Urinary microbiome in non-muscle invasive bladder cancer: impact of sample types and sex differences.
Author(s): Kang C, Lee J, Baek MG, Kim NE, Son H, Yoo S, Yi H
Publication: BMC Microbiol, 2025, Vol. 25, Page 623
PubMed ID: 41039241 PubMed Review Paper? No
Purpose of Paper
This paper compared the microbiome of case-matched specimens from the bladder mucosa and from urine collected midstream and via a catheter; Patients with (35) and without (15) bladder cancer were included in the study. Potential effects of patient sex, collection method/specimen type and bladder cancer on the microbiome were investigated.
Conclusion of Paper
An average of 197379, 182561, and 95765 reads were obtained from urine specimens collected midstream, via catheter and from bladder mucosal specimens, respectively;60918 reads were obtained in negative controls (wipe down of surgical instruments and reagents). The genera Escherichia and Stenotrophomonas accounted for approximately 60% of the reads, with the remaining 40% of reads from Rhodococcus, Streptococcus, and Pseudomonas. Most of the reads from negative controls were Escherichia, with the most diversity (lowest percentage of Escherichia) in the specimen cup/lid, saline and Assayassure. In principal component analysis (PCA), the microbiome clustered by sample type (urine versus tissue), and while there were significant differences in Beta diversity among the three sample types (P=0.000999 in the specimens from men, P=0.01598 in the specimens from women), there was no difference in Beta diversity between the two urine types. Mucosal specimens had more Rhodococcus, Rhizobium, and Pseudomonas, and midstream urine tended to have more Proteus and Providencia and less Comamonas and Fusimonas than urine collected via catheter. There was no difference in Beta diversity between urine specimens from men and women when collected via a catheter, but Beta-diversity did differ between men and women when urine was collected midstream (P=0.004995), with more Bacteroides (P= 0.0949) and Bifidobacterium (P = 0.0781) and fewer Ralstonia (P = 0.0186) in midstream urine specimens of women than men. Because of the absence of sex based-differences, urine collected using a catheter was used to assess cancer-associated differences in the microbiome. In urine collected from men Beta-diversity did not differ between specimens from patients with and without cancer (P=0.1319), but analysis with ANCOM-BC found significantly more Curvibacter in the specimens from patients with cancer than those without (P=0.035). In urine collected from women via a catheter, there was a non-significant trend toward more Curvibacter in urine from patients with cancer than those without. No clinical markers were found that correlated with Curvibacter levels, but when classified based on severity and reoccurrence risk ordinal logistic regression analysis found that Curvibacter increased with increasing severity (P=0.042) or risk for reoccurrence (P=0.036).
Studies
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Study Purpose
This study compared the microbiome of case-matched specimens from the bladder mucosa and from urine collected midstream and via a catheter; Patients with (35) and without (15) bladder cancer were included in the study. Potential effects of patient sex, collection method/specimen type and bladder cancer on the microbiome were investigated. Midstream clean-catch urine was collected from 50 patients the day before transurethral resection of a bladder tumor (35 patients diagnosed with bladder cancer and 15 without; 42 men and 8 women). During surgery, case-matched urine was collected via a catheter and bladder mucosal tissue specimens >3 cm from the tumor were procured. Urine (collected mid-stream pre-surgery and via a catheter during surgery) were preserved with AssayAssure RNA-stabilizing reagent and refrigerated until analysis. Tissue specimens were stored at -80°C until analysis. Urine was centrifuged at 14 g for 10 min, the supernatant was filtered through a syringe filter (pore size: 5 μm) and bacterial cells were concentrated using a Vivaspin 20 ultrafiltration device at 5,000 g. DNA was extracted from bacterial cells using the FastDNA SPIN Kit for Soil. The V3-V4 hypervariable region of the bacterial 16 S rRNA gene was PCR amplified and sequenced using an Illumina MiSeq v3 instrument. Details of tissue specimen preparation and analysis were not provided and are assumed to be similar to urine. Negative controls were represented by a wipe down of instruments (forceps, cystoscope, swab kit, irrigation inlet/outlet, external sheath, bridge, obturator, sealing cap, gloves, catheter, IV set, specimen containers) and reagents (lidocaine jar, MT buffer, saline, and Assayassure). Sequencing reads were processed using the QIIME2 and the Alpha-diversity (Chao1, ACE, Shannon, and Simpson indices) and Beta-diversity (Bray-Curtis distance) were assessed.
Summary of Findings:
An average of 197379, 182561, and 95765 reads were obtained from urine specimens collected midstream, via catheter and from bladder mucosal specimens, respectively;60918 reads were obtained in negative controls (wipe down of surgical instruments and reagents). The genera Escherichia and Stenotrophomonas accounted for approximately 60% of the reads, with the remaining 40% of reads from Rhodococcus, Streptococcus, and Pseudomonas. Most of the reads from negative controls were Escherichia, with the most diversity (lowest percentage of Escherichia) in the specimen cup/lid, saline and Assayassure. In principal component analysis (PCA), the microbiome clustered by sample type (urine versus tissue), and while there were significant differences in Beta diversity among the three sample types (P=0.000999 in the specimens from men, P=0.01598 in the specimens from women), there was no difference in Beta diversity between the two urine types. Mucosal specimens had more Rhodococcus, Rhizobium, and Pseudomonas, and midstream urine tended to have more Proteus and Providencia and less Comamonas and Fusimonas than urine collected via catheter. There was no difference in Beta diversity between urine specimens from men and women when collected via a catheter, but Beta-diversity did differ between men and women when urine was collected midstream (P=0.004995), with more Bacteroides (P= 0.0949) and Bifidobacterium (P = 0.0781) and fewer Ralstonia (P = 0.0186) in midstream urine specimens of women than men. Because of the absence of sex based-differences, urine collected using a catheter was used to assess cancer-associated differences in the microbiome. In urine collected from men Beta-diversity did not differ between specimens from patients with and without cancer (P=0.1319), but analysis with ANCOM-BC found significantly more Curvibacter in the specimens from patients with cancer than those without (P=0.035). In urine collected from women via a catheter, there was a non-significant trend toward more Curvibacter in urine from patients with cancer than those without. No clinical markers were found that correlated with Curvibacter levels, but when classified based on severity and reoccurrence risk ordinal logistic regression analysis found that Curvibacter increased with increasing severity (P=0.042) or risk for reoccurrence (P=0.036).
Biospecimens
Preservative Types
- Other Preservative
- Frozen
Diagnoses:
- Neoplastic - Benign
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform Cell count/volume Next generation sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Patient gender Female
Male
Preaquisition Diagnosis/ patient condition Bladder cancer
Non-cancerous bladder mass
Biospecimen Acquisition Method of fluid acquisition Catheterized urine
Voided urine (spot collection)
Biospecimen Acquisition Biospecimen location Bladder mucosa
Urine