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Antibacterial and Hemolytic Activity of Crotalus Triseriatus and Crotalus Ravus Venom

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Rattlesnakes have venoms with a complex toxin mixture comprised of polypeptides and proteins. Previous studies have shown that some of these polypeptides are of high value for the development of new medical treatments. The aim of the present study is to evaluate, in vitro, the antibacterial and hemolytic activity of Crotalus triseriatus and Crotalus ravus venoms. A direct field search was conducted to obtain Crotalus triseriatus and Crotalus ravus venom samples. These were evaluated to determine their antibacterial activity against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa through the techniques of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC). Hemolytic activity was also determined. Antibacterial activity was determined for treatments (Crotalus triseriatus 2) CT2 and (Crotalus ravus 3) CR3, obtaining a Minimum Inhibitory Concentration of 50 µg/mL and a Minimum Bactericidal Concentration of 100 µg/mL against Pseudomonas aeruginosa. CT1 (Crotalus triseriatus 1), CT2, and CR3 presented hemolytic activity; on the other hand, Crotalus ravus 4 (CR4) did not show hemolytic activity. The results of the present study indicate for the first time that Crotalus triseriatus and Crotalus ravus venoms contain some bioactive compounds with bactericidal activity against Pseudomonas aeruginosa which could be used as alternative treatment in diseases caused by this pathogenic bacterium.
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animals
Article
Antibacterial and Hemolytic Activity of Crotalus
triseriatus and Crotalus ravus Venom
Adrian Zaragoza-Bastida 1, Saudy Consepcion Flores-Aguilar 2, Liliana Mireya Aguilar-Castro 2,
Ana Lizet Morales-Ubaldo 1, Benjamín Valladares-Carranza 3, Lenin Rangel-López 1,
Agustín Olmedo-Juárez 4, Carla E. Rosenfeld-Miranda 5and Nallely Rivero-Pérez 1, *
1Universidad Autónoma del Estado de Hidalgo, Área Académica de Medicina Veterinaria y Zootecnia,
Instituto de Ciencias Agropecuarias, Rancho Universitario Av. Universidad km 1, EX-Hda de Aquetzalpa,
Tulancingo, Hidalgo 43600, Mexico; adrian_zaragoza@uaeh.edu.mx (A.Z.-B.);
ubaldolizet8@gmail.com (A.L.M.-U.); ralolenin@gmail.com (L.R.-L.)
2
Universidad Aut
ó
noma del Estado de Hidalgo,
Á
rea Acad
é
mica de Biolog
í
a, Instituto de Ciencias B
á
sicas e
Ingeniería. Carretera Pachuca-Tulancingo S/N Int. 22 Colonia Carboneras, Mineral de la Reforma,
Hidalgo 42180, Mexico; saudy.fa@gmail.com (S.C.F.-A.); profe_3192@uaeh.edu.mx (L.M.A.-C.)
3Facultad de Medicina Veterinaria y Zootecnia Universidad Autónoma del Estado de México,
El Cerrillo Piedras Blancas, Toluca 50295, Mexico; benvac2004@yahoo.com.mx
4Centro Nacional de Investigación Disciplinaria en Salud Animal e Inocuidad (CENID SAI-INIFAP),
Carretera Federal Cuernavaca-Cuautla No. 8534 /Col. Progreso, Jiutepec 62550, Morelos, Mexico;
aolmedoj@gmail.com
5Facultad de Ciencias Veterinarias, Universidad Austral de Chile, Isla Teja s/n, Casilla 567, Valdivia, Chile;
crosenfe@uach.cl
*Correspondence: nallely_rivero@uaeh.edu.mx; Tel.: +52-771-717-2000
Received: 14 January 2020; Accepted: 7 February 2020; Published: 11 February 2020


Simple Summary:
Rattlesnakes (Crotalus ravus and Crotalus triseriatus) have some compounds that
resemble polypeptides and proteins in their venoms which can be used in therapeutic treatment
as antibacterial compounds. The aim of the present study is to evaluate the antibacterial and
hemolytic activity of two rattlesnake venoms. The results of the present study indicate that the
evaluated venoms have bactericidal activity against Pseudomonas aeruginosa, an important bacterium
that aects animals and humans, thereby providing a new and ecient treatment alternative against
this pathogenic bacterium.
Abstract:
Rattlesnakes have venoms with a complex toxin mixture comprised of polypeptides and
proteins. Previous studies have shown that some of these polypeptides are of high value for the
development of new medical treatments. The aim of the present study is to evaluate,
in vitro
,
the antibacterial and hemolytic activity of Crotalus triseriatus and Crotalus ravus venoms.
A direct
field search was conducted to obtain Crotalus triseriatus and Crotalus ravus venom samples. These
were evaluated to determine their antibacterial activity against Escherichia coli,Staphylococcus aureus
and Pseudomonas aeruginosa through the techniques of Minimum Inhibitory Concentration (MIC) and
Minimum Bactericidal Concentration (MBC). Hemolytic activity was also determined. Antibacterial
activity was determined for treatments (Crotalus triseriatus 2) CT2 and (Crotalus ravus 3) CR3, obtaining
a Minimum Inhibitory Concentration of 50
µ
g/mL and a Minimum Bactericidal Concentration of
100 µg/mL
against Pseudomonas aeruginosa. CT1 (Crotalus triseriatus 1), CT2, and CR3 presented
hemolytic activity; on the other hand, Crotalus ravus 4 (CR4) did not show hemolytic activity.
The results
of the present study indicate for the first time that Crotalus triseriatus and Crotalus ravus
venoms contain some bioactive compounds with bactericidal activity against Pseudomonas aeruginosa
which could be used as alternative treatment in diseases caused by this pathogenic bacterium.
Animals 2020,10, 281; doi:10.3390/ani10020281 www.mdpi.com/journal/animals
Animals 2020,10, 281 2 of 9
Keywords:
Crotalus ravus; Crotalus triseriatus; venom; antibacterial activity; Pseudomonas aeruginosa;
hemolytic activity
1. Introduction
Rattlesnakes are a species widely distributed through Mexico, occupying practically the whole
territory. There exists a great variety of these species, among them, are Crotalus triseriatus, distributed
in the States of Veracruz, Puebla, Tlaxcala, M
é
xico, Morelos, and Michoac
á
n and Crotalus ravus, which
occupies the States of Morelos, M
é
xico, Puebla, Tlaxcala, Guerrero, Oaxaca, and Hidalgo. These species
are primarily recognized for their characteristic hemotoxic venoms [13].
Crotalid venoms are comprised mainly of enzymes that cause severe local inflammation, necrosis,
hemorrhagic syndromes, and neurological manifestations. These responses would typically help rapid
prey subjugation or capture, as well as serve as a defense mechanism [4].
Animal venoms, including that of snakes, are complex mixtures of bioactive compounds that
contain large amounts of proteins, peptides, and small molecules that can be considered for use in a
wide range of medical applications [5,6].
There are several examples in the development of treatments derived from snake venom
compounds. One of the most widely known is Capoten
®
, a hypotensive agent, used for the treatment
of congestive heart failure, diabetic nephropathy, and heart attacks. Another known example is
Viprinex®, developed to treat acute strokes [7,8].
Aside from their qualities as potential therapeutic agents, venoms are currently considered as
possible sources of molecules with antibacterial activity [
9
]. This, in fact, has a great impact on public
health especially due to the increase of antibacterial resistant bacteria.
In 2017 the World Health Organization (WHO) compiled a list of antibiotic-resistant priority
pathogens, among which, were the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa
bacteria resistant to carbapenems, and Staphylococcus aureus resistant to methicillin and vancomycin [
10
].
Due to the increased antibiotic resistance found in these pathogens, the aim of the present study was to
evaluate,
in vitro
, the antibacterial and hemolytic response of Crotalus triseriatus and Crotalus ravus
venoms on bacteria of public health importance.
2. Material and Methods
2.1. Field Sampling
Two field outings were carried out per month during each of the months of August, September,
October and November 2018 in the state of Hidalgo, Mexico; covering the municipalities of Acatl
á
n,
Almoloya, Cuautepec de Hinojosa, Mineral del Chico, Mineral del Monte, Santiago Tulantepec,
Singuilucan, Tula de Allende and Zacualtipán.
A direct search was conducted according to the methodology described by McDiarmid et al. in
2012. The rattlesnakes were trapped in accordance with the ocial norms for wildlife protection
(NOM-059-SEMARNAT-2010) established by the government of Mexico and with a scientific collecting
permit issued by General Directorate of Wildlife of the Secretariat of Environment and Natural
Resources of Mexico (Oce NSGPA/SGVS/003613/18) [11,12].
2.2. Obtaining Venom Samples
Four samples were collected in the field, two of them belonging to the species Crotalus triseriatus
(CT1 and CT2) and the remaining from the species Crotalus ravus (CR3 and CR4). A record of each
individual was noted.
Once the samples were obtained, they were subjected to a lyophilization process and kept at
70 C until further evaluation.
Animals 2020,10, 281 3 of 9
2.3. Antibacterial Activity
The venom’s antibacterial activity was determined through the Minimum Inhibitory Concentration
(MIC) and the Minimum Bactericidal Concentration (MBC) procedures, in accordance with the CLSI
guidelines and with the standards published by Olmedo-Ju
á
rez et al., in 2019 and by Morales-Ubaldo
et al., in 2020 [1315].
Escherichia coli ATCC
35218
,Pseudomonas aeruginosa ATCC
9027
, and Staphylococcus aureus ATCC
6538
strains were used to perform the evaluation. These samples were the same which were reactivated
from cryopreservation in Müller–Hinton agar (BD Bioxon, Heidelberg, Germany) through simple strain
technique to obtain isolated colonies. A Gram staining was performed to corroborate their morphology.
Once the purity was confirmed, one colony of each strain was inoculated in nutritive broth
(BD Bioxon), and incubated under constant agitation at 70 rpm for 24 h at 37
C. The bacterial cell
suspension was adjusted to a 0.5 McFarland (Remel, R20421, Kansas, U.S.A.) standard (approximately
1.5 ×106Colony Forming Units (CFU) per mL).
2.3.1. Minimal Inhibitory Concentration (MIC)
Micro-dilution was used to determine the MIC, evaluating dierent venom concentrations (100,
50, 25, 12.5, 6.25, 3.12, 1.56, 0.78 µg/mL).
In a sterile 96- well plate, 100
µ
L of each venom concentrations were added along with 10
µ
L of
bacterial cell suspension previously adjusted to a 0.5 McFarland standard. The plates were incubated
at 37
C for 24 h at 70 rpm. Kanamycin (AppliChem 4K10421, Darmstadt, Germany) was used as a
positive control (128 to 1
µ
g/mL) and nutritive broth as the negative control. Treatments were evaluated
by triplicate.
After incubation 20
µ
L of a 0.04% (w/v) p-iodonitrotetrazolium (Sigma-Aldrich I8377, Missouri,
U.S.A.) solution was added into each well and incubated for 30 min. The MIC was determined by the
concentration at which the solution turned to a pinkish color.
2.3.2. Minimal Bactericidal Concentration (MBC)
After incubation and previous addition of p-iodonitrotetrazolium, 5
µ
L from each well was
inoculated in Müller–Hinton agar (BD Bioxon) and incubated at 37
C for 24 h. The MBC was
considered as the lowest concentration where no visible growth of the bacteria was observed on
the plates.
2.4. Indirect Hemolytic Activity
In accordance with the protocols described by Pirela et al., in 2006 with modifications, the venom’s
indirect hemolytic activity was evaluated [
16
]. A donor donkey blood sample was collected. The blood
sample was stored in 10 mL sodium citrate (3.2%) tubes (DB Vacutainer) and in 3 mL EDTA (10.8 mg)
tubes (BD Vacutainer).
Blood agar was used (Merck
©
, Darmstadt, Germany). To obtain plates with 8% blood
concentration, 250 mL of agar base was prepared, and 20 mL of blood was added.
One hundred micrograms (100
µ
g) of each treatment were weighed out (lyophilized venom) and
reconstituted in 1 mL of nutritive broth (BD Bioxon). Dilutions were made (100, 50, 25, 12.5, 6.25,
3.12 µg/mL) from this concentrated solution for further evaluation.
Four wells were made (6 mm diameter) on the plate’s surface. Twenty micrograms (20
µ
L) were
added of each concentrate to be evaluated. Treatments were performed by triplicate. Tween 80 at
100% (Sigma-Aldrich) and nutritive broth (BD Bioxon) were used as positive and negative controls,
respectively. Plates were incubated for 24 h at 37
C. Once the incubation period elapsed, hemolysis
halos were measured (mm).
Animals 2020,10, 281 4 of 9
2.5. Statistical Analysis.
Obtained data were analyzed using two-way variance analysis (ANOVA) and a means comparison
by Tukey at a significance level of 0.05% through Minitab 18 statistical package [17].
3. Results and Discussion
3.1. Individuals Data
A record of each individual was made with the following information: length, weight, age,
and gender
(Table 1). The characteristics of the rattlesnakes in the study coincided with those reported
by Campbell and Lamar in 2004 [1], as seen in Figure 1.
Table 1. Individual data of trapped rattlesnakes in Hidalgo State.
Species Species Characteristics Individual
Identification Gender Age Length (cm) Weight (g)
Crotalus
triseriatus
Triangular head
8–10 rattles
Postocular strip
CT1 Male Adult 37 210
CT2 Male Adult 25 175
Crotalus
ravus
Triangular head
Thin rattle
Symmetric scales in head
CR3 Male Adult 25 180
CR4 Male Adult 25 175
Animals 2020, 10, 281 4 of 9
Obtained data were analyzed using two-way variance analysis (ANOVA) and a means
comparison by Tukey at a significance level of 0.05% through Minitab 18 statistical package [17].
3. Results and Discussion
3.1. Individuals Data
A record of each individual was made with the following information: length, weight, age, and
gender (Table 1). The characteristics of the rattlesnakes in the study coincided with those reported by
Campbell and Lamar in 2004 [1], as seen in Figure 1.
Table 1. Individual data of trapped rattlesnakes in Hidalgo State.
Species Species Characteristics Individual
Identification Gender Age Length (cm) Weight (g)
Crotalus
triseriatus
Triangular head
8-10 rattles
Postocular strip
CT1 Male Adult 37 210
CT2 Male Adult 25 175
Crotalus
ravus
Triangular head
Thin rattle
Symmetric scales in head
CR3 Male Adult 25 180
CR4 Male Adult 25 175
(a) Crotalus triseriatus (b) Crotalus ravus
Figure 1. Captured species (a) Crotalus triseriatus and (b) Crotalus ravus.
3.2. Antibacterial Activity
A MIC of 50 µg/mL and an MBC of 100 µg /mL were determined as effective for treatments CT2
and CR3 over P. aeruginosa (Table 2, Figure 2). Nevertheless, antibacterial activity was not detected
for E. coli and S. aureus.
Table 2. Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) of
Crotalus triseriatus and Crotalus ravus venoms.
Evaluated Bacteria Evaluated Treatments µg/mL (MIC/MBC) Controls (MIC/MBC)
CT1 CT2 CR3 CR4 Nutritive Broth Kanamycin g/mL)
E. coli - -
-
- - 2/4
P. aeruginosa - 50
a
/100
A
50
a
/100
A
- - 16
b
/64
B
S. aureus - - - - - 1/4
CT1 Crotalus triseriatus 1, CT2 Crotalus triseriatus 2, CR3 Crotalus ravus 3, CR4 Crotalus ravus 4
a,b
Different small letters indicate significant statistical differences between MIC (p < 0.05)
A,B
Different
capital letters indicate significant statistical differences between MBC (p < 0.05)
Figure 1. Captured species (a)Crotalus triseriatus and (b)Crotalus ravus.
3.2. Antibacterial Activity
A MIC of 50
µ
g/mL and an MBC of 100
µ
g/mL were determined as eective for treatments CT2
and CR3 over P. aeruginosa (Table 2, Figure 2). Nevertheless, antibacterial activity was not detected for
E. coli and S. aureus.
Table 2.
Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) of
Crotalus triseriatus and Crotalus ravus venoms.
Evaluated
Bacteria
Evaluated Treatments µg/mL (MIC/MBC) Controls (MIC/MBC)
CT1 CT2 CR3 CR4 Nutritive Broth Kanamycin (µg/mL)
E. coli - - - - - 2/4
P. aeruginosa -50 a/100 A50 a/100 A- - 16 b/64 B
S. aureus - - - - - 1/4
CT1 Crotalus triseriatus 1, CT2 Crotalus triseriatus 2, CR3 Crotalus ravus 3,CR4 Crotalus ravus 4
a,b
Dierent small letters
indicate significant statistical dierences between MIC (p<0.05)
A,B
Dierent capital letters indicate significant
statistical dierences between MBC (p<0.05)
Animals 2020,10, 281 5 of 9
Animals 2020, 10, 281 5 of 9
(a) MIC of venoms (b) MBC of CT2
Figure 2. Antibacterial activity of rattlesnake’s venoms against P. aeruginosa: (a) columns 1–3, CT1
from 100 at 0.78 µg/mL, columns 4–6, CR4 from 100 at 0.78 µg/mL, columns 7–9, CT2 from 100 at 0.78
µg/mL, columns 10–12, CR3 from 100 at 0.78 µg/mL. The MIC value is read at the minimal
concentration in which the color changes to pink; (b) Plate with P. aeruginosa + CT2 in Müller–Hinton
agar; A CT2 to 100 µg/mL, B CT2 to 50 µg/mL , C CT2 to 25 µg/mL, D CT2 to 12.5 µg/mL, E CT2 to
6.25 µg/mL, F CT2 to 12.5 µg/mL, G CT2 to 6.25 µg/mL, H CT2 to 0.78 µg/mL. The MBC is read to the
lowest concentration where no visible growth of the bacteria
It was determined that the antibacterial response seen in treatments CT2 and CR3 were
bactericidal, since the relation between MIC and MBC is less than 4, in accordance with González-
Alamilla et al., in 2019 [18].
Boda et al., in 2019 evaluated the antibacterial activity of eleven crude venoms from different
snake species including Crotalus atrox and Crotalus polystictus against Staphylococcus aureus, Escherichia
coli and Pseudomonas aeruginosa among others, at varied concentrations of 500 to 1.95 µg/mL,
determining a MIC and MBC of 125 and 500 µg/mL against S. aureus for Crotalus atrox and Crotalus
polystictus, respectively. In the present study, antibacterial activity was not found for S. aureus and E.
coli but was determined for Pseudomonas aeruginosa, obtaining a MIC of 50 and a MBC of 100 µg/mL
for C. triseriatus and C. ravus (CT2 and CR3). According to Boda et al., 2019, the antibacterial activity
of venoms from viperid species is probably due to their content of proteins with proteolytic activity
[19].
Samy et al., in 2014, evaluated CaTx-II a toxin isolated from Crotalus adamanteus venom,
determining a MIC of 7.8 µg/mL for S. aureus and 62.5 to 125 µg/mL for P. aeruginosa. Oguiura et al.,
in 2011, evaluated crotamine, a myotoxin from Crotalus durissus venom against different bacteria
strains which included E. coli, S. aureus, and P. aeruginosa. They report a MIC of 100 µg/mL for E. coli
and >200 µg/mL for the other two [20,21], a contrast with the results obtained in our present study
since the antibacterial activity was not determined for E. coli or S. aureus. Since it was determined that
a MIC of 50 µg/mL from C. triseriatus and C. ravus (CT2 and CR3) occurred in crude venom, the
activity could be attributed to the presence of these bioactive compounds in the venom of the
individuals used for this evaluation since both compounds were isolated from snake venom of the
same genus (Crotalus).
Although the aim of the study did not include identifying the venom’s active mechanism, it has
been reported that phospholipase A
2
(CaTx-II) interacts with lipopolysaccharide (LPS), particularly
with lipid A, a Gram-negative bacteria component, causing membrane permeabilization. Crotamine
also has effects over some bacteria through membrane permeabilization, so it could be suggested that
CT2 and CR3 treatments antibacterial activity is related to this mechanism [21,22].
In this respect, the efficiency of these compounds, specially phospholipase A
2
against antibiotic-
resistant bacteria, holds promise for biotechnological applications, in this case, new medical
treatment alternatives, however, it should be understood there are different antibacterial activity
mechanisms from venom-based drugs [23,24].
Figure 2.
Antibacterial activity of rattlesnake’s venoms against P. aeruginosa: (
a
) columns 1–3, CT1 from
100 at 0.78
µ
g/mL, columns 4–6, CR4 from 100 at 0.78
µ
g/mL, columns 7–9, CT2 from 100 at 0.78
µ
g/mL,
columns 10–12, CR3 from 100 at 0.78
µ
g/mL. The MIC value is read at the minimal concentration in
which the color changes to pink; (
b
) Plate with P. aeruginosa +CT2 in Müller–Hinton agar;
A
CT2 to
100 µg/mL
,
B
CT2 to 50
µ
g/mL,
C
CT2 to 25
µ
g/mL,
D
CT2 to 12.5
µ
g/mL,
E
CT2 to 6.25
µ
g/mL,
F
CT2
to 12.5
µ
g/mL,
G
CT2 to 6.25
µ
g/mL,
H
CT2 to 0.78
µ
g/mL. The MBC is read to the lowest concentration
where no visible growth of the bacteria.
It was determined that the antibacterial response seen in treatments CT2 and CR3 were bactericidal,
since the relation between MIC and MBC is less than 4, in accordance with Gonz
á
lez-Alamilla et al.,
in 2019 [18].
Boda et al., in 2019 evaluated the antibacterial activity of eleven crude venoms from dierent snake
species including Crotalus atrox and Crotalus polystictus against Staphylococcus aureus, Escherichia coli
and Pseudomonas aeruginosa among others, at varied concentrations of 500 to 1.95
µ
g/mL, determining
a MIC and MBC of 125 and 500
µ
g/mL against S. aureus for Crotalus atrox and Crotalus polystictus,
respectively. In the present study, antibacterial activity was not found for S. aureus and E. coli but was
determined for Pseudomonas aeruginosa, obtaining a MIC of 50 and a MBC of 100
µ
g/mL for C. triseriatus
and C. ravus (CT2 and CR3). According to Boda et al., 2019, the antibacterial activity of venoms from
viperid species is probably due to their content of proteins with proteolytic activity [19].
Samy et al., in 2014, evaluated CaTx-II a toxin isolated from Crotalus adamanteus venom, determining
a MIC of 7.8
µ
g/mL for S. aureus and 62.5 to 125
µ
g/mL for P. aeruginosa. Oguiura et al., in 2011,
evaluated crotamine, a myotoxin from Crotalus durissus venom against dierent bacteria strains which
included E. coli,S. aureus, and P. aeruginosa. They report a MIC of 100
µ
g/mL for E. coli and
>200 µg/mL
for the other two [
20
,
21
], a contrast with the results obtained in our present study since the antibacterial
activity was not determined for E. coli or S. aureus. Since it was determined that a MIC of 50
µ
g/mL
from C. triseriatus and C. ravus (CT2 and CR3) occurred in crude venom, the activity could be attributed
to the presence of these bioactive compounds in the venom of the individuals used for this evaluation
since both compounds were isolated from snake venom of the same genus (Crotalus).
Although the aim of the study did not include identifying the venom’s active mechanism, it has
been reported that phospholipase A
2
(CaTx-II) interacts with lipopolysaccharide (LPS), particularly
with lipid A, a Gram-negative bacteria component, causing membrane permeabilization. Crotamine
also has eects over some bacteria through membrane permeabilization, so it could be suggested that
CT2 and CR3 treatments antibacterial activity is related to this mechanism [21,22].
In this respect, the eciency of these compounds, specially phospholipase A
2
against
antibiotic-resistant bacteria, holds promise for biotechnological applications, in this case, new medical
treatment alternatives, however, it should be understood there are dierent antibacterial activity
mechanisms from venom-based drugs [23,24].
Animals 2020,10, 281 6 of 9
In accordance with WHO, P. aeruginosa actually is in the critical priority group of the list of
antibiotic-resistant pathogens. WHO has been expressing its interest by promoting the research and
development of new antibiotics for this bacterium [
10
]. These results obtained herein show that CT2
and CR3 treatments demonstrated bactericidal activity against this pathogen showing its importance,
since rattlesnake venoms or compounds thereof could be used to develop eective therapeutic agents
to treat infections caused by P. aeruginosa.
3.3. Hemolytic Activity
With respect to the hemolysis produced, the generated halos showed significant statistical
dierences between them (p<0.05) (Table 3). It was observed that CT1, CT2, and CR3 showed the
highest hemolytic potential and there were no statistically significant dierences between them at
100 µg/mL concentration compared with the other treatments (Figure 3).
Table 3. Hemolysis halos generated by C. triseriatus and C. ravus venoms.
Concentration
(µg/mL)
Evaluated Treatments
CT1 CT2 CR3 CR4 Nutritive Broth Tween 80
100 18.67 ±1.53 a,A,* 17.00 ±1.00 a,A 18.67 ±1.15 a,A, * 0.0 b
0.00
20.33
±
0.58 *
50 15.00 ±0.0 b,B 12.33 ±0.58 c,B 16.67 ±0.58 a,B 0.0 d
25 13.67 ±0.58 a,B,C 13.67 ±1.15 a,B 13.33 ±0.58 a,C 0.0 b
12.5 12.00 ±1.00 a,C 10.00 ±0.00 a,b,C 10.33 ±0.58 b,D 0.0 c
6.25 8.67 ±0.58 a,D 8.33 ±0.58 a,C 7.33 ±0.58 a,E 0.0 b
3.12 0.00 ±0.00 a,E 0.00 ±0.00 a,D 0.00 ±0.00 a,F 0.0 a
a,b,c
Dierent letters indicate significant statistical dierences between treatments (p<0.05).
A,B,C
Dierent letters
indicate significant statistical dierences between concentrations (p<0.05). * No statistical dierences between
treatments (p>0.05). CT1 Crotalus triseriatus 1, CT2 Crotalus triseriatus 2,CR3 Crotalus ravus 3, and CR4 Crotalus
ravus 4.
Animals 2020, 10, 281 6 of 9
In accordance with WHO, P. aeruginosa actually is in the critical priority group of the list of
antibiotic-resistant pathogens. WHO has been expressing its interest by promoting the research and
development of new antibiotics for this bacterium [10]. These results obtained herein show that CT2
and CR3 treatments demonstrated bactericidal activity against this pathogen showing its importance,
since rattlesnake venoms or compounds thereof could be used to develop effective therapeutic agents
to treat infections caused by P. aeruginosa.
3.3. Hemolytic Activity
With respect to the hemolysis produced, the generated halos showed significant statistical
differences between them (p < 0.05) (Table 3). It was observed that CT1, CT2, and CR3 showed the
highest hemolytic potential and there were no statistically significant differences between them at
100 µg/mL concentration compared with the other treatments (Figure 3).
Table 3. Hemolysis halos generated by C. triseriatus and C. ravus venoms.
Concentration
(µg/mL)
Evaluated Treatments
CT1 CT2 CR3 CR4 Nutritive Broth Tween 80
100 18.67 ± 1.53
a,A,
* 17.00 ± 1.00
a,A
18.67 ± 1.15
a,A,
* 0.0
b
0.00 20.33 ± 0.58 *
50 15.00 ± 0.0
b,B
12.33 ± 0.58
c,B
16.67 ± 0.58
a,B
0.0
d
25 13.67 ± 0.58
a,B,C
13.67 ± 1.15
a,B
13.33 ± 0.58
a,C
0.0
b
12.5 12.00 ± 1.00
a,C
10.00 ± 0.00
a,b,C
10.33 ± 0.58
b,D
0.0
c
6.25 8.67 ± 0.58
a,D
8.33 ± 0.58
a,C
7.33 ± 0.58
a,E
0.0
b
3.12 0.00 ± 0.00
a,E
0.00 ±0.00
a,D
0.00 ± 0.00
a,F
0.0
a
a,b,c
Different letters indicate significant statistical differences between treatments (p < 0.05).
A,B,C
Different letters indicate significant statistical differences between concentrations (p < 0.05). * No
statistical differences between treatments (p > 0.05). CT1 Crotalus triseriatus 1, CT2 Crotalus triseriatus
2, CR3 Crotalus ravus 3, and CR4 Crotalus ravus 4.
(a) Controls (b) Venoms
Figure 3. Indirect hemolytic activity of the rattlesnake’s venoms: (a) hemolytic activity of controls,
positive Tween 80, negative nutritive broth; (b) hemolytic activity of venoms A, B, C 100 µg/mL of
CT1; D, E, F 100 µg/mL of CT2.
Maas-Rodríguez et al., in 2014 [25] evaluated the hemolytic activity of Crotalus molossus venom
(C. molossus molossus and C. molossus nigrescens) at a 50 µg/mL concentration. In the present study,
hemolytic halos were measured over different periods of time, (1, 2, 3, and 14 h). The results obtained
showed that at 14 h halos generated measured 19.2 ± 1.5 and 17.00 ± 1.2 mm for C. molossus molossus
and C. m. molossus nigrescens, respectively, whereas at the same concentration over a longer period of
time (24 h) with the venoms of C. triseriatus and C. ravus generated smaller halos 15.00 ± 0.0 (CT1),
12.33 ± 0.58 (CT2), 16.67 ± 0.58 (CR3), and 0.00 ± 0.00 (CR4), showing the hemolytic potential of these
species is lower.
Figure 3.
Indirect hemolytic activity of the rattlesnake’s venoms: (
a
) hemolytic activity of controls,
positive Tween 80, negative nutritive broth; (
b
) hemolytic activity of venoms
A
,
B
,
C
100
µ
g/mL of CT1;
D,E,F100 µg/mL of CT2.
Mac
í
as-Rodr
í
guez et al., in 2014 [
25
] evaluated the hemolytic activity of Crotalus molossus venom
(C. molossus molossus and C. molossus nigrescens) at a 50
µ
g/mL concentration. In the present study,
hemolytic halos were measured over dierent periods of time, (1, 2, 3, and 14 h). The results obtained
showed that at 14 h halos generated measured 19.2
±
1.5 and 17.00
±
1.2 mm for C. molossus molossus
and C. m. molossus nigrescens, respectively, whereas at the same concentration over a longer period of
time (24 h) with the venoms of C. triseriatus and C. ravus generated smaller halos 15.00
±
0.0 (CT1),
12.33
±
0.58 (CT2), 16.67
±
0.58 (CR3), and 0.00
±
0.00 (CR4), showing the hemolytic potential of these
species is lower.
Animals 2020,10, 281 7 of 9
On the other hand, Pirela et al., in 2006, determined that the indirect hemolytic dose of Crotalus
durissus cumanensis venom to produce a 20 mm hemolytic halo was 379.51
±
67.67
µ
g of venom [
16
]. In
a similar study, Dos Santos et al. in 1993 obtained a dose of approximately 310
µ
g for the white venom
and 350
µ
g for the yellow venom of Crotalus durissus ruruima to produce hemolytic halos of
20 mm [26]
.
With respect to Crotalus triseriatus and Crotalus ravus venoms, an average of
18.67 ±1.53 mm
was
obtained at 100
µ
g/mL concentration of venom. Although there are no equivalent values in the
measurements of hemolytic halos, the venom of C.triseriatus and C. ravus have close values in the
measure of their halos in comparison with the other studies and in a lower venom concentration.
In accordance with Mac
í
as-Rodr
í
guez et al., in 2014 [
27
], during the fall months, there exists a high
proteomic concentration in rattlesnake venom. C. ravus and C. triseriatus were sampled in September
and November, respectively, months which correspond to the autumn, while the individuals sampled
by Pirela et al. in 2006 [
16
] were sampled in May, June, and July, months that have been shown to have
decreased protein concentration. On the other hand, in 2010 Chippaux et al., [
6
] reported that the
species C. durissus durissus and C. durissus terrificus have myotoxic and neurotoxic venoms compared to
other species of the genus Crotalus, which mostly have hemotoxic and histologic venoms [
28
]. Therefore,
due to this, in C. triseriatus and C. ravus, the highest concentration evaluated in this study (100
µ
g/mL)
was enough to produce halos with measurements similar to those of the aforementioned study.
Treatment CR4, characterized by its transparent color, did not show hemolytic activity.
This variation in color has been observed in other viperids. In the study carried out by
Macías-Rodríguez et al.,
in 2014 [
25
], C. molossus presented a yellowish venom which turned out
to be more hemolytic than Crotalus tigris venom, which was transparent in appearance, similar to
C. ravus (CR4). Gal
á
n et al., in 2004 [
29
], reported that yellowish venoms have greater toxicity compared
to white venoms. Lourenço et al., in 2013 [
30
], reported that the yellow coloration of the venom is due
to the presence of crotamine, a myotoxin from rattlesnakes.
Snake venom complexity produces a source of bioactive molecules with dierent activities.
The results
obtained in this study confirm rattlesnake’s crude venom contains compounds that could
be used as therapeutic models, in this case, molecules with antibacterial activity. Although the venom
cannot be used directly due to its high toxicity, some of its compounds will serve as prototypes for the
development of new drugs.
4. Conclusions
Until today, there are no studies reporting on the antibacterial and hemolytic activity of the
venoms of C. triseriatus and C. ravus. The results of the present study indicate that both rattlesnakes
produce venoms rich in bioactive compounds with a bactericidal eect against Pseudomonas aeruginosa.
These compounds could also serve as new antimicrobial drugs for the treatment of diseases caused by
this bacterium; however, the isolation, identification, and evaluation of these molecules is necessary
since it could present hemolytic activity.
Author Contributions:
Conceptualization and Methodology, N.R.-P. and A.Z.-B.; Validation and Formal analysis,
A.Z.-B., A.L.M.-U. and L.M.A.-C.; Investigation and Resources, N.R.-P., A.Z.-B. and L.R.-L.; Data curation, S.C.F.-A.,
C.E.R.-M. and A.O.-J.; Writing—original draft preparation, S.C.F.-A., A.L.M.-U and N.R.-P.; Writing—review and
editing, A.Z.-B., B.V.-C., A.O.-J. and L.R.-L.; Supervision, N.R.-P., C.E.R.-M. and A.Z.-B.; Project administration,
N.R.-P. and A.Z.-B.; Funding acquisition, N.R.-P., A.Z.-B. and L.M.A.-C. All authors have read and agreed to the
published version of the manuscript.
Funding:
This research did not receive any specific grant from funding agencies in the public, commercial, or
not-for-profit sectors.
Acknowledgments:
The authors would like to acknowledge to the Universidad Aut
ó
noma del Estado de Hidalgo
(UAEH) by for the support provided for carrying out the study in its facilities.
Conflicts of Interest: The authors declare no conflicts of interest.
Animals 2020,10, 281 8 of 9
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2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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