Broad Antibacterial Activity of Bothrops jararaca Venom against Bacterial Clinical Isolates

Purpose: To evaluate the antibacterial activity of Bothrops jararaca venom against bacterial clinical isolates. Methods: Antibacterial activity of Bothrops jararaca venom was evaluated through agar diffusion method against the following bacteria: Acinetobacter baumannii, Oxacillinase-producing Acinetobacter baummanii, extended-spectrum β-lactamase-producing (ESBL) Enterobacter aerogenes, Escherichia coli, Escherichia coli ESBL, Klebsiella pneumoniae, Klebsiella pneumoniae ESBL, Proteus mirabilis, Pseudomonas aeruginosa, metallo β-lactamase-producing Pseudomonas aeruginosa, Staphylococcus aureus, oxacillin resistant Staphylococus aureus (ORSA), Staphylococcus epidermidis, and oxacillin resistant Staphylococus epidermidis. Minimum inhibitory concentration was determined through microdilution plate protocol. Results: The venom presented antibacterial activity against all tested bacteria. More pronounced results were observed to Grampositive bacteria, especially against ORSA. Conclusion: The present study evidenced the great antibacterial potential of Bothrops jararaca venom showing promising results even with resistant bacterial clinical isolates.


Introduction
Increasing prevalence of multiresistant bacteria, mainly related to hospital environment, is a public health problem and elicits a negative economic impact due to increasing time of hospitalization, which brings morbidity and high costs associated with increased number of pharmacological approaches trying to counteract multiresistant bacteria and restore health [1] [2].The inadequate use of antimicrobial and recurrent bacterial infections generates resistant strain [3].Considering that bacteria becomes resistant in about 10 hours through mutations and that it takes about 10 to 24 years to develop new drugs, research focusing on compounds with promisor antibacterial activity is warranted [4].
In this context, venom from venomous animals is a rich source of protein and non-protein compounds of pharmacological interest [5].The antimicrobial activity of the Bothrops jararaca (Taxonomy ID: 8724) crude venom was showed against Staphylococcus aureus [6] and the isolated peptide from B. jararaca (Pep5Bj) inhibited the growth of different fungi (Fusarium oxysporum, Colletotrichum lindemuthianum, Candida albicans and Saccharomyces cerevisiae) [7].Considering the relevance of the topic, we conducted a study on the antibacterial potential of Bothrops jararaca crude venom against bacterial clinical isolates.

Protein Quantification of B. jararaca Venom
Crude venom was lyophilized in order to prevent proteolysis [8] and was kept at −20˚C.Protein content was quantified through Bradford method using albumin bovine serum as standard [9].Lyophilized venom was suspended in NaCl 0.9%, under laminar flow, and filtrated using a 0.20 µm membrane (Sartorius Stedim Biotech, Göttingen, LS, Germany) in order to obtain a concentrated solution containing 2 mg/mL of protein.New solutions were prepared daily.
In order to allow bacterial growing, clinical isolates were kept in blood agar at 37˚C, for 24 hours, in aerobic conditions.ATCC and NEWP strains were recovered in nutritive broth and cultured in Petri dishes containing Müeller-Hinton agar.After growing, bacterial colonies were collected and suspended in 5 mL de NaCl 0.9%.Turbidity was adjusted to 0.5 in McFarland scale, which corresponds to 2 × 10 8 UFC/mL [1].Antibacterial activity assay was performed after 24 hour after the turbidity adjust.
Diffusion disks containing 10 µL of crude snake venom solutions in a concentration range of 2 mg/mL, 500 µg/mL, 250 µg/mL, 100 µg/mL, 50 µg/mL, and 25 µg/mL was added to petri dishes with above cited bacteria strains.Negative control was NaCl 0.9% and positive controls were disks containing imipenem (10 µg/mL), vancomycin (30 µg/mL), and polymyxin B (300 µg/mL) to enterobacteria, Pseudomonas aeruginosa and Staphylococcus aureus, respectively.Dishes were incubated at 37˚C, for 24 hours, as recommended by Clinical Laboratory Standards Institute [10].Antibacterial activity was considered in the presence of an inhibition zone around the diffusion disk, measured in millimeters.Each condition was tested in triplicate.

Statistical Analysis
Statistical analysis was performed using One-Way ANOVA followed by Tukey post hoc test.Results presenting a p < 0.05 were considered as statistically significant.

Broad Antibacterial Activity of Crude B. jararaca Venom
The protein content of the crude venom was 245 µg/mL.The present work evidenced that crude B. jararaca venom presented antibacterial activity against all clinical isolates, ATCC and NEWP bacterial strains tested, as shown in the Table 1 (images with the agar diffusion test are shown in supplementary material).The magnitude of inhibition zones showed that better results were obtained against Gram-positive bacteria, especially oxacillin resistant Staphylococcus aureus.Hence, MIC assay was performed only for Gram-positive bacteria (Table 2).

Discussion
The major finding of this work was to evidence the wide spectrum antibacterial activity of crude venom isolated from B. jararaca.For the first time, it was evidenced that crude snake venom was effective against both Grampositive and Gram-negative bacteria from commercial sources and clinical isolates presenting resistance mechanisms.It highlights that it might be considered a promisor source to be explored by pharmaceutical industry.
To the best our knowledge, it is the first time that crude venom from B. jararaca presents wide spectrum antibacterial activity, being effective against all Gram-positive and Gram-negative tested strains, even those expressing resistance mechanisms.In the past, venom from B. jararaca (0.8 mg/mL) inhibited bacterial growth of Eubacterium lentum, Peptoestreptococcus anaerobius, Porphyromonas gingivalis, Prevotella intermedia, Propionibacterium acnes, Pseudomonas aeruginosa, Salmonella typhimurium, Staphylococcus aureus and Staphylococcus epidermidis.However, it failed against Bacteroides fragilis, Eikenella corrondes, Enterococcus faecalis, Escherichia coli and Streptococcus mutans [5].Moreover, Ferreira (2007), using the high concentration of 5 mg/mL demonstrated that B. jararaca venom presents antibacterial activity only against Staphylococcus aureus.However, in that study, author used a conservative criterion since it was considered positive for antibacterial activity inhibition zones ≥ 15 mm.It is important to point that other bacteria also presented inhibition zones, namely Enterobacter cloacae (6 mm), Enterococcus faecalis (13 mm), Escherichia coli (5 mm), Pseudomonas
It is important to refer that the present study is a screening since crude venom was used.Regarding promising results obtained, next steps involve working with different isolated fractions from B. jararaca in order to elucidate which fraction are responsible/more effective to the antibacterial activity.Considering known compounds present in the bothropic venom, the enzyme LAAO is frequently associated to antibacterial activity [16].LAAO is an oxidoreductase involved in the stereospecific oxidative desamination of L-amino acids, producing ammonia and hydrogen peroxide which can cross membranes and induce the oxidation of macromolecules such as protein, lipids and DNA [5] [16]- [19].

Conclusion
In summary, the present work evidenced that B. jararaca venom is a promising source from antibiotics development research.Its antibacterial activity is warranted to be explored considering the good results even with clinical isolates presenting resistant mechanisms.However, additional studies are needed in order to elucidate which substance in this complex matrix is in charge of antibacterial activity.

Table 1 .
Inhibition zones around diffusion disks containing antibiotic (positive control) or B. jararaca crude venom, indicating antibacterial activity.Results are expressed in mm of diameter (mean of 3 independent experiments).

Table 2 .
MIC from crude venom of B. jararaca against Gram-positive clinical isolates.