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Toucan Vd. 29, No . 9, pp. 1129-1141.

1991 Papmoo PRr plc


Printed in Croat Britain.

ANTIBACTERIAL EFFECTS OF DIFFERENT SNAKE VENOMS:


PURIFICATION AND CHARACTERIZATION OF
ANTIBACTERIAL PROTEINS FROM PSEUDECHIS AUSTRALIS
(AUSTRALIAN KING BROWN OR MULGA SNAKE) VENOM

BRADLEY G. STILFS, Fxwrlcls W. SEXTON and ScoTr A. WSINSTI~IV


Department of Toxinology, Pathophysiology Division, United States Army Medical Research Institute of
Infectious Diseases, Frederick, MD 21702-SOl l, U .S.A .

(Received 4 February 1991 ; accepted 12 April 1991)

B. G. STU.ES, F. W. SEXTON and S. A. WEINSTEIN. Antibacterial effects of


differentsnake venoms : purification andcharacterization ofantibacterial proteins
from Pseudechis atrstralis (Australian king brown or mulga snake) venom.
Toxicon, 29, 1129-1141, 1991 .-Venoms from 30 different snake species
were tested in a disc diffusion assay for antibacterial effects against gram-
positive and gram-negative bacteria . A number of venoms gave a zone of
inhibition against both groups of bacteria, including Aeromonas hydrophila, an
important pathogen of reptiles and amphibians . Two antibacterial components
from the venom of an Australian elapid, Pseudechis australis (Australian king
brown or mulga snake) were purified to homogeneity. The proteins, designated
LAO1 and LA02, had potent antibacterial properties associated with L-amino
acid oxidase activity . Both had native and subunit mol. wts of 142,000 and
56,000, respectively . Antibacterial activity correlated with enzymatic activity
and was eliminated with catalase . LAO1 and LA02 had 244 and 113 units of
L-amino acid oxidase activity/mg protein, respectively . Compared to
tetracycline, a drug of choice for Aeromonas infections in humans, reptiles and
amphibians, the in vitro antibacterial effects of LAO1 and LA02 were
respectively 70 and 17 .5 times more effective (on a molar basis) .

INTRODUCTION

SNAKE venoms contain many proteinaceous components which incapacitate and digest
prey . Neurotoxins (pre- and post-synaptic), cytotoxins, myotoxins, professes and
nucleases are found in varying quantities in snake venoms, depending on the species. Most
venomous snakes belong to two taxonomic families, the Elapidae (cobras, mambas, kraits,
coral snakes and sea snakes), and Viperidae, which include the pit vipers (rattlesnakes,
copperheads, cottonmouths) and true vipers (European viper, gaboon viper, puff adder) .
Previous investigators have isolated and characterized a large number of bioactive
components from snake venoms, however, no systematic search for antibacterial com-
ponents has been described. One isolated report showed that direct lytic factor from a

1129
1130 B. G . STILES et al.

cobra venom (Hemachatus haemachatus) had antibacterial effects against Staphylococcus


aureus and Escherichia cola (ALOOF-Hmscs et al., 1968). This group of basic, low
molecular weight proteins disrupts phospholipid membranes and is found in many elapid
venoms such as those of Naja sp . and H. haemachatus ((~tv et al., 1987).
Antibacterial effects of viperid venoms have also been described (GLASER, 1948 ;
SxwRlvFS, 1970). Bactericidal effects were observed with two different rattlesnake venoms
on gram-positive Sarcina species, but there was little effect against Bacillus subtilis, E. cola,
or S. aureus (GLASER, 1948) . Another report showed that L-amino acid oxidase, purified
from the venom of Crotalus attamanteus (eastern diamondback rattlesnake), had anti-
bacterial activity against various gram-negative micro-organisms (SKARNE3, 1970). Anti-
bacterial components present in snake venoms may protect the host after eating prey
contaminated with pathogens like Aeromonas (T~IOMAS et al., 1979).
The L-amino acid oxidase converts L-amino acids into keto acids, ammonia and
hydrogen peroxide. Catalase effectively prevents the antibacterial effects of L-amino acid
oxidase from C. adamanteus, suggesting that hydrogen peroxide is the bioactive product
of the enzymatic reaction ($KARNES, 1970). The only other venom component known to
have antimicrobial properties is from the common honey bee, Apis mellijera (F~xxEL
et al., 1968). This venom contains mellitin, a 2800 mol. wt peptide, which is more active
against gram-positive than gram-negative bacteria .
We describe here the antibacterial properties of 30 different snake venoms and the
purification and characterization of two antibacterial proteins purified from the venom of
Pseudechis australis (the mulga or king brown snake) .

MATERIALS AND METHODS


Venovn, magairtins and purified i-amfiw acid oxidase
All lyophilized venoms were obtained commercially (Sigma, St Louis, MO, U.S .A.) except Crotalus alma,
C. scutulatur sctrtulatus. C. adammtteus and Echis carinatus, which were extracted from long-term captive
specimens. Venom from the long-term captive specimens was collected manually, immediately frozen, lyophilized
and then stored at 4°C over desicc8nt in the dark . The L-amino acid oxidase purified from the venom of C. atrox
and magainin peptides I and II were obtained commercially (Sigma), reconstituted with phosphate buffered
saline, pH 7 .2 (PBS), and stored at -40°C .

Antibacterial assays
Bacteria were obtained from the American Type Culture Collation (ATCC, Rockville, MD, U .S .A .) and
grown in IO ml nutrient broth tubes, as recommended by ATCC. Bacteria used in this study include Pseudo-
morrar aerttglnosa no. 27853 (blood isolate) ; Escherlchia cola no. 25922 (clinical isolate) ; Staphylococcus aureus
no . 29213 (wound isolate); Bacillus subtAit no. 6051 (type strain); Aerontortas hydrophlla strains no . 7965 (source
unknown); no . 7966 (type strain isolated from milk); no. 43408 (frog, nasal ulcer) ; no . 43409 (frog, skin ulcer) ;
no. 43414 (snake, stomach afar); A . veronll no . 35622 (human, diarrheal stool); and A . sobrla no . 9071 (frog,
hemorrhagic septicemia). A disc diffusion assay (BeuOe et al., 1966) was used with the following madifiartions :
bacteria (200 ltl of a 0 .1 A6ao culture containing 1 .75 x (0" colony forming units (CFU)/ml were spread onto
I S ml nutrient agar plates (90 mm diameter). Sterile paper discs (7 mm diameter) were they plead onto the agar
surface and IS pl (188 ~ of venom was added per disc . Plates were incubated at either 25, 30 or 37°C,
depending on the optimum growth temperature recommended by ATCC for each bacterial strain. After 18 hr,
the diameters of inhibition zones were recorded in mm minus the disc diameter.
Antibacterial effects of r.-amino acid oxidase 1 (LAOI) or 2 (LA02) purified from P. australis venom and
~-a~+±+no acid oxidase from C . atrox venom wem also tested in nutrient broth cultures . Solutions containing
LA01, LA02 or L-amino acid oxidase from C. alma and purified Aspergildts nager c8talaae (Calbiochem, San
Diego, CA, U.S .A .) were filter sterilized with a 0.22 pm membrane (Millipore, Bedford, MA, U.S .A .), and a
total volume of 100 pl was added per nutrient broth tube (10 ml) . Tubes were inoculated with 0.5 ml of a 0.075
d~ inoculum (7 .5 x los CFU/ml) and the turbidity maaaured hourly .
Antibacterial Effects of Snake Venoms 1131

Pwifecation of P. suatralis antibacterial comparanta


Crude P. australis venom (30mg) was dissolved in 0.6 ml of 0.05 M Tris-HCl buffer, pH 7.2, containing 0.7 M
NaCI (buffer n. The sample was filtered through a 0.22 ran membrane and O.S ml of filtrate was injected into a
Superose-12 (molecular sieve) column (1 cm x 30 cm) attached to a Fast Protein Liquid Chromatography System
(FPLC, Pharmacia, Piecataway, NJ, U.S.A .) . The column was equilibrated with buffer I at 1 ml/min, and i ml
fractions were collected. Column eluant was monitored at 280 nm and fractions with antibacterial effects against
A. hydrophila 7965 were pooled, dialysed against deionized water, and lyophiliad. The sample was dissolved in
650 i~l of 0.05 M 2[N-morpholino] ethane sulfonic acid buffer, pH 5.6 (MES), and injected into a Mono S (cation
exchange) column (1 cm x 10 cm) equilibrated with the same buffer. Proteins were eluted with a linear NaCI
gradient (0.0-1 .0 M) in MES and frictions assayed for antibacterial activity. Active fractions were pooled,
dialysed against PBS, lyophilized and finally reconatitutod with 1 ml of sterile, deionized water.

Protein dEterminations
Protein content was determined by the bicinchoninic acid assay (S~artt et al., 1985) from Pierce Chemicals,
Rockford, IL, U.S .A. with bovine serum albumin as a standard.

Polyacrylarnidt gel electrophorcals


Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with a 4% slacker and 10%
separating gel was run in a Mini Protean II electrophoresis unit (Bio-Rad, Richmond, CA, U.S .A.), according to
the technique of L~r~aal (1970). Prnatained mol. wt standards (14,000-2(10,000) were purchased from Amer-
aham (Ameraham, U.K .). Precast 4-30% polyacrylamide gradient gels (Pharmacia) were run for 20 hr at 150
volts in 80 mM boric acid-90 mM Tria buffer, pH 7.3 . Protein standards were ferritin (440,000), catalane
(232,000), and bovine serum albumin (67,000) (Pharmacia) . Proteins were stainod with 0.025% Coomaanie blue
R-250 dissolved in methanol :water:glacial acetic acid (5:4a) .

liHzyme ~Y
The t.-amino acid oxidase activities of purified LAOI, LA02 and the t:amino acid oxidase from C. atrox were
determined at 30°C with an oxygen sensor (Yellow Springs Instrument Co . Inc., Yellow Springs, OH, U.S.A.),
according to the procedure of Ctrnrt et al., (1968) .

Lethal potency rkterminatlons


Lethal potency of crude P. australls venom was determined by i.p. injection of Swiss-Webstet mice (male,
18-20 g) in four groups of four mice per group. Doses were derived from a 1 mg/ml solution of lyophilized
venom roconstituted in PBS, pH 7.2. The ~z was calculated by the Spearman-Karber method (W.H .O., 1981).

Protease assay
Protease activity in venom was measured with a commercial casein substrate (Bio-Rad). Casein was
incorporated into agarwe, wells were punched, and an appropriate venom dilution (0.125-1 .0 mg/ml in PBS)
was applied. A trypsin solution, 232 units/mg stock (Millipore Corp ., Freehold, NJ, U.S .A .), was used as a
positive control (S pg/asaay) and PHS as a negative control.

RESULTS

Antibacterial effects of crude venoms


Table 1 summarizes the antibacterial activity of venoms from 30 different snake species.
The venoms from five species of African and Asian cobras (Naja sp.) showed strong
antibacterial effects, especially against A. hydrophila 7965. Noticeable exceptions were
venoms from one Asian cobra (N. n. oxiana) and one African cobra (N. melanoleuca),
which lacked any antibacterial activity . Venom from the distinctive African elapid,
H. haemachatus, had a moderate effect against A . hydrophila 7965. Two Australian elapid
venoms (Notschis scutatus scutatus and P. australis), also had potent antibacterial activity .
1132 B. G. STILES et al.

TABLE I . AIVT>eACIEwAL EPPECI3 OF D~HNT 3NAtE VENOMS (188 hg~DL1C) WITH A DDC DII'RU310N A.SSAY~

Venom P. aengirtosa S. aumv E. coli A . hydrophila B. sebtilis

Elapidae
.lcanthophis ontarcticus (Australian death adder) 0 0 0 0 I
Bengares tnelticlnctes (Many banded kraft) 1 0 0 1
i?endroasptr angesticeps (Eastern green mamba) 0 0 0 0 2
Den~oaspis jarrusonU (Jameaon's mamba) 0 0 0 0 1
ikndroaspis polylepis (Black mamba) 0 0 0 0 0
1)crtdroaspir vbidis (Western green mamba) 0 0 0 0 0
Enhydrina schirtosa (Beaked sea snake) 0 0 0 0 0
Hemachates hacnwchates (Ringhals spitting cobra) 0 2 0 4 1
Hydrophis cyanocinctlis (Hliu-banded sea snake) 0 0 0 0 0
Latkaeda latkaedato (Common sea kraft) 0 0 0 0 0
l aticauda semijasciata (Handed sea kraft) 0 0 0 0 1
Micrurur julvies (Eastern coral snake) 0 I 0 0 1
Naja haje hajc (Egyptian cobra) 0 4 0 7 3
Naja naja afro (Chinese cobra) 0 3 0 6 1
Naja rmlanoleeca (Black forest cobra) 0 0 0 0 0
Naja mossmnbica (Mozambique spitting cobra) 0 3 0 5 2
Naja nigricoUis (Black-necked spitting cobra) 0 5 0 6 1
Naja mues (Yellow or cape cobra) 0 2 0 6 1
Naja naja oxiana (Oxus cobra) 0 0 0 0 I
Notechis scutates scetates (Australian tiger snake) 1 7 0 10 3
Psetudcchis oestrous (King brown or mulga snake) 0 7 1 I1 5
Viperidae
.lgkistrodon contortrix contortrix (Southern
copperhead)
Bitit arictans (Puff adder)
Crotahrs atrox (western diamondback
rattlesnake) 3 8 1 11 3
Crotahts scetelates scetelates (Mojave
rattlesnake) 2 9 1 1I 5
Crotahts adammttees (Eastern diamondback
rattlesnake) 2 8 1 12 5
Crotales dertrses terrlJices (South American
rattlesnake or Cascabel) 0 0 0 0 1
Echis casinates (Saw-sealod or carpet viper) 4 8 l 12 5
TrLntreserus okbtavenrir(IÜmehabu) 1 0 0 0 0
Pipera russelli (Russell's viper) 1 0 0 0 1

rI'he values given represent a venom inhibition zone in mm, minus the 7 mm diameter of the disc, alkr an
l8 hr incubation.
The bacterial inoculum per plate contained 3.5 x 10' colony forming units which were spread onto the agar
surface wiW a sterile glass rod. Sterile paper discs (7 mm diameter) were placed onto the agar surface and I S ltl of
venom (188 Rg) added.

Venoms from several species of sea snakes (Enhydrina schistose, Hydrophis cyanocinctus
and Laticauda sp.) and the coral snake, Micrurus julvius fulvies, were uniformly negative .
None of the elapid venoms tested had any significant effects against P. aeruginosa or
E. coli and only moderate effects were seen with B. subtilis. Of the gram-positive and
gram-negative bacteria tested, S. aureus and A . hydrophila were most susceptible to elapid
venoms . A broader spectrum of activity was seen with some viperid venoms, including
those of rattlesnakes (Crotalus sp.) and Echfs carinates . The susceptibility of the bacteria
tested against viperid venoms was as follows; A. hydrophila > S. aureus > B, subtilis >
P. aeruginosa > E. coli (Table 1). Pseudechis oestrous venom was chosen for additional
studies since this elapid species is known to feed largely upon frogs, a potential source of
A. hydrophila which is a major pathogen of amphibians and reptiles.
(p) o .e

o .~
a

os

(g) o.zso

ozz5

o.zoo

0 .175

0 .1 b0

a 0.126

0.100

0.07b

0.050

0.026

6 1016 z0 25 90 96 ,0 46
FRAC110N NUMBER
Fra. 1 . Ptramtcanox of P. auttrdLt LAOI ~ IA02 e1r rror~e emus crtxonuroaiurttr erro
cn~noN rxcx~cta c~auroax~rft~r .
(A). Molecular sieve chromatography of P. aurtrdis crude venom . The pear with antibacterial
activity v indicated by an asterisk (') . (H). Canon exchange proSle of pooled P, aurtrdLs
antibacterial fractions from molecular sieve chromatography . Peaks with antibacterial activity
were eluted with a 0.0-1 .0 M NaCI gradient (%H) and sre labeled as LAOI and LA02.
113 4 B. G . STILES et al.

Ptir~cation of P. australis antibacterial components


Two steps were required to purify the antibacterial components of P. australis venom to
homogeneity. Figure 1 A shows the chromatographic profile obtained from fractionating
30 mg of P. australis venom on a molecular sieve column . Antibacterial activity was found
in the first eluted peak contained in fractions 13-15. Interestingly the antibacterial activity
of venoms from N. n. atra, N. nigricollis and N. haje haje were located in fractions 12-14.
Fractions containing antibacterial activity also exhibited a yellow color which is
commonly associated in snake venoms with the FAD prosthetic group found in L-amino
acid oxidase (KOANALIK and MASTER, 1964; Tu, 1977; ZELLER, 1948) . Canon exchange
chromatography of P. atrstralis venom fractions with antibacterial activity resolved three
peaks (Fig. 1B) . Only two peaks had antibacterial activity and were designated LAO1,
which did not bind to the column, and LA02, which eluted with 100 mM NaCI (Fig. 1B).
The LAO1 and LA02 represented 2.6% and 0.25% of total venom protein, respectively.
Both proteins were homogeneous and had native mol . wts of 142,000, based on gradient
gel electrophoresis, and subunits of 56,000 in SDS-PAGE . The marine Ln~ of P. australis
crude venom was 0.57 mg/kg. Neither LAO1 nor LA02 were lethal in mice (up to
2.75 mg/kg), nor did they have any proteolytic activity.

Antibacterial effects and enzymatic activity ofpur~d LAOI and LA02


The antibacterial effects of LAO1 and LA02 are shown in Table 2 and Fig . 2A-C.
Variation was noted in susceptibilities between different strains of A. hydrophila. There
was a slight antibacterial effect against A. sobria 9071, but not A . veronü 35622 . Neither
venom from the common honey bee, Apis mellifera (187 fig), nor 7.5 hg ofmagainin I or II
isolated originally from skin extracts of the African clawed frog, Xenopus laevis, had any
antibacterial effects against A. hydrophila 7965. Magainin I or II had no effect on other
A. hydrophila strains . However, 18 pg of L-amino acid oxidase from C. atrox venom gave a

TAHtE 2 . AxnaACrewAi ta+t+ects oF LAO1 Axn 2 AGAn~sr B . subtüir, S. a~eua AND Acromonas sp. ox rntramv~r
AGAa

Inhibition Zones of LAO1 and LA02 (mm)"

Protein A. hydrophila A. hydrophila A. hydrophila A . so~bria B. subtüir S. aureus


(Pg) ATCC ~ 7%S ATCC ~ 43409 ATCC ~ 43414 ATCC ~ 9071 ATCC ~ 6031 ATCC
# 29213

S .0 8/6/ND 10/6 3/0 2/1 4/2 4/3


2 .3 8/S/ND 8/4 2/0 2/0 2/0 3/2
1 .23 S/3/15 6/2 1/0 1/0 l/0 2/1
0 .62 4/2/14 S/2 O/0 0/0 0/0 1/0
0 .31 3/0/12 3/0 0/0 0/0 0/0 0/0
0 .16 0/0/8 0/0 0/0 0/0 0/0 0/0
0/O8 0/0/6 0/0 0/0 0/0 0/0 0/0
0 .04 0/0/1 0/0 0/0 0/0 0/0 0/0
o .oz o/o/o o/o o/o o/o o/o o/o

'Bacterial inoculum (3 .3 x 10" colony forming units) for each plate was spread onto the nutrient agar surface
with a sterile glass rod. The inhibition zones of LAOI and LA02 are listed in the first and second positions,
respectively (e.g. (0/6 = zones of inhibition of 10 mm for LAOI and 6 mm for LA02). Values do not include the
7 mm diameter of the disc . Tetracycline susceptibility was only tested with A. hydrophila ATCC no. 7%S between
concentrations of 1 .25 pg/disc and 0.02 Pg/disc. Tetracycline zones of inhibition are recorded under A . hydrophila
ATCC no. 7%Sin the third position (e.g . S/3/1 S ~ zones of inhibition of 5 mm for LAOI, 3 mm for LA02, and
15 mm for tetracycline . ND = Not determined .
Antibacterial Effects of Snake Venoms 1135

F~a. 2. (A,B)

significant zone of inhibition (6 mm diameter). Molar comparisons of tetracycline


(0.14 nmoles) with LA02 (0.008 nmoles) and LAO1 (0.002 nmoles) showed that 17.5-70-
fold more tetracycline was necessary for a 3 mm zone of inhibition (Table 2). After 48 hr,
the inhibition zones of LAO1 or LA02 did not contain any resistant colonies unlike those
found within the tetracycline inhibition zone (Fig. 2D and E).
1136 B. G. STILES et al.

Fia. 2, (C,D)

Antibacterial activity correlated with ~-amino acid oxidase activity . LAO1 and LA02
had 244 and 113 enzymatic units/mg protein, respectively . LAO1 showed consistently
higher antibacterial activity than LA02 (Table 2). Crotalas atrox venom L-amino acid
oxidase contained 6I enzymatic units/mg protein, which was lower than either P. australis
enzyme. The C. atrox enzyme concomitantly had less antibacterial effects (18 Fig = 6 mm
inhibition zone) compared to either LAO1 or LA02 (Table 2}.
Antibacterial Effects of Snake Vcnoms l 137

FIC . 2 . ZONES of INI~ITION EY LAO1, LA02 AND TETRACYCLINE AGAINST .! . hydrophila 7965 .
(A). Disc no. 1 contains 5 pg of LAOI . Proceeding counterclockwise, each disc contains 2.5 pg,
1 .25 l+g, 0 .62 Rg, 0.31 pg, 0.16 l+g, 0 .08 Kg, 0 .04 pg and 0 .02 pg LAO 1 /disc. (B). Disc no . 1 contains
5 pg of LA02. Proceeding counterclockwise, each disc contains 2 .5 Pg, 1 .25 Pg, 0 .62 kg, 0.31 pg,
0 .16 Pg, 0.08 hg, 0 .04 pg and 0 .02 pg LA02/disc . (C) . Disc no . 1 contains 1 .25 pg of tetracycline.
Proceeding counterclockwise, each disc contains 0.62 pg, 0.31 Pg, 0.16 Rg, 0 .08 hg, 0 .04 ug, 0.02 pg
0 .01 !+g and 0 .005 Rg tetracycline/disc. (D) . Resistant colonies within the tetracycline zone of
inhibition after 48 hr at 1 .25 pg tetracycline/disc (labeled as 1) and clockwise is a disc containing
0 .62 pg tetracycline. (E) . Lack of resistant colonies within the LAOI zone of inhibiton after 48 hr
at 5 hg LAOI/disc (labeled as I) and clockwise is a disc containing 2 .5 pg LAOI .

Inhibition of growth in broth cultures was also observed with LAO1, LA02 and
C. atrox L-amino acid oxidase (Tables 3 and 4). Broth cultures were used to ascertain if
hydrogen peroxide, one of the products from L-amino acid oxidase reactions, was the
actual inhibitory component. Titration studies with LAO1 and A . hydrophila 7965 showed
an effective bactericidal concentration in broth between 0 .75-1 .5 leg/ml (Table 3). Catalase
added to culture tubes containing 0.5 pgJml of LAO1 and A . hydrophila 7965 prevented

TABLE 3 . ANTIewcrERIwL EFmcIS oF LAOI AGAINST d . hydrophila ATCC No . 7965 uv aROTx cuLTURE

Hourly turbidimetric readings"

LAOI (pg/ml) 0 1 2 4 6 20

6 .0 0.005/0.010 0 .010/0 .010 0 .008/0 .010 0.008/0 .010 0.008/0.010 0.005/0.005


3 .0 0.010J0.010 0 .010J0 .010 0 .009/0 .010 0 .009/O .OlO 0.008/0.008 0.005/0.005
Ls o.olo/o.olo o.olo/o .olo o.olo/o .olo o .olo/o.olo 0.008/o.oos o.oos/o.oos
0 .75 0.010/0.010 0 .010/0 .010 0 .0t0/0 .010 0.010/0 .012 0.008/0.010 0.005/0.032
0 .38 0.010/0.010 0 .010J0 .012 0 .012/0 .012 0.010/0 .010 0.008/0.008 0.120/0.120
O .19 0.010/0.010 0 .012/0 .012 0 .018/0 .025 0.045/0 .050 0.055/0.062 0.145/0.150
0 .09 0.010/0.010 0 .015/0 .015 0 .027/0 .029 0.050/0 .059 0.065/0.065 0.143/0.145
Untreated 0.010/0.010 0 .018/0 .018 0 .037/0 .040 0 .052/0 .060 0.064/0.070 0.130/0.145

"Turbidimetric readings were taken every hour at .lam. The inoculum per tube (two tubes per LAOI
oonocntration) consisted of 3.2 x lo" colony forming units from logarithmic phase grown cells.
1138 H. G. STILES et al.

TearE 4. Et~r+ecrs a~F c~ret.~ ox LAOI/LA02/C. atrox L-~imvo Acro oxm~se Acrtvrrsr sa~sr A. hydrophila
796s nv anm~rrr ctn.ivae

Hourly turbidimetric readings'


Time (lu) Untreatedt LAOI/LA02/Atrox$ Catalane+LAOI/LA02/Atrox~ Catalase~j
0 o.aos o.olo/o.ololo.oos o.ols/o.ols/o.olo o.olo
1 o.ols o.012/0.012/0.010 0.020/o.ols/o.olg o.ols
2 0.026 0.012/0.014/0.010 0.038/0.03s/0.040 0.03s
3 0.04s 0.010/0.010/O.OIS O.OS2/0.04BJ0.04s O.OS2
4 O.OS2 0.008/0.010/0.03s 0.060/O.OSS/0.060 0.060
s o.oss o.012/o.ols/o.oso 0.060/o.o6z/o.o60 0.060
6 0.060 0.009/o.o3s/o.oso o.o6s/o.mo/o.o60 0.060
20 o.14g o.ogs/o.ll2/0.122 o.lso/o.l20/0 .130 o.lag
'Turbidimetric readings were taken every hour at Ate. The inoculum per 10 ml nutrient broth tube oonninted
of 3.2 x 10' colony forming units from logarithmic phase grown cells.
tUntreated tubes contained bacterial inoculum only.
$Turbidimetric headings are in groups of three (e .g . At 2 hr, readings for LAOI (O.s ~g/ml) = 0.012, LA02
(1 ~g/ml) = 0.014, and Atrox (2 Pg/mp - 0.010). Atrox = C. atrox r.-amino acid oxidase.
¢'I'urbidimetric readings are in groups of three as described above. Heaides containing inaculum and LAOI
(O.s trg/ml), LA02 (1 pg/ml) or Atrox (2 pg/ml), each tube contained 2000 units of catalane.
~jThe tube contained inaculum and 2000 units of catalane.

the antibacterial effect of LAO1 (Table 4). Similar results were seen with LA02 and the
L-amino acid oxidase from C. atrox.

DISCUSSION
This report shows that several different snake venoms have antibacterial effects against
gram-positive and gram-negative bacteria. The more active venoms were from Asian and
African cobras (Naja sp.), Australian elapids (Notschis scetatus seetates and Pseedeehis
aestralis), and North American rattlesnakes (Crotales sp.). There has been a single report
of elapid antibacterial properties associated with direct lytic factor ( ~ 7000 mol. wt) from
H. haemachates venom (ALOOF-HnzscH et al., 1968). Otu molecular sieve chromatography
experiments revealed antibacterial activity from N. n. atra, N. nigricollis, N. h. haje and
P. aestralis elapid venoms only in yellow, high mol. wt fractions ( > 100,000). With the
physical and enzymatic characteristics of the antibacterial proteins isolated in the present
study, we conclude that the majority of antibacterial effects seen in elapid venoms
examined wen due to L-amino acid oxidase ( ~ 140,000 mol. wt) and not direct lytic
factor .
Antibacterial effects of viperid venoms have also been studied (GLAS>~t, 1948; SxwItrTrs,
1970). One study showed minimal effects of venoms from Crotales mitchellü pyrrhus
(South-western speckled rattlesnake) and C. rober (red diamondback rattlesnake) against
E. coli, S. aureus and B. sebtilis (GL~t, 1948). We observed markod effects with three
out of four Crotales venoms against S. aerees and B. sebtilis, but little effect on E. coli.
Interestingly venom from the highly toxic South American rattlesnake, C. derisses terrifi-
cws, was negative in our assay. The venom sample was from a geographic race which
produces a white venom, suggesting lack of L-amino acid oxidase. We suspect that yellow
venom from a different C. d. terrifitces population would have antibacterial effects.
The yellow color in venom is commonly associated with the FAD prosthetic group of
L-amino acid oxidase ('I~r, 1977). D>bnTltov and ICwNIwexAIt (1968) showed that L-amino
Anübactcrial E8'ects of Snake Venoms 1139

acid oxidase was present in greater quantity in yellow than white venoms of Vipers
russelli. Yellow venom from Vipers ammodytes had 200 times more L-amino acid oxidase
than white venoms from the same species (KORNALIK and M~sret, 1964). Two lightly
yellow venoms from N. melanoleuca and N. n. oxiana were negative in our antibacterial
assay while five more markedly yellow venoms, from Naja sp., were positive .
Another report described the antibacterial effects of L-amino acid oxidase from
C. odamanteus venom against various gram-negative bacteria (SKARNE4, 1970). Escherichia
coli was the most resistant bacterium tested requiring a 50 ~g/ml concentra-
tion of enzyme to get aII LD Sp dose (SR.ARNE3, 1970). An effective Ln~ dose of
C. adamareteus enzyme for P. aeruginosa was 3.5 pgJml. This correlates with our findings
showing increased susceptibility of P. aeruginosa, compared to E. coli, using viperid
venoms . Little or no effect was seen with elapid venoms tested against P. aeruginosa or
E. coli.
Initial fractionation of P. australis venom on a molecular sieve column yielded three
successive, yellow fractions corresponding to antibacterial activity. A subsequent cation
exchange step resolved two proteins, LAO1 and LA02, with antibacterial properties .
Both had z-amino acid oxidase activity and identical native and subunit mol. wts. The
native mol, wts of LAO1 or LA02 (142,000) were very similar to the 140,000 determina-
tion for C. adamanteus venom t-amino acid oxidase (D~cox and RAW7TCH, 1969).
Purified preparations of LAO1 were noticeably yellow while LA021acked any color. The
perceptible color may be attributed to the ten-fold higher protein concentration of LAO1 .
Antibacterial effects of LAO1 and LA02 correlated well, but not perfectly, with the
enzymatic activity of each protein. For instance, there is a four-fold difference between
LAO1 and LA02 in antibacterial activity (disc diffusion), yet only a two-fold difference in
enzymatic activity . Discrepancies between the biological and enzymatic activities of LAO1
and LA02 may be due to professes produced by metabolically active bacteria . Since both
proteins have different elution profiles on cation exchange chromatography, the amino
acid sequences probably differ and render LA02, which has less antibacterial activity
relative to enzymatic activity, more susceptible to professes. The minimum effective
bactericidal dose of LAO1 for A. hydrophila 7965 in broth was between 0.75 and
1 .5 pg/ml.
There have been no previous reports characterizing an L-amino acid oxidase from
P. australis but isoenzymes of L-amino acid oxidase are common in other snake venoms .
As many as 18 isofonms of the enzyme have been found in C. adamanteus venom (HwFs
and WELLNSR, 1969). Our study showed that C. adamanteers venom contained significant
antibacterial activity. As reported with C. adamanteus t,-amino acid oxidase (Sx~xrrt s,
1970), catalase also neutralized the antibacterial effects of LAO1 and LA02 . Some of the
viperid venoms produced larger inhibition zones relative to elapid venoms. This may be
due to differences in z-amino acid oxidase concentration, enzymatic activity of the
r.-amino acid oxidase and/or there may be another undescribed antibacterial component.
The concentration and/or activity of z-amino acid oxidase present in nonreacting venoms
is probably lower than the reactive venoms .
Antibacterial effxts of snake venoms against A. hydrophila, a major pathogen of
reptiles and amphibians (Fsz~snni et al., 1972; Gmas et al., 1971 ; Huts et al., 1983),
have not been reported previously . Aeromonas species are also opportunistic human
pathogens, causing wound infections and diarrheal disease (HicxMwx-BRENNER et al.,
1987; PITARANGSI et al., 1982). We observed antibacterial effects against different strains of
A . hydrophila and A. sobria isolated from cutaneous lesions of frogs and a boa constrictor
1140 B . G. STILES et al.

with a septic stomach ulcer. Frogs can be a reservoir for A. hydrophila (H>Rn et al., 1983)
and comprise a significant part of the P. australis diet (Coc~et, 1983; SHINE and
CovACEVICH, 1983). An A. veronü strain, isolated from a human diarrheal stool specimen,
was not affected by either LAO1 or LA02. Obviously, there is variation in the LAO1 and
LA02 susceptibility among different species and strains of Aerornoreas.
Antibacterial effects of LAO1 and LA02 were alsô seen with a S. aureus wound isolate
and a spore forming B. st~btilis. It is noteworthy that S. aureus and A. hydrophila, which
were the most sensitive bacteria tested with LAO1 and LA02, also produce catalase
during growth (ICRIEG et al., 1984), but obviously not in sufficient quantities to counteract
the bactericidal quantities of H20Z produced by LAO1 or LA02.
The function of L-amino acid oxidases in snake venoms is poorly understood. Some
authors suggest that it may serve a digestive function (MINTON and MiN~roN, 1980). While
we and others have demonstrated an antibacterial property associated with snake venom
L-amino acid oxidase, the biological significance remains unclear.

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