ORAL MICROBIOTA OF BRAZILIAN CAPTIVE SNAKES

The present work aimed to determine the oral microbiotic composition of snakes from São José do Rio Preto city, São Paulo State, Brazil. Ten snake species, comprising the families Boidae, Colubridae, Elapidae and Viperidae, were submitted to microbiological examination of their oral cavity, which indicated positivity for all buccal samples. Gram-negative bacilli, gram-negative cocci bacilli, grampositive bacilli and gram-positive cocci were isolated from the snakes. Among isolated bacterium species, the occurrence of coagulase-negative staphylococci in the buccal cavity of Crotalus durissus (Viperiade), Eunectes murinus (Boidae), Mastigodryas bifossatus (Colubridae) and Bacillus subtilis, common to oral cavity of Bothrops alternatus (Viperidae) and Phalotris mertensi (Colubridae), was detected. It was observed higher diversity of isolated bacteria from the oral cavity of Micrurus frontalis (Elapidae) and Philodryas nattereri (Colubridae), as well as the prevalence of gram-positive baccillus and gram-positive cocci. The composition of the oral microbiota of the studied snakes, with or without inoculating fangs, is diverse and also related to the formation of abscesses at the bite site in the victims of the ophidian accidents, and to pathogenic processes in the snakes that host these microorganisms.


INTRODUCTION
Studies on oral microbiota of snakes are rare in Brazil and the existing reports are mainly on the Bothrops genus, which is involved in most of the reported accidents (1)(2)(3)(4)(5).Envenomation caused by this snake genus is characterized by inflammation and tissue destruction that leads to infection and abscess formation (6)(7)(8)(9).
Among snakes that contribute to accidents in Brazil, four genera -Micrurus (with proteroglyph dentition); Bothrops, Crotalus and Lachesis (10,6) (with solenoglyph dentition) -are considered common.However, some snakes with opistoglyph and aglyph dentition, considered non-venomous, can also cause injuries at the bite site and, consequently, complications that determine the seriousness of the accidents (11).
Currently, several hypotheses try to explain infectious origins in lesions caused by ophidian accidents.In one of these theories, bacteria -from the venom, mouth and fangs of snakes -are involved (8,(11)(12)(13)(14).Related research has been showing a strong relationship between microorganisms present in abscesses or in patient lesions (1,2) and the ones from snake oral cavities (10,11,13).
In the present work, oral microbiota composition of snakes from the municipality of São José do Rio Preto, state of São Paulo, Brazil, was determined.

MATERIALS AND METHODS
All the analyzed snakes were from the municipality of São José do Rio Preto, state of São Paulo, and were maintained in captivity at the Animal House of São Paulo State University, São José do Rio Preto campus.
The collection of buccal samples (teeth and mucous membrane) was performed after the animal had been immobilized.Its oral cavity was exposed and a swab, previously moistened in TSB (tryptic soy broth), was introduced.The samples were placed onto plates with blood agar (Difco, USA) and incubated at 36°C, for 24 hours, in the Microbiology Laboratory of São José do Rio Preto Medical School, FAMERP.
Gram staining was performed on all different types of colonies that had grown on the agar surface.Afterwards, each colony was isolated in brain heart infusion (BHI) agar Only one collected sample from each snake was used for the microbiological study.
Bacteria isolated from snakes, classified according to genus and species, were analyzed by means of qualitative parameters for each buccal sample.

RESULTS
The test was positive for all buccal samples from animals that represented the following families: Boidae, Colubridae, Elapidae and Viperidae.Gram-negative bacilli (GNB), gram-positive bacilli (GPB), gram-positive cocci (GPC) and gram-negative cocci bacilli (GNCB) were isolated from each oral cavity.Higher bacterial diversity was found in the oral cavities of M. frontalis and P. nattereri (Table 1
In the current study, concerning the Viperidae snakes, Bacillus subtilis was isolated from B. alternatus, Yersinia enterocolitica from B. pauloensis and a coagulasenegative staphylococcus from C. durissus.Draper et al. (7), studying the microbiota from oral cavities of healthy and sick snakes, found gram-positive bacteria in healthy animals, whereas the sick ones presented, most frequently, Pseudomanas, Providencia and gram-negative bacilli.
In the current study, gram-positive bacilli were common to all oral cavities of the following snake families: Boidae, Colubridae, Elapidae and Viperidae.
These results reinforce what has been described in the literature (3, 12-14, 16, 22, 23), regarding different countries, according to the diversity of the oral microbiota of the snakes, which can be considered nonspecific and associated with the environment, with animal feeding habits and seasonality (11,20).