Urinary microbiota in patients with prostate cancer and benign prostatic hyperplasia

DNA extraction

Samples were melted to liquid at room temperature (15–25°C) and stored on ice for DNA extraction. QIAamp DNA Mini Kit (Qiagen, USA) and distilled water were equilibrated to room temperature before DNA extraction. Twenty µl QIAGEN Protease was pipetted into the bottom of a 1.5 ml micro-centrifuge tube, and then a 200 µl sample (urine, EPS and seminal fluid) was added to the tube. Following this, 200 µl of Lysis buffer was added to the sample and mixed for 15 s, and the mixture was incubated at 56°C for 10 min in a water bath. Two hundred µl of absolute ethanol was pipetted into the mixture and mixed well. Following brief centrifugation, the mixture was carefully applied to a QIAamp Mini spin column without wetting the rim. On the other hand, following 1-min-long centrifugation at 8000 rpm, 50 µl of buffer AW1 was carefully added to the column without wetting the rim. An additional 1-min-long centrifugation at 8000 rpm was performed and the column was placed in a clean 2 ml collection tube. Five hundred µl buffer AW2 was added to the column and was centrifuged for 3 min at 14,000 rpm. Subsequently the column was kept in a clean 1.5 ml tube, 20 µl Elution buffer was added, and it was incubated at room temperature for 1 min. DNA was collected from the columns by centrifuging at 8000 rpm for 1 min. The DNA samples collected were subpackaged and stored at –20°C.

Polymerase chain reaction amplification

The concentrations of DNA in the samples were measured by ultraviolet spectrophotometer (Thermo Electron Corporation, USA). All DNA samples were amplified in the V3 regions of the 16S rRNA using universal bacterial primers (341F:5’-GTATTACCGCGGCTGCTGG-3’; 534R:5’-ACTCCTACGGGAGGCAGCAG-3’) with a 40-bp GC clamp (5’-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGG-3’) attached to the 5’ ends of the reserve primers (534R) for DGGE analysis. The PCR mixture comprised 1 µl of Bestar Taq DNA Polymerase (2.5 U/µl, DBI Bioscience, Shanghai, China), 5 µl of deoxynucleoside triphosphates (dNTPs, 2 mM each), 5 µl of 10× Bestar Taq Buffer, 1 µl of each primer (10 µM) and 2 µl of extracted bacterial DNA (∼60 ng). The total PCR reaction volume was 50 µl. The protocol for the PCR procedure was the touchdown protocol as described by Muyzer et al. [8] as follows: 94°C for 5 min; 20 cycles of touchdown PCR: denaturation at 95°C for 30 s, annealing at 65°C for 30 s with the temperature decreasing by 0.5°C/cycle until the annealing temperature was 55°C, and extension at 72°C for 30 s; 20 cycles of PCR: 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s; final extension at 72°C for 5 min; 4°C hold. Polymerase chain reaction (PCR) was performed in a thermal cycler PCR system (BBI, Canada) and PCR products were checked by electrophoresis on 1.0% (w/v) agarose gel (Biowest Regular Agarose G-10, Spain). Electrophoresis was performed at a voltage of 100 V for 20 min with 1× TAE buffer, and visualized by ethidium bromide staining using a gel imaging system (Bio-Rad Laboratories Gel-Doc 2000, Oslo, Norway). All PCR products were stored at –20°C before DGGE electrophoresis.

Denaturing gradient gel electrophoresis

Denaturing gradient gel electrophoresis analysis was performed for the EPS specimens of prostate cancer and BPH, using the Bio-Rad Dcode mutation detection system (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions. DNA fragments with different sequences were separated in 8% polyacrylamide (acrylamide: bisacrylamide = 37.5: 1; w/v) gels in 1× TAE buffer with 200 ng of each PCR product. A 30–60% denaturant gradient (100% denaturant was 7 mol/l urea and 40% (v/v) deionized formamide) was applied in the DGGE electrophoresis, which was performed in 1×TAE buffer at 180 V at 60°C for 4 h. Subsequently, the gels were washed with ultrapure water and stained with 5% Goldview dye for 30 min and photographed. DGGE graphs were digitized by Quantity One Analysis software (Gene Genius).

Denaturing gradient gel electrophoresis band sequencing and phylogenetic analysis

Denaturing gradient gel electrophoresis bands (#5, #6, #8, #10, #13, #15, #16, #17, #19) excised from the gel under UV and the DNA fragments were collected and purified with DNA Gel Extraction Kit (SK1135, Sangon, Shanghai, China) according to the manufacturer's instructions. The purified DNA was used as templates for PCR with the primers mentioned above. The PCR products were ligated into the pUCm − T vector (Sangon, Shanghai, China) and competent E. coli DH5α cells (Sangon) were transformed. The recombinant cells were selected and cultured overnight at 37°C. Subsequently, bacteria were collected, and DNA with the plasmid was extracted using the UNIQ-10 column kit (Sangon) following the kit instructions. Clones located in the same position as the original DGGE bands were sequenced (Sangon, Shanghai, China).

Similarity searches with obtained sequences were performed online based on the NCBI GenBank database (http://www.ncbi.nlm.nih.gov) BLAST. We compared the 16S rDNA sequences of the selected bands and their identity was arrived at (by comparing with the NCBI GenBank database) when the similarity of the sequence was above 97%. Based on the similarity search results, a phylogenetic tree was constructed by the neighbor-joining method with the MEGA 5 software (version 5.05). The consistency of the relationship on the tree was assessed by bootstrap resampling. Bootstrapping was performed for 1000 trials in accordance with the Clustal X program [19].

qPCR assay for Escherichia coli and Enterococcus

Each DNA sample in this study was amplified with primers (VS8F: 5’-GGCGGATTAGACTTCGGCTA-3’, VS9R:5’-CGTTTTGGCACTATTTGCCC-3’) for E. coli and primers (Ent1F: 5’-TACTGACAAACCATTCATGATG-3’, Ent2R:5’-AACTTCGTCACCAACGCGAAC-3’) for Enterococcus. Each PCR reaction mix consisted of the following in a total volume of 20 µl: 10 µl of Bestar SybrGreen qPCR mastermix (DBI Bioscience, Shanghai, China), 0.5 µl of each PCR primer (10 µM), 2.5 µl of DNA template and 6.5 µl of distilled water. Polymerase chain reaction conditions employed were as follows: initial denaturation at 95°C for 2 min; 40 cycles for PCR reaction of 95°C for 10 s, 55°C for 30 s and 72°C for 30 s; melting curve at 65°C for 5 s and 60 cycles from 65°C to 95°C (10 s/cycle, 0.5°C/cycle).

A DNA sample with the maximum concentration was diluted 10, 10², 10³, 10⁴, 10⁵ times and amplified under the same PCR conditions for the standard curves. All the samples were amplified in triplicate using Thermal Cycler (CFX-Touch, BIO RAD) and the Ct values were compared with the standard curve to obtain the relative quantity of E. coli and Enterococcus.

Polymerase chain reaction efficiency of 16s RNA

The total DNA of each specimen was extracted successfully and PCR assays for 16sRNA gene fragments of bacteria were performed using bacterial universal primers 341F and 534R. The amplified fragments of PCR were approximately 230 bp, as shown in Figures 1 and ​and2.2. Based on the results obtained, it is evident that 16s RNA gene fragments of bacteria in the specimens had good amplification efficiency.

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Figure 1

Gel profiles of the V3 region amplified by PCR from the extracted DNA from subjects with prostate cancer

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Figure 2

Gel profiles of the V3 region amplified by PCR from the extracted DNA from subjects with BPH

Denaturing gradient gel electrophoresis analysis and sequencing results

Polymerase chain reaction-denaturing gradient gel electrophoresis fingerprinting analysis of patients with BPH and prostate cancer for predominant bacteria was used to identify EPS microbiota (Figure 3). The extracted DNA was amplified with universal primers targeting the hypervariable V3 region of the 16S rRNA gene. Based on PCR-DGGE fingerprinting analysis, the diversity of microbiota structures was presented with separated bands of different DNA sequences, as shown in Figure 3, which showed that there were about 20 discernible bands.

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Figure 3

Gel profiles of the V3 region amplified by polymerase chain reaction from bands #5, #6, #8, #10, #13, #15, #16, #17 and #19. The lengths of fragments were approximately 190 bp (BPH (A2) and prostate cancer (B-2))

According to the DGGE profile of EPS, several bands of the prostate cancer group were discovered to be distinct from those present in the BPH group. These distinct bands, namely #5, #6, #8, #10, #13, #15, #16, #17 and #19, were identified as significant variables in discriminating the prostate cancer group from the BPH group. These distinct bands were sequenced to determine the exact microorganisms that were present in the urinary system during prostate cancer. The bands (#5, #6, #8, #13, #16, #17, and #19) that were brighter in the prostate cancer and bands (#10 and #15) in the BPH groups were amplified, and the lengths of these fragments were about 190 bp (Figure 4).

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Figure 4

DGGE profile of EPS obtained from subjects with BPH (A2) and prostate cancer (B-2)

The sequences of prominent bands #5, #6, #8, #13, #16, #17, and #19 in the prostate cancer group matched with Bacteroidetes bacteria, Alphaproteobacteria, Firmicutes bacteria, Lachnospiraceae, Propionicimonas, Sphingomonas, Ochrobactrum and bands #10 and #15 of BPH matched with Eubacterium and Defluviicoccus species respectively. It is evident from these results that there were almost no significant differences in the diversity of bacteria between the groups except for the fact that seven kinds of microbes in the prostate cancer group were distinctly increased in quantity compared with those in the BPH group. However, Eubacterium and Defluviicoccus were present in lower amounts in the prostate cancer group compared with the BPH group.

Phylogenetic analysis

Sequences of the bands were blasted in the NCBI database and a sequence having above 97% similarity was regarded as the same. Every sequence of the band examined showed about 97% similarity. Among these strains, the one that showed the highest similarity of the same category was selected. Based on these results, the phylogenetic tree was created by MEGA 5.05 (Figure 5).

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Figure 5

Phylogenetic tree of distinguished strains in prostate cancer group and BPH group

As shown in the phylogenetic tree (Figure 5), most of the strains were uncultured and values on the branches of the tree showed a high confidence coefficient and close genetic relationships.