Informed written consent to obtain mid-stream voided urine specimens and immediately thereafter a urine specimen from the bladder at the time of cystoscopy was obtained individuals undergoing surveillance cystoscopy for a prior history of superficial UC defined as non-muscle invasive bladder cancer (IRB# 15–1986). Inclusion criteria were 1) History of non-muscle invasive bladder cancer undergoing routine surveillance cystoscopy; 2) > 180 days since last intra-vesical therapy; 3) > 180 days since last urethral instrumentation; 4) No current symptoms or signs of an active urinary tract infection; 5) > 18 years of age; 6) No history of urinary tract stones and 7) No nephrostomy or bladder catheter in place. Exclusion criteria were 1) Failure to satisfy inclusion criteria and 2) Inability to provide informed consent.
Standard technique for mid-stream collection was employed for men and women. Cystoscopy was then performed. Intraurethral lubricant jelly was used for all subjects for comfort and safety. No subject received periprocedural antibiotics. Immediately after insertion of the cystoscope a 20 cc aliquot of urine was removed. Urine samples were stored within 90 min of collection at − 80 °C until analysis. Clinical data was collected including demographics and recent antibiotic use.
Prior to DNA extraction, 1–1.5 ml of urine was thawed and centrifuged at 20,000 x g for 3 min. The supernatants were discarded and the pellets were resuspended in 400 μL of Buffer ATL (Qiagen, CA) and treated with 25 μL of lysozyme solution (50 mg/mL) (Sigma Aldridge, MO). Samples were heated for 5 min at 70 °C and cooled before loading on the EZ1 Advanced (Qiagen) for DNA extraction by using the EZ1 DSP Virus kit (Qiagen). Samples were then cleaned and concentrated using the DNeasy PowerClean Cleanup Kit (Qiagen). Sequencing was prepared using a Nextera XT kit according to the Illumina 16S Metagenomic Sequencing Library Preparation protocol for analysis of hypervariable regions V3-V4. The locus specific sequences (Illumina, CA) using standard IUPAC nucleotide nomenclature were: 16S Amplicon PCR Forward Primer = 5′- GACTACHVGGGTATCTAATCC -3′, 16S Amplicon PCR Reverse Primer = 5′- CCTACGGGNGGCWGCAG − 3′.For normalization, each library was quantified with the KAPA Library Quantification Kit (Kapa Biosystems, MA). The libraries were sequenced on the Illumina MiSeq with paired-end 300 bp reads.
Three control samples were included; Staphylococcus aureus (Strain NCTC 8532, ATCC, VA), Escherichia coli (Strain NCTC 9001, ATCC, VA) and a “blank” control of 400 μL of buffer ATL which underwent the same DNA extraction process as above. Quantification of the library of the negative control was below the recommended concentration of 4 nM.
Qiime 1.9 was used to demultiplex fastq files, for open-reference Operational Taxonomic Unit (OTU) picking and building the phylogenetic tree using the open reference method [18]. The OTU table and the tree were then imported into R packages phyloseq [19] (version 1.19.1) and vegan (version 2.4–3) for analysis. Similar to our previous microbiome study, OTUs that were not seen more than three times in at least 20% of the samples were removed [20]. Samples with low read counts (< 5,000) and subjects with unpaired samples (i.e. not having both a voided and a cystoscopy sample) were removed. Rarefaction was performed on the remaining samples to be able to compare species richness.
Alpha diversity measures (i.e., observed species, Shannon, Simpson, Fisher) and beta diversity measures (unweighted UniFrac Distance, weighted UniFrac Distance, Bray-Curtis, and Jensen-Shannon divergence) were evaluated between voided samples and those collected by cystoscopy.
The 16S rRNA sequence data is deposited in NCBI Sequence Read Archive (SRA), PRJNA600265.
