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Kaltume Umar Hambali,1 Emmanuel Eilu,1 Sunil Kumar,1 Abdullateef Opeyemi Afolabi,1 Naheem Adekilekun Tijani,1 Yusuf Olusola Faseun,1 Martin Odoki,1 Christine Gechemba Mokaya,1 Danladi Makeri,1 Shango Patience Emmanuel Jakheng,1 Vidya Sankarapandian,1 Rasheed Omotayo Adeyemo,2 Taofeek Tope Adegboyega,2 Ismail Abiola Adebayo,3 Ibrahim Ntulume,4 Saheed Adekunle Akinola2 1Department of Medical Microbiology and Immunology, Kampala International University-Western Campus, Ishaka-Bushenyi, Uganda; 2Department of Microbiology and Parasitology, School of Medicine and Pharmacy, College of Medicine and Health Sciences, University of Rwanda, Butare, Rwanda; 3Department of Medical Biochemistry, Molecular Biology and Genetics, School of Medicine and Pharmacy, College of Medicine and Health Sciences, University of Rwanda, Butare, Rwanda; 4School of Biosecurity Biotechnical and Laboratory Sciences, College of Medicine and Veterinary Medicine, Makerere University, Kampala, UgandaCorrespondence: Saheed Adekunle Akinola, Email seddorf2003@gmail.comBackground: The concerning frequency of K. pneumoniae in various recreational settings, is noteworthy, especially regarding multi-drug resistant (MDR) strains. This superbug is linked to the rapid spread of plasmids carrying these resistance genes. The objective of this study was to evaluate the spatiotemporal prevalence of MDR-K. pneumoniae in the Kitagata hot spring, Southwestern Uganda.Methods: A laboratory-based descriptive longitudinal study was conducted between May and July 2023. During rainy and dry seasons, we collected eighty water samples in the morning and evening from the hot spring. The temperature at each point was measured prior to sample collection, and two samples were obtained at varying depths. 5 mL of each homogenized sample were pre-enriched in brain heart infusion broth, and subsequently in both blood and violet red bile agar. The Kirby-Bauer disk diffusion method was performed, followed by the detection of carbapenemase (CR) and extended-spectrum β-lactamase (ESBL) production. Polymerase chain reaction showed resistance genes viz. blaTEM, blaCTX-M and blaKPC. Data were analyzed using SPSS-20 to obtain chi-square tests and regression analysis.Results: K. pneumoniae accounted for 30.0% of isolates obtained from Kitagata hot springs, with all isolates classified as multi-drug resistant. All isolates were resistant to ampicillin, rifampicin, ceftazidime, and azithromycin (79.2%). Additionally, 95.8% of isolates harbored blaTEM gene alone and both blaTEM and blaCTX genes, followed by blaKPC alone (33.3%), with 25% harboring all three resistance genes. During the dry season, K. pneumoniae had a higher prevalence (35.0%) compared to the wet season (25.0%). The prevalence of MDR-K. pneumoniae significantly increased over the course of the study. The presence of the three studied resistance genes in the isolates showed a positive correlation with the second phase of sample collection and the dry season but exhibited a negative correlation with temperature, except for isolates harboring either blaTEM alone or blaTEM+KPC+CTX genes.Conclusion: Kitagata hot spring serves as a hotspot for continuous dissemination and acquisition of MDR-K. pneumoniae harboring resistance genes that encode for ESBL and CR production. The healthcare sector ought to implement an ongoing monitoring and surveillance system as well as robust antimicrobial resistance stewardship programs aimed at delivering health education to the community.Keywords: antimicrobial resistomes, one health, first generation genomics, Kitagata healing hot spring, MDR-K. pneumonia