Open Veterinary Journal, (2026), Vol. 16(4): 2014-2020

Research Article

10.5455/OVJ.2026.v16.i4.5

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Bacterial diversity in certain captive snake species in Bulgaria: A One Health challenge – pilot study

Betina Boneva-Marutsova

Department of Veterinary Microbiology, Infectious and Parasitic Diseases, Faculty of Veterinary Medicine, Trakia University, Stara Zagora, Bulgaria

*Corresponding Author: Betina Boneva-Marutsova. Department of Veterinary Microbiology, Infectious and Parasitic Diseases, Faculty of Veterinary Medicine, Trakia University, Stara Zagora, Bulgaria. Email:betina.boneva [at] trakia-uni.bg

Submitted: 10/12/2025 Revised: 10/03/2026 Accepted: 25/03/2026 Published: 30/04/2026

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Abstract

Background: Snakes are becoming increasingly popular companion animals in Bulgaria, yet their potential as zoonotic pathogen reservoirs remains underexplored.

Aim: This study examined the presence of bacteria in captive pet snakes and assessed the public health risks associated with their handling.

Methods: Bacteriological analysis was conducted on 29 snake samples from pythons, boas, corn snakes, and one venomous species. Each individual was nurtured in a carefully controlled environment, receiving a specialized diet primarily consisting of frozen rodents. This approach ensures optimal health and well-being. Standard microbiological techniques were used for bacterial isolation and identification, and the results were confirmed using the Vitek 2 Compact System.

Results: Salmonella enterica (group B) was isolated from eight samples, indicating a notable prevalence of this pathogen among the examined snakes. Several other bacterial genera were identified, including Pseudomonas spp., Staphylococcus spp., Proteus spp., Enterobacteriaceae spp., Sphingomonas paucimobilis, Enterococcus spp., Bacillus spp., Achromobacter denitrificans, Citrobacter koseri, and Klebsiella pneumoniae.

Conclusion: The study highlights the potential zoonotic risks associated with snake keeping, particularly when reptiles come in direct contact or are exposed to contaminated environments. Food sources can act as potential transmission mechanisms for microbial contamination. It is essential to adopt strict hygiene practices, conduct regular monitoring of snake health, and implement appropriate feeding and cleaning protocols to minimize the risk of bacterial transmission between snakes and humans.

Keywords: Bacterial diversity, Captive pet snakes, One Health, Vitek 2.

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Introduction

In recent decades, the growing interest in reptiles, particularly snakes, as pets has led to a significant increase in their popularity across various demographics, ranging from casual hobbyists to professional researchers. These animals are appreciated not only for their unique behaviors and diverse appearances in so-called designer morphs but also for their critical roles in ecological systems. However, the surge in reptile ownership brings with it important health considerations, especially concerning zoonotic diseases that can affect humans. Reptiles carry a variety of bacterial pathogens, often as asymptomatic carriers, thereby posing potential health risks to humans (Mermin et al., 2004; Warwick et al., 2013). The ability of reptiles to disseminate pathogenic microorganisms without manifesting clinical signs complicates the awareness and management of health risks associated with their care. A deeper understanding of the oral and cloacal microbiota in snakes is essential for evaluating the effects of these pathogens on animal health and public safety. Some research indicates that the snake’s diet and associated flora significantly influence the composition of their microbiota (Song et al., 2025). It has been suggested that the cloacal flora from the prey of snakes may be transferred to the snakes during feeding. This transmission could alter the microbial composition of snakes, affecting their digestion and overall health (Mermin et al., 2004; Whiley et al., 2017). Given the critical importance of bacterial interactions, studies that thoroughly characterize the microbial diversity and distribution in reptiles are lacking (Sodagari et al., 2020). Gram-positive bacteria, such as Staphylococcus and Bacillus spp., dominate the microbiota of healthy snakes (Atravia-Leon et al., 2017; Young Yusty and Prescilla-Ledezma, 2025). Gram-negative pathogens such as Pseudomonas aeruginosa, Providencia rettgeri, and Stenotrophomonas maltophilia are often found in snakes suffering from stomatitis (Lin and Tsai, 2023). Members of the Enterobacteriaceae family have also been implicated in human respiratory diseases (Pasmans et al., 2020). Salmonella spp. are among the most concerning pathogens linked to reptiles, especially because of their severe health impacts on humans, particularly those in vulnerable populations (Mermin et al., 2004; Lin and Tsai, 2023). Although reptile-associated salmonellosis represents a small percentage of overall salmonellosis cases in Europe and the USA, its clinical course can be markedly severe, especially in young children, the elderly, and pregnant women, contributing to numerous disease outbreaks globally (Meletiadis et al., 2022; Pees et al., 2023). The presence of opportunistic pathogens, such as Pseudomonas spp., Staphylococcus spp., and Proteus spp., further emphasizes the need for careful management and awareness regarding the risks posed by these reptiles (Jacobson, 2007).

Therefore, as the trend of keeping reptiles continues to rise, it becomes increasingly critical to educate potential owners about the associated health risks. Effective hygiene practices, proper handling techniques, and regular veterinary care are vital strategies for mitigating the risk of zoonotic infections. Consequently, promoting responsible ownership and facilitating the dissemination of accurate information are essential for ensuring safe coexistence between humans and reptiles (Pasmans et al., 2017). Therefore, fostering responsible reptile ownership and sharing accurate, evidence-based information are crucial for achieving a safe coexistence between humans and reptiles.

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Materials and Methods

Fecal swab samples were collected from the cloaca of 29 healthy adults of different snake species (Table 1). Snakes were kept as pets in five different private terrariums in Bulgaria. Every individual is raised in their unique environment. All animals included in the study (n=29) were clinically healthy adult snakes, as determined by a routine physical examination conducted before sampling. Health status was assessed based on the following criteria: normal body condition, absence of visible lesions or ectoparasites, normal posture and behavior, and standard coloration of the oral and cloacal mucosae. At the time of examination, no signs of respiratory, gastrointestinal, or neurological disease were observed.

Table 1. Types of snake species.

Samples were collected using sterile cotton swabs under aseptic conditions. Swabs were gently inserted into the cloaca and rotated to ensure adequate contact with the mucosal surface. After collection, each swab was placed into a sterile transport medium and labeled. The samples were stored at 4°C and transported to the microbiology laboratory within 24 hours for immediate processing and bacteriological analysis.

Body weight and sex were not recorded, as these factors were not considered relevant to this study’s objectives.

Each snake was sampled individually with new sterile swabs and gloves to reduce the risk of cross-contamination. All sampling instruments and work surfaces between each animal were disinfected. Samples were processed separately in the laboratory following standard aseptic techniques.

All samples were cultured in liquid enrichment media (Tryptic Soy Broth and Selenite Broth HiMedia™) and on solid media using the four-quadrant streak plate method (Blood Agar Base with 5% defibrinated ovine blood and MacConkey agar, HiMedia™) (Sanders, 2012; Seth-Smith et al., 2025). The incubation process typically occurs at 37°C for 24 hours (Mariam, 2021). After this incubation, an automated biochemical profiling by Vitek 2 Compact system (bioMérieux, France) was used for more precise and rapid identification (Funke and Funke-Kissling, 2004; Boneva-Marutsova et al., 2025).

Data analysis

Statistical analysis was conducted using IBM^® SPSS^® version 26.0. Data were summarized using descriptive statistics, and the prevalence of each bacterial species was calculated as the proportion of positive samples, expressed as percentages with 95% confidence intervals (95% CI). The Shapiro–Wilk test was used to assess the normality of data distribution. Non-parametric statistics were used due to the non-normal distribution and small sample size (n=29), presenting results as median and interquartile range (IQR). The confidence intervals for prevalence estimates were calculated using the Wilson binomial method, suitable for small sample sizes. Sampling followed a convenience approach, and each sample was independently analyzed. This study focused on descriptive statistics rather than inferential hypothesis testing due to its exploratory nature and limited sample size.

Ethical approval

No ethical review and approval were required for the animal study, as we are presenting the results of laboratory diagnostic tests. We have obtained the consent of the animals’ owners to publish the results for scientific purposes.

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Results

In this study, the prevalence of various bacterial species was summarized using descriptive statistics. The frequency of positive samples varied considerably among the different microorganism species (Table 2 and Fig. 1). Due to the small sample size (n=29) and the variability observed among bacterial groups, the median was employed as the primary measure of central tendency. Overall, the prevalence values exhibited a wide distribution across the identified species. Staphylococcus species had the highest prevalence at 62.07% (95% CI: 44.1–80.1), followed by Enterococcus species at 44.83% (95% CI: 26.6–63.0). The overall median prevalence across all bacterial groups was 27.59%, indicating that approximately one-quarter to one-third of the samples contained 50% of the detected taxa. The IQR, which extended from 20.69% to 31.03%, indicates a moderate dispersion around the median.

Table 2. Prevalence of isolated bacterial species from samples (n=29).

Fig. 1. Isolated bacterial species, % (blue – Gram-positive bacteria; red–Gram negative bacteria).

Several bacterial groups, including Salmonella species (27.59%, 95% CI: 12.8–42.3), Enterobacteriaceae (27.59%, 95% CI: 12.8–42.3), and Pseudomonas species (27.59%, 95% CI: 12.8–42.3), exhibited prevalence values that matched the median. A slightly higher prevalence of 31.03% (95% CI: 15.1–46.9) was observed for Bacillus species.

Lower prevalence values were noted for opportunistic Gram-negative bacteria, such as Achromobacter denitrificans, Sphingomonas paucimobilis, Citrobacter koseri, and Klebsiella pneumoniae, each detected in 20.69% of the samples. These species shared identical 95% CIs ranging from 6.7% to 34.6%, indicating comparable detection levels across the sampled population. This finding highlights the relatively high level of asymptomatic carriage in snakes and the potential public health risk associated with reptile handling.

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Discussion

The results of this study show that a substantial proportion of the examined snakes (59.4%) were carriers of potentially pathogenic bacteria. These results are consistent with those of other studies reporting complex polymicrobial microbiota in domestic snakes (Krishnankutty et al., 2018; Su et al., 2023).

Our findings reveal a diverse spectrum of bacterial isolates: the most common genera were Staphylococcus spp. (62%), Enterococcus spp. (44.83%), and Bacillus spp. (31.03%), alongside Salmonella spp., members of Enterobacteriaceae, and Pseudomonas spp. (27.59%). This finding is consistent with international data indicating that between 30% and 80% of reptiles are chronic carriers of Salmonella spp. (Pees et al., 2023; Muslin et al., 2025). Salmonellosis outbreaks have been linked to a wide range of sources, including contaminated food, water, and contact with reptiles, particularly pet snakes and turtles (Muslin et al., 2025). These events highlight the importance of hygiene and public awareness among reptile owners and individuals in close contact with such animals.

The isolation of S. enterica group B in 15.6% of the samples aligns with the reported prevalence values (Mermin et al., 2004; Chen et al., 2010). Several authors and health institutions have emphasized that contact with reptiles, including snakes, is associated with an increased risk of salmonellosis, especially in children, the elderly, and immunocompromised patients (Marin et al., 2018; Varela et al., 2022; Waltenburg et al., 2022). Our results, demonstrating the simultaneous presence of Salmonella spp. and Pseudomonas spp., confirm the view that snakes kept as pets or in zoos can act as a reservoir and mechanical vector of zoonotic pathogens, including multidrug-resistant strains (Ebani, 2017; Marin et al., 2018; Dégi et al., 2023). When studying bacteria in snakes, it is important to recognize that not all bacteria are harmful; many coexist without causing disease. Some of them probably represent normal or conditionally normal flora adapted to the specific physiology and ecology of reptiles. A meta-analysis of the gut microbiota in reptiles revealed a limited “core” of bacterial taxa and a significant influence of both the environment and the host on the microbiome composition (Hoffbeck et al., 2023). In this context, the bacteria isolated by us should be interpreted both as an indicator of bacterial contamination and as part of the captive snakes’ natural microbiome profile.

Another factor that may influence bacterial colonization in snakes is related to food sources, specifically frozen rodents. Some researches indicate that 8%–10% of commercially supplied frozen rodent packages for reptile feeding contain Salmonella spp. (Marin et al., 2018). Frozen feeder rodents should be recognized as a significant reservoir of pathogens that can colonize reptiles' gastrointestinal tracts without causing any clinical signs (Marin et al., 2018; Shivambu et al., 2023; Muslin et al., 2025). The isolation of Salmonella spp. (27.59%) and other members of the Enterobacteriaceae family, such as C. koseri and K. pneumoniae, is essential from a public health perspective. A recent systematic review and meta-analysis revealed that the overall prevalence of Salmonella in reptiles is high, often exceeding 50% in snakes, which confirms their role as a reservoir for the bacteria (Muslin et al., 2025). Additionally, a review by Pees et al. (2023)highlights that Salmonella in reptiles is considered part of the normal intestinal microbiota, with continuous and intermittent shedding into the environment, posing significant zoonotic potential.

Citrobacter koseri and K. pneumoniae are well-described opportunistic pathogens associated with urinary tract infections, respiratory tract infections, neonatal meningitis, and nosocomial outbreaks in humans. Their isolation from snakes raises the question of the transfer of antimicrobial-resistant strains from animals to humans, especially in the context of multidrug-resistant enterobacteria (Marin et al., 2018; Su et al., 2023; Dégi et al., 2023; Marques et al., 2025).

The isolation of A. denitrificans, S. paucimobilis, and other less common genera aligns with observations that the reptile microbiome contains a variety of opportunistic bacteria, each with different potential for pathogenicity (Artavia-León et al., 2017; Lin and Tsai, 2023; Marques et al., 2025). Sphingomonas paucimobilis and related species have been identified as the causative agents of nosocomial infections in immunocompromised patients. Although these bacteria are found in the environment, they can also colonize animals without causing any clinical symptoms. Several studies have highlighted the potential role of snake oral microbiota as a reservoir for multidrug-resistant Gram-negative pathogens (Chuang et al., 2022; Marques et al., 2025; Young Yusty and Prescilla-Ledezma, 2025). Recent high-throughput 16S rRNA sequencing studies of snake oral cavities have shown a predominance of gram-negative bacteria, such as Pseudomonas, Aeromonas, and Enterobacteriaceae. Although Gram-positive cocci are less abundant, they remain stable microbiome components (Pasmans et al., 2020; Hoffbeck et al., 2023). High frequencies of Staphylococcus spp. (62%) and Enterococcus spp. (44.83%) were found, which aligns with the existing literature. In certain species, especially those kept in captivity, Gram-positive bacteria—including coliforms and coagulase-negative staphylococci—can dominate the oral cavity (Dehghani et al., 2016; Chuang et al., 2022). This prevalence is likely linked to human exposure and contact with feed, substrate, and surfaces in the artificial environment. Non-venomous snakes can pose significant risks to humans even without venom due to their potential to carry harmful pathogens. These pathogens can cause serious infections and diseases, underscoring that snakes are not only venomous. Understanding the ecological roles and risks associated with both venomous and non-venomous species is crucial for effectively addressing public health and safety concerns.

Preventive measurement

Numerous studies and guidelines emphasize the importance of maintaining good hygiene practices, especially in environments where there is a risk of exposure to certain animals or pathogens. Key recommendations from various sources highlight the need to thoroughly wash hands after any contact with animals or their habitats. This simple yet effective practice helps prevent the transmission of potential infectious diseases. Avoiding unnecessary contact with snakes, particularly in areas such as kitchens where food is prepared, is also advisable, as this can lead to contamination and health risks. Proper storage of pet food is equally important; keeping it in secure containers can prevent the introduction of pests and reduce the likelihood of animal interactions. Furthermore, keeping children away from terrariums or habitats containing reptiles is essential to mitigate the potential dangers associated with these animals. Collectively, these guidelines stress the importance of vigilance and education to ensure personal safety and public health (European Food Safety Authority, 2019; Centers for Disease Control and Prevention, 2025).

Sample recommendations for snake owners:

• Wash hands with soap and warm water after handling snakes, enclosures, equipment, or feeding rodents.
• Terrariums and cleaning products should be kept away from kitchens/food preparation areas.
• Be careful with children and immunocompromised individuals.
• Use individual equipment (brushes, buckets) for terrariums and regularly disinfect them.
• Store and thaw rodents that are feeding separately from human food, and be sure to clean and disinfect surfaces afterward.
• Wear disposable gloves when cleaning enclosures or handling snakes.
• Seek veterinary advice for unusual and suspicious behavior or suspected illness.

Read about potential zoonotic risks when purchasing/adopting an animal.

Limitations and future directions

The absence of antibiograms, molecular typing of the isolates, and possible cross-contamination are limitations of the current analysis, which would allow a more precise assessment of the zoonotic risk and the potential for the spread of resistant clones. Given the increasing number of data on multidrug-resistant strains of Salmonella, Citrobacter, and Klebsiella isolated from both humans and animals, future studies should include a systematic analysis of antibiotic susceptibility and genetic determinants of resistance (Marin et al., 2018; Dégi et al., 2023; Hoffbeck et al., 2023; Pees et al., 2023; Lamichhane et al., 2024).

Due to the limited quantity of feces obtained during the initial examination, a larger sample was collected after defecation for further testing, specifically for parasitological examination (Abdisa, 2018).

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Conclusion

Non-venomous snakes can be almost as dangerous as venomous ones, even without venom, because they can carry harmful pathogens to humans. Domestic snakes, particularly the genus Salmonella, are increasingly recognized as a significant reservoir of pathogenic bacteria with well-documented zoonotic potential. These bacteria can cause serious gastrointestinal diseases in humans, especially in vulnerable populations, such as children and immunocompromised individuals.

One potential route of transmission is through food practices, particularly the feeding of frozen rodents that may contain these pathogens. Without adequate hygiene practices, handling and preparing such food products can create opportunities for cross-contamination and subsequent infections.

It is imperative to implement strict hygiene protocols to mitigate the risk of transmission and protect the health of both humans and animals. This includes regular cleaning and disinfection of snake habitats, proper hand washing after handling snakes or their food, and snake owners’ education on safe handling practices.

Additionally, public health campaigns targeting pet owners could play a crucial role in raising awareness about the risks associated with pet snakes and the importance of preventive measures. We can help reduce the incidence of zoonotic infections associated with these fascinating reptiles by promoting a better understanding of the potential health effects and necessary precautions.

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Acknowledgments

None.

Conflicts of interest

The author has no conflicts of interest to declare.

Funding

This research received no external funding.

Author contributions

One author for this study. Conceptualization, Methodology, Investigation, Writing-original draft, Writing-review & Editing: B. B-M.

Data availability

Data supporting the findings are available from the corresponding author upon reasonable request.

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