9 9.3±0.5
Elapidae
Ophiophcus 'nmuzh King
Bimgæus camåås Malay an/blue krait 6.3±0.5 7.3±0.5
Bungo•u.ffasc.idus Banded krait 7.24:
A.Wa kaouz/ia. Monocled
A.Wa siamengis Indochinese spitting cobra
Ncja sum afrcma Equatorial spitting cobra
Hydrophiidae
Enhyöirn schistosa Common sea snake
Control
Vanc(xnycin 14.3±0.9 15.0±1.4 15.0±0.0
Methicillin 15.0±0.0
The values are prsented as Mean±SD (n = 4). •me inhibiti(Y1 z(nes were aftu• 18 h of incubation at 3 70C and the irülibition zone diameta•s include
the 4 rmn diameters of the wells containing either 30 of crude venc.xn (1 mg mL-l) or antibi(lic (30 mL-1). Bacterial inoculurn of 105 cm mL-1
(AEÜÜ = 0.1) is added pa plate. SA: Sqhylæoccus aueus•, PA: ærugimsa. EC: Escherichia coli. -: No activity

TQ

KC CR

c)

Fig. l: Antibacterial assays of crude venom using the hole-plate method. (a) SA ATCC29213, (b) SA ATCC25923 and
(c) SA ATCC4330(). SA: S. aureus. KC: O. hannah (King Cobra); CR: C. rhodostoma; T S: T. sumafr•anus•,
BC: B. candidus•, BF: B. Jhsciatus•, NN: N. kaouthia•, Van, Vancomycin; Met, Methicillin. A vancomycin disc
(30 pg mL¯l) was also used against SA ATCC29213, as sh0Mff1 in (1 a) in cornparison '*Vith diluted
vancomycin solution (30 gg
The diluted vancomycin showed higher inhibition diameter than the vancomycin disc
screened in
study exhibits no antibacterial activ ity.
The venom of the single Ilydrophiidae species
 Thc
crude venoms also have lower antibacterial
a.ctivity as compared to that of the convent.ional
antibiotics, as shown by the larger inhibition zones of
vancomycin and methicillin.
Figure 1 a-c shovvred t.he ant.ibact.erial assays of the
various crude venoms test.ed against. the t.hree strains of
 J. Apphed sci., 10 (19).• 2328-2332, 2010

Table 2: MIC vahnes of C. rinias•toma, O. fr»vnJ1 and ant.ibiciics

Minimum inhibituy strains of Gram-positive S. aureus to the crude venoms
MIC (pg nL-l) and the resistance of the myo strains of Gram negative
Bacta•ial strains C. rhe:Ostoma O. /y:znrnh Vancunycin Mdhicillin bact.eria against the venoms.
SA ATCC,2.5923 125 250 Similar patterns of sFEcies specific
SA ATCC29213

250 250
SA ATCC433(X) 125 500 susceptibility arid resistance against Gram negative have
MIC was detamined as the lowest concentration of venom which gave a
250

also been noted in omuraprm, a peptide isolated from the

ddectable inhibitiuu zme ana ISh of incubati(Y1 at 37 oc. The valor-ns were

diluted in the range of l, 0.5, 0.25. 0.125, 0.0625 and 0.03125 mg mL-L v enorrl of Oxyuranuy microlepidotus. Orn•wapnn is
Each

concentration was added to effective

well. Bacterial inoculurn against
of 103 cfu mL-l (Aaa = 0.1) is added per plate. SA. Sqhyl«occus amus•
Gram positive bacteria such
S. aureus ATCC29213, ATCC25923 and ATCC43300 using as
the hole-plate method. In all frree assays shown, it could Bacillus megaterium and S. wagneri, but shows
resistance when tested against Gram negative bacteria
be seen
and other Gram positive bacteria such as B. thuringiensis,
venoms from Calloselasma rhodostoma and S. aureus and Streptococcus clavuligerus (Nair ef al.,
Ophiophagus hannah demonstrat.ed clear inhibit.ion
2007). The resistance against Gram negative bacteria
zones that were significantly larger than the other crude could possibly be due the outer membrane of the bacterim
venoms screened 'I'he outer membrane of Gram negative bacteria has
Since the lipopolysaccharides (LPS). Charges on the LPS molecule
crude venoms can affect the uptake of antimicrobial peptide and such
from Calloselasma similar mechanism might have resulted in the resistance
rhodosloma and Ophiophagus hannah demonstrat.ed thewe observed among the Gram negative bactena tested in
most significant inhibition zones against the three strains this screening (Devine and Hancæk, 2W2). It has also
of S. aureus, including the lvfRSA strain, these two been observed
venoms •were selected for NflC determination. Table 2 Gram negative bacteria, when treated
shows the NflC values of Calloselama rhodostoma and with EDI'A that disrupts the outer membrane, become
Ophiophagus hannah. The N'flC values of both venoms susceptible to antibacterial agents such as bronchochin
were found to (Gao et al.. 1999).
within the range of 125-250 gg mL- Nevertheless, this preliminary screening is by no
When tested against S. aureus ATCC25923 and rn eans representative arid conclusive of the resistance of
API'CC43300, Calloselasma rhodostoma demonstrated Gram negative bacteria against snake venoms, as the
nurn ber of bacterial strams used is verv rninirnal-
lower
Moreover, PLA? and cathelicidin previously isolated from
as compared to Ophiophagus hannah. The other snake venoms were found to be effective against
MIC values of these bevo venorns were found to be muchGram
higher than that of the antibiotics.
negative bacteria such
bacterial membrane
DISCUSSION as
Burkholderia
One of the main patterns that can be identified in thispseudomallei and E. coli (Samy et al., 2006, \Vang et al.,
preliminary screening is that the Viperidae venoms 2008), cont.radict.ing our findings from t.his screening.
exhibited larger inhibition zones than Elapidae venoms, Species specific susceptibility also could not be identified
from this screening as only one t.YFR of Gram positive
with the exception of Ophiophagus hannah. This pattern bacterium has
is very similar to the pattern found when enzymatic used.
properties of Viperidae and Elapidae venoms were While it has been observed with electron rnicroscopy
cornpared which sh0',ved that venorrls of Viperidae that the antibacterial mechanism of snake venoms
species had higher enzymatic activities than venoms of involves cell surface membrane blebbing and subsequent
Elapidae species, with the exception of Ophiophagus cell contents leakage (Nair et al., 2007', Samy et al., 2008)
the exact mechanism of the cell membrane disruption
hannah venom, an Elapidae venom that showed high
leading to cell death is st.ill very much unclear. Snake
enzynatic activities (Kocholaty et al-, 1971). The results
venom
of t.his preliminary screening, t.herefore, highly suggest. antimicrobial mechanism is complex and is
that the antibacterial activitv of snake venoms is due to affected by factors such

enzymat.ic components. is also in tandem with ot.her charge of the protein, as amino acid sequence, net
three-dimensional süucture,
findings, which have
shos.vn
and salinity of the
that snake venom environment (Nair et al., 2007). Hence, the different
antimicrobial activity is due to erizymes such as proteins/peptides from different SIRCies of snakes
P (Samy et al., 2008). can have varied mechanusms of cell membrane
Another pattem that can be identified from this disruption, resulting in the differences of
preliminary screening is the susceptibility of the free susceptibility among the different bacterial strains.
 J. Apphed sci., 10 (19).• 2328-2332, 2010

S'Vhile the WC values obtained from this screen-mg Nair, D.G., B.G. Fry, P. Alewood, P.P. Kumar and
is many times higher than that of the conventional

R M. Kini, 2007. Antimicrobial activity of omwapnn-,

antibiotics, it has been sh0'„sm in a comparison study that
a new member of the waprin farnily of snake venom
purified LA AO exhibited significa.nt.ly higher inhibition
proteins. Biochem. J. , 402: 93-1 ()4.
than crude venoms (Samy et aZ., 2C07)- The
Samy, R.P , A. Pachiappan, P. Gopalakrishnakone,
exhibited by fractions obtained from each stage of
M.M. 'l*hwin and Y.E. Hian et al.- 2006. In vitro
purification also increases (Samy et al.. 20CE).
ant.imicrobial activit.y of nat.ural toxins and animal
In summary, we have successfully established the
venoms tested against Burkholderia pseudomallei.
potential of antimicrobial activity using snake venom and
BMC Infect Dis.. 6:
00.
have
also
identified two
promising venoms,
samy,
R.P., P. Gopalak,ishnakone, lvf.M. Thwiru
Calloselasma rhodostoma and Ophiophagug hannah,
T.K.V. Chow, 1-1. Bow, E.H. Yap and T,W.J, Thong,
which show the highest inhibition. Further study is in the
2007. Antibact.erial activit.y of snake, scorpion a.nd
progress
to
purify
the
active
antimicrobial
bee venoms: A comparison with purified venom
proteins/peptides from these two venoms, as well as to
phospholipase A2 enzymes. J. Applied Microbiol.,
screen a larger number of bact.eria t.o characterize the
1 02: 650-659.
susceptibility of these fivo venoms more accurately.
Samy, R.P., P.
Gopalakäishnakone, II. Bow and
V.'I'.K. Chow,
Purification, chamcterization and
ACKNO'„VLEDGMENTS bactericidal activities of basic phospholipase AZ from
the venom of Agkistrodon halys (Chinese pallas).

Authors 'yvould like to thank Ministry of Scierlce,

Biochimie, 90: 1.372-1388.
Technology and Innovation (MOST I), Malaysia for
Shivik, J.A.,
Are vultures birds and do snakes have
funding under EScience grant 02-02-1 (PSF0033 and
venom, because of macro-and microscavenger
Monash Univ ersit.y Sunway Carnpus for funding urider
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