Antimicrobial activity of Amazonian plant species against the causative agents of secondary infection in snakebites

Riverine communities in the Brazilian Amazon region use a variety of plants to treat snakebites. These plants can be effective against secondary infections, one of the main complications of snakebites. The aim of this study was to determine whether plants traditionally used to treat snakebites in the Brazilian Amazon may also have antimicrobial and antioxidant activities, and if so, which classes of chemicals may be responsible for these activities. Aqueous extracts of nine plants were tested in microdilution assays and the more active were prepared using solvents (hexane, methanol and water) and decoction, and nine assays were performed. Assays to determine the antioxidant activity of the most active species were carried out, as well as phytochemistry studies to determine the active components of this species. Bellucia dichotoma exhibited the greatest antimicrobial potential, particularly the hexane, methanol and decoction extracts. In comparative TLC, extracts of this species showed characteristics of terpenoids, compounds with double bonds and flavonoids. In 1H NMR, characteristic signals of sterols such as β-sitosterol, stigmasterols or triterpenes were observed, as well as signals indicating the presence of aromatic hydrogens, characteristic of aromatic substances, and sugars. The methanol extracts and decoction were considered active in the antioxidant assay.


Introduction
In the Amazon region, snakebites continue to be a public health concern. The long distances that must be covered between the accident site and the nearest medical center where specialized care can be provided, Revista Fitos the unreliable transport (very often by river) and the frequent lack of snake antivenom reflect the distressing reality faced by patients, who may have to wait from six to more than twelve hours for medical care and may only receive a smaller dose of the antivenom than recommended or even none at all, with the consequent risk of death [1][2][3] .
Among the snakebite envenomations reported in the region, those caused by Bothrops atrox (known locally as jararaca, jararacuçu, jararaca-do-norte, jararaca-de-rabo-branco, surucucurana and surucucu-dobarranco) are the most common [1,4] . Care is required when using popular names to report snakebites by B.
atrox as the term surucucu is frequently also used for Lachesis muta, whose bite causes similar symptoms but requires different treatment [5][6][7] .
The treatment recommended by the World Health Organization is snake antivenom, which often does not reach the patient in a satisfactory condition for several reasons, such as problems with distribution and storage in remote areas and insufficient training of healthcare staff in these areas to ensure ideal management of snakebite cases [8] . Furthermore, snake antivenom neutralizes mainly the systemic effects of the bite (such as hemorrhage, myolysis, hemolysis, paralysis, kidney injuries and abnormal coagulation) but not the local effects (such as intense pain, edema, blisters, ecchymosis and muscle necrosis), leaving the door open for complications and sequelae in the affected limb [2,9] .
One of the main clinical complications caused by snakebites is secondary infection, which worsens the clinical picture, leading to abscesses, infectious cellulitis, necrotizing fasciitis, gangrene, sepsis and toxic shock syndrome, in turn very often leading to limb amputation or loss of limb function [10][11][12] . Secondary infection can be monomicrobial or polymicrobial, and the microorganisms that cause the infection, which are inoculated at the time of the snakebite, may come from the oral cavity of the snake or the victim's skin. The damaged wound tissue itself may act as a source for bacterial colonization and may result in complications in the affected limb [13] .
As a result of the difficulties they face, the Amazonian population, particularly the riverine population, which lives far from major centers and is most affected, ends up using alternative or complementary treatments such as those based on plants in the form of infusions, tinctures and other herbal preparations to treat snakebites [1,32,33] . An ethnobotanical study by Moura et al. [9] of healers and inhabitants in areas with a high incidence of snakebite envenomations in the Santarém-PA region identified 24 plants used to treat snakebites. In vitro and in vivo tests of some of these plants showed that they can potentially be used to treat the effects of Bothrops atrox and Bothrops jararaca venoms [34,35,9,[36][37][38] . The discovery of a plant species, or even a combination of plant species, that could block the local effects of snake venom and inhibit or reduce growth of the microorganisms responsible for secondary infection would be a major step forward as such plants could be used to complement conventional treatment. In this study we evaluated the potential antimicrobial activity of aqueous extracts of nine plants used in folk medicine (TABLE 2) that have one or more anti-snakebite properties.

Plant material
The plant species studied were collected in the Santarém region (PA) close to the Vila de São Pedro and lyophilized [9,36] .

Antimicrobial assay
Minimum inhibition concentration of the extracts was determined by the broth microdilution method (Mueller-Hinton Broth) and spectrophotometry at 625 nm. Each assay was performed in triplicate and expressed as mean ± standard deviation of the mean. The means were compared by two-way ANOVA followed by the post hoc Tukey test using p<0.05. The following microorganism strains were used for the assays: AB

Phytochemical study of Bellucia dichotoma
The extracts were prepared from 675 g of bark powder. Of the 675 g, 275 g were used for decoction with a bark mass-to-water volume ratio of 1:10 following the protocol described by Mourão et al. [35] and 400 g were

Antioxidant activity
The antioxidant activity of the Bellucia dichotoma extracts was determined by two methods: (i) measurement of the extract's ability to sequester Fe 3+ free radicals and (ii) the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. In both cases the results were expressed as ascorbic acid equivalents [39] .

Results and Discussion
Of the nine plant species investigated (  (GRAPHS 1, 2, 3). The means were compared by two-way ANOVA followed by Dunnett's post hoc test using p<0.05. The results in Graphs 1 to 4 are expressed as mean absorbance at 586 nm: the greater the value of absorbance, the greater the turbidity due to microbial growth, and the smaller the value of absorbance, the greater the growth inhibition caused by the extract.
New extracts of Bellucia dichotoma and Connarus favosus were prepared, this time with solvents of increasing polarity (hexane, methanol and water) and tea (decoction), and then used in new antimicrobial activity assays (GRAPH 4). Shows that the best results for antimicrobial inhibition were achieved with the hexane and methanol extracts and decoction (tea) of B. dichotoma.
In CTLC, the hexane extract exhibited purple spots when developed with ceric sulfate and lilac stains when developed with anisaldehyde, both indicative of terpenoids. When developed with iodine, the extract showed characteristics of double bonds, and when developed with NP-PEG at 365 nm there was an increase in the fluorescence intensity of spots characteristic of flavonoids.   The antimicrobial activity of nine plant species used in folk medicine to treat snakebites was tested against gram-positive and gram-negative strains of bacteria and two yeasts that can be found in the oral cavity of snakes and in abscesses caused by snakebites.
Although snake venom itself has antimicrobial properties, the bacterial microbiota in the oral cavity of snakes varies. Some studies have shown that the composition of this microbiota reflects the fecal flora of the snake's prey, generally rodents, amphibians, reptiles and small birds, as these normally defecate when they are being ingested [40,41,11,42,43,12,44] . According to other studies, the composition of the oral microbiota in snakes is related to the species of snake rather than its diet. In addition to suggesting that bacteria can play an important role in the production of enzymes for snakes to digest their prey, these studies showed that adult individuals had the same microbiota composition as neonates before their first meal and that, in captivity, different species of snake fed the same food in the same environment had different oral microbiota [45] .
Interestingly, snakes of the species Bothrops insularis, which are endemic to the isolated Queimada Grande Island in Brazil and have distinct eating habits, have very similar microbiota to that found in Bothrops sp. species on the mainland [23] .
Many cases of secondary infection can be attributed to the oral microbiota of snakes. A particular cause for concern is the recent publication of studies describing bacterial strains isolated from snakes or snakebites that are resistant to the antibiotics normally prescribed in such cases [17,46] .
Brazilian researchers have suggested various measures to address the problem of envenomation by animals (such as snakes and scorpions) in the Amazon region, including support for studies on the use of phytomedicines as alternative or complementary therapies [8] .  [32][33][34][35]9,[36][37][38] . In the present study we sought to evaluate plant species that not only show activity against the Bothrops venom, but also can act on bacterial strains that cause secondary infection in snakebite envenomations.
After antimicrobial screening assays with nine species whose anti-snakebite potential has been tested previously in other studies (Aniba fragrans (synonym: A. parviflora) Bellucia dichotoma is a tree from the family Melastomataceae, which is native to, but not endemic in, Brazil and is found in the states of Amazonas, Amapá, Pará and Rondônia in areas with a human presence and terra firme forests [47] . It is known popularly in the region as muúba, araçá-de-anta or goiaba-de-anta.
According to Moura et al. [9] , it is used in the form of tea (decoction) by riverine communities to treat snakebites. The tea is prepared in a ratio of 100 g of plant extract to 1L of water, of which 150 mL is drunk immediately after the victim is bitten and the remainder is drunk three times a day thereafter. The antisnakebite properties of the aqueous extract have been demonstrated in studies by Moura et al., who found that it can inhibit local effects, such as edema and the hemorrhagic and clotting activities induced by Bothrops atrox venom, can inactivate phospholipase A2 in the venom and is an antioxidant [35,9,37] .
The CTLC and NMR assays yielded results suggestive of the presence of terpenoids such as sterols (possibly β-sitosterol), triterpenes and flavonoids in Bellucia dichotoma extracts prepared with hexane. Using colorimetric assays of the aqueous extract in an earlier study, Moura et al. [34] , also identified terpenoids and flavonoids in B. dichotoma. These classes of substances were observed in various studies of species of the family Melastomataceae [48][49][50] and may be associated with the antimicrobial and antioxidant properties of extracts from these plants.
According to some studies, the ability of triterpenes and flavonoids to inhibit microorganisms such as Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae can be explained by the fact that these classes of metabolites can damage and disrupt the bacterial cell membrane and interfere with cell metabolism, cellular biosynthesis of essential components and the cell cycle [51,52] .
In the assessment of antioxidant activity, the methanol extract and decoction (the most polar of the four preparations used here) had the greatest activity. Antioxidant activity plays an important role in healing as although generation of ROS (reactive oxygen species) is essential in the initial stages of wound healing (e.g., abscesses caused by snakebites) and the removal of microorganisms, excessive production of these oxidants can delay wound healing and cause greater tissue damage [53] . The antioxidant activity of the extracts studied here may thus help to restore tissue integrity.