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Reptiles appear to be an important vector for Gram-negative pathogens, therefore, they are epidemiologically relevant. However, the composition of reptilian microbiota has been poorly recognized so far. The majority of studies concern exotic reptiles as asymptomatic carriers of Salmonella serovars. Studies of other intestinal bacteria of reptiles are rare. Only recently, the microbiota of free-living European reptiles have been investigated, however, on the basis of small samples, mainly in protected areas. Here, we aim to investigate cloacal Gram-negative microbiota of free-living Natrix natrix. Snakes (N = 45) used in the study were collected in Kraków (Poland) and its vicinity. Nineteen species of Gram-negative bacteria were isolated. The most common species were: Aeromonas hydrophila, Morganella morganii, Proteus vulgaris, Salmonella spp. The bacteria prevalent in N. natrix cloacal swabs are likely to represent the natural intestinal Gram-negative microbiota of the examined snakes. Importantly, the identified bacteria are pathogenic to humans, which clearly highlights the epidemiological potential of free-living N. natrix. The risk of infection is high for immunocompromised humans, children (under 5 years old), elderly persons, and pregnant women. Our study provides the largest dataset on intestinal Gram-negative microbiota of wild snakes. The presence of multiple human pathogens determined by us calls for the necessity of further studies on reptile-transmitted bacteria in anthropogenic environments. [ABSTRACT FROM AUTHOR]