Negative-control-anchored urinary microbiome profiling with absolute 16S quantification : a pilot study in newly diagnosed, treatment-naive bladder cancer and healthy individuals

Accetto, Tomaž (avtor), Strašek Smrdel, Katja (avtor), Taskovska, Milena (avtor), Starčič Erjavec, Marjanca (avtor), Smrkolj, Tomaž (avtor), Seme, Katja (avtor), Erdani-Kreft, Mateja (avtor)

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.

• Jezik: Angleški jezik
• Ključne besede: bladder cancer, midstream urine, microbiota, urinary tract infection, next-generation sequencing, nonmetric multidimensional scaling, absolute quantification, negative controls, low biomass
• Vrsta gradiva: Članek v reviji
• Tipologija: 1.01 - Izvirni znanstveni članek
• Organizacija: BF - Biotehniška fakulteta ; MF - Medicinska fakulteta
• Status publikacije: Objavljeno
• Različica publikacije: Objavljena publikacija
• Leto izida: 2026
• Št. strani: 8 str.
• Številčenje: Vol. 373, art. fnag020
• PID: 20.500.12556/RUL-185282
• UDK: 579:616.6
• ISSN pri članku: 0378-1097
• DOI: 10.1093/femsle/fnag020
• COBISS.SI-ID: 272226819
• Datum objave v RUL: 30.07.2026

Gradivo je del revije

• Naslov: FEMS microbiology letters
• Skrajšan naslov: FEMS microbiol. lett.
• Založnik: Oxford University Press
• ISSN: 0378-1097
• COBISS.SI-ID: 25455104

Licence

• Licenca: CC BY 4.0, Creative Commons Priznanje avtorstva 4.0 Mednarodna
• Povezava: http://creativecommons.org/licenses/by/4.0/deed.sl
• Opis: To je standardna licenca Creative Commons, ki daje uporabnikom največ možnosti za nadaljnjo uporabo dela, pri čemer morajo navesti avtorja.

Sekundarni jezik

• Jezik: Slovenski jezik
• Ključne besede: rak mehurja, srednji tok urina, mikrobiota