Detection Of Escherichia Coli DNA From Interstitial Cystitis Bladder Biopsies Provides Little Evidence Of A Causal Microbe
R Dixon, M Agarwal
bacteria, bladder biopsies, dna, interstitial cystitis, pcr
R Dixon, M Agarwal. Detection Of Escherichia Coli DNA From Interstitial Cystitis Bladder Biopsies Provides Little Evidence Of A Causal Microbe. The Internet Journal of Urology. 2009 Volume 7 Number 1.
The aetiology of interstitial cystitis in patients remains unknown and despite many investigations no single micro-organism has been implicated. We have studied the possible role of bacteria in interstitial cystitis by investigating PCR amplified DNA sequences from 33 individually well-documented interstitial cystitis patients. The frozen or archived bladder biopsy specimens have been examined for the presence of bacterial DNA by the amplification of 16S rRNA and subsequent RFLP of cloned DNA fragments. Bacterial DNA was detected from 21/33 (64%) of frozen or archived bladder biopsy samples. RFLP analysis of cloned DNA strongly resembled the fragment profile of
The aetiology of interstitial cystitis (IC) in patients continues to elude us and despite intensive research, the possible causative agent of the disease and its ideal treatment remains unknown. Acute onset with exacerbations and remissions, a previous history of urinary tract infections, virtual confinement of the disease to female patients in which the short urethra allows easy access to bacteria, all strongly suggest an infective aetiology 
Investigations into the aetiology of IC using patient groups has remained limited, often due to insufficient frozen biopsy specimens and to date, studies have been small and conclusions difficult to make. In the present study, we are attempting to detect DNA from any bacteria present not only from frozen biopsies but a larger number of material preserved in paraffin wax.
Materials and Methods
Thirty-three bladder biopsy specimens (including 29 paraffin-embedded and 4 fresh frozen specimens) were collected from patients with a clinical diagnosis of IC fulfilling (National Institute of Diabetes and Digestive and Kidney diseases - NIDDK) criteria  with some modifications (criteria based on cystometrogram were not strictly adhered to, as urodynamic studies were not performed in all the patients . Five ‘normal’ bladder biopsy specimens from patients with previous bladder tumours, three biopsy specimens from patients with chronic cystitis and one from acute cystitis were included in the study as controls. One ‘normal’ bladder biopsy specimen was spiked with
Five m thick tissue sections were cut from the paraffin blocks. The first 5 sections from the blocks were discarded to ensure that the outer surface remained free of exterior contamination. A total of 5 thin sections were collected in an Eppendorf tube without de-waxing. The cutting knife was changed frequently and all surfaces coming into the contact with the specimen wiped clean with alcohol between each specimen and between first 5 discarded sections and the 5 sections used for DNA extraction. Pre-treatment of all specimens with lysozyme and lysostaphin preceded DNA extraction which was carried out by the methods described previously . DNA was isolated from all specimens with the
All specimens were amplified for the presence of a 97 bp sequence of the mitochondrial d loop region specific only to humans  with human placental DNA as a control . Amplification reactions were performed in Eppendorf tubes kept on ice. 5 l of 10 PCR buffer (500 mM KCl, 100 mM Tris-Cl, 15 mM MgCl2, 0.1%[wt/vol.] gelatin [pH 8.3] ), 8 l of deoxynucleoside triphosphate mixture - 0.02 moles each of dATP [10 mM], dCTP [10 mM], dGTP [10 mM], and dTTP [10 mM], 50 pmol each of M1 and M2 primers (M1 - 5 CGC CCT TAC ACA AAA TGA CAT CAA 3- i.d. 13232, M2 - 5 GTG TGG TTG GTT GAT GCC GA 3- i.d. 13286,  distilled water and 2 l of template DNA were added to the reaction mixture. These components were added together to give a final reaction volume of 50l and tubes were sealed with an overlay of 40 l of autoclaved mineral oil.
All 33 specimens from individually documented IC patients produced the anticipated amplification products of 97 bp from the mammalian mitochondrial region (data not shown).
PCR-amplified sequences of bacterial 16S rRNA products resulted in 21 out of 33 (64 %) biopsy samples from IC patients producing the expected sized amplification band (1156bp). The bands obtained from each of the 21 biopsies were subjected to restriction fragment length polymorphism (RFLP) analysis and compared with RFLP obtained from reference
Bacterial DNA was detected in 21/33 (64%) of bladder biopsy samples from well-documented IC patients and 10 of these strongly suggested
Typical gel of restriction analysis of PCR products cut with
lane 1 100bp ladder;
lane 2 negative control (no DNA added);
lane 3 sample 20 - IC patient DNA
lane 4 sample 24 – non-IC control
lane 5 sample 58 - non-IC control
lane 6 sample 34 - non-IC control UTI
lane 7 sample 69 - IC patient (cloned DNA)
lane 8 sample 76 - IC patient (cloned DNA)
lane 9 sample 11 - non-IC patient spiked with
lane 10 sample 63 - IC patient DNA
lane 11 sample 4 - IC patient (cloned DNA)
lane 12 sample 22 - non-IC patient specimen
Over the past 25 years the perception of IC has changed from a psychosomatic disorder to one as a distinct disease entity. Significant advances have been made by the application of molecular biology to the detection of microbes involved in the pathogenesis of IC and whilst pre-molecular approaches focused on culture, serologic and microscopic methods for detection of microbes, the molecular approach allows the direct detection of both prokaryotic and eukaryotic DNA. Amplification of human-specific sequences (97bp product) of mitochondrial DNA from clinical biopsies in the present study, served to provide an estimate of the general state of DNA preservation in the specimens, test for absence of PCR inhibitors as well as control for PCR-negative amplifications of bacterial 16S rDNA..
In the present study, bacterial DNA could be detected by amplification in 21/33 (64%) of bladder biopsy samples from IC patients and cloned DNA from at least 5 of these confirmed that a profile identical to
Other investigators have used amplified 16S rDNA analysis as a tool for the potential identification of bacterial pathogens in IC biopsy specimens. Our findings are consistent with the molecular study of Keay
The interpretation of results on IC biopsy samples from frozen and archival samples in our present study is difficult for two major reasons: Firstly in the light of results from ‘normal’ controls. We failed to find alternative controls to address this problem and to validate the presence of bacterial DNA in the IC group of patients. Secondly, the other major issue was the authenticity of DNA from archival tissue. Most biopsies are probably unavoidably contaminated by bacteria and DNA at the point of collection and although it is possible to ensure the sterility of forceps we were unable to ensure that amplifiable DNA was avoided .
The possible role of bacteria in the pathogenesis of IC has been investigated by DNA -based techniques in the present study. Bladder biopsies from both IC and control patients appeared to reveal putative