Interstitial cystitis (IC), a chronic inflammation of the bladder of unknown etiology, is characterized by bladder pain, urinary frequency, and urgency. While first recognized in 1907, Hunner fully did not describe the urothelial ulcers seen in the more severe cases until 1915. IC has often been associated with other systemic diseases, including lupus erythematosus, rheumatoid arthritis, and polyarteritis. More than 30% of patients with IC have a history of allergic conditions. The diagnostic triad is sterile urine, chronic unexplained irritable voiding symptoms which are often associated with premicturitional suprapubic pain and a history of dyspareunia, urothelial glomerulations or ulcerative disruption of the urothelium (Hunner’s ulcer) at cystoscopy.

Interstitial cystitis has been subdivided into early and late phases. In the early phase, the bladder capacity is greater than 450 cc; irritative voiding symptoms are moderate and cystoscopy reveals only glomerulations which have pinpointed petechial hemorrhagic lesions within the urothelium. Average age of these patients is 38 years old. In the classical late phase, the bladder capacity is reduced, i.e. is less than 450 cc; voiding symptoms are severe and cystoscopy reveals Hunner’s ulcer and urothelial fissures. These patients are older and the average age is 57 years old. Biopsy of the bladder reveals pancystitis with transmural inflammation and fibrosis. Marked submucosal edema and vasodilatation are evident. Mast cells in the submucosa, especially in the muscle layers, are strongly suggestive of IC. The ratio of affected women to men is 9 to 1. IC has a rapid onset with its peak incidence in patients in their 30s and 40s. The syndrome waxes and wanes for 30 years or longer and can be debilitating; IC patients report less life satisfaction than patients with end-stage renal disease (Trifillis, 1995). It is estimated that 20,000 to 90,000 women have this condition in the United States. Hypotheses about the etiology of IC include neuropathic processes, abnormal inflammatory or immune states, chronic infection by difficult-to-culture organisms and defects in the epithelial barrier of the bladder ( Messing, 1994).

At the time of by cystoscopic diagnosis, bacteriuria is not substantiated by routine microbiological cultures of multiple specimens. A number of investigators have suspected an infectious etiology and used special techniques for unusual organisms such as mycoplasma, chlamydia, anaerobic bacteria, fungi and viruses. No microorganism has been convincingly incriminated as a cause of IC, and most authorities have questioned the role of infectious agents in its etiology because of negative routine culture. Present knowledge of urinary tract infection (UTI) and ecological microbiology may yield hints for appropriate techniques useful in the search for microbes in the pathogenesis of IC.

First, bacteria may be present in or on the bladder mucosa but not in the urine. In women, bacterial entry into the bladder is relatively frequent; more than 99% of organisms are cleared with the next voiding. Remaining bacteria may attach to the mucosa and secrete exopolysaccharides which surround the enlarging colonies and create a microenvironment to protect the bacteria from mechanical dislodgment, phagocytosis, and even antibiotic effects. In 1985, Hultgren et al., made observations in an experimental animal model of UTI that 60% of mice with Escherichia coli adherent to bladder mucosa have sterile urine. The work of Elliott et al. (1986) also supported this hypothesis. They obtained urine and bladder biopsies from 25 patients with chronic UTI, who had urinary symptoms one week prior to biopsy and no antibiotics for one month. At the time of the biopsy, 13 patients did not have bacteriuria and none of them has more than 10³ bacterial colonies per ml. By scanning electron microscopy, 12 of 13 biopsies had visible bacteria adherent to the bladder mucosa. The investigators isolated organisms from the bladder biopsies of 7 of these 12 patients; E. coli and Proteus mirabilis were prominent. Warren et al., (1994) demonstrated that human renal and bladder epithelial cells internalize bacteria.

Second, animal work indicated that certain bacterial constituents such as bacterial endotoxin and P fimbriae in the absence of whole organisms could elicit urinary inflammation. Bacterial endotoxin and P fimbriae (an adhesin of E. coli for uroepithelial cells) each alone cause inflammation following bladder instillation (Warren 33). Third, bladders of IC patients may contain viable but non-culturable organisms. In low-nutrient fluids, bacteria are dormant, only infrequently dividing. Culture techniques depending upon actively growing organisms are inadequate for discovery of these microbes. Human pathogens, including E. coli, Klebsiella, Salmonella, Vibrio and Legionella species are capable of this type of dormancy. Permeability is consistent with an occult bladder infection. Bacterial cystitis is also associated with glycosaminoglycan deficiencies, widened tight junctions, increased permeability and circulating antibodies to Tamm-Horsfall protein (urinary slime). A microbe may cause a breech of the epithelial barrier by disrupting the glycosaminoglycan or by widening a tight junction. Fourth, these bacteria may not grow very well due to possible local mucosal IgA inhibition on the bladder surface.

It has also been proposed that IC may be the consequence of a host immunological response such as autoimmunity. A microbe may possess a surface component that is antigenically similar to a structure on human bladder epithelial cells. The inflammatory response to the microbial antigen cross-reacts with the host antigen and elicit an inflammatory reaction. Alternatively anti-idiotype antibodies may bear the internal image of the original microbial antigen and therefore bind to a receptor which may be present on bladder cells.

In the meantime, the pathogenesis of another inflammatory disease, chronic gastritis which has been attributed to Helicobacter pylori, is solved by discovery. Chronic gastritis is a syndrome characterized by persistent pain in a lumenal organ in which epithelial damage, inflammatory response and ulceration are present. Special culture and staining techniques have helped distinguish this organism as the apparent cause of many cases of gastritis (Weiss, 1994). Is IC caused by the similar stealthy pathogens?

Studies of bacterial urinary tract infection have prompted an interest in IC (Reid, 1987). The epithelium may be a critical site of pathogenesis because the hallmarks of the disease are visible epithelial defects, i.e., Hunner’s ulcers and epithelial ruptures. Areas denuded of epithelium are commonly seen and defects in epithelial permeability are characteristic. Therefore, epithelial cells may be targets for initiating agents and inflammatory effects of IC and have been useful for studies of the pathogenesis of this disease (Trifillis, 1995). Bladder epithelium can be cultured for microorganisms. Special culture techniques should be considered, as the microorganisms may be fastidious.

Other techniques may be employed to discover microbes. The polymerase chain reaction (PCR) is a useful method to identify non-cultivable bacteria (Warren, 1994). The PCR technique devised by Kary Mullis in the mid-1980s has revolutionized molecular genetics by making possible a whole new approach to the study and analysis of genes. The PCR exploits certain features of DNA replication. DNA polymerase uses single-stranded DNA as a template to synthesis of a complementary new strand by specifically or randomly designed primers. Taq polymerase, purified from Thermus aquaticus, has a temperature optimum of 72° C and is reasonably stable at temperatures as high as 94° C. At high temperatures, most of double-strand structure of DNA is denatured and becomes single-strand structure which can easily be accessed a single addition of the enzyme by Taq polymerase. Taq polymerase can be added just once at the beginning of a reaction and will remain active through a complete set of many amplification cycles resulting in the general 2^n-3 to 2ⁿ copies ot the target sequences. n is number of cycles. This development has allowed the automation of the PCR through the use of thermal cyclers, heating blocks that can be programmed to carry out the time and temperature cycles for a PCR (Watson, 1993).

In numerous reports, PCR has been applied to the detection and identification of specific bacterial pathogens. Often, the target genes are known to be involved in the pathogenicity or virulence of the pathogen. The use of universal PCR primers targeting conserved bacterial DNA regions for the purpose of DNA sequencing has also been described. These primers and probes have been designed for the detection of bacteria in blood, CSF, and other normally sterile body fluids. In fact, universal bacterial primers have been tested for the purpose of detection of major bacterial species causing septicemia and meningitis and also to diagnose common contamination of clinical samples (Greisen et al., 1994). Detection of occult bacteria in IC may be accomplished by using PCR (Domingue et al., 1995).

Which regions or genes should we choose to detect IC by PCR? Ribosomal RNAs maintain a high degree of structural conservation. (Banks et al., 1993). There are conserved domains in the 16S ribosomal RNA (rRNA) genes common to various bacterial species. All rRNAs appear to be identical (or very nearly so) in function, for all are involved in the production of protein. The overall three-dimensional rRNA structure that corresponds to this function shows only minor, but in some senses highly significant, variation. While mutations occur at the same rate of any other genes on the bacterial chromosome, rRNAs are fixed at a slow and somewhat constant rate. The approach employed in the current study that synthetic nucleotides can serve as PCR primers to these regions and can be used to detect unknown fastidious bacteria from IC patients’ biopsies.

PCR amplification unknown DNA via the primers of conservative 16S rRNA genes can be a potential efficient diagnostic method. In 1994, Weiss et al., showed that 94% sensitivity and 100% specificity by PCR on the biopsy specimens for the diagnosis of H. pylori infection. The commercially immunoassay only has 94% sensitivity and 98% specificity. It was highly reliable for the detection of infection H. pylori which was presented in the population of chronic active gastritis. To improve the clinical application of PCR, Greisen et al., (1994) designed a set of broad-range PCR primers for 16S rRNA gene. The first series (universal Gram-positive and Gram-negative oligonucleotide probes) correctly identified the majority of the 102 bacterial species tested. The second series included seven probes which detected DNA from the major bacterial causes of meningitis: N. menigitidis, H. influenzae, S. pneumoniae, S. agalactiae, E. coli and other enteric bacteria, L. monocytogenes and Staphylococcus aureus. The third series consisted of five probes for species or genera which were commonly considered contaminants: Bacillus, Corynebacterium, Propionibacterium and coagulase-negative Staphylococcus spp. Besides PCR amplification by conservative rRNA primers, we already have the sizable rRNA gene database server to access.

The first complete 16S rRNA sequence was determined in 1978. Since then, the number of complete 16S-like rRNA sequences has reached over 2,200 (compilation as of August 1993) in GenBank (Gutell, 1994). This molecule has been the focus of many phylogenetic studies. The more highly conserved regions can be used to compare distantly related organisms, and the more rapidly evolving segments provide useful data for closely related species. Thus, the 16S rRNA gene has frequently been referred to as a "molecular chronometer" used to reflect the evolutionary distance between two bacteria over time. Actually, all species could be compared through a single type of molecule with a highly conserved function. Indeed, the whole microbial phylogenetic superstructure rested almost entirely on studies of rRNA sequences (Singer et al., 1991).

Sequence comparison of 16S rRNA from bacteria is one of the most powerful and precise methods for determining phylogenetic relationships. Sequences of 16S rRNA are used in many basic investigations of phylogenetic relationships, secondary structures and bacterial diversity from a specific environment. rRNA has been used to develop identification systems for mycoplasma (Bascunana; Pewtterson, 1994). Partial 16S rRNA sequences have been used in a number of phylogenetic studies of the Genus Campylobacter of organisms and in the development of rapid identification methodology (Trust et al., 1994).

Dr. Domingue (Department of Microbiology and Urology, Tulane University Medical Center) has hypothesized that IC could be caused by some difficult-to-culture bacteria (Domingue, 1995). In order to evaluate this hypothesis, Drs. Domingue and Johnston designed newly methodology that combined 16S rRNA primer PCR techniques and sequencing. Unknown DNA was amplified from IC patients’ biopsies by 16S rRNA universal primers. There were two strategies to sequence these unknown DNA. One was directly to sequence PCR amplified DNA; another was to clone these PCR products into pUC18 vectors and sequence PCR products on pUC18-IC clones. Dr. Domingue chose the later method, because of several reasons. First, amount of IC patients’ biopsies was limited. Cloning would store these DNA and enable us avoid taking sample from the same patient again. Second, the PCR products could be heterogeneous. It could reserve the variety of PCR products to construct a library of these DNA domains.

In this thesis, the amplified DNA was subjected to sequence analysis to determine whether and which microbial sequences were present. To sequence efficiently, Dr. Johnston developed a method to purify the biotinylated, single-strand template DNA by magnetic beads. To compare the similarity of these clones’ sequences to reported 16S rRNA genes, these sequences were compared to the BLAST (Basic Local Alignment Search Tool) network service (Blaster) in National Center for Biotechnology Information (NCBI) of NIH (Altschul et al., 1990) and RDP (ribosomal database project) from University of Illinois (Maidak et al., 1994).

This a newly developed protocol can be a convenient approaching method to diagnosis IC patients. This new method possess the advantages of time-saving, no special techniques or equipment required and high specificity. The results reveal various species of bacteria. Meanwhile, which database is more reliable and which bacterium is a infectious agents of IC are questions that need to be resolved. In the future, we may contribute to developing effective diagnosis and treatment for IC patients.