James L. Duncan

EVALUATION OF THE BACTERIAL FLORA OF THE PROSTATE USING A 16S rRNA GENE BASED POLYMERASE CHAIN REACTION

PURPOSE The role of bacteria in the chronic pelvic pain syndrome (nonbacterial prostatitis and prostatodynia) is controversial and difficult to assess because the bacterial flora of the prostate is not well defined. Polymerase chain reaction (PCR) is a highly sensitive molecular method of bacterial detection. It confirms the sterility of tissue with a high level of confidence and detects small numbers of microbial agents that may represent pathogens. We performed PCR to determine bacterial colonization of the prostate in presumably healthy men and in those undergoing simple or radical prostatectomy. MATERIALS AND METHODS We analyzed 28 prostate samples from 18 organ donors from whom prostate tissue was obtained under sterile surgical conditions at organ withdrawal, 14 sterile surgical prostate specimens from 7 patients undergoing radical prostatectomy for prostate cancer who previously underwent transrectal biopsy and 6 sterile surgical specimens from 2 men who underwent simple prostatectomy for benign prostatic hyperplasia (BPH), including 1 with an indwelling catheter for several weeks. For PCR we used 2 sets of primers to detect bacterial 16S rRNA gene sequences. Normal prostate tissue seeded in vitro with known numbers of Escherichia coli was used to assess the sensitivity of PCR. RESULTS Only 3 of the 28 organ donor samples had histological signs of minimal inflammation and all other samples appeared to be normal without evidence of inflammatory reaction. All of these samples were PCR negative. Of several PCR control reactions the mixture of prostate tissue seeded with known numbers of E. coli demonstrated the high sensitivity of the assay, allowing the detection of as few as 6 bacteria in the presence of 25 mg. of prostate tissue. A focal and heterogeneous distribution of inflammation and infection was noted in the 14 radical prostatectomy specimens. In the prostate cancer and BPH groups there was a strong association of inflammation with positive PCR findings. Of 11 samples 3 without but all 9 with inflammation were PCR positive. CONCLUSIONS PCR is a highly sensitive method for detecting bacteria in the prostate. In our study negative PCR reactions in the prostate tissue of apparently healthy men made the presence of normal bacterial flora in the prostate extremely unlikely. The presence of bacteria and/or inflammation in radical prostatectomy specimens was found to be a localized process. Concordance between inflammation and positive PCR results in simple and radical prostatectomy specimens suggests that bacteria may frequently have a role in histologically inflammatory prostatitis.

Approximate dimensions of membrane lesions produced by streptolysin S and streptolysin O

Membrane lesions produced by the streptococcal membranolysins streptolysin S and streptolysin O were investigated. Escape of labeled marker molecules of various sizes from resealed sheep erythrocyte ghosts treated with the toxins for 30 min allowed estimation of the sizes of the primary channels formed. Streptolysin S formed lesions ranging in size up to 45 A in diameter, and even high toxin concentrations did not result in larger channels. The lesions produced by streptolysin O exceeded 128 A in diameter. Kinetics experiments demonstrated that the primary streptolysin O lesions were formed rapidly (1-2 min), but release of marker molecules from streptolysin S-treated vesicles began only after a 5-15-min lag period. Label release from large unilamellar liposomes treated with streptolysin S suggested that membrane fluidity does not affect the size of the streptolysin S lesions.

Psychosocial variables affect the quality of life of men diagnosed with chronic prostatitis/chronic pelvic pain syndrome

To examine interactions between demographic, pain, urinary, psychological and environmental predictors of quality of life (QOL) in men with chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS).

Host pathogenesis in urinary tract infections.

Urinary tract infections (UTIs) are the result of an interaction between bacterial virulence and host defense factors that compete to invade or protect the host, respectively. Research over the past 30 years has demonstrated that vaginal colonization with uropathogens precedes most UTIs. Receptivity of the vaginal mucosa for uropathogens is an essential initial step in vaginal mucosa colonization. When vaginal and buccal epithelial cells were collected from patients susceptible to reinfection and compared with such cells obtained from controls resistant to UTIs, the strains that caused cystitis adhered much more avidly to the epithelial cells from susceptible women. These genotypic traits for epithelial cell receptivity may be a major susceptibility factor in UTIs. The presence or absence of blood group determinants on the surface of uroepithelial cells may influence an individuals susceptibility to UTIs. The protective effect in women with the secretor phenotype may be due to fucosylated structures at the cell surface which decrease the availability of putative receptors for Escherichia coli. Susceptibility among women who do not secrete blood group antigens may be due to specific E. coli-binding glycolipids that are absent in women who secrete blood group antigens. Recent studies have shown that the vaginal fluid, which forms an interface between uropathogens and epithelial cells, also influences vaginal colonizations.

Intraorifice sealing of gutta-percha obturated root canals to prevent coronal microleakage

A study was conducted to evaluate Cavit, Intermediate Restorative Material, and Super-EBA as intraorifice filling materials to prevent coronal microleakage. Root canal instrumentation and obturation was done on 74 extracted single-rooted teeth. Three and one-half millimeters of the gutta-percha was removed from the coronal aspect of the root canal and replaced with one of the three filling materials. The teeth were suspended in scintillation vials containing trypticase soy broth, and human saliva was added to the pulp chambers. Microbial penetration was detected as an increase in turbidity of the broth corresponding to bacterial growth. At the end of 90 days, the results showed that 15% of the Cavit-filled orifices leaked, whereas 35% of the Intermediate Restorative Material and Super-EBA-filled orifices leaked. The gutta-percha obturated root canals that received an intraorifice filling material leaked significantly less than the obturated, unsealed control group--all of which leaked in < 49 days.

Do infectious agents cause interstitial cystitis

The possibility that infectious agents play a role in the etiology of interstitial cystitis (IC) has been investigated for a number of years. Early studies were directed toward attempts to cultivate bacteria and fungi on routine culture media and microscopic examinations of urine or bladder tissue specimens for the presence of microorganisms. In more recent years, this approach has been expanded to include sophisticated culture techniques to search for the presence of fastidious and unusual organisms that would not be detected by routine culture methods. Similarly, the presence of viruses has been sought by incubating specimens from interstitial cystitis patients in mammalian cell cultures to detect cytopathic effects. None of these approaches has provided convincing evidence that micro-organisms or viruses are associated with IC. The latest attempts to search for the presence of bacteria have made use of the polymerase chain reaction (PCR) to amplify bacterial 16S rRNA genes that would be present if bacteria were present in bladder tissue or urine of IC patients. This approach allows bacteria to be detected and even identified without culture. However, the results from the great majority of bladder biopsy samples analyzed by these molecular techniques have been negative. PCR strategies have also been used to search for the presence of certain viruses in IC specimens, again without success. At this time, the results from laboratory culture, light and electron microscopy, and various molecular strategies to detect micro-organisms and viruses in IC specimens all argue against an infectious etiology for IC.

Mannose-Sensitive Adherence of Escherichia Coli to Epithelial Cells from Women with Recurrent Urinary Tract Infections

The effect of D-mannose on adherence of 73 Escherichia coli strains to vaginal and buccal epithelial cells from women with recurrent urinary tract infections, and on agglutination of human and guinea pig erythrocytes was tested. Urinary, vaginal or anal isolates from women with such infections were used. Of the strains 66 (90 per cent) demonstrated adherence to epithelial cells. D-mannose inhibited completely the adherence of 25 strains (42 per cent) that adhered to vaginal cells and inhibited an additional 11 strains (18 per cent) by at least 50 per cent. Similar results were obtained with buccal cells. The inhibitory effect of D-mannose was similar regardless of the origin of the strains. Hemagglutination frequently was inhibited by D-mannose but no consistent association between hemagglutination, and epithelial cell adherence and the effect of D-mannose was observed. The results suggest that mannose-sensitive as well as mannose-resistant adhesins frequently mediate Escherichia coli adherence to vaginal epithelial cells, and may contribute to vaginal colonization and cystitis.

Mannose‐sensitive haemagglutination in the absence of piliation in Escherichia coli

The relationship between type 1 pilus structure and the mannose‐sensitive adhesin was investigated by analysing the properties of an 11.2 kb fragment of DNA derived from the chromosomal pil region of a type 1 piliated uropathogenic strain of Escherichia coli. The recombinant plasmids pHA9 and pSJH9, containing the cloned fragment, conferred a mannose‐sensitive haemagglutination (MSHA)‐positive but non‐piliated phenotype on recipient cells of ORN104. Most of the DNA sequences homologous to the pilA and hyp genes were not present in the 11.2kb insert, and the genetic information necessary for MSHA in the absence of piliation spanned a 6.5 kb region of the cloned fragment. The polypeptides expressed by pSJH9 were examined in minicells and Tn 1000 insertions in three genes encoding proteins of molecular weights 90 kD, 29 kD and 17kD abolished the MSHA phenotype.

Blood Group Antigen Expression on Vaginal and Buccal Epithelial Cells and Mucus in Secretor and Nonsecretor Women

Blood group antigens on epithelial cells may influence bacterial adherence to mucosal surfaces. In the urinary tract the presence of these genetically determined carbohydrate structures may affect bacterial colonization of the vaginal mucosa and subsequent urinary tract infection. In previous studies the detection of ABH and Lewis antigen expression and distribution in tissues have made use of semiquantitative immunohistochemical staining techniques. To determine the pattern and intensity of blood group antigens on epithelial cells and in the mucus overlying them, we developed quantitative immunoassays that use monoclonal antibodies to detect changes in the expression and intensity of ABH and Lewis antigens on cells and in mucus. Vaginal and buccal cell specimens from 23 healthy women (15 secretors and 8 nonsecretors) with no history of urinary tract infections and known blood group types were analyzed for the expression of these antigenic determinants. The profile of ABH antigen expression was generally consistent with the ABO phenotype of the individual and appeared to be influenced by the secretor status; the levels of A, B and H determinants were higher for secretors than nonsecretors. Lewis antigens were detected on vaginal and buccal cells, and mucus. Le(a) and Le(x) antigen expression was greater on cells and mucus from nonsecretors, whereas the expression of Le(b) and Le(y) was greater on cells and mucus from secretors. Variability in antigen expression was observed among individuals with the same blood type and secretor status. The patterns of antigen expression were similar for the vaginal and buccal cell, and mucus samples of an individual but the amount of antigen generally differed for the various samples. These findings document the variation of blood group antigen expression on vaginal epithelial cells and mucus, which may have a significant role in susceptibility to urinary tract infections in women.

Relatedness of Escherichia coli colonizing women longitudinally.

PURPOSE The longitudinal colonization patterns by Escherichia coli of the vaginal introitus and urinary tract were investigated. MATERIALS AND METHODS Cultures of the vaginal introitus and midstream urine were collected once a week for 12 consecutive weeks from five women with (patients) and five without (controls) a history of urinary tract infection (UTI). RESULTS A total of 63 E. coli isolates was obtained from the 10 women, 26 from controls and 37 from patients. The bacterial counts of E. coli present in control individuals were uniformly low, < or = 200 E. coli/mL. The numbers in patients were higher and more variable, reaching > 10(5)/mL in urine and vaginal specimens. In 16 instances, E. coli was present in the urine and the vaginal introitus concurrently (matched isolates). Random amplified polymorphic DNA (RAPD) fingerprinting was used to characterize all matched E. coli isolates. Concurrent vaginal and urinary tract colonization was more common in the patient population, and usually, the same E. coli strain was present at both sites; only 15% of the matched isolates represented different strains. The RAPD fingerprinting was also carried out on selected isolates recovered from four patients and three control individuals over the 12-week study period. Colonization of the vaginal introitus and urinary tract in these individuals varied over time. Generally, however, a predominant E. coli strain was present in the vaginal milieu, urinary tract, or both, either continuously (for as long as 9 consecutive weeks in one patient) or intermittently. CONCLUSION The results support the concept that the vaginal mucosa acts as reservoir of E. coli which may enter the urinary tract.