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Acta Scientiarum 

 

http://www.uem.br/acta 
ISSN printed: 1679-9283 
ISSN on-line: 1807-863X 
Doi: 10.4025/actascibiolsci.v39i3.33826 

 

Acta Scientiarum. Biological Sciences 

Maringá, v. 39, n. 3, p. 309-319, July-Sept., 2017 

Antibacterial activity of different types of snake venom from the 

Viperidae family against Staphylococcus aureus 

Isabela Nascimento Canhas

1

, Luiz Guilherme Dias Heneine

2

, Thaís Fraga

1

, Débora Cristina 

Sampaio de Assis

3

, Márcia Helena Borges

2

, Edmar Chartone-Souza

1

 and Andréa Maria Amaral 

Nascimento

1

*

 

1

Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais; Av. Antônio Carlos, 6627, 31270-901, 

Belo Horizonte, Minas Gerais, Brazil. 

2

Departamento de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias, Belo Horizonte, Minas Gerais, 

Brazil. 

3

Departamento de Tecnologia e Inspeção de Produtos de Origem Animal, Escola de Veterinária, Universidade Federal de Minas Gerais; 

Belo Horizonte, Minas Gerais, Brazil. *Author for correspondence. E-mail: amaral@ufmg.br 

ABSTRACT. Toxins and venoms produced by living organisms have exhibited a variety of biological 

activities against microorganisms. In this study, we tested seven snake venoms from the family Viperidae 
for antibacterial activity and the activities of reversal of antibiotic resistance and inhibition of biofilm 
formation against 22 clinical isolates of Staphylococcus aureus.  Bothrops moojeni venom exhibited anti 
staphylococcal activity with the lowest mean value of minimum inhibitory concentration (MIC). 
Moreover, reversal of antibiotic resistance was observed for combinations of B. moojeni venom (½ x MIC) 
and norfloxacin or ampicillin (both ½ x MIC) for 86.4% and 50% of the isolates, respectively. B. moojeni 
venom alone at ½ MIC inhibited 90% of biofilm formation, whereas in combination with ciprofloxacin, 
both  at  ½  MIC,  a  reduction  on  the  NorA  efflux pump activity was observed. The detection of in vitro 
mutants colonies of S. aureus resistant to B. moojeni venom was low and they did not survive. A 
phospholipase A2 was purified from the venom of B. moojeni and displayed anti-staphylococcal activity 
when tested alone or in combination with ciprofloxacin. The results presented here will contribute to the 
search for new antimicrobial agents against resistant S. aureus.

 

Keywords: Bothrops moojeni, bacteria, antibiotic-resistance, NorA efflux pump, biofilm. 

Atividade antibacteriana de diferentes tipos de veneno de serpentes da família Viperidae 
contra Staphylococcus aureus 

RESUMO. Toxinas e venenos exibem uma variedade de atividades biológicas contra micro-organismos. 
Neste estudo, investigou-se a atividade de sete venenos de serpentes, da família Viperidae, sobre o 
crescimento de Staphylococcus aureus, na reversão fenotípica da resistência a antibióticos e inibição de 
formação de biofilme contra 22 isolados clínicos de S. aureus. O veneno de Bothrops moojeni apresentou a 
menor média de concentração inibitória mínima (CIM). Além disso, observou-se reversão da resistência a 
antibióticos para combinações do veneno de B. moojeni (½ x CIM) e norfloxacina ou ampicilina (ambos ½ x 
CIM) para 86,4% e 50% dos isolados, respectivamente. O veneno de B. moojeni na concentração de 

½ CIM 

inibiu 90% de formação de biofilme, enquanto ele em combinação com ciprofloxacina, ambos na 
concentração de ½ CIM, diminuiu a atividade da bomba de efluxo NorA. A detecção in vitro de colônias 
mutantes de S. aureus resistente ao veneno de B. moojeni foi baixa e eles não sobreviveram. Uma fosfolipase 
A2 purificada a partir do veneno de B. moojeni exibiu atividade antibacteriana quando testada sozinha ou em 
combinação com ciprofloxacina. Os dados obtidos poderão contribuir para a pesquisa de novos agentes 
antimicrobianos contra S. aureus

Palavras-chave: Bothrops moojeni, bactéria, resistência, antibiótico, bomba de efluxo NorA, biofilme. 

Introduction 

Since the late 1940s, bacterial resistance to 

antibiotics has drastically increased worldwide. It 

negatively affects the treatment of all infectious 

diseases, and is a major cause of mortality and 

morbidity, significantly increasing the cost of health 

care (Martinez et al., 2009). S. aureus can be 

responsible for both local and generalized infections, 

and is naturally susceptible to nearly all antibiotics 
that have been developed (Chambers & DeLeo, 
2009). The emergence of strains of S. aureus resistant 
to penicillin, methicillin, vancomycin and linezolid 
was reported soon after their clinical use (North & 
Christie, 1946; Labischinski, Ehlert, & Berger-Bächi, 
1998; Tsiodras, et al., 2001). Multiple drug resistant 
S.  aureus   is   one   the   most  common  nosocomial 

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310 

Canhas et al. 

Acta Scientiarum. Biological Sciences 

Maringá, v. 39, n. 3, p. 309-319, July-Sept., 2017 

pathogens worldwide and is of great concern to the 
global health community. The NorA efflux pump is 
one of the major contributors to the resistance of S. 
aureus
, promoting extrusion of chemically unrelated 
compounds, such as ethidium bromide, quaternary 
amine compounds, chloramphenicol and 
fluoroquinolones from the cell (Costa, Viveiros, 
Amaral, & Couto, 2013). Moreover, S. aureus has the 
ability to form biofilm, which prevents antibiotics 
from accessing bacterial cells, thereby contributing 
to its success as a human pathogen (McCarthy et al., 
2015). 

As multidrug resistance is an increasing problem, 

it highlights the urgent need for new antibiotics and 
treatment strategies. The discovery of chemically 
diverse and relatively non-toxic antimicrobials from 
different natural sources shows the promise that 
natural products have as a source of new 
antimicrobial drugs (Lima et al., 2005; Abreu, 
McBain, & Simões, 2012). A promising strategy to 
restore antibiotic effectiveness against pathogens has 
been the use of a combination of two or more 
antibiotics or of antibiotics and natural products, and 
the identification of efflux pump and biofilm 
inhibitors (Braga et al., 2005; Credito, Lin, & 
Appelbaum, 2007; Nascimento, Brandão, Oliveira, 
Fortes, & Chartone-Souza, 2007). 

Snake venom, a complex mixture of proteins and 

peptides with potential biological activity, could lead 
to the development of new drugs with therapeutic 
significance (Ferreira et al., 2011; Vyas, Brahmbhatt, 
Bhatt, & Parmar, 2013). Snake venoms of the family 
Viperidae in particular are an important source of 
peptides, but they remain underexplored (Ferreira et 
al. 2011). Studies have already demonstrated that 
snake venoms of Bothrops jararaca (Ciscotto et al., 
2009),  B. leucurus (Nunes et al. 2011) B. marajoensis 
(Costa-Torres et al., 2010), Bothrops alternatus 
(Bustillo et al., 2008) Crotalus adamanteus (Samy, et 
al., 2014), C. durissuscumanensis (Vargas et al., 2012) 
and  Porthidium nasutum (Vargas et al., 2013) have 
antibacterial activity.  

The aim of this study was to evaluate the 

potential antibacterial activity of crude snake venom 
from snake species of the genera Bothrops
Bothropoides and Rhinocerophis of the family Viperidae 
on  S. aureus isolates. We also investigated the 
antibacterial activity of Bothrops moojeni venom, its 
interaction with antibiotics, as well as its inhibitory 
effect on biofilm formation and the NorA efflux 
pump. 

Material and methods 

Bacterial strains 

Antibacterial activity and synergistic effects were 

evaluated against 22 clinical isolates of methicillin-

resistant S. aureus (MRSA) and methicillin-sensitive 

S. aureus (MSSA) (Braga et al., 2005), and S. aureus 

strain RN 7044 carrying the plasmid pWBG32 

which encodes the NorA efflux pump (Pillai, Pillai, 

Shankel, & Mitscher, 2001). In addition, S. aureus 

ATCC 25923 was included as a negative control. 

The bacteria were cultured in Müller-Hinton 

(MHB; Difco Laboratories, Detroit, Michigan) or 

Luria-Bertani (LB; Difco Laboratories, Detroit, 

Michigan) broths at 37º C for 24 hours.  

Venoms and antibiotics 

Venoms from Bothropoides erythromelas

Bothropoides jararaca,  Bothropoides neuwiedi,  Bothrops 

atrox,  Bothrops jararacussu,  Bothrops moojeni and 

Rhinocerophis alternatus were kindly donated by the 

Serpentarium of Fundação Ezequiel Dias, which is a 

recipient of the authorization n° 117521 from the 

brazilian institute of environment and renewable 

natural resources (IBAMA) to work with the wild 

fauna under the category 20.45 (scientific animal 

husbandry of the wild fauna for research purposes). 

Initially, crude venom was dissolved in ammonium 

acetate buffer (0.2 M, pH 8) in order to make stock 

solutions of 20 mg mL

-1

 that were centrifuged at 

10,000 x g for 10 minuntes at 4º C. The supernatant 

was aliquoted and then stored at -20° C until use.  

The following antibiotics were used in this 

study: amoxicillin/clavulanate (Glaxo Smith Kline, 

Brentford, Middlesex, UK), and ampicillin, 

ciprofloxacin, levofloxacin, norfloxacin, and 

ofloxacin (Sigma Chemical Co. St. Louis, MO, 

USA). Beta-lactams were chosen for having an 

effect on a wide range of infectious agents, whereas 

fluoroquinolones are widely used against multi-

resistant cocci infections. 

Fractionation of Bothrops moojeni venom 

Crude venom of B. moojeni was dissolved in 0.2 

M ammonium acetate pH 8 at a concentration of 50 

mg mL

-1

 and centrifuged for 10 min at 10,000 g at  

4° C. The supernatant was removed and subjected to 

gel filtration. Fractionation was performed on an 

Akta Purifier System using the chromatographic 

column Sephacryl S-100 XK 16/60, both from GE 

Healthcare (Uppsala, Sweden), with 0.2 M 

ammonium acetate pH 8 as elution buffer in a 1 mL 

min

-1

 flow. Aliquots of 2 mL were collected, pooled 

into six major fractions according to their elution 

time, lyophilized and resuspended in ultrapure 

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Maringá, v. 39, n. 3, p. 309-319, July-Sept., 2017 

water. The fraction with antibacterial activity was 
subjected to high performance liquid 

chromatography, using a μRPC C2/C18 ST 4.6/100 

reverse phase column (GE Healthcare, Uppsala, 

Sweden), previously equilibrated with 0.1% 

trifluoroacetic acid (TFA). Elution of the unbound 

sample was carried out for 2 column volumes with 

0.1 % TFA (buffer A). The bound sample was eluted 

under a linear gradient from 0 to 80% acetonitrile 

(buffer B) added to buffer A at a flow rate of 0.7 ml 

min

-1

.  

Amino acid sequencing 

Intact protein (20 μg) was solubilized in 

acetonitrile/water solution (1:1) and submitted to 

Edman degradation using a Shimadzu PPSQ-21A 

automated protein sequencer. The resulting 

sequence was compared with the sequences of other 

related proteins in the SWISS-PROT/TREMBL 

data bases using the programs FASTA 3 

(http://www.ebi.ac.uk/Tools/services/web/toolresult.

ebi?jobId=fasta) and BLAST (http://blast.ncbi.nlm. 

nih.gov/Blast). Later, sequences were aligned using 

the  program  Mafft  (http://mafft.cbrc.jp/alignment/ 

software/). 

 

Determination of the minimum inhibitory concentrations 
(MIC) 

Determination of MIC was performed by the 

broth dilution method in accordance with the 

Clinical and Laboratory Standards Institute (CLSI, 

2016) and using MHB with an inoculum of 

approximately 10

5

 colony-forming units per 

milliliter (CFU mL

-1

). The MHB was 

supplemented with serial antibiotic concentrations 

ranging from 0.0612 to 1,024 μg mL

-1

 and venoms at 

concentrations from 2 to 1,024. To evaluate the 

effect of venoms in combination with antibiotics, 

increasing concentrations (with a 2-fold step, i.e., 

0.0612, 0.125, …, 1024 μg mL

-1

) of these antibiotics 

were added to MHB containing venom at 1/2 × 

MIC. MICs were interpreted as the lowest 

concentration of antibiotics or venoms that inhibited 

visible growth after 24 hours of incubation at 37° C. 

To evaluate the effect of B. moojeni venom as a 

resistance-modifying agent, crude venom in 

combination with antibiotics in different 

concentrations (½, ¼, ⅛  x  MIC)  were  used. 
Cultures that contained neither venom nor 

antibiotics were included as controls; all tests were 

carried out in duplicate. The MIC was defined as 

the lowest concentration that completely suppressed 

visible growth after 24 hours of incubation at 37º C. 

The bactericidal concentration was the lowest 

concentration at which bacteria failed to grow in 
MHB and after plating onto Muller-Hinton agar 

(Smith-Palmer, Stewart, & Fyfe, 1998). 

Survival curves 

Growth curves for S. aureus RN 7044 were 

determined by the use of the dilution tube method 

with 1×10

5

 CFU mL

-1

 as standard inoculum in the 

presence of B. moojeni venom at concentrations of ½ 

and 1 x MIC, in MBH and in combination with ½ x 

MIC of ciprofloxacin. A tube containing only MHB 

was inoculated and included as a control. Tubes 

were incubated at 37º C for 24 hours. At different 

times (3, 6, 9 12, 24, 36 hours), the optical density 

(OD) of each culture was read at 600 nm using a 

NanoDrop Spectrophotometer (NanoDrop 

Technologies) for CFU mL

-1

 determination. 

Bacterial density (CFU mL

-1

) was then determined. 

Monitoring of ethidium bromide efflux 

S. aureus RN 7044 was cultured with aeration in 

MHB at 37° C up to OD600 = 1.8. The culture was 

further incubated for 30 min at 37° C in the absence 

or presence of B. moojeni venom after which 20 μg 

mL

-1

 ethidium bromide were added and the cells 

were again incubated for 30 min at 37° C. The cells 

were then collected by centrifugation (5,000g) and 

washed twice with 20 mmol L

-1

 HEPES–NaOH 

(pH 7) buffer, followed by resuspension in the same 

buffer at a final OD269 = 0.1. Fluorescence of 

ethidium bromide was measured using a Varian 

fluorescence reader (Cary Eclipse Fluorescence 

Spectrophotometer, Palo Alto, CA, USA) with 

excitation at 269 nm and emission at 600 nm for 1 

hour. Readings were taken with the fluorescence 

reader set at high sensitivity every minute for the 
first 5 min, then every 5 min until the end of 90 

min. 

Biofilm inhibition assays 

The effect of B. moojeni venom on the biofilm 

formed by S. aureus RN7044 was tested according to 

Pimenta, Martino, Bouder, Hulen, & Bligh (2003) 

with modifications. Briefly, S. aureus was grown in 

LB medium at 37° C for 24 hours. Afterwards, 0.1 

mL of culture containing approximately 10

5

 CFU 

mL

-1

 were transferred to a polystyrene 96-well 

microplate containing either LB medium or LB 

medium supplemented with the B. moojeni venom at 

the concentrations of ½, ¼, ⅛ x MIC, and then 

incubated at 37° C for 24 hours. Following the 

incubation period, the suspension cultures were 

discarded, the plate was washed three times with 

distilled water and the biofilms were stained with 

0.1% crystal violet for 30 minutes at room 

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Canhas et al. 

Acta Scientiarum. Biological Sciences 

Maringá, v. 39, n. 3, p. 309-319, July-Sept., 2017 

temperature. Extra dye was then removed by five 
washes with distilled water. The dye retained by the 

cells of the biofilm was dissolved with 120 μL of 1% 

(w/v) sodium dodecyl sulfate. The results were 

recorded as absorbance at 595 nm to quantify total 

biofilm mass. 

Detection of mutants resistant to Bothrops moojeni venom 

Spontaneous mutants of S. aureus RN 7044 

resistant to B. moojeni venom were obtained as 

described previously (Szybalsky & Bryson 1952). 

Twenty milliliters of culture in MBH was grown for 

24 hours at 37° C, centrifuged at 3,000 x g for 20 
min at 4º C and resuspended in 10

-1

 of the initial 

volume in saline (0.9% NaCl). After this, 0.1 mL 

was spread onto a gradient plate supplemented with 

venom at concentrations of 2, 5, and 10 x MIC and 

incubated at 37° C. The results were read at 24, 36, 

72 and 96 hours. Mutant colonies were considered 

those that had grown beyond the edge of confluent 

growth.

 

Determination of the fractional inhibitory concentration 

The fractional inhibitory concentration (FIC) 

index is frequently used to assess the drug 

interactions (Mackay et al. 2000). The indices were 

calculated as follows: FIC of drug A = MIC drug A 

+ venom/ MIC drug A alone. The interpretation 

was made as follows: synergy (≤0.5), indifference 

(>0.5 to 4), and antagonism (>4). 

Statistical analysis 

Mann-Whitney U test at R platform was used to 

determine significant differences between venom´s 

MIC, with 5% of significance. 

Results and discussion 

In this study, the susceptibility of S. aureus to 

seven snake venoms from the family Viperidae 
(Bothropoides erythromelas, B. jararaca, B. neuwiedi, B. 
atrox, B. jararacussu, B. moojeni 
and  Rhinocerophis 
alternatus
) was determined. MICs of the crude snake 
venoms are shown in Table 1. Although all the 
venoms came from snakes of the same family, great 
variation in MIC (from 2 to >1,024 μg mL

-1

) was 

observed against S. aureus. Moreover, no significant 
difference (p>0.05) was observed among the MICs 
of different venoms, except for the Bothropoides 
erythromelas
 and B. jararaca venoms. However, the 
mean MIC of B. moojeni venom was the lowest, 
being chosen for further studies.  

For all venoms tested, MICs were greater than 

1,024 μg mL

-1

 for ten out of the 22 S. aureus  isolates, 

whereas the remaining isolates had MICs >1,024 μg 
mL

-1

 against at least one of the venoms. B. jararaca 

showed weak antibacterial activity against all clinical 
isolates and the reference strain (ATCC 25923) with 
MIC >1,024 μg mL

-1

. Similar results were obtained 

with B. erythromelas venom. In contrast, S. aureus RN 
7044 was more sensitive to venoms of B. atrox and B. 
neuwiedi
. Moreover, our data revealed that the type 
ATCC strain was less sensitive than the S. aureus 
isolates. Interestingly, minimum bactericidal 
concentrations (MBCs) correlated with the MICs 
for all venoms tested. 

There are several reports in the literature on the 

antibacterial activity of snake venom against gram-
positive and gram-negative bacteria (Lu et al., 2002; 
Stábeli et al., 2004; Klein et al., 2015), including 
Bacillus subtilis,  Sarcina spp., Escherichia coli and S. 
aureus
. A previous study with B. marajoensis venom 
(also of the family Viperidae), revealed that it was 
able to inhibit the growth of P. aeruginosaS. aureus 
and  Candida albicans, thereby demonstrating an 
antifungal effect is also present in some venoms 
(Costa-Torres et al., 2010). 

The MICs of six antibiotics belonging to two 

classes are shown in Table 2. Overall, the isolates 
were resistant to three of the six antibiotics tested. 
The isolates exhibited resistance to β-lactams, with 
the highest frequency of resistance to ampicillin 
(100%) and amoxicillin/clavulanate (63.6%).  The 
increase in the frequency of isolates of S. aureus 
resistant to β-lactam antibiotics has been reported in 
the literature since the beginning of its clinical use in 
1940 (Livermore, 2000). Alzolibani et al. (2012) 
found 96.7% ampicillin resistance in clinical isolates 
of S. aureus, which agrees with the data obtained in 
our study.  

Among the quinolones of clinical use, the 

isolates were resistant to only ofloxacin (45.4%). It 
should be noted that all the isolates were susceptible 
to ciprofloxacin, levofloxacin and norfloxacin. 
Similarly, Kowalski et al. (2003) suggest that S. 
aureus
 had increased susceptibility to fourth 
generation fluoroquinolones. In contrast, other 
studies revealed high frequency of resistance to 
ciprofloxacin (Alzolibani et al., 2012; Flamm et al., 
2012; Kwak et al., 2013). A possible explanation for 
this discrepancy could be that the isolates tested in 
this study were collected in the late 70s and early 80s 
prior to the introduction of fluoroquinolones to 
clinical use. 

 

 

 

 

 

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Table 1. Minimum inhibitory concentration (MIC) of snake venom from the Viperidae family against clinical isolates and strains of 
Staphylococcus aureus 

Isolate/Strain 

MIC (μg mL

-1

Bothropoides

erythromelas 

Bothropoides 

jararaca 

Bothropoides

neuwiedi 

Bothrops

atrox 

Bothrops

jararacussu 

Bothrops 

moojeni 

Rhinocerophis

alternatus 

RN 

7044 

32 

512 

2 2 8 4 4 

ATCC 25923 

>1,024 

>1,024 

64 

64 

64 

128 

64 

>1,024 >1,024 >1,024 >1,024 >1,024 >1,024 >1,024 

8 >1,024 

>1,024 

64 

64 

64 

64 

10 >1,024 

>1,024 

64 

64 

64 

>1,024 

64 

12 

>1,024 >1,024 >1,024 >1,024 >1,024 >1,024 >1,024 

17 512 

>1,024 

64 

64 

64 

64 

22 

>1,024 >1,024 >1,024 >1,024 >1,024 >1,024 >1,024 

23 

>1,024 >1,024 >1,024 >1,024 >1,024 >1,024 >1,024 

25 >1,024 

>1,024 

64 

64 

64 

>1,024 

64 

29 >1,024 

>1,024 

>1,024 

>1,024 

>1,024 

>1,024 

30 >1,024 

>1,024 

>1,024 

>1,024 

>1,024 

>1,024 

31 

>1,024 >1,024 >1,024 >1,024 >1,024 >1,024 >1,024 

34 

>1,024 >1,024 >1,024 >1,024 >1,024 >1,024 >1,024 

37 

>1,024 >1,024 >1,024 >1,024 >1,024 >1,024 >1,024 

40 

>1,024 >1,024 >1,024 >1,024 >1,024 >1,024 >1,024 

41 

>1,024 >1,024 >1,024 >1,024 >1,024 >1,024 >1,024 

42 >1,024 

>1,024 

64 

64 

64 

64 

44 >1,024 

>1,024 

>1,024 

>1,024 

>1,024 

128 

>1,024 

48 

>1,024 >1,024 >1,024 >1,024 >1,024 >1,024 >1,024 

49 >1,024 

>1,024 

64 

64 

>1,024 

256 

64 

50 >1,024 

>1,024 

64 

64 

64 

128 

64 

52 512 

>1,024 

64 

64 

64 

16 

64 

54 >1,024 

>1,024 

64 

64 

>1,024 

64 

Table 2. Minimum inhibitory concentration (MIC) of antibiotics alone, and in combination with ½ x MIC of Bothrops moojeni

Isolate/Strain 

Amoxicillin/ 

Clavulanate 

Ampicillin Ciprofloxacin 

Levofloxacin 

Norfloxacin  Ofloxacin 

MIC MIC

a

 MIC MIC

a

 MIC MIC

a

 MIC MIC

a

 MIC MIC

a

 MIC MIC

a

 

RN 

7044  4  2  0.5 0.25 128 64 16 16 256 128 64  64 

ATCC 

25923 

0.0612 

0.0612 0.5 0.25  1  0.5 0.5 0.25 0.5 0.25 0.5 0.25 

7* 128 

128 

256 

128 

0.0612 

0.0612 

0.125 

0.125 

0.5 

0.25 

0.125 

0.125 

0.5 0.5 0.5 0.25  2  1  1 0.5  1  0.5 0.5 0.5 

10 

128 128  128  128  0.25 0.25 

0.125 

0.125 0.5  0.5 0.125 0.125 

12 128 

128 

128 

128 

0.0612 

0.0612 

0.0612 

0.0612 

0.5 

0.25 

0.125 

0.125 

17 

128 

128 16  8  1  0.5 0.5 0.5 0.5 0.25 0.25 0.25 

22 128 

128 

256 

128 

0.0612 

0.0612 

0.5 

0.5 

0.5 

0.25 

0.25 

0.25 

23*+ 

1,024 

1,024 512  256 0.125 

0.0612 

0.125 

0.125 0.5  0.25 0.25 0.25 

25 

0.5 

0.5 

0,5 

0.25 

2 1 

0.5 

0.5 2 1 8 4 

29 64 

64 

128 

128 

0.25 

0.25 

0.0612 

0.0612 

0.5 

0.25 

0.0612 

0.0612 

30 

0.5 

0.5 2  2 0.5 

0.25 

0.5 

0.25 2  1 0.5 0.5 

31 128 

128 

64 

64 

0.125 

0.125 

0.125 

0.125 

0.5 

0.25 

0.125 

0.125 

34 256 

256 

128 

128 

0.0612 

0.0612 

0.0612 

0.0612 

0.5 

0.25 

37*+ 256 

256 

64 

64 

0,5 

0,25 

0.0612 

0.0612 

0.5 

16 

40*+  256 

128 

256 

128 

0.125 

0.125 

0.125 

0.125 

4 2 4 2

41 

512 

512 

256 

256 

0.0612 

0.0612 

0.0612 

0.0612 

1 0.5 8  4 

42 

0.5 

0.25 

0.5 

0.25 

1 1 

0.25 

0.25 

4 2 4 4 

44* 256 

256 

256 

128 

0.0612 

0.0612 

0.0612 

0.0612 

0.5 

16 

16 

48 

128 

128 

256 

256 

0.0612 

0.0612 

0.0612 

0.0612 

1 1 8 8 

49 

0.5 

0.25 

0.0612 

0.0612 

1 0.5 

0.125 

0.125 

2 1 8 4 

50 

0.5 0.25 0.5 0.25  1  0.5 0.5 0.25  4  2  0.25 0.25 

52 

0.5 0.5 0.125 

0.125 2  1 0.125 

0.125 4  2  0.25 0.25 

54* 

0.5 

0.25 

1 0.5 2 1 1 

0.5 4 2 4 2

Critical point for determination of drugs resistance in 

μg/mL for S. aureus according to CLSI (2016): Amoxicillin-Clavulanate ≥ 8; Ampicillin ≥ 0.25; Ciprofloxacin ≥ 4; Levofloxacin 

≥ 4; Norfloxacin ≥ 16; Ofloxacin ≥ 4.

 

*β-lactamase positivos; + Methicillin-resistant S. aureus (MRSA). 

a

 Antibiotic concentration in combination with B. moojeni venom 

that prevented the development of turbidity (i.e., growth).  

Results highlighted in bold refers to reversal of resistance

.

 

 

The capacity of B. moojeni venom to enhance the 

activity of the tested antibiotics was also investigated. 

The MICs of antibiotics and B. moojeni venom 

individually and in combination are shown in Table 

2. A synergic effect (FIC index of 0.5) was observed 

between  B. moojeni venom and all antibiotics 

investigated. The combinations of the B. moojeni 

venom with, ampicillin, amoxicillin/clavulanate, 

ciprofloxacin, levofloxacin, norfloxacinin and 

ofloxacin achieved synergy of 50%, 22.7%, 45.5%, 

18.2%, 86.4% and 31.8%, respectively, in the S. 

aureus clinical isolates. Moreover, no antagonistic 

action was found.

 

These results suggest that B. 

moojeni venom increased the antibacterial activity of 

these antibiotics against S. aureus. It should be noted 

that five of the six β-lactamase positive isolates were 

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inhibited by the combination of ampicillin and B. 
moojeni
 venom.  Additionally, the reduction of the 

MIC of ofloxacin, when in combination with B. 

moojeni venom, for the isolate 40 (β-lactamase 

positive, methicillin-resistant S. aureus-MRSA), and 

the isolate 54 (β-lactamase positive) led to the 

reversal of ofloxacin resistance in these isolates.  

Previous studies have reported antimicrobial 

peptides in various animal venoms, which are 
traditionally associated with defense mechanisms, 
such as antibacterial activity (Jenssen, Hamill, & 
Hancock, 2006; Wang, Li, & Wang, 2009). 
Moreover, other studies have shown that the 
fluoroquinolones, erythromycin and rifampicin had 
their effects enhanced by antimicrobial peptides, 
demonstrating synergistic action (Ulvatne, 
Karoliussen, Stiberg, Rekdal, & Svendsen, 2001; 
Fehri, Wróblewski, & Blanchard, 2007).  

Among the fractions of B. moojeni venom 

obtained by gel filtration chromatography, the 
fraction BmooIV (Figure 1A) was the only one that 
exhibited antibacterial activity when tested against S. 
aureus
 RN 7044 (Figure 2A). The antibacterial 
activity of BmooIV was detected in the amount of 
1.14 mg mL

-1

 of protein. To investigate whether 

BmooIV also had the ability to enhance the 
susceptibility of Saureus RN 7044 to ciprofloxacin it 
was tested in combination with this antibiotic, both 
in ½ x MIC concentration. The result indicated the 
reversal of phenotypic resistance to ciprofloxacin of 
S. aureus RN 7044 

The fraction BmooIV was lyophilized and 

subjected to a high-performance liquid 
chromatography (HPLC) on the μRPC C2/C18 
reverse phase column (Figure 1B). The fraction 
obtained from reverse phase named Bmoo-SII 
presented antibacterial activity when tested against S. 
aureus
 RN 7044. It also enhanced the susceptibility of 
S. aureus RN 7044 to ciprofloxacin, when tested in 
combination, both at ½ x MIC concentration (Figure 
2B).  

The comparison of 50 amino acids residues from 

Bmoo-SII placed it in the phospholipase A

superfamily with high identity to Myotoxin II from 
B. moojeni and basic phospholipase A

2

 from B. 

moojeni and B. asper (Figure 3). Silveira et al. (2013) 
characterized the phospholipase A

2

 (PLA

2

) from B. 

moojeni. PLA

was first purified and characterized 

from cobra venom and later from rattlesnake 
venom. They are small, secreted proteins of 14–18 
kDa that usually contain 6 to 8 disulfide bonds. 
Queiroz et al. (2011) compared their results with 
other studies (Soares et al. 1998; Soares et al., 2000; 

Borja-Oliveira et al., 2007; Calgarotto, et al., 2008; 
Santos-Filho et al., 2008), which reported isoforms 
of PLA

2

 myotoxin of B. moojeni with molecular 

weight ranging between 13,400 Da to 16,500 Da. 
Thus, we were able to infer from the amino acids 
residues obtained in our analysis that Bmoo-SII has 
a molecular weight within this range. 

 

 

Figure 1. A. Chromatographic profile of Bothrops moojeni venom 
separation in gel filtration Sephacryl-S100 XK 16/60 column. The 
pointer indicates the fraction named BmooIV. B 
Chromatographic profile of BmooIV in μRPC C2-C18 the 
reverse phase column. The highest peak is Bmoo-SII. 

PLA

plays important roles in cellular signaling 

and metabolism. It also participates in the first line 
of antimicrobial defense (Nevalainen, Graham, & 
Scott, 2008; Dennis, Cao, Hsu, Magrioti, & 
Kokotos, 2011). The bactericidal action of PLA

2

 

depends on whether the bacteria are gram-positive 
or gram-negative. In general, PLA

2

 hydrolyses the 

phospholipid membrane of the bacteria cell causing 
death to both gram-positive as gram-negative 
bacteria (Nevalainen et al., 2008). In vitro studies by 
Grönroos, Laine, Janssen, Egmond, & Nevalainen, 
2001, showed that PLA

2

s from groups IIA and V 

were found to kill both methicillin-resistant 
staphylococci and vancomycin-resistant enterococci. 
Snake venom from the family Viperidae possesses 
PLA

2

 from group IIA (Dennis et al., 2011). The 

efficiency of PLA

against antibiotic-resistant bacteria 

is an important property that holds promise for 
biotechnological applications. 

 

 

 

 

 

 

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Figure 2. AActivity of BmooIV against Staphylococcus aureus RN 7044. 1- BmooIV (1.14 mg mL

-1

 – 1 x MIC); 2- Bothrops moojeni crude 

venom (1 x MIC); 3- BmooIV (0.57 mg mL

-1

 – ½ x MIC); 4- BmooIV (0.57 mg mL

-1

 – ½ x MIC) in combination with ciprofloxacin (64 

μg mL

-1

 – ½ x MIC); 5- Ciprofloxacin (64 μg mL

-1

 – ½ x MIC); and 6- Elution buffer 0.2 M ammonium acetate pH 8. B.

 Antibacterial 

activity of Bmoo-SII against S. aureus RN 7044. 1- Elution buffer 0.2 M ammonium acetate pH 8; 2- Bmoo-SII (50 μL – ½ x MIC) in 
combination with ciprofloxacin (64 μg mL

-1

 – ½ x MIC); and 3- Bmoo-SII (100 μL – 1 x MIC). 

 

 

Figure 3. Alignment of Bmoo-SII with other phospholipase A

2

-like sequences.  

 

Survival kinetics were evaluated for S. aureus 

RN7044, for which synergistic activity had been 

observed (Figure 4). A bactericidal profile was 
observed at sub-inhibitory concentrations of 

ciprofloxacin (½ x MIC) and B. moojeni venom (½ x 

MIC) and of B. moojeni crude venom alone (1 x 

MIC). It should be noted that B. moojeni venom or 

ciprofloxacin, when tested individually in sub-MIC 

concentrations, allowed bacterial growth similar to 

that of the control, although there was a longer lag 

phase in these concentrations.  

The possible inhibitory action to the efflux 

pump of S. aureus RN 7044 by B. moojeni venom was 

evaluated by the loss of fluorescence (Figure 5). In 

the control culture, a greater reduction in the 

fluorescence between 10 and 20 minutes due to the 

higher extrusion of ethidium bromide by the NorA 

efflux pump was observed. Cultures exposed to B. 

moojeni venom (½ x MIC) presented similar results 
to the control. However, when B. moojeni venom 

was used in combination with ciprofloxacin (both ½ 

x MIC) a slower descent in fluorescence was 

noticed, indicating a reduction in pump activity. 

Although the crude venom and ciprofloxacin (both 

½ x MIC) by themselves were not effective 

antibacterials, they can reverse the resistance by 

blocking the NorA efflux pump. Previous studies 

have identified products from natural sources as 

inhibitors of the S. aureus NorA efflux pump, which 

co-administered with fluoroquinolone can 

potentiate its antibacterial activity (Marquez et al., 

2005). 

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Figure 4. Survival curves of Staphylococcus aureus RN 7044 alone 
(control), in the presence of ciprofloxacin (½ MIC) alone, in the 
presence of Bothrops

 

moojeni venom (½ and 1 x MIC) alone, and in 

the presence of the combination of the two (½ MIC). 

 

 

Figure 5. Measurement of active efflux of ethidium bromide in 
Staphylococcus aureus RN 7044 with excitation at 269 nm and 
emission at 600 nm. 

To date, most studies of the activity of snake 

venom on bacteria have focused on their bactericidal 
and bacteriostatic effects. In this study, we evaluated 
the influence of B. moojeni venom as a therapeutic 
agent for biofilm formation by S. aureus RN 7044. 
When tested with ½ x MIC, B. moojeni venom was 
able to inhibit 90% of biofilm formation, without 
affecting bacterial growth. Recently, Klein et al. 
(2015) demonstrated for the first time that a lectin 
purified from the venom of Bothrops jararacussu 
disrupts staphylococcal biofilms. These findings are 
of interest because nosocomial infections involving 
the formation of biofilm caused by S. aureus are 
often difficult to treat with antibiotics.  

Considering the relevance of resistant mutants in 

the search for potential antimicrobial agents against 

S. aureus, we investigated the occurrence of mutants 

in a gradient plate. At concentrations of 2, 5 and 10 x 

MIC of B. moojeni venom, a small number of mutant 

colonies was detected after 48 hours of incubation 

(Figure. 6). The relatively low frequency of the 

spontaneous emergence of mutants resistant to B. 

moojeni in the population of S. aureus RN 7044, as 

well as their inability to grow after consecutive 

subcultures on non-selective medium, is relevant 
since the loss of viability of the mutant S. aureus and 

the inhibitory power of B. moojeni venom can be 

explored further with the aim of possible 

biotechnological application. 

 

 

Figure 6. Mutants on Müller-Hinton agar gradient plate with 
Bothrops moojeni venom (10 x MIC) after 48 hours of incubation at 
37º C. 

The results show that B. moojeni venom and its 

bioactive constituent (phospholipase A

2

) possess 

strong antimicrobial activity against S. aureus and its 

biofilm formation. The fact that B. moojeni venom 

displayed greater potency than other venoms was 

not investigated, but may be due to the presence of 

the enzyme PLA

2

.  B. moojeni venom enhanced 

antibiotic susceptibility, mostly in combination with 

norfloxacin. The effects of B. moojeni venom on the 

biofilm formation, efflux pump, and occurrence of 

mutants of S. aureus all promise to be useful in the 

search for antibacterial agents against drug resistant 

S. aureus

Acknowledgements 

The authors acknowledge financial support from 

Fundação de Amparo à Pesquisa do Estado de Minas 
Gerais
 (FAPEMIG), Conselho Nacional de 

Desenvolvimento Científico e Tecnológico (CNPq) and 

Coordenação de Aperfeiçoamento de Pessoal de Nível 
Superior
 (CAPES). We thank Mr. Rômulo Righi de 

Toledo from the Serpentarium for providing the 

venoms. 

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Received on September 3, 2016. 

Accepted on June 12, 2017. 

 

 

License information: This is an open-access article distributed under the terms of the 
Creative Commons Attribution License, which permits unrestricted use, distribution, 
and reproduction in any medium, provided the original work is properly cited. 

 

  

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