Characterization of urinary microbiome dysbiosis in prostate cancer and benign prostatic hyperplasia: A metagenomics study from North-Eastern Algeria

Technical Summary: Characterization of Urinary Microbiome Dysbiosis in Prostate Cancer and Benign Prostatic Hyperplasia

Problem Statement
Prostate cancer (PCa) remains a leading cause of cancer-related mortality in men, while benign prostatic hyperplasia (BPH) significantly impacts quality of life. Chronic inflammation is a recognized contributor to prostate carcinogenesis, and emerging evidence suggests the gut microbiome influences systemic inflammation and hormonal regulation. However, the urinary tract, previously considered sterile, has been underexplored as a source of microbial biomarkers for prostate diseases. To date, no studies had investigated urinary microbiome profiles in PCa and BPH patients within the Algerian population, leaving a gap in understanding regional microbial dynamics and potential non-invasive diagnostic markers.

Methodology
This retrospective pilot study analyzed urine samples from 68 male participants in North-Eastern Algeria, categorized into three groups: 22 patients with PCa (11 receiving chemotherapy), 32 patients with BPH, and 14 healthy controls. All participants were over 40 years old.

• Sample Processing: Urine samples (minimum 10 mL) were centrifuged, and total DNA was extracted from the pellet using a customized protocol adapted for the EZ1 Advanced XL instrument (Qiagen).
• Sequencing: Targeted metagenomic analysis was performed via 16S rRNA gene sequencing of the V3–V4 hypervariable regions on an Illumina MiSeq platform (2×300 bp paired-end).
• Bioinformatics: Raw sequences were processed using the DADA2 pipeline for quality filtering, chimera removal, and error correction. Taxonomic classification was performed against the SILVA reference database (v2022) to generate Amplicon Sequence Variant (ASV) tables.
• Statistical Analysis: Microbiome data were analyzed using R (phyloseq, decontam packages). Alpha diversity was assessed via the Shannon and Inverse Simpson indices. Beta diversity was evaluated using Principal Coordinates Analysis (PCoA) based on Bray-Curtis dissimilarity, with PERMANOVA testing for significance while adjusting for age as a covariate. Differential abundance was tested using Kruskal-Wallis tests with Benjamini-Hochberg correction.

Key Results
The study identified 2,916 ASVs across the cohort, revealing distinct microbial signatures across the three groups:

• Diversity Patterns: BPH samples exhibited the highest alpha diversity, followed by PCa, with controls showing the lowest diversity (Shannon index, p = 0.001). Beta diversity analysis confirmed distinct clustering of PCa samples (p = 0.001), indicating reduced intra-group variability compared to the more heterogeneous BPH and control groups.
• Taxonomic Composition:
  • PCa: Urine was dominated by Streptococcus (13.45%), Gardnerella (10.80%), and Klebsiella (10.48%). At the species level, Gardnerella vaginalis and Streptococcus mitis were prominent.
  • BPH: Samples showed higher abundances of pro-inflammatory anaerobic or microaerophilic bacteria, including Fusobacterium (16.4% at genus level), Prevotella (10.7%), and Campylobacter (7.43%).
  • Controls: While less diverse, controls showed enrichment of Escherichia-Shigella (p < 0.0001) compared to disease groups.
• Chemotherapy Impact: Within the PCa group, chemotherapy induced profound dysbiosis. Post-chemotherapy samples showed a significant increase in Proteobacteria (56.2% vs. 29.8% in untreated) and a depletion of Firmicutes and Actinobacteria. Beneficial taxa such as Lactobacillus crispatus and Corynebacterium amycolatum were significantly reduced, while uropathogenic species like Escherichia coli and Enterococcus faecalis, and opportunistic bacteria like Finegoldia magna and Prevotella bivia, were enriched.
• Confounding Factors: While age differed significantly between groups, multivariate analysis indicated that diagnosis (PCa vs. BPH vs. Control) was the primary driver of microbiome composition, not age.

Significance and Claims
The authors claim this study provides the first regional overview of urinary microbiome dynamics in prostate disease within North African populations. Key contributions include:

1. Identification of Biomarkers: The study identifies distinct microbial signatures for PCa (enriched in Gardnerella and Klebsiella) and BPH (enriched in anaerobic pro-inflammatory taxa like Fusobacterium and Prevotella), suggesting the urinary microbiome as a promising non-invasive biomarker for disease stratification.
2. Chemotherapy Effects: The research highlights that chemotherapy significantly destabilizes the urinary ecosystem, depleting protective bacteria and increasing the abundance of uropathogens, which may compromise mucosal integrity and increase infection risk.
3. Therapeutic Implications: The authors propose that microbiota-based interventions, specifically probiotic supplementation to restore beneficial taxa like Lactobacillus, could help restore microbial balance and support urogenital health in prostate disease management.

Limitations
The authors acknowledge several limitations: a relatively small sample size (particularly in the PCa and control groups), the lack of longitudinal data to track microbiome changes before and after chemotherapy, the use of partial 16S rDNA sequencing which limits species-level resolution, and the absence of follow-up data on BPH patients who may have progressed to PCa. Despite these constraints, the study establishes a foundational link between urinary dysbiosis and prostate pathology in this specific demographic.