20.500.12556/RUL-185282 Negative-control-anchored urinary microbiome profiling with absolute 16S quantification a pilot study in newly diagnosed, treatment-naive bladder cancer and healthy individuals Recent studies utilizing 16S rRNA amplicon sequencing have challenged the notion of urine sterility, yet urine is a low-biomass specimen in which apparent community profiles can be strongly influenced by background signal from reagents and processing. To address this interpretability gap, we integrate culture-independent absolute 16S rRNA gene quantification with urinary 16S amplicon sequencing in a negative-control-anchored workflow. Bacterial load provides a biomass-aware quality control gate that defines interpretable low-biomass thresholds and objective exclusion criteria. As a pilot application, we compared midstream urine collected prior to instrumentation from healthy volunteers and newly diagnosed bladder cancer (BC) patients, quality filtering retained 29 controls and 5 BC cases. Samples > 106 copies/ml typically produced > 10 000 reads; near 105 copies/mlread counts dropped sharply yetremained distinguishable from background. Thirteen negative controls (V3–V4 PCR and stabilization buffer; median 90, mean 124 reads) supported excluding samples with < 1000 reads. Median bacterial load was lower in BC than in controls (7.0 × 103 vs 1.07 × 106 copies/ml), although not significant in this underpowered cohort (P = 0.07). This cohort-size-independent framework enables load-based triage for sequencing, reduces backgrounddriven over-interpretation in low-biomass urine datasets, and supports modeling bacterial load as a covariate or stratifier in future studies of the bladder cancer microbiome. bladder cancer midstream urine microbiota urinary tract infection next-generation sequencing nonmetric multidimensional scaling absolute quantification negative controls low biomass rak mehurja srednji tok urina mikrobiota true false true Angleški jezik Slovenski jezik Članek v reviji 2026-07-30 12:41:54 2026-07-30 12:41:56 2026-08-07 13:34:51 0000-00-00 00:00:00 2026 0 0 8 str. art. fnag020 Vol. 373 2026 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 579:616.6 0378-1097 10.1093/femsle/fnag020 272226819 RAZ_Accetto_Tomaz_2026.pdf RAZ_Accetto_Tomaz_2026.pdf 1 9C063499DA5FA27245611676CD1CE3A1 fa0c56544cb52af3c21ec333eb9137d4e4b27e1a56bb9e4ca4a96f138ff2882c 2431b4b2-90a8-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=243933 https://academic.oup.com/femsle/article/doi/10.1093/femsle/fnag020/8488588 0 79170022-8c04-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=243652 Biotehniška fakulteta Medicinska fakulteta 0 0 0