Introduction: To investigate the presence of dysbiosis in patients with naive bladder cancer. Methods: Twelve male patients with non-invasive bladder cancer and twelve age-matched healthy males had midstream urine and tissue samples taken. A history of endourological interventions was determined as an exclusion criterion, ensuring that the study was designed solely with naïve participants. The bacterial 16s ribosomal RNA V3-V4 regions were used to examine urine and tissue samples. We compared the microbiota composition of the bladder cancer and control groups. Results:Escherichia Shigella (p < 0.001), Staphylococcus (p < 0.001), Delftia (p < 0.001), Acinetobacter (p < 0.001), Corynebacterium (p < 0.001), and Enhydrobacter (p < 0.001) were abundant in bladder cancer tissue samples. Escherichia Shigella (p < 0.001), Ureaplasma (p < 0.001), Lactobacillus (p = 0.005), Stenotrophomonas (p < 0.001), Streptococcus (p < 0.001), Corynebacterium (p < 0.001), and Prevotella (p = 0.039) were abundant in bladder cancer urine samples. Midstream urine has a sensitivity of 83% for detecting dysbiotic bacteria in cancer tissue. Conclusions: Our research is the first microbiota study of bladder cancer done with naive patients who have never had an endourological intervention. Escherichia Shigella, Staphylococcus, Acinetobacter, Enhydrobacter, Delftia, Corynebacterium, and Pseudomonas were detected as dysbiotic bacteria in bladder cancer. The sensitivity of the midstream urine sample in detecting dysbiosis in tissue is 83%.

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