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

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.

• Language: English
• Keywords: bladder cancer, midstream urine, microbiota, urinary tract infection, next-generation sequencing, nonmetric multidimensional scaling, absolute quantification, negative controls, low biomass
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: BF - Biotechnical Faculty; MF - Faculty of Medicine
• Publication status: Published
• Publication version: Version of Record
• Year: 2026
• Number of pages: 8 str.
• Numbering: Vol. 373, art. fnag020
• PID: 20.500.12556/RUL-185282
• UDC: 579:616.6
• ISSN on article: 0378-1097
• DOI: 10.1093/femsle/fnag020
• COBISS.SI-ID: 272226819
• Publication date in RUL: 30.07.2026

Record is a part of a journal

• Title: FEMS microbiology letters
• Shortened title: FEMS microbiol. lett.
• Publisher: Oxford University Press
• ISSN: 0378-1097
• COBISS.SI-ID: 25455104

Licences

• License: CC BY 4.0, Creative Commons Attribution 4.0 International
• Link: http://creativecommons.org/licenses/by/4.0/
• Description: This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.

Secondary language

• Language: Slovenian
• Keywords: rak mehurja, srednji tok urina, mikrobiota