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POLYMERASE CHAIN REACTION AMPLIFICATION OF BACTERIAL 16S rRNA GENES IN INTERSTITIAL CYSTITIS AND CONTROL PATIENT BLADDER BIOPSIES
Vol. 159,280-283, 1% Printed in U S A .
POLYMERASE CHAIN REACTION AMPLIFICATION OF BACTERIAL 16s rRNA GENES IN INTERSTITIAL CYSTITIS AND CONTROL PATIENT BLADDER BIOPSIES S. m y , * C.-0. ZHANG, B. R. BALDWIN, S. C. JACOBS AND J. W. WARREN From the Division of Infectious Diseases, Department of Medicine, the Department of Molecular and Cellular Biology, and the Division of Umbgy, Department of Surgery, University of Maryland School of Medicine, and the Research Service, Department of Veterans m a i m Medical Center, Baltimore, Maryland
ABSTRACT
Purpose: Several characteristics of the chronic bladder disease called interstitial cystitis (IC) suggest an infectious etiology. However, a single causative organism has not been convincingly cultured in vitro, and DNA for a variety of microorganisms has been found inconsistently in bladder biopsies from IC patients. We therefore looked for a possible bacterial cause for IC by using a sensitive nested PCR assay on cystoscopic bladder biopsy specimens obtained from IC patients and controls. Materials and Methods: Bladder biopsies were obtained a t C ~ S ~ ~ S C O Pfrom Y 6 IC patients and 6 controls. DNA was extracted from these specimens and PCR with 2-round amplification performed using nested primers from a highly conserved region of the bacterial 16s rRNA gene. Amplified DNA was purified and sequenced using the Sequenase PCR Product Sequencing Kit, and the sequences obtained were compared with bacterial rRNA gene sequences recorded in GenBank. Results: Biopsy specimens from all 6 patients and 6 controls were positive by PCR for DNA encoding bacterial 16s rRNA. Sequence data indicated a predominant microorganism in 10 of the 12 specimens, with >95% homology to DNA from several different genera of bacteria including Acinetobacter, Propwnobacterium, Salmonella, and Escherichia. None of the organisms identified by PCR had been cultured from tissue or urine obtained simultaneously from these persons, using sensitive culture techniques. Conclusions: These data indicate no difference between IC patients and controls in the proportion of bladder biopsies with PCR positivity or the typeb) of organism present, providing additional evidence that IC is not associated with infection by a particular type of bacterium. KEY WORDS:cystitis, polymerase chain reaction
Interstitial cystitis (IC) is a chronic bladder disease of a possible cause for IC, the prevalence of microorganisms unknown etiology. Several characteristics of IC suggest (including bacteria, viruses, and fungi) was significantly that it may be caused by an infectious organism.' This greater in urine of IC patients (6/11)than in urine of disease, which usually has a rapid onset,2ssgenerally OC- controls (0/7)(p <0.05).7 These findings led to the hypothC u r s in otherwise healthy individuals. Ninety percent Of I c esis that abnormalities of the bladder epithelium may patients are women, which parallels the epidemiolom of predispose IC patients to colonization with a variety of bacterial tract i n f e c t h ~Indeed, .~ Patients with microorganisms, but did not provide evidence that IC was IC are twice as likely to have a history of urinary tract caused by an infectious agent. infection controls, and 10 to 12 times as l*eb to have a %o other labratofies subsequently from Of tract Idammastudies that used polperase (PCR) technoltory changes compatible with infection are also sometimes for evidence of ribosomal DNA in Ic seen on histopathology, including the presence of IgA de- ogy to patient biopsy specimens?*9However, these studies yielded posits in bladder epithelium.' ~ w ~to identify ~ t san infectious etiologyfor IC have not confliding results on the possibility of a bacterial cause for yet yielded definitive results. In our own initial studies, we Ic. In the first report, 4 of 14 (29%)IC patients, but 0 of 15 obtained urine and bladder tissue at cystoscopy from 11 IC had evidence bY nested PCR for the Presence Of patients and 7 controls and cultured these specimens for a bacterial 16s r m A genes; 2 of the amplified segments bore variety of fastidious and nonfastidious bacteria, mycobac- greatest homology to Escherichia coli and the other 2 were teria, fungi and viruses; in addition, special staining tech- similar to members of the genus Pseudomonas. The second niques were used to examine biopsy specimens and cyto- report, however, found no evidence for bacterial DNA in spun urine, and tissue sections and outgrowths of biopsy specimens from 11 IC patients and 3 controls when explanted bladder celle were examined by electron micros- PCR with a single set of primers was used, a procedure that copy. Although no single microorganism was identified as is likely to be less sensitive than the nested method. Because of these reported contradictory findings, we proF p t a d for publication Auguet 8,1997. R e q u a for reprints: VA Medical Center. Rm. 3Bl&I, 10 N. ceeded to estimate the sensitivity of single versus nested Greene St., B a l k - MD from 21201. the National and primer PCR, and then used the more sensitive of these methSupportad b of adb%-, National ~ d b ofhHealth ( ~ 0 1 0 d S to detect bacterial 16s rRNA gene in bladder biopsies :#%81 and a pilot grant from the Interstitial Cystitis Association. obtained at cystoscopy from 6 IC patients and 6 controls.
=
280
281
POLYMERASE CHAIN REACTION AMPLIFICATION MATERIALS
AND METHODS
Lkoxyribonucleic mid sequencing. Amplified DNA was puPatients and biopsy specimens. Bladder biopsies were ob- rified by electrophoresis using a 1.4% NuSieve GTG gel (FMC tained at cystoscopy from inflamed areas in the bladder wall BioProducts, Rockland, ME)and the Wizard PCR DNA puof 6 patients with IC and areas of normal bladder wall in 6 rification resin kit (Promega, Madison, WI)according to the controls. The controls were women who were scheduled to manufacturer's instructions, and stored at -2OC. The gene undergo cystoscopy for other urological diseases (benign fragments were sequenced directly using the Sequenase Verstricture of the ureteropelvic junction, glomerulosclerosis of sion 2.0 PCR Product Sequencing Kit (Amersham, Arlingtnn the kidney, caliceal diverticulum with stones, ureteral endo- Heights, IL) and the primers 3 and 4 (as used in the PCR metriosis, bladder papillary adenocarcinoma and renal cell reaction). carcinoma). Each IC patient and control was required to have no known bacterial urinary tract infection for at least three RESULTS months and no antibiotic use for at least one month prior to We determined the sensitivity of both the nested PCR cystoscopy. Cystoscopy was performed under general anesthesia using nonbacteriostatic normal saline as a bladder method for amplification of bacterial 16s rRNA genes develirrigant, and cold cup biopsies were obtained as previously oped by Domingue et al8, as well as another method that described? Specimens used for PCR analysis were trans- used primers and single round amplification similar to the ported from the operating room on saline-soaked Telfa (Ken- method reported by Haarala et aLg, by amplifying DNA exdall Co., Boston, MA) in a sealed sterile container at room tracted from a known quantity of colony forming units of temperature; immediately upon arrival in the laboratory, Escherichia coli (strain Y1090r-). The nested PCR method these specimens were placed into a separate sterile container proved to be very sensitive, allowing detection of rRNA genes from a dilution of purified bacterial DNA containing the and frozen dry at -80C. Deoxyribonucleic acid extraction and PCR amplification. amount of DNA extracted from approximately 0.5 colony Biopsy specimens were homogenized using a sterile dispos- forming units of bacteria after the second round of amplification (fig. 1).In contrast, we could not detect DNA from able tissue grinder (Sage Products, Inc., Crystal Lake, IL), fewer than 5 x 10' colony forming units of bacteria by using and the homogenized tissue was then suspended in 0.4 ml. the amplification method with a single set of primers (data digestion buffer (100 mM. NaCl, 10 mM. Tris-HC1 pH 8.0,25 not shown). mM. EDTA pH 8.0,0.5% SDS, and 0.1 mg./ml. proteinase K) Only the nested PCR method was therefore considered to and incubated at 50C overnight. DNA was extracted from be of sufficient sensitivity for amplification of bacterial DNA these specimens by mixing with an equal volume of phenol/ from IC patient and control bladder biopsy specimens. A chloroformlisoamyl alcohol (25/24/1) and centrifuging 1700 negative control (containing all elements of the PCR reaction xg at 4C for 10 minutes. The DNA was precipitated from the mixture except DNA from a tissue specimen) and a positive aqueous layer by adding 0.5 volume of 7.5 M. ammonium control (containing E. coli DNA instead of DNA from the acetate and 2.0 volume of absolute ethanol a t -8OC for 30 tissue specimen) were run with each set of samples. Results minutes and centrifuging 1700 X g at 4C for 10 minutes. of these experiments indicated the presence of a band of the Following a rinse with 70% ethanol, the pellet was lyophi- appropriate size for the bacterial rRNA gene fragment in all lized, resuspended in 20 p1. TE buffer (10 mM. Tris-HC1 pH twelve biopsy specimens (6IC and 6 control patient samples) 8.0,l.O mM. EDTA pH 8.0), and incubated at 65C for 1hour. (fig.2). Purified DNA specimens were stored at -2OC. Sequence analysis of DNA amplified from one IC patient PCR amplification to a m p w the bacterial 16s rRNA gene biopsy and one control biopsy each appeared to contain genes was carried out using a single set of primers, andlor using from more than one bacterium, and the organisms present in nested primers. The nested primer PCR used to generate most these specimens were therefore unable to be directly identiof the data for this report was performed according to the fied. DNA from the remaining 5 IC patients and 5 controls method previously published by Domingue et a18 To prevent represented a predominant organism. We compared the secontamination bv exogenous DNA. 5 units Thenus ~UatiCus quences obtained from these amplified DNA segments with DNA polymeras;! (AmiliTaq PolGerase, Perkin Elmer, Foster City, CA) were preincubated with 0.2 units bovine pancreatic DNase I (BoehringerMannheim, Indianapolis, IN) for 1hour at 2nd run 1st run 37C, and the DNase then heat inactivated at 95C for 10 minutes. Each DNA specimen was then mixed with 0.2 mM. dNTPs, 1 j g . of each primer, and 5 units DNaSe-treated AmpliTaq polymerase in PCR buffer (50 mM. KCl, 1.5 mM. MgCl,, and 10 mM. Tris-HC1 pH 8.3), and amplification performed using a Perkin Elmer DNA Thermal Cycler p n grammed for 95C denaturation, 58C annealing, and 72C extension temperatures (first 3 cycles had extended denaturation and 0.5kb annealing times). A single round of ampWcation was used for the single primer set method, and two rounds of amplification were used for the nested PCR. The primers used for the single primer set method were 64W-TACCTI'G'ITACGACT1'-3'] and 65[5'-'ITAGATACCCTDGTAGTCC-3' where D equals G or A or TI. Primers used for the 6rst round of the nested a m p w a a m m m E I m a m c n ~ tion were 1 A [ 5 I - C A C A A G C ~ ~ A ~ A ~ ~ ' I and T-3'1 E g Mc M * 0 9e g E x M - me
-
5 [ 5 ' - C C T A C G G Y T A C C T C A C " - 3 ' where Y equals c off l ; t h ~ e u s e d f o r t h e s w e r e 3 [ 5 ' cGCGAAGAACCTl'ACCT-3'] and 45'-GCGGAXmTK'I'G A C G G G c a m m T A - 3 ' where K equals G or T].Following PCR amplification, appm-bly 204b of the -donmixture was run on a 1%agarose/ethidium bromide el,and the P=* pair h g m e n t detected by UV fluorescence. ence of a 467
-
%-?%2%5!y%2 7 7 ~ ~ ~ ~ FIG.1. Polymerase chain, reaction amplification of bacterial 16s
rRNA genes using nested m e ? and two run (round)mpMebon. Follom?~~ second ofam~Mabon,467 pair was evident m samples eontamipg DNA from as few as 6 X lo-' m l o ~ forming unita O ~ Emli . ( s t n u n ~ l m - F~U ) . 1eR lane e b o w ~locatmn of DNA standards,w i t h labeling of 500 bp standard for refemma
282
POLYMERASE CHAIN REACTION AMPLIFICATION
by cystoscopic biopsy from IC or control patients commonly contain bacterial DNA detectable by nested PCR. The discrepancy between our findings and the observations of H m a l a et al. may be explained by the relative insensitivity of PCR Using a single set of primers for DNA encoding bacterial rRNA as compared with the nested PCR method used by Domingue et al. and US. The differences between our findings and those of 0.5kb Domingue et al., however, are more difficult to explain. While our study found bacterial DNA in both Ic and control specimens, their data suggested that bacterial DNA was present almost exclusively in IC specimens, albeit from a minority of patients. Another difference between our studies is the types of organisms identified (Pseudomonas sp. and E. coli in their study, and Acinetobacter sp., Pmpwnobacterium sp., SalmoFIG 2. Amplification of bacterial 16s rRNA nee in bladder bi- nella sp. and E. coli in ours). The two studies, however, are ops specimens from IC and control patients. X n g nested primers anBtwo round amplification d u r e as in fig. 1, a 467 base pair similar in one respect: the bacteria identified by PCR in both frapent was amplified in 6 IC and 6 control patient biopsy investigations are not fastidious and, if alive, should have speamens. been (but were not) culturable. Our findings suggest two hypotheses. The first is that all or most women, with or without IC, have nonculturable or nonviable bacteria on or in bladder mucosa. We are unaware of sequencesfrom various bacterial speciesrecorded in the Gen- other data suggesting this concept. Moreover, were this to be Bank database, and found evidence for the presence of DNA the case, these data indicate that women with IC would not with >95% homology to several bacteria, as shown in the differ from women without IC in this respect. The second hypothesis is that nonculturable or nonviable table. None of the organisms identified by PCR was previously cultured from a duplicate tissue specimen or urine bacteria contaminated the bladder specimens. A review of obtained at C~S~QSCOPYfrom these patients, even though we our methods for collecting and processing of specimens might used a very sensitive culture method that detected as few as be instructive. Copious povidone iodine preparation of the 1 organism per biopsy specimen, or 10 organism per ml. of perineum resulted in 0/7 urine specimens from controls being culture positive for bacteria, even using methods sensitive urine.7 enough to grow organisms at concentrations as low as 10 bacteria per ml. of urine.7 However, cystoscopic biopsy was DISCUSSION performed after collection of urine by catheterization, and That IC might be an infectious disease is not an unreason- could have introduced organisms into the bladder. Indeed, able hypothesis, given its epidemiology and the tissue intlam- three of the bladder specimens, when homogenized and culmatory changes sometimes seen with this disorder. However, tured, grew very small numbers of organisms (though differour previous study that used very sensitive culture tech- ent species than those identified by PCR).7 This finding is niques to look for a variety of microorganisms in both urine consistent with small numbers of live organisms contaminatand bladder tissue of IC patients did not find evidence for an ing the cystoscope or biopsy forceps. It is also possible that bacteria killed or damaged by povidone iodine could have infectious etiol~gy.~ In planning our previous studies, we had hypothesized contaminated bladder specimens during insertion, removal, that a method such as PCR might be too sensitive to search or subsequent manipulation of the cystoscope. In addition, for a bacterial c a w for IC, and would amplie DNA from specimens were obtained with cold cup biopsy forceps that urethral organisms introduced by the cystoscope. We there- were routinely hand cleaned with a soap solution (Endozyme fore concentrated on techniques that could identify replicat- solution, Ruhof Corp., Valley Stream, NY), washed additioning microorganisms. However, the reports of Domingue et al. ally in a commercial washer, then decontaminated in Cidex and Haarala et al. indicated that all of their control speci- solution (Johnson & Johnson, Arlington, TX) over several mens were negative for bacterial DNA by PCR. Although hours; conceivably, bacteria killed by this method of cleaning Haarala et al. also did not find evidence for bacterial DNA in could remain on the forceps and their DNA could have been IC specimens, the finding by Domingue of DNA from various amplified by the PCR procedure. We favor the second hypothesis and feel that the bladder bacteria in bladder tissue from a significantly greater percentage of IC patients than controls suggested that nested specimens obtained from IC and control women were probaPCR might pmve useful to identify a bacterial cause for IC. bly contaminated with nonviable organisms a t the time of Because we determined that this method was indeed sufii- cystoscopy, from the urethra, the cystoscope sheath, the CYSciently sensitive to detect bacterial DNA from less than one toscope, or even the biopsy forceps. Once obtained, the specE. coli bacterium, we proceeded to examine bladder biopsy imens were immediately placed in sterile sealed containers; tissue fiom both IC and control patients. at opening they were processed in parallel with negative Our findings indicate that bladder tissue specimens ob- controls which remained negative, suggesting it is unlikely that the processing procedure itself caused contamination with bacterial DNA. Identity of bacteria present in IC and control bladder biopsies Although the three reported studies (by Domingue et al., Haarala et al., and this report) that sought bacterial DNA in IC Contml IC patient bladder tissue by PCR have reached different Aeinetobeeter sp. Propionobacterium sp. E. coli (strain #1)* specific conclusions, one observation is consistent: no single Propionobncterium sp. E. coli (strain #2)* E. wli (etraint l P microorganism appears to be associated with IC. Our own E. coli (sbain#2P E. coli (strain #2)* data further indicate that a very sensitive method for the selmonella sp. E.coli (strain #2)* detection of bacteria such as nested PCR may be inapproprimixed mixed Taro sbainsof E.ooli ware wident by idantid base pair changesat speci6c ate and unhelpful for determining whether bacterial DNA is allsk. present in tissue specimens obtained at cystoscopy. Methods
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i POLYMERASE CHAIN REACTION AMPLIFICATION
aimed a t detection of tissue-associated bacteria, such as in situ hybridization, may be more helpful for future studies. The authors gratefully acknowledge Edward W. Campbell, Jr., M.D.., Michael J. Naslund, M.D., Ms. Linda Home and Terry Wilson, R.N., for their assistance with patient enrollment and specimen acquisition, and Michael Donnenberg, M.D. for helpful discussions. REFERENCES
1. Warren, J. W.: Interstitial cystitis as an infectious disease. Urol. Clinics North Am., 21: 31,1994. 2. Messing, E. M. and Stamey, T. A.: Interstitial cystitis: early diagnosis, pathology, and treatment. Urology, 12: 381,1978. 3. Oravisto, K J.: Epidemiology of interstitial cystitis. Ann. Chir. Gynaecol. Fenn., 64: 75,1975. 4. Warren, J. W.: Clinical presentation and epidemiologyof urinary
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tract infections. In: Urinary Tract Infections, Mobley, H. L. T. and Warren, J. W., eds. Washington, D. C., ASM Press, 1996. 5. Held, P. J., Hanno, P. M. and Wein, A. J.: Epidemiology of interstitial cystitis: 2. In: Interstitial Cystitis, Hanno, P. M., Staskin, D. R., Krane, R. J. and Wein, A. J., eds. London, Springer-Verlag, 1990. 6. Said, J. W, Van de Velde, R. and Gillespie, L.: Immunopathology of interstitial cystitis. Mod.Pathol., 2 593,1989. 7. Keay, S.,Schwalbe, R. S., Trifillis, A. L., Lovchik, J. C., Jacob, S.and Warren, J. W.: A prospective study of microorganisms in urine and bladder biopsies from interstitial cystitis patients and controls. Urology, 45: 223,1995. 8. Domingue, G. J., Ghoniem, G. M.,Bost, K L., Fermin, C. and Human, L. G.: Dormant microbes in interstitial cystitis. J. Urol., 153: 1321,1995. 9. Haarala, M.,Jalava, J., Laato, M.,Kiilholma, P., Nurmi, M.and Alanen, A: Absence of bacterial DNA in the bladder of patients with interstitial cystitis. J. Urol., 166: 1843,1996.
POLYMERASE CHAIN REACTION AMPLIFICATION OF BACTERIAL 16s rRNA GENES IN INTERSTITIAL CYSTITIS AND CONTROL PATIENT BLADDER BIOPSIES S. m y , * C.-0. ZHANG, B. R. BALDWIN, S. C. JACOBS AND J. W. WARREN From the Division of Infectious Diseases, Department of Medicine, the Department of Molecular and Cellular Biology, and the Division of Umbgy, Department of Surgery, University of Maryland School of Medicine, and the Research Service, Department of Veterans m a i m Medical Center, Baltimore, Maryland
ABSTRACT
Purpose: Several characteristics of the chronic bladder disease called interstitial cystitis (IC) suggest an infectious etiology. However, a single causative organism has not been convincingly cultured in vitro, and DNA for a variety of microorganisms has been found inconsistently in bladder biopsies from IC patients. We therefore looked for a possible bacterial cause for IC by using a sensitive nested PCR assay on cystoscopic bladder biopsy specimens obtained from IC patients and controls. Materials and Methods: Bladder biopsies were obtained a t C ~ S ~ ~ S C O Pfrom Y 6 IC patients and 6 controls. DNA was extracted from these specimens and PCR with 2-round amplification performed using nested primers from a highly conserved region of the bacterial 16s rRNA gene. Amplified DNA was purified and sequenced using the Sequenase PCR Product Sequencing Kit, and the sequences obtained were compared with bacterial rRNA gene sequences recorded in GenBank. Results: Biopsy specimens from all 6 patients and 6 controls were positive by PCR for DNA encoding bacterial 16s rRNA. Sequence data indicated a predominant microorganism in 10 of the 12 specimens, with >95% homology to DNA from several different genera of bacteria including Acinetobacter, Propwnobacterium, Salmonella, and Escherichia. None of the organisms identified by PCR had been cultured from tissue or urine obtained simultaneously from these persons, using sensitive culture techniques. Conclusions: These data indicate no difference between IC patients and controls in the proportion of bladder biopsies with PCR positivity or the typeb) of organism present, providing additional evidence that IC is not associated with infection by a particular type of bacterium. KEY WORDS:cystitis, polymerase chain reaction
Interstitial cystitis (IC) is a chronic bladder disease of a possible cause for IC, the prevalence of microorganisms unknown etiology. Several characteristics of IC suggest (including bacteria, viruses, and fungi) was significantly that it may be caused by an infectious organism.' This greater in urine of IC patients (6/11)than in urine of disease, which usually has a rapid onset,2ssgenerally OC- controls (0/7)(p <0.05).7 These findings led to the hypothC u r s in otherwise healthy individuals. Ninety percent Of I c esis that abnormalities of the bladder epithelium may patients are women, which parallels the epidemiolom of predispose IC patients to colonization with a variety of bacterial tract i n f e c t h ~Indeed, .~ Patients with microorganisms, but did not provide evidence that IC was IC are twice as likely to have a history of urinary tract caused by an infectious agent. infection controls, and 10 to 12 times as l*eb to have a %o other labratofies subsequently from Of tract Idammastudies that used polperase (PCR) technoltory changes compatible with infection are also sometimes for evidence of ribosomal DNA in Ic seen on histopathology, including the presence of IgA de- ogy to patient biopsy specimens?*9However, these studies yielded posits in bladder epithelium.' ~ w ~to identify ~ t san infectious etiologyfor IC have not confliding results on the possibility of a bacterial cause for yet yielded definitive results. In our own initial studies, we Ic. In the first report, 4 of 14 (29%)IC patients, but 0 of 15 obtained urine and bladder tissue at cystoscopy from 11 IC had evidence bY nested PCR for the Presence Of patients and 7 controls and cultured these specimens for a bacterial 16s r m A genes; 2 of the amplified segments bore variety of fastidious and nonfastidious bacteria, mycobac- greatest homology to Escherichia coli and the other 2 were teria, fungi and viruses; in addition, special staining tech- similar to members of the genus Pseudomonas. The second niques were used to examine biopsy specimens and cyto- report, however, found no evidence for bacterial DNA in spun urine, and tissue sections and outgrowths of biopsy specimens from 11 IC patients and 3 controls when explanted bladder celle were examined by electron micros- PCR with a single set of primers was used, a procedure that copy. Although no single microorganism was identified as is likely to be less sensitive than the nested method. Because of these reported contradictory findings, we proF p t a d for publication Auguet 8,1997. R e q u a for reprints: VA Medical Center. Rm. 3Bl&I, 10 N. ceeded to estimate the sensitivity of single versus nested Greene St., B a l k - MD from 21201. the National and primer PCR, and then used the more sensitive of these methSupportad b of adb%-, National ~ d b ofhHealth ( ~ 0 1 0 d S to detect bacterial 16s rRNA gene in bladder biopsies :#%81 and a pilot grant from the Interstitial Cystitis Association. obtained at cystoscopy from 6 IC patients and 6 controls.
=
280
281
POLYMERASE CHAIN REACTION AMPLIFICATION MATERIALS
AND METHODS
Lkoxyribonucleic mid sequencing. Amplified DNA was puPatients and biopsy specimens. Bladder biopsies were ob- rified by electrophoresis using a 1.4% NuSieve GTG gel (FMC tained at cystoscopy from inflamed areas in the bladder wall BioProducts, Rockland, ME)and the Wizard PCR DNA puof 6 patients with IC and areas of normal bladder wall in 6 rification resin kit (Promega, Madison, WI)according to the controls. The controls were women who were scheduled to manufacturer's instructions, and stored at -2OC. The gene undergo cystoscopy for other urological diseases (benign fragments were sequenced directly using the Sequenase Verstricture of the ureteropelvic junction, glomerulosclerosis of sion 2.0 PCR Product Sequencing Kit (Amersham, Arlingtnn the kidney, caliceal diverticulum with stones, ureteral endo- Heights, IL) and the primers 3 and 4 (as used in the PCR metriosis, bladder papillary adenocarcinoma and renal cell reaction). carcinoma). Each IC patient and control was required to have no known bacterial urinary tract infection for at least three RESULTS months and no antibiotic use for at least one month prior to We determined the sensitivity of both the nested PCR cystoscopy. Cystoscopy was performed under general anesthesia using nonbacteriostatic normal saline as a bladder method for amplification of bacterial 16s rRNA genes develirrigant, and cold cup biopsies were obtained as previously oped by Domingue et al8, as well as another method that described? Specimens used for PCR analysis were trans- used primers and single round amplification similar to the ported from the operating room on saline-soaked Telfa (Ken- method reported by Haarala et aLg, by amplifying DNA exdall Co., Boston, MA) in a sealed sterile container at room tracted from a known quantity of colony forming units of temperature; immediately upon arrival in the laboratory, Escherichia coli (strain Y1090r-). The nested PCR method these specimens were placed into a separate sterile container proved to be very sensitive, allowing detection of rRNA genes from a dilution of purified bacterial DNA containing the and frozen dry at -80C. Deoxyribonucleic acid extraction and PCR amplification. amount of DNA extracted from approximately 0.5 colony Biopsy specimens were homogenized using a sterile dispos- forming units of bacteria after the second round of amplification (fig. 1).In contrast, we could not detect DNA from able tissue grinder (Sage Products, Inc., Crystal Lake, IL), fewer than 5 x 10' colony forming units of bacteria by using and the homogenized tissue was then suspended in 0.4 ml. the amplification method with a single set of primers (data digestion buffer (100 mM. NaCl, 10 mM. Tris-HC1 pH 8.0,25 not shown). mM. EDTA pH 8.0,0.5% SDS, and 0.1 mg./ml. proteinase K) Only the nested PCR method was therefore considered to and incubated at 50C overnight. DNA was extracted from be of sufficient sensitivity for amplification of bacterial DNA these specimens by mixing with an equal volume of phenol/ from IC patient and control bladder biopsy specimens. A chloroformlisoamyl alcohol (25/24/1) and centrifuging 1700 negative control (containing all elements of the PCR reaction xg at 4C for 10 minutes. The DNA was precipitated from the mixture except DNA from a tissue specimen) and a positive aqueous layer by adding 0.5 volume of 7.5 M. ammonium control (containing E. coli DNA instead of DNA from the acetate and 2.0 volume of absolute ethanol a t -8OC for 30 tissue specimen) were run with each set of samples. Results minutes and centrifuging 1700 X g at 4C for 10 minutes. of these experiments indicated the presence of a band of the Following a rinse with 70% ethanol, the pellet was lyophi- appropriate size for the bacterial rRNA gene fragment in all lized, resuspended in 20 p1. TE buffer (10 mM. Tris-HC1 pH twelve biopsy specimens (6IC and 6 control patient samples) 8.0,l.O mM. EDTA pH 8.0), and incubated at 65C for 1hour. (fig.2). Purified DNA specimens were stored at -2OC. Sequence analysis of DNA amplified from one IC patient PCR amplification to a m p w the bacterial 16s rRNA gene biopsy and one control biopsy each appeared to contain genes was carried out using a single set of primers, andlor using from more than one bacterium, and the organisms present in nested primers. The nested primer PCR used to generate most these specimens were therefore unable to be directly identiof the data for this report was performed according to the fied. DNA from the remaining 5 IC patients and 5 controls method previously published by Domingue et a18 To prevent represented a predominant organism. We compared the secontamination bv exogenous DNA. 5 units Thenus ~UatiCus quences obtained from these amplified DNA segments with DNA polymeras;! (AmiliTaq PolGerase, Perkin Elmer, Foster City, CA) were preincubated with 0.2 units bovine pancreatic DNase I (BoehringerMannheim, Indianapolis, IN) for 1hour at 2nd run 1st run 37C, and the DNase then heat inactivated at 95C for 10 minutes. Each DNA specimen was then mixed with 0.2 mM. dNTPs, 1 j g . of each primer, and 5 units DNaSe-treated AmpliTaq polymerase in PCR buffer (50 mM. KCl, 1.5 mM. MgCl,, and 10 mM. Tris-HC1 pH 8.3), and amplification performed using a Perkin Elmer DNA Thermal Cycler p n grammed for 95C denaturation, 58C annealing, and 72C extension temperatures (first 3 cycles had extended denaturation and 0.5kb annealing times). A single round of ampWcation was used for the single primer set method, and two rounds of amplification were used for the nested PCR. The primers used for the single primer set method were 64W-TACCTI'G'ITACGACT1'-3'] and 65[5'-'ITAGATACCCTDGTAGTCC-3' where D equals G or A or TI. Primers used for the 6rst round of the nested a m p w a a m m m E I m a m c n ~ tion were 1 A [ 5 I - C A C A A G C ~ ~ A ~ A ~ ~ ' I and T-3'1 E g Mc M * 0 9e g E x M - me
-
5 [ 5 ' - C C T A C G G Y T A C C T C A C " - 3 ' where Y equals c off l ; t h ~ e u s e d f o r t h e s w e r e 3 [ 5 ' cGCGAAGAACCTl'ACCT-3'] and 45'-GCGGAXmTK'I'G A C G G G c a m m T A - 3 ' where K equals G or T].Following PCR amplification, appm-bly 204b of the -donmixture was run on a 1%agarose/ethidium bromide el,and the P=* pair h g m e n t detected by UV fluorescence. ence of a 467
-
%-?%2%5!y%2 7 7 ~ ~ ~ ~ FIG.1. Polymerase chain, reaction amplification of bacterial 16s
rRNA genes using nested m e ? and two run (round)mpMebon. Follom?~~ second ofam~Mabon,467 pair was evident m samples eontamipg DNA from as few as 6 X lo-' m l o ~ forming unita O ~ Emli . ( s t n u n ~ l m - F~U ) . 1eR lane e b o w ~locatmn of DNA standards,w i t h labeling of 500 bp standard for refemma
282
POLYMERASE CHAIN REACTION AMPLIFICATION
by cystoscopic biopsy from IC or control patients commonly contain bacterial DNA detectable by nested PCR. The discrepancy between our findings and the observations of H m a l a et al. may be explained by the relative insensitivity of PCR Using a single set of primers for DNA encoding bacterial rRNA as compared with the nested PCR method used by Domingue et al. and US. The differences between our findings and those of 0.5kb Domingue et al., however, are more difficult to explain. While our study found bacterial DNA in both Ic and control specimens, their data suggested that bacterial DNA was present almost exclusively in IC specimens, albeit from a minority of patients. Another difference between our studies is the types of organisms identified (Pseudomonas sp. and E. coli in their study, and Acinetobacter sp., Pmpwnobacterium sp., SalmoFIG 2. Amplification of bacterial 16s rRNA nee in bladder bi- nella sp. and E. coli in ours). The two studies, however, are ops specimens from IC and control patients. X n g nested primers anBtwo round amplification d u r e as in fig. 1, a 467 base pair similar in one respect: the bacteria identified by PCR in both frapent was amplified in 6 IC and 6 control patient biopsy investigations are not fastidious and, if alive, should have speamens. been (but were not) culturable. Our findings suggest two hypotheses. The first is that all or most women, with or without IC, have nonculturable or nonviable bacteria on or in bladder mucosa. We are unaware of sequencesfrom various bacterial speciesrecorded in the Gen- other data suggesting this concept. Moreover, were this to be Bank database, and found evidence for the presence of DNA the case, these data indicate that women with IC would not with >95% homology to several bacteria, as shown in the differ from women without IC in this respect. The second hypothesis is that nonculturable or nonviable table. None of the organisms identified by PCR was previously cultured from a duplicate tissue specimen or urine bacteria contaminated the bladder specimens. A review of obtained at C~S~QSCOPYfrom these patients, even though we our methods for collecting and processing of specimens might used a very sensitive culture method that detected as few as be instructive. Copious povidone iodine preparation of the 1 organism per biopsy specimen, or 10 organism per ml. of perineum resulted in 0/7 urine specimens from controls being culture positive for bacteria, even using methods sensitive urine.7 enough to grow organisms at concentrations as low as 10 bacteria per ml. of urine.7 However, cystoscopic biopsy was DISCUSSION performed after collection of urine by catheterization, and That IC might be an infectious disease is not an unreason- could have introduced organisms into the bladder. Indeed, able hypothesis, given its epidemiology and the tissue intlam- three of the bladder specimens, when homogenized and culmatory changes sometimes seen with this disorder. However, tured, grew very small numbers of organisms (though differour previous study that used very sensitive culture tech- ent species than those identified by PCR).7 This finding is niques to look for a variety of microorganisms in both urine consistent with small numbers of live organisms contaminatand bladder tissue of IC patients did not find evidence for an ing the cystoscope or biopsy forceps. It is also possible that bacteria killed or damaged by povidone iodine could have infectious etiol~gy.~ In planning our previous studies, we had hypothesized contaminated bladder specimens during insertion, removal, that a method such as PCR might be too sensitive to search or subsequent manipulation of the cystoscope. In addition, for a bacterial c a w for IC, and would amplie DNA from specimens were obtained with cold cup biopsy forceps that urethral organisms introduced by the cystoscope. We there- were routinely hand cleaned with a soap solution (Endozyme fore concentrated on techniques that could identify replicat- solution, Ruhof Corp., Valley Stream, NY), washed additioning microorganisms. However, the reports of Domingue et al. ally in a commercial washer, then decontaminated in Cidex and Haarala et al. indicated that all of their control speci- solution (Johnson & Johnson, Arlington, TX) over several mens were negative for bacterial DNA by PCR. Although hours; conceivably, bacteria killed by this method of cleaning Haarala et al. also did not find evidence for bacterial DNA in could remain on the forceps and their DNA could have been IC specimens, the finding by Domingue of DNA from various amplified by the PCR procedure. We favor the second hypothesis and feel that the bladder bacteria in bladder tissue from a significantly greater percentage of IC patients than controls suggested that nested specimens obtained from IC and control women were probaPCR might pmve useful to identify a bacterial cause for IC. bly contaminated with nonviable organisms a t the time of Because we determined that this method was indeed sufii- cystoscopy, from the urethra, the cystoscope sheath, the CYSciently sensitive to detect bacterial DNA from less than one toscope, or even the biopsy forceps. Once obtained, the specE. coli bacterium, we proceeded to examine bladder biopsy imens were immediately placed in sterile sealed containers; tissue fiom both IC and control patients. at opening they were processed in parallel with negative Our findings indicate that bladder tissue specimens ob- controls which remained negative, suggesting it is unlikely that the processing procedure itself caused contamination with bacterial DNA. Identity of bacteria present in IC and control bladder biopsies Although the three reported studies (by Domingue et al., Haarala et al., and this report) that sought bacterial DNA in IC Contml IC patient bladder tissue by PCR have reached different Aeinetobeeter sp. Propionobacterium sp. E. coli (strain #1)* specific conclusions, one observation is consistent: no single Propionobncterium sp. E. coli (strain #2)* E. wli (etraint l P microorganism appears to be associated with IC. Our own E. coli (sbain#2P E. coli (strain #2)* data further indicate that a very sensitive method for the selmonella sp. E.coli (strain #2)* detection of bacteria such as nested PCR may be inapproprimixed mixed Taro sbainsof E.ooli ware wident by idantid base pair changesat speci6c ate and unhelpful for determining whether bacterial DNA is allsk. present in tissue specimens obtained at cystoscopy. Methods
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aimed a t detection of tissue-associated bacteria, such as in situ hybridization, may be more helpful for future studies. The authors gratefully acknowledge Edward W. Campbell, Jr., M.D.., Michael J. Naslund, M.D., Ms. Linda Home and Terry Wilson, R.N., for their assistance with patient enrollment and specimen acquisition, and Michael Donnenberg, M.D. for helpful discussions. REFERENCES
1. Warren, J. W.: Interstitial cystitis as an infectious disease. Urol. Clinics North Am., 21: 31,1994. 2. Messing, E. M. and Stamey, T. A.: Interstitial cystitis: early diagnosis, pathology, and treatment. Urology, 12: 381,1978. 3. Oravisto, K J.: Epidemiology of interstitial cystitis. Ann. Chir. Gynaecol. Fenn., 64: 75,1975. 4. Warren, J. W.: Clinical presentation and epidemiologyof urinary
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tract infections. In: Urinary Tract Infections, Mobley, H. L. T. and Warren, J. W., eds. Washington, D. C., ASM Press, 1996. 5. Held, P. J., Hanno, P. M. and Wein, A. J.: Epidemiology of interstitial cystitis: 2. In: Interstitial Cystitis, Hanno, P. M., Staskin, D. R., Krane, R. J. and Wein, A. J., eds. London, Springer-Verlag, 1990. 6. Said, J. W, Van de Velde, R. and Gillespie, L.: Immunopathology of interstitial cystitis. Mod.Pathol., 2 593,1989. 7. Keay, S.,Schwalbe, R. S., Trifillis, A. L., Lovchik, J. C., Jacob, S.and Warren, J. W.: A prospective study of microorganisms in urine and bladder biopsies from interstitial cystitis patients and controls. Urology, 45: 223,1995. 8. Domingue, G. J., Ghoniem, G. M.,Bost, K L., Fermin, C. and Human, L. G.: Dormant microbes in interstitial cystitis. J. Urol., 153: 1321,1995. 9. Haarala, M.,Jalava, J., Laato, M.,Kiilholma, P., Nurmi, M.and Alanen, A: Absence of bacterial DNA in the bladder of patients with interstitial cystitis. J. Urol., 166: 1843,1996.