Oral bacterial flora of Indian cobra (Naja naja) and their antibiotic susceptibilities

Objectives The objective of the present work was to examine the bacterial flora associated with the oral cavity of Indian cobra and to study their antibiogram. Methods Oral swabs, collected from six healthy (4 males and 2 females) adult cobra, were subjected to microbiological examination through differential media. A total of 74 isolates which demonstrated noticeable colony characters were studied with different biochemical tests. The strains that showed distinctive colonies, morphology and biochemical parameters were additionally subjected to phylogenetic characterization using 16S rRNA gene sequences. Further, the isolates were subjected to antimicrobial susceptibility testing using ICOSA-20-plus and ICOSA-20-minus. Results Microscopic examination of the oral cavity of Indian cobra revealed the dominance of Gram-negative bacteria over Gram-positive. The oral microflora constituted of bacteria such as Salmonella sp. (S. typhi, S. paratyphi A); Pseudomonas sp. (P. aeruginosa, P. fluorescence); Proteus sp. (P. mirabilis, P. penneri, P. vulgaris); E. coli; Morganella sp.; Citrobacter sp. (C. diversus, C. freundii); Aeromonas sp. (A. hydrophila, A. salmonicida); Enterobacter sp. (E. aerogens); Acinetobacter sp. (A. baumannii); Neisseria sp.; Serratia sp.; Bacillus sp. (B. cereus, B. megatarium, B. atrophaeus and B. weihenstephanensis); Enterococcus sp. (E. faecalis, E. faecium); Staphylococcus sp. (S. aureus, S. epidermidis); Alcaligenes sp.; Chryseobacterium sp. and Micrococcus sp. Most of the isolates were resistant towards antibiotics such as Penicillin, Cefpodoxime, Amoxyclav, Co-Trimoxazole, Ticarcillin, Erythromycin and Nalidixic acid while sensitive towards Ciprofloxacin, Gentamicin, Ofloxacin, Sparfloxacin, Tobromycin, Ceftriaxone, Tetracycline, Novobiocin and Imipenem. Conclusions The secondary complications of the snake bite victims should be managed with appropriate antibiotics after proper examination of the bacterial flora from the wound sites.


Introduction
Snakes are distributed throughout the world and considered as threat to public health. Recent surveys reported 1220000e5500000 snakebite cases per annum globally, out of which 125000 cases lead to death or disability. An estimated 4 million cases occur annually in Asia, most being in southeastern parts [1]. India registers about 200000 snakebite cases annually but the fatality rate is not exactly known. The number of deaths varies from 1000 to 50000 as reported by different Government agencies. The variation is due to the fact that most victims of snakebite opt for village-based traditional therapists, not government hospitals. This massive statistical discrepancy has significant and urgent consequences. Mohapatra et al. [2], estimated 123000 snakebite deaths from 6671 randomly selected areas during the period 2001e2003. In India, the annual snakebite deaths were highest in the states of Uttar Pradesh (8700), Andhra Pradesh (5200) and Bihar (4500). Odisha, the eastern coastal state, registers a death rate of 5.6 per 100000 cases. People in rural areas, primarily farmers, laborers and their family members, when affected by snakebites, not always have treatment available.
In the Indian subcontinent, almost all snakebite deaths have traditionally been attributed to the big four snakes, consisting of the Russell's viper, Indian cobra, sawscaled viper, and the common krait. "Naja naja" (Linnaeus, 1758), commonly known as cobra and seen in large numbers in Odisha, is a potentially harmful snake as it inhabits around human habitations, paddy fields, bushy forests both in rural and even urbanized areas [3]. Fifty percent of snakebite deaths in Odisha is due to cobra bite and has later complication like local necrosis and sloughing of skin which takes several months to recover [3]. This extensive necrosis may be due to both venom and the contaminated microflora. Hence, the aim of the present study was to examine the associated bacteria from the oral cavity of healthy Indian cobra and study of their antibiogram.

Collection of snakes
All the snakes used in this study ( were later released back into the wild. The snakes were transferred separately in cloth bags and locked within a ventilated box. They were not given any food, drugs or antibiotics. The mouth swabs were taken after 7 day of capture. Physically inactive (unhealthy) snakes and snakes too small to produce a satisfactory oral swab were excluded from the study. The snakes were released back to the wild immediately after processing.

Swabbing procedure
The mouth of the snakes were opened by experts with the help of sterile mouth gags to facilitate swabbing of the oral cavity. Two oropharyngeal swab samples were collected from each snake using sterile cotton tipped swab sticks. Swabs were taken by rotating the cotton tip on the floor of the oral cavity and spread immediately on

Bacterial identification
The isolated strains were first identified based on their colony morphology and Gram character. Further, the strains were subjected to different biochemical characters viz. Catalase, Oxidase, Motility test, Indole, Methyl red, Voges Proskauer, Citrate, utilization of sugars and production of H 2 S in Triple sugar iron agar slant [4]. Growth of the bacteria were checked at different NaCl concentrations, temperature and pH ranges, fermentation of sugars such as arabinose, mannitol, xylose, glucose, lactose, citrate and utilization of amino acids arginine and lysine decarboxylase test. The production of extracellular enzymes namely caseinase, protease, gelatinase and lipase was studied [4].

16S rRNA gene sequencing of the isolates
Few isolates were subjected to 16S rRNA gene sequencing based on distinctive colonies, morphology and biochemical parameters. The isolates were sub-cultured from À80 C in 25% glycerol on MHA agar. Phylogenetic characterization of the isolates was carried out using 16S rRNA gene sequences amplified using with three universal

Antibiotic sensitivity assay
The isolates were tested with two groups of antibiotics: ICOSA-20-plus and ICOSA-20-minus (Himedia, India

Results and discussion
The bacteria associated with the oral cavity of N. naja were successfully isolated and characterized. The mouth cavity was shown to harbor diverse and abundant bacterial communities. A total of ninety-five colonies were isolated out of which seventy-four demonstrated noticeable colony characters and were selected for different biochemical tests. All the isolates were grouped into different Genera and species based on their similarities among biochemical features. Most of the bacteria were Gramnegative, motile with the presence of flagella. A total of 57 isolates were identified to 20 species with 18 genera while 11 isolates remained unidentified ( Fig. 1).
Among Gram-negative members, Pseudomonas and Proteus were the dominant genera followed by Salmonella, Morganella and Aeromonas. Among others, E.
coli and Acinetobacter species have proportionate distribution. Alcaligenes, Citrobacter, Enterobacter, Chryseobacterium and Serratia sp. were the minor components (Table 2). Among Gram-positive members, Bacillus and Staphylococcus dominated over other bacteria such as Enterococcus and Micrococcus (Table 3). All strains were subjected to different sugar fermentation test to identify species level (Table 4).
Strains such as 24N3, 28L2, 40X2, and 59N3 were further studied for molecular characterization by 16S rRNA sequences. These strains were selected as they showed changeable characters with repeated experiments as well as certain peculiar characteristics. Strain numbers 24N3, 28L2 and 59N3 were identified as Bacillus sp.
of which 59N3 was further confirmed up to species level by BLAST analysis of the 16S rRNA gene sequence that showed 99% similarity with Bacillus atrophaeus.
Morganella sp. Other two strains, 24N3 and 28L2, were closest to Bacillus weihenstephanensis ( Fig. 2). Similarly strain number 40X2 is further confirmed up to species level by BLAST analysis of the 16S rRNA gene sequence that showed 99% similarity with Proteus mirabilis.
The oropharynx of the Chinese cobra contained a wide range of bacteria (10 aerobic Gram-positive species, 20 aerobic Gram-negative species and 14 anaerobic species) [6]. Among Gram-negative bacteria, Morganella morganii was the commonest pathogen. Other important Gram-negative pathogens included Aeromonas hydrophila and Proteus species. Enterococcus faecalis and coagulase-negative Staphylococci were the commonest Gram-positive isolates. Various anaerobic Clostridium species were also recorded. Lam et al. [7] studied the oral bacterial flora of the same two species (N. atra and Cryptelytrops albolabris) from the same locality. Nevertheless, the most common aerobic Gram-positive bacteria were Enterococcus faecalis, Tsukamurella species and coagulase-negative Staphylococcus. A total of 41 aerobic Gram-negative bacteria species were cultured from these two species of snakes, with Morganella morganii, Pseudomonas aeruginosa and Stenotrophomonas maltophilia being the most common. Among anaerobic bacteria, the most common isolates were Clostridium  bifermentans, Clostridium baratii/sardiniense and Clostridium perfringens. Recently, Shaikh et al. [8] studied cultivable oral bacterial flora of important venomous snakes of India where Indian cobra was included (N ¼ 5). These authors reported 27 aerobic Gram-positive and 60 aerobic Gram-negative bacteria mostly dominated by enterobacteriaceae. Results of the present study matches well with Shaikh et al. [8].
Chinese cobra harbored more bacteria in the oral cavity compared to both venomous (C. albolabris) and non-venomous snakes in terms of total number of species, both pathological and non-pathological [7]. The diversity of the oral microbiota of the snakes can be considered as nonspecific and associated with the environment, animal feeding habits and seasonality [9,10]. Clostridium was the dominant genera in the Chinese cobra. However, Clostridium species were not recorded during the present study as we did not perform anaerobic cultures. The genus Neisseria is a Gram-negative coccus which mainly includes non-pathogenic species such as N. sicca and N. flavescens, These are the common members of the oral bacterial community of humans [11]. The same could not be identified in the present study, due to their complex biochemical and physiological properties. Moreover, Acinetobacter baumannii, Aeromonas hydophilla, Citrobacter diversus, C. freundii, Enterococcus faecalis, Enterobacter aerogens, Escherichia coli, Morganella sp., Proteus mirabilis, P. vulgaris, Pseudomonas aeruginosa, Serratia sp. and Staphylococcus aureus were some of the important pathogens identified in the present study and these bacteria had also been recovered from cobra bite wounds [10,12].
In India, doctors usually prescribe broad spectrum antibiotics which results a low incidence of wound infection after cobra bites. However, use of antibiotics in the management of snakebite has been criticized by many researchers [13,14]. In the