Long-term disability after cerebral ischemic stroke following a Bothrops atrox snakebite in the Brazilian Amazon.

Bothrops atrox envenomations in the Brazilian Amazon are responsible for a number of local and systemic effects. Among these, stroke presents the worst prognosis for the patient since it may evolve into disabilities and/or premature death. This complication is caused by coagulation disorders and generates hemorrhagic and thrombotic conditions. This study presents a case report of a 54-year-old female patient who presented extensive cerebral ischemia after a B. atrox envenomation that occurred in the state of Amazonas, Brazil. The patient was hospitalized for 102 days, which included a stay in the intensive care unit. Clinical and laboratory findings indicated a thrombogenic coagulopathy. On discharge, the patient had no verbal response, partial motor response, and right hemiplegia. The assessment carried out four years after discharge evidenced incapacitation, global aphasia and bilateral lower and upper limbs showed hypotrophy with a global decrease in strength. Ischemic stroke is a possible complication of B. atrox snakebites even after antivenom treatment, with the potential to cause debilitating long-term consequences.

Long-term hospital care needs after Bothrops atrox envenomation with hemorrhagic stroke in the Brazilian Amazon: 'From social to physical death' - A case report.

Hemorrhagic stroke is a severe complication reported in cases of Bothrops atrox snakebite envenomation. We report an unusual case of a patient who evolved with an intracranial hemorrhagic stroke and was in a coma for more than five years in a tertiary hospital located in Manaus, Amazonas. 52-year-old man, carpenter, resident in the rural area of the municipality of Tabatinga, located 1106 km from Manaus, capital of Amazonas, Brazil, victim of an accident involving Bothrops atrox evolution with cardiorespiratory arrest, acute kidney injury and hemorrhagic stroke. After 43 days of hospitalization in the ICU, he was transferred to the ward, without contact with the environment and family, sent for home treatment, however, without acceptance by family members. During a long hospital stay for a period of 6 years, totally dependent on special care, in a flexed position, using a tracheostomy and mechanical ventilation, diagnosed and treated for hospital infections throughout his hospitalization, he died due to bacterial pneumonia. Losses of autonomy can result in an individual being completely disconnected from social life - a "social death before physical death".

Bothrops atrox venom: Biochemical properties and cellular phenotypes of three highly toxic classes of toxins.

Snake venoms have a complex mixture of compounds that are conserved across species and act synergistically, triggering severe local and systemic effects. Identification of the toxin classes that are most damaging to cell homeostasis would be a powerful approach to focus on the main activities that underpin envenomation. Here, we focus on the venom of Bothrops atrox, snake responsible for most of the accidents in Amazon region of South America. We identified the key cytotoxic toxin fractions from B. atrox venom and mapped their biochemical properties, protein composition and cell damage. Five fractions were obtained by mass exclusion chromatography and contained either a single class of enzymatic activity (i.e., L-amino acid oxidases or Hyaluronidases) or different activities co-distributed in two or more protein fractions (e.g., Metalloproteinases, Serine Proteases, or Phospholipases A2). Only three protein fractions reduced cell viability of primary human cells. Strikingly, such activity is accompanied by disruption of cell attachment to substratum and to neighbouring cells. Such strong perturbation of morphological cell features indicates likely defects in tissue integrity in vivo. Mass spectrometry identified the main classes of toxins that contribute to these phenotypes. We provide here a strategy for the selection of key cytotoxic proteins for targeted investigation of their mechanism of action and potential synergism during snakebite envenomation. Our data highlights putative toxins (or combinations of) that may be the focus of future therapeutic interference.

Simultaneous identification of three clinically relevant peruvian pit vipers by multiplex loop-mediated isothermal amplification (mLAMP).

Previous knowledge about the taxonomic distribution of venomous snake species is very useful for epidemiological aspects of ophidism. Here, we sought to develop an assay for the differential identification of clinically relevant snakes in Peru: Bothrops atrox, Lachesis muta, and Crotalus durissus using a multiplex loop-mediated isothermal amplification (mLAMP) assay. For this, DNA was extracted from the shed snake skins and the mitochondrial genes Cytb, COI, and 12S rRNA were amplified and further sequenced, for the design of mLAMP reaction primers. For each snake species the forward and reverse primers, internal forward and reverse primers, and the loop primers were obtained, bearing the latter different fluorophores for product identification. Finally, the reaction was standardized in the presence of all primer sets, and an optimal amount of low molecular weight polyethyleneimine. The precipitated products were observed in a UV light transilluminator, finding a differential fluorescence according to the DNA used, with a detection limit to the naked eye in the range of 0.2-25 ng of DNA, within 30 min. This study is the first report on the use of mLAMP technology for the identification of venomous snakes.

Bothrops atrox mice experimental envenoming treatment using light-emitting diode (led) as an adjunct therapy to conventional serum therapy.

The use of anti-venom is one of the main control measures for snakebite envenoming when applied immediately after the snakebite. Systemic effects of the envenoming are usually reversed; however, neutralization of local effects is hardly achieved. The need for adjuvant therapies associated with serum therapy can improve the treatment for local effects of envenoming, with greater effectiveness in preventing or delaying the progression of damage, reducing the clinical signs and symptoms of victims of snakebites. The purpose of the study was to evaluate the photobiomodulation therapy using LED and/or dexamethasone associated with conventional serum therapy for the treatment of local damage caused by Bothrops atrox envenomation in a murine model. For this, experimental envenoming was carried out in the gastrocnemius muscle of male Swiss mice weighing 18 to 22 g divided into 8 groups of animals, distributed in groups non-treat, treated with anti-bothropic serum, dexamethasone, and LED, or the associated treatments, by intramuscular inoculation of 50 µg of venom or sterile PBS (control). After 30 min, the proposed treatments were administered alone or in combination. After 3 h, blood and muscle samples were collected for myotoxicity, cytotoxicity, histological analysis, and IL-1ß assays. The evaluation of the treatment alone showed that serum therapy is not effective for the treatment of local damage and photobiomodulation demonstrated to be an effective therapy to reduce leukocyte infiltration, hemorrhage, and myotoxicity in experimental envenoming; dexamethasone proved to be a good resource for the treatment of the inflammatory process reducing the leukocyte infiltration. The association of serum therapy, LED, and dexamethasone was the best treatment to reduce the local effects caused by Bothrops atrox venom. All in all, the association of photobiomodulation therapy using LED with conventional serum therapy and the anti-inflammatory drug is the best treatment for reducing the undesirable local effects caused by snakebite accidents involving B. atrox species.

[Determination of the species origin and thrombin-like enzyme content of Bothrops atrox venom by ultra-high performance liquid chromatography-tandem mass spectrometry based on marker peptide].

Snake venom thrombin drugs are hemostatic drugs prepared from Agkistrodon halys venom, and the main active ingredients are snake venom thrombin-like enzymes (svTLEs). The svTLEs derived from different snake species differ in their structures, hemostatic mechanisms, and pharmacological effects. Therefore, accurate identification of the source of snake venom species and determination of the svTLE content are essential to ensure the quality of these products. Based on proteomics technology, the marker peptides of svTLEs from Bothrops atrox were screened with species specificity for the first time in this study, and an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for species identification and determination of the svTLE content of Bothrops atrox was established. After reductive alkylation and trypsin enzymolysis of the purified svTLE from Bothrops atrox, enzymatic peptide fragments were obtained and determined by easy-nano liquid chromatography-quadrupole/electrostatic field orbitrap high resolution mass spectrometry (Nano LC-Q-Exactive-MS). The mass spectrum data were analyzed by Proteome Discoverer 2.2 software. The maker peptide "EAYNGLPAK", which characterized the svTLE from Bothrops atrox, was finally screened and validated by comparison of the basic local alignment search tool (BLAST) with the NCBI and UniProt databases. For the marker peptide, the enzymolysis temperature, enzymolysis time and amount of enzyme for the sample preparation were optimized. The optimized enzymolysis conditions were as follows: enzymolysis temperature, 37 ℃; enzymolysis time, 4 h; and amount of enzyme, 10 µL. A qualitative and quantitative detection method based on UHPLC-MS/MS was established by optimizing the chromatographic and mass spectrometric conditions. Accordingly, 20 mg of the evenly mixed sample was weighed and placed in a 100 mL volumetric flask. Then, 25 mmol/L ammonium bicarbonate solution was added to dissolve the sample, and the solution was diluted to the scale. Precisely 1.00 mL of the solution was extracted; subsequently, addition of 10 µL trypsin solution was added, followed by shaking, and the mixture was placed in an incubator for 4 h to induce enzymolysis at a constant temperature of 37 ℃. The mixture was subsequently removed from the incubator, cooled to ambient temperature, centrifuged at 12000 r/min for 10 min, and analyzed by LC-MS. Separation was performed on the UPLC system with a Thermo Hypersil GOLD C18 column (100 mm×2.1 mm, 3.0 µm) under the gradient elution of acetonitrile containing 0.1% (v/v) acetic acid and water containing 0.1%(v/v) acetic acid, at a flow rate of 0.3 mL/min, column temperature of 30 ℃, and injection volume of 2 µL. The maker peptides were determined in the electrospray positive ionization (ESI+) and multiple reaction monitoring (MRM) modes using the external standard curve method. The detection ions were m/z 481.9> 315.2 and 481.9> 485.2. There was a good linear relationship between the mass concentration of the marker peptide and the chromatographic peak area in the range of 2.5-30 ng/mL, and the correlation coefficient (r) was 0.9996, The limit of detection (S/N=3) and limit of quantification (S/N=10) were 2.5 mg/kg and 6.25 mg/kg, respectively. At spiked levels of 40, 80, and 120 mg/kg, the recoveries of the marker peptides were 95.5%-101.9%, while the relative standard deviations (RSDs) of the results for parallel analyses at various spiked levels were 1.1%-3.2%. The developed method is simple, rapid, sensitive, and specific, and it can be used for the identification of Bothrops atrox venom species and determination of the svTLE content. The findings of this study would help ensure the quality of hemocoagulase products from the relevant source and provide a reference for the quality control of other snake venom products.

Experimental Bothropsatrox Envenomation: Blood Plasma Proteome Effects after Local Tissue Damage and Perspectives on Thromboinflammation.

The clinical manifestations of Bothrops atrox envenoming involve local and systemic changes, among which edema requires substantial attention due to its ability to progress to compartmental syndromes and sometimes cause tissue loss and amputations. However, the impact of edema on the poisoned body's system has not been explored. Thus, the present study aimed to explore the systemic pathological and inflammatory events that are altered by intraplantar injection of B. atrox venom in a mouse model through hematologic, lipidic, and shotgun proteomics analysis. Plasma samples collected showed a greater abundance of proteins related to complement, coagulation, lipid system, platelet and neutrophil degranulation, and pathways related to cell death and ischemic tolerance. Interestingly, some proteins, in particular, Prdx2 (peroxiredoxin 2), Hba (hemoglobin subunit alpha), and F9 (Factor IX), increased according to the amount of venom injected. Our findings support that B. atrox venom activates multiple blood systems that are involved in thromboinflammation, an observation that may have implications for the pathophysiological progression of envenomations. Furthermore, we report for the first time a potential role of Prdx2, Hba, and F9 as potential markers of the severity of edema/inflammation in mice caused by B. atrox.

Functional, immunological characterization, and anticancer activity of BaMtx: A new Lys49- PLA2 homologue isolated from the venom of Peruvian Bothrops atrox snake (Serpentes: Viperidae).

Bothorps atrox is responsible for most of the ophidism cases in Perú. As part of the envenoming, myotoxicity is one of the most recurrent and destructive effects. In this study, a myotoxin, named BaMtx, was purified from B. atrox venom to elucidate its biological, immunological, and molecular characteristics. BaMtx was purified using CM-Sephadex-C-25 ion-exchange resin and SDS-PAGE analysis showed a unique protein band of 13 kDa or 24 kDa under reducing or non-reducing conditions, respectively. cDNA sequence codified a 122-aa mature protein with high homology with other Lys49-PLA2s; modeled structure showed a N-terminal helix, a ß-wing region, and a C-terminal random coil. This protein has a poor phospholipase A2 enzymatic activity. BaMtx has myotoxic (DMM = 12.30 ± 0.95 µg) and edema-forming (DEM = 26.00 ± 1.15 µg) activities. Rabbit immunization with purified enzyme produced anti-BaMtx antibodies that reduced 50.28 ± 10.15% of myotoxic activity and showed significant cross-reactivity against B. brazili and B pictus venoms. On the other hand, BaMtx exhibits mild anti-proliferative and anti-migratory effects on breast cancer cells, affecting the ROS and NADH levels, which may reduce mitochondrial respiration. These results contribute to the understanding of B. atrox Lys49-PLA2 effects and establish the anticancer potential de BaMtx.

Characteristics of Snakebite-Related Infection in French Guiana.

Wound infection is frequently reported following snakebite (SB). This study is retrospective. It was conducted in the emergency department and the Intensive Care Unit (ICU) of Cayenne General Hospital between 1 January 2016 and 31 July 2021. We included 172 consecutive patients hospitalized for SB envenoming. All patients were monitored for wound infection. Sixty-three patients received antibiotics at admission (36.6%). The main antibiotic used was amoxicillin-clavulanate (92.1%). Wound infection was recorded in 55 cases (32%). It was 19% in grade 1, 35% in grade 2, and 53% in grade 3. It included abscess (69.1%), necrotizing fasciitis (16.4%), and cellulitis (21.8%). The time from SB to wound infection was 6 days (IQR: 3-8). The main isolated microorganisms were A. hydrophila and M. morganii (37.5% and 18.8% of isolated organisms). Surgery was required in 48 patients (28.1%), and a necrosectomy was performed on 16 of them (33.3%). The independent factors associated with snakebite-associated infection were necrosis (p < 0.001, OR 13.15, 95% CI: 4.04-42.84), thrombocytopenia (p = 0.002, OR: 3.37, 95% CI: 1.59-7.16), and rhabdomyolysis (p = 0.046, OR: 2.29, 95% CI: 1.02-5.19). In conclusion, wound infection following SB is frequent, mainly in grade 2 and 3 envenomed patients, especially those with necrosis, thrombocytopenia, and rhabdomyolysis. The main involved bacteria are A. hydrophila and M. morganii.

A rational in silico approach to identify inhibitors of Batroxrhagin from Bothrops atrox.

Bothrops atrox venom comprises several types of bioactive molecules, enzymatic and non-enzymatic, among those, Batroxrhagin is the most predominant SVMP P-III enzyme, which are responsible for induction of local and systemic hemorrhage and muscle fibers damage, impairing regeneration. Due to great difficulties in establishing an antibothropic drug, new strategies must be addressed to achieve a more effective and efficient treatment. There are no studies of specific catalytic inhibitors of Batroxrhagin. However, there are in vitro studies that have described similar metalloprotease inhibitors. The inhibitor batimastat was used as a leading compound for the search and selection of similar candidates. This molecule is widely cited as a metalloprotease inhibitor and as an antimetastatic. In addition to batimastat-like molecules, four other reported metalloprotease inhibitors were included to compose the study's positive control group. Hence, 580 molecules were tested. The three-dimensional structure of B. atrox Batroxrhagin was predicted based on homologous structures using Modeller 9.20. Molecular docking calculation was performed using Autodock 4.2 and molecular surfaces and interactions were analyzed using Biovia/Discovery Studio 2017. Among 576 molecules, 42 similar to batismast resulted in a better energy of interaction than all positive controls, including batimastat itself. The batimastat-like molecules with lowest energy and positive controls were subjected to molecular dynamics for 30 ns in Gromacs 2019.4. This batimastat-like molecule produced better stability among all the Batroxrhagin-ligand complexes analyzed. Overall, the proposed compounds present justifiable evidence for future in vitro tests aiming to inhibit Batroxrhagin. Communicated by Ramaswamy H. Sarma.

Observation of Bothrops atrox Snake Envenoming Blister Formation from Five Patients: Pathophysiological Insights.

In the Brazilian Amazon, Bothrops atrox snakebites are frequent, and patients develop tissue damage with blisters sometimes observed in the proximity of the wound. Antivenoms do not seem to impact blister formation, raising questions regarding the mechanisms underlying blister formation. Here, we launched a clinical and laboratory-based study including five patients who followed and were treated by the standard clinical protocols. Blister fluids were collected for proteomic analyses and molecular assessment of the presence of venom and antivenom. Although this was a small patient sample, there appeared to be a correlation between the time of blister appearance (shorter) and the amount of venom present in the serum (higher). Of particular interest was the biochemical identification of both venom and antivenom in all blister fluids. From the proteomic analysis of the blister fluids, all were observed to be a rich source of damage-associated molecular patterns (DAMPs), immunomodulators, and matrix metalloproteinase-9 (MMP-9), suggesting that the mechanisms by which blisters are formed includes the toxins very early in envenomation and continue even after antivenom treatment, due to the pro-inflammatory molecules generated by the toxins in the first moments after envenomings, indicating the need for local treatments with anti-inflammatory drugs plus toxin inhibitors to prevent the severity of the wounds.

Individual Variability in Bothropsatrox Snakes Collected from Different Habitats in the Brazilian Amazon: New Findings on Venom Composition and Functionality.

Differences in snake venom composition occur across all taxonomic levels and it has been argued that this variation represents an adaptation that has evolved to facilitate the capture and digestion of prey and evasion of predators. Bothrops atrox is a terrestrial pitviper that is distributed across the Amazon region, where it occupies different habitats. Using statistical analyses and functional assays that incorporate individual variation, we analyzed the individual venom variability in B. atrox snakes from four different habitats (forest, pasture, degraded area, and floodplain) in and around the Amazon River in Brazil. We observed venom differentiation between spatially distinct B. atrox individuals from the different habitats, with venom variation due to both common (high abundance) and rare (low abundance) proteins. Moreover, differences in the composition of the venoms resulted in individual variability in functionality and heterogeneity in the lethality to mammals and birds, particularly among the floodplain snakes. Taken together, the data obtained from individual venoms of B. atrox snakes, captured in different habitats from the Brazilian Amazon, support the hypothesis that the differential distribution of protein isoforms results in functional distinctiveness and the ability of snakes with different venoms to have variable toxic effects on different prey.

Antimicrobial peptidomes of Bothrops atrox and Bothrops jararacussu snake venoms.

The worrisome emergence of pathogens resistant to conventional drugs has stimulated the search for new classes of antimicrobial and antiparasitic agents from natural sources. Antimicrobial peptides (AMPs), acting through mechanisms that do not rely on the interaction with a specific receptor, provide new possibilities for the development of drugs against resistant organisms. This study sought to purify and proteomically characterize the antimicrobial and antiparasitic peptidomes of B. atrox and B. jararacussu snake venoms against Gram-positive (Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus-MRSA), Gram-negative (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae) bacteria, and the protozoan parasites Leishmania amazonensis and Plasmodium falciparum (clone W2, resistant to chloroquine). To this end, B. atrox and B. jararacussu venom peptides were purified by combination of 3 kDa cut-off Amicon® ultracentrifugal filters and reverse-phase high-performance liquid chromatography, and then identified by electrospray-ionization Ion-Trap/Time-of-Flight mass spectrometry. Fourteen distinct peptides, with masses ranging from 443.17 to 1383.73 Da and primary structure between 3 and 13 amino acid residues, were sequenced. Among them, 13 contained unique sequences, including 4 novel bradykinin-potentiating-like peptides (BPPs), and a snake venom metalloproteinase tripeptide inhibitor (SVMPi). Although commonly found in Viperidae venoms, except for Bax-12, the BPPs and SVMPi here reported had not been described in B. atrox and B. jararacussu venoms. Among the novel peptides, some exhibited bactericidal activity towards P. aeruginosa and S. aureus, had low hemolytic effect, and were devoid of antiparasitic activity. The identified novel antimicrobial peptides may be relevant in the development of new drugs for the management of multidrug-resistant Gram-negative and Gram-positive bacteria.

Use of freeze-dried trivalent antivenom to neutralize the toxic activities of Bothrops atrox snake venoms from the Amazon.

In Brazil, antivenom for snakebite is currently formulated in liquid form and requires storage at 4 °C. Here, a new freeze-dried trivalent antivenom, which would enable cold-chain free storage, was determined to have efficacy in neutralizing the biological activities of Bothrops atrox venoms from Manaus (Brazil) and Leticia (Colombia), exhibiting an efficacy similar to those of currently available liquid Bothrops antivenoms. These results indicate that freeze-dried trivalent antivenom may be beneficial for applications in the Brazilian and Colombian Amazon regions.

Identification of a novel filovirus in a common lancehead (Bothrops atrox (Linnaeus, 1758)).

I performed metaviromic analysis of publicly available RNA-seq data from reptiles to understand the diversity of filoviruses (family Filoviridae). I identified a coding-complete sequence of a filovirus from the common lancehead (Bothrops atrox (Linnaeus, 1758)), tentatively named Tapajós virus (TAPV). Although the genome organization of TAPV is similar to mammalian filoviruses, our phylogenetic analysis showed that TAPV forms a cluster with a fish filovirus. However, TAPV is still distantly related to all the known filoviruses, suggesting that TAPV can be assigned as a species of a novel genus in Filoviridae. To our knowledge, this is the first report identifying a filovirus in reptiles, and thus contributes to a deeper understanding of the diversity and evolution of filoviruses.

Phytochemical composition, antisnake venom and antibacterial activities of ethanolic extract of Aegiphila integrifolia (Jacq) Moldenke leaves.

Snakebites are considered a major neglected tropical disease, resulting in around 100,000 deaths per year. The recommended treatment by the WHO is serotherapy, which has limited effectiveness against the toxins involved in local tissue damage. In some countries, patients use plants from folk medicines as antivenoms. Aegiphila species are common plants from the Brazilian Amazon and are used to treat snakebites. In this study, leaves from Aegiphila integrifolia (Jacq) Moldenke were collected from Roraima state, Brazil and its ethanolic extract was evaluated through in vitro and in vivo experiments to verify their antiophidic activity against Bothrops atrox crude venom. The isolated compounds from A. integrifolia were analyzed and the chemical structures were elucidated on the basis of infrared, ultraviolet, mass, 1H and 1³C NMR spectrometry data. Among the described compounds, lupeol (7), betulinic acid (1), ß-sitosterol (6), stigmasterol (5), mannitol (4), and the flavonoids, pectolinarigenin (2) and hispidulin (3), were identified. The ethanolic extract and flavonoids (2 and 3) partially inhibited the proteolytic, phospholipase A2 and hyaluronidase activities of B. atrox venom, and the skin hemorrhage induced by this venom in mice. Antimicrobial activity against different bacteria was evaluated and the extract partially inhibited bacterial growth. Thus, taken together, A. integrifolia ethanolic extract has promising use as an antiophidic and antimicrobial.

Failure of a Mexican antivenom on recovery from snakebite-related coagulopathy in French Guiana.

INTRODUCTION: In French Guiana, most snakebites are caused by crotalids, with the main signs being tissue damage and bleeding due to venom-induced coagulopathy. Since December 2014 the Western Guiana Hospital (WGH) has used Antivipmyn Tri TM, a Mexican polyvalent antivenom. The aim of the study was to assess its benefit on the correction of snakebite-related coagulopathy. METHODS: This retrospective study included patients hospitalized at the WGH with snakebite and a coagulopathy defined by: a prothrombin rate (PR) lower than 45%, an activated partial thromboplastin time ratio (aPTTr) greater than 2 or a fibrinogen lower than 100 mg.dL-1. The antivenom group included patients receiving Antivipmyn Tri TM from December 2014 to September 2017. The control group included patients admitted between January 2013 and November 2014 (when antivenom was unavailable) or admitted between December 2014 and September 2017 during times of antivenom shortage. We graphically compared the time courses of PR, aPTTr and fibrinogen between groups. Other endpoints were the length of hospital stay and the need for surgery or dialysis. RESULTS: 84 patients were included: 42 in the antivenom group, 42 in the control group. Both groups were similar for age, sex-ratio, proportion of bleedings, necrosis, and severity. Most patients in the antivenom group received 3 vials. There were no significant differences in recovery of PR, aPTTr and fibrinogen through the first 24 h. Fibrinogen declined again in the control group at 30 h and showed a slower rise to normal concentration. There were no significant differences in any secondary endpoint. CONCLUSION: Antivipmyn Tri TM as currently used did not show any benefit in recovery from coagulopathy.

Individual variability in bothrops atrox snakes collected from different habitats in the Brazilian Amazon: new findings on venom composition and functionality

Differences in snake venom composition occur across all taxonomic levels and it has been argued that this variation represents an adaptation that has evolved to facilitate the capture and digestion of prey and evasion of predators. Bothrops atrox is a terrestrial pitviper that is distributed across the Amazon region, where it occupies different habitats. Using statistical analyses and functional assays that incorporate individual variation, we analyzed the individual venom variability in B. atrox snakes from four different habitats (forest, pasture, degraded area, and floodplain) in and around the Amazon River in Brazil. We observed venom differentiation between spatially distinct B. atrox individuals from the different habitats, with venom variation due to both common (high abundance) and rare (low abundance) proteins. Moreover, differences in the composition of the venoms resulted in individual variability in functionality and heterogeneity in the lethality to mammals and birds, particularly among the floodplain snakes. Taken together, the data obtained from individual venoms of B. atrox snakes, captured in different habitats from the Brazilian Amazon, support the hypothesis that the differential distribution of protein isoforms results in functional distinctiveness and the ability of snakes with different venoms to have variable toxic effects on different prey.

Observation of Bothrops atrox snake envenoming blister formation from five patients: pathophysiological insights

In the Brazilian Amazon, Bothrops atrox snakebites are frequent, and patients develop tissue damage with blisters sometimes observed in the proximity of the wound. Antivenoms do not seem to impact blister formation, raising questions regarding the mechanisms underlying blister formation. Here, we launched a clinical and laboratory-based study including five patients who followed and were treated by the standard clinical protocols. Blister fluids were collected for proteomic analyses and molecular assessment of the presence of venom and antivenom. Although this was a small patient sample, there appeared to be a correlation between the time of blister appearance (shorter) and the amount of venom present in the serum (higher). Of particular interest was the biochemical identification of both venom and antivenom in all blister fluids. From the proteomic analysis of the blister fluids, all were observed to be a rich source of damage-associated molecular patterns (DAMPs), immunomodulators, and matrix metalloproteinase-9 (MMP-9), suggesting that the mechanisms by which blisters are formed includes the toxins very early in envenomation and continue even after antivenom treatment, due to the pro-inflammatory molecules generated by the toxins in the first moments after envenomings, indicating the need for local treatments with anti-inflammatory drugs plus toxin inhibitors to prevent the severity of the wounds.

Antimicrobial peptidomes of Bothrops atrox and Bothrops jararacussu snake venoms

The worrisome emergence of pathogens resistant to conventional drugs has stimulated the search for new classes of antimicrobial and antiparasitic agents from natural sources. Antimicrobial peptides (AMPs), acting through mechanisms that do not rely on the interaction with a specific receptor, provide new possibilities for the development of drugs against resistant organisms. This study sought to purify and proteomically characterize the antimicrobial and antiparasitic peptidomes of B. atrox and B. jararacussu snake venoms against Gram-positive (Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus—MRSA), Gram-negative (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae) bacteria, and the protozoan parasites Leishmania amazonensis and Plasmodium falciparum (clone W2, resistant to chloroquine). To this end, B. atrox and B. jararacussu venom peptides were purified by combination of 3 kDa cut-off Amicon® ultracentrifugal filters and reverse-phase high-performance liquid chromatography, and then identified by electrospray-ionization Ion-Trap/Time-of-Flight mass spectrometry. Fourteen distinct peptides, with masses ranging from 443.17 to 1383.73 Da and primary structure between 3 and 13 amino acid residues, were sequenced. Among them, 13 contained unique sequences, including 4 novel bradykinin-potentiating-like peptides (BPPs), and a snake venom metalloproteinase tripeptide inhibitor (SVMPi). Although commonly found in Viperidae venoms, except for Bax-12, the BPPs and SVMPi here reported had not been described in B. atrox and B. jararacussu venoms. Among the novel peptides, some exhibited bactericidal activity towards P. aeruginosa and S. aureus, had low hemolytic effect, and were devoid of antiparasitic activity. The identified novel antimicrobial peptides may be relevant in the development of new drugs for the management of multidrug-resistant Gram-negative and Gram-positive bacteria.