First Report of Kocuria kristinae in the Skin of a Cuban Boa (Epicrates angulifer)

**Background:** The Cuban boa (Epicrates angulifer) is a non-venomous snake native to Cuba, increasingly kept as a pet and in zoos. Snakes can act as reservoirs of pathogenic bacteria, including multidrug-resistant strains, posing risks to both animal and human health. Kocuria kristinae is a Gram-positive, facultative anaerobic bacterium commonly found on the skin, oral cavities, and urinary tracts of mammals, including humans, and is associated with infections in immunocompromised patients. To date, K. kristinae has not been reported in snakes. This study aims to describe the first isolation of K. kristinae from a skin lesion of a Cuban boa.

**Methods:** A four-year-old female Cuban boa from a zoological park in northern Portugal presented with an area of dry scales and extensive skin changes. A skin sample was collected using a sterile AMIES swab under manual restraint, without sedation, following European Animal Welfare Directives. Standard laboratory methods were used for bacterial isolation, purification, and identification. Selective and differential media (Chromogenic Coliform Agar, MacConkey Agar, Baird Parker Agar, Glutamate Starch Phenol Red Agar, Mannitol Salt Agar) were used. Pure cultures were identified using the automated Vitek 2 Compact system with GP and GN identification cards. Antimicrobial susceptibility testing (AST) was performed using Vitek 2 AST-GP80 (Gram-positive) and AST-GN97 (Gram-negative) cards. Thirteen antibiotics from eight classes were tested on Gram-positive isolates: aminoglycosides (gentamicin, kanamycin, neomycin), fluoroquinolones (enrofloxacin, marbofloxacin, pradofloxacin), macrolides (erythromycin), lincosamides (clindamycin), tetracyclines (doxycycline, tetracycline), nitrofurans (nitrofurantoin), phenicols (chloramphenicol), and sulphamides (trimethoprim/sulphamethoxazole). Six antibiotics from two classes were tested on Gram-negative isolates: beta-lactams (ceftazidime, ceftriaxone, piperacillin) and fluoroquinolones (danofloxacin, enrofloxacin, marbofloxacin).

**Key Results:** Bacteriological analysis identified one Gram-positive bacterium, Kocuria kristinae, and two Gram-negative bacteria, Stenotrophomonas maltophilia and Aeromonas hydrophila. K. kristinae was positive for arginine dihydrolase type 1, leucine arylamidase, L-proline arylaminase, L-pyrrolidonyl arylamidase, alanine arylamidase, and tyrosine arylamidase. The antimicrobial susceptibility profile of K. kristinae showed: gentamicin MIC ≤0.5 (susceptible), kanamycin MIC 32 (resistant), neomycin MIC 16 (intermediate), enrofloxacin MIC 2 (intermediate), marbofloxacin MIC 2 (intermediate), pradofloxacin MIC ≥4 (resistant), erythromycin MIC ≥8 (resistant), clindamycin MIC ≥4 (resistant), doxycycline MIC 8 (intermediate), tetracycline MIC ≥16 (resistant), nitrofurantoin MIC 128 (resistant), chloramphenicol MIC 16 (intermediate), trimethoprim/sulphamethoxazole MIC ≥320 (resistant). The isolate was resistant to seven classes of antibiotics (aminoglycosides, fluoroquinolones, macrolides, lincosamides, tetracyclines, nitrofurans, and sulphamides) and was considered multidrug-resistant (resistant to at least one agent in three or more antimicrobial categories). Both Gram-negative isolates (S. maltophilia and A. hydrophila) showed resistance to all tested beta-lactams (ceftazidime, ceftriaxone, piperacillin) and susceptibility to all tested fluoroquinolones (danofloxacin, enrofloxacin, marbofloxacin).

**Clinical Implications:** This is the first report of K. kristinae in a snake, specifically from a skin lesion of a captive Cuban boa. The multidrug-resistant profile of this isolate, along with the co-isolation of other opportunistic pathogens (S. maltophilia and A. hydrophila), highlights the potential for snakes to harbor and transmit antibiotic-resistant bacteria. The findings emphasize the need for antimicrobial resistance surveillance in zoological parks and the importance of a One Health approach integrating human, animal, and environmental health. Laboratory diagnostics are crucial for identifying emerging pathogens and guiding appropriate treatment. The possibility of transmission from animal keepers, food, or terrarium surfaces should not be ruled out. Further genetic characterization of resistance determinants is warranted to understand resistance mechanisms and mitigate the spread of multidrug-resistant bacteria.