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Bacterial Infection in Prostatodynia
0022-5347/95/1544-1376$03.00/0
Vol. 154, 1378-1381, October 1995 Printed in U . S A
THE JOURNAL OF UROWGY Copyright 0 1995 by
h l E R l C A N UROLAXICALASSOCIATION, I N C
BACTERIAL INFECTION IN PROSTATODYNIA JOSHUA E. LOWENTRITT, KAZUYA KAWAHARA,* LISET G. HUMAN, WAYNE J. G. HELLSTROM AND GERALD J. DOMINGUE From the Departments of Urology and Microbiology and Immunology, Tulane University School of Medicine, New Orleans, Louisiana
ABSTRACT
Purpose: We investigated a possible bacterial etiology for prostatodynia. Materials and Methods: We evaluated segmented urine specimens from 22 patients and 16 controls by bacteriological localization studies. Immunological studies were performed on patient and control sera. Results: Nine patients had positive cultures from prostatic secretions. When compared to controls, this novel finding was statistically significant (p <0.025). Coagulase-negative staphylococci were the most common isolates (68%).No humoral ( 1 6 )immune differences were found between patients and controls. Conclusions: In a subset of prostatodynia patients bacteria may have a n etiological role. Antibiotic treatment demonstrated clinical efficacy. KEYWORDS:prostate, infection, prostatitis, Staphylococcus
Prostatodynia is a commonly diagnosed and poorly treated urological syndrome. In a large study of 597 prostatitis patients nearly a third were diagnosed with prostatodynia, which is a significant fraction of the urological patient population.' Clinically prostatodynia frustrates the patient and physician due to its chronicity and resistance to therapy. The syndrome is usually characterized by persistent perineal pain, and functional and somatic urological complaints, including abnormal urine flow, frequency, urgency and dysuria. The diagnosis is differentiated from other common prostatitis syndromes by a lack of prostatic inflammation and no Significant leukocytes or bacteria in the expressed prostatic secretions.2 However, the etiology of prostatodynia is unknown despite these distinct diagnostic criteria. Urodynamic research by Barbalias3.4 and Hellstrom~e t al, and Meares6 established that prostatodynia patients have an acquired functional disorder of the bladder neck and prostatic urethra, causing an intraprostatic reflux of urine and resulting in "chemical prostatitis."7 Blacklocks and Millerg suggested that prostatodynia is causally associated with psychological factors, including anxiety states and excessive tension or stress. However, no definitive pathophysiological agent or mechanism has been clearly established. To evaluate a possible infectious etiology of prostatodynia we studied segmented urine specimens and sera from a series of patients. In this prospective nonrandomized bacteriological, clinical and immunological study we compared nonblinded patient findings to those of a normal control group. We did not standardize empirical antibiotic treatment regimens in this study although we analyzed treatment data, and performed microbiological and symptomological evaluations for therapeutic efficacy. MATERIALS AND METHODS
Patient populations. During 3 years 26 symptomatic patients with a clinical diagnosis of prostatodynia were referred to our institution for study. Most patients had been treated by many urologists and had completed multiple courses of different antibiotics before being examined a t our clinic. We Accepted for publication March 31. 1995. Supported in part by a fellowship from the American Foundation for Urologic Diseases, Inc. and instltutional grants from Tulane University School of Medicine. * Current address: Kagoshima IJniversity School of Medicine, Kagoshima, 890, Japan.
later confirmed each clinical diagnosis by microscopic examination of prostatic secretions, particularly the concentration of white blood cells per high power field. Investigators who study prostatodynia disagree regarding the precise concentration delimiting normal from excessive white blood cells per high power field in the expressed prostatic ~ e c r e t i o n . ~ . ~ We used the level of 20 white blood cells per high power field in expressed prostatic secretions as an exclusionary upper limit to separate patients with a noninflammatory prostatitis syndrome (prostatodynia) from inflammatory disorders, chiefly nonbacterial prostatitis and chronic bacterial prostatitis. Four patients referred with symptoms suggestive of prostatodynia who had greater than 20 white blood cells per high power field in expressed prostatic secretions were excluded from the study and further analysis. Thus, 22 patients with the clinical diagnosis of prostatodynia confirmed by microscopy were included. Controls included 16 asymptomatic men with no recent genitourinary complaints. Clinical evaluations. Patients and controls were evaluated by the same physician for routine bacteriological localization studies, as described by Meares and Stamey.10 The urethral urine, midstream bladder urine, expressed prostatic secretion by prostate massage and post-massage urine were collected, examined microscopically and cultured bacteriologically. Venous blood specimens for immunological studies were allowed to clot and were centrifuged. Serum was stored a t -70C in small aliquots. Serum specimens were thawed no more than twice before immunological study. Bacteriological methods. Quantitative routine cultures were performed on urine, and centrifuged urine sediments were inoculated into tryptose broth to detect colony counts fewer than 100 colony-forming units per ml. Standard microscopy was accompanied by fluorescent acridine orange staining for bacterial, leukocytic and cellular nucleic acids. All cultured bacteria were identified by biotyping. Extractions of bacterial proteins. To determine possible immune responses to solubilized bacterial antigens, a protocol was adapted from Bhaduri." Bacteria isolated from patients were cultured in 1,600 ml. tryptose broth overnight, collected by centrifugation, washed twice in phosphate b d ered saline and resuspended in 25 ml. cold acetone. After 1 hour of incubation with agitation at OC cells were collected and excess acetone w a s removed over a nitrogen stream. Cells were resuspended in 10 ml. I(.; sodium dodecyl sulfate a t room temperature for 15 minutes. The mixture was cen-
1378
1379
BACTERIAL INFECTION IN PROSTATODYNIA
trifuged, the supernatant was removed and proteinase inhibitors (0.5 mg./ml. phenylmethylsulfonylfluoride and 1.0 mgJ ml.knzamidine) were added. The supernatant was fdtered by ultrafiltration over a nitrogen stream, dialyzed with 3 changes of 0.01 M. phosphate buffered saline (pH 7.2)for 72 hours at 4C and stored at 4C. Protein concentration was determined by a modified Lowry assay. Indirect enzyme-linked immunosorbent assay. Our indirect enzymelinked immunosorbent assay method was adapted from that of ShortWe et a1.12 Binding and diluting buffers were identical: 0.01 M. phosphate buffered saline (pH 7.2) with 0.05% polysorbate 20, 1% bovine serum albumin and 0.05% sodium azide. The washing buffer was 0.01 M. phosphate buffered saline (pH 7.2)with 0.5% polysorbate 20 and 0.01% sodium azide. Briefly, in each well of a U-bottom microtiter enzyme-linked immunosorbent assay plate 50 pg. solubilized bacterial antigen were suspended in the binding buffer and incubated for 1 hour at 37C, and wells were washed 3 times with washing buffer. Serum (100pl.) from a patient or control was serially diluted in diluting buffer in each well and incubated for 2 hours at 37C and wells were washed as noted previously. Goat anti-human IgG horseradish peroxidase (100pl.) was diluted in diluting buffer to 15 units per ml. and incubated for 2 hours at 37C, and wells were washed 5 times as noted previously. "hen 100 p,l. substrate solution were added to each well and allowed to develop for 1 hour in a dark chamber at room temperature. Absorbance was read at 410 nm. and the titer was read as the reciprocal of the highest dilution producing an absorbance greater than 0.100. Whole cell enzyme-linked immunosorbent assay. Whole cell enzyme-linked immunosorbent assay protocols were adapted from those of Henricksen et al.13 Using bacteria cultured from patients, and sera from patients and controls assays were performed for humoral (IgG) immune responses to the organisms isolated. Briefly, bacteria were cultured in Luria broth overnight. Cells were collectedby centrhgation, washed in phosphate buffered saline with divalent cations (phosphate buffered saline, calcium and magnesium) and adjusted to standard optical density. A solution of los cells per ml. (900 4.)and 100 4. serially diluted patient or control serum with 5% bovine serum albumin was inoculated in microcentrifuge tubes for 1 hour at 37C.Cells were washed twice with phosphate buffered saline, resuspended in 1 ml. phosphate buffered saline with 0.25% bovine serum albumin containing a 1:1,000dilution of goat anti-human IgG horseradish peroxidase conjugate and absorbed for 1 hour at 37C. Cells were washed 3 times, resuspended in 50 pl. phosphate buffered saline, transferred to a modified flabbottom micm titer enzyme-linked immunoaorbent assay plate and 100 pl. substrate were added. After developing in a dark chamber for 1 hour at mom temperature absorbance was read at 410 nm. Titers were defined as the reciprocal of the highest dilution with a minimum absorbance of 0.200.
TABLE1. Comparison of patient and contrul subject ages, presenting complaints, micmscopy and culture of segmented urine specimens with localization studies characteristic PLe. Controls 22
No. subjects No. specimens
Age: Mean
f SD
h P No. plun complaint 1%)
46
16 16
40.1 2 10.6 31.4 f 10.7 21-62 20-60 20 (89)
No. umdynamic complaint (8) No. expressed pmtatic -tiom with 0 to 10 white blood cells/high power field (%): Cdtllre-p.*
cultureneg. No. expressed prostatic secretion with 11 to 20
white blood cells/hiph power field (46): Culture-p. CUltllre-neK.
No. dtllre-p&. expressed pmntatic secretion (%)t * chi-squareanalysie, p <0.001.
9 (41)
1 (6)
blood cells per high power field) of which 7 were expressed prostatic secretion culture-negative (table 1). In addition, of these 8 patients 3 had culture-negative results in all 4 urine segments; the diagnosis could have been uonbacterial prostatitis or prostatodynia depending on the level of white blood cells per high power field used as an exclueionary limit between inilammatory and noninflammatory prostatic seamtions. Thus, our study confirms that of Meares that the diagnostic distinction between culture-negative p r o s t a h dynia and nonbacterial prostatitis is not obvious in some patients.6 B m ~ When ~ routine . bacterial localization criteria were used on the bacterial counts from segmented urine cultures, 9 of the 22 patients had culture-positive expressed prostatic secretions (less than lo6 colony-forming units per ml.).l0 In 15 specimens from these 9 patients 22 bacterial isolates were localized to the prostatic aecretions. As shown in table 2, eoagulase-negative staphylococci predominated (68%), particularly Staphylococcus epidermidis and Staph. haemolyticus. A known uropathogen, Staph. saprophyticus, was also isolated. Other isolates included the gram-negative rods, Escherichia coli and Klebsiella pneumoniae, and grampositive organisms, streptucmci, enterococci and diphtheroids. Mean number of bacteria isolated per expressed prostatic secretion culture-positive specimen was 13,500colony-forming units per ml. (range 200 to 50,000, table 2). These generally low numbers are consistent with other studies suggesting that coagulase-negative staphyloeocei species grow more slowly in urine than other uropathogens, such as the gramnegative rods.14 It follows that low colony counts of these organisms may represent significant infection rather than commensal growth or contamination. Unfortunately most routine clinical microbiology laboratories would report these RESULTS results as commensals, attributing no significance to their Patient population. Summary patient and control data are isolation. We do not support this interpretation in this shown in table 1. The patient population, mean age and unique patient population. range, and presenting complaints are consistent with previPatient group prostatic bacteriology becomes significant OUS studies on prostatodynia.6 The patients were generally when compared to that of the control group (table 1). Young to middle-aged men. The most common complaint was Whereas 41% of the patients had positive expressed prostatic an intermittent, often severe pain localized to 1 or more areas secretion cultures despite multiple previous courses of anti(penis, testis, perineum, suprapubic area and/or lower back). biotics, only 6% of control cultures were positive. The 1 conMore than half had 1 or more nonspecific d y n a m i c com- trol subject with a positive expressed prostatic secretion culPlaints (frequency, urgency, nacturia and/or dysuria). ture was totally asymptomatic and had no history of Meares demonstrated that a subset of patients diagnosed genitourinary disease.This difference in frequency is statisW h prostatodynia (25%) had moderately elevated (11to 15 tically Signiscant (p <0.025),supporting the premise of a role white blood cells per high power field) inflammatory cells in for bacteria in wme prostatodynia patients. Immunological studies. We assayed patient and control the expressed prostatic secretion.6 In our study 8 cases (36%) had moderately elevated inflammatory cells (11ta 20 white sera with bacterial antigens derived from organisms cultured
1380
BACTERIAL INFECTION IN PROSTATODYNIA
TABLE2. Quantitative exoressed orostatic secretion bacterial cultures and concentration of inflammatory cells fronl specimens of expressed prostatic secretion cn culture-positiue patients F't-Age
Date
White Blood Cellsl Hieh Power Field
-
Bactena Isolated*
3,700 Staph epidermidis (30001 1,000 Staph haemolyticus (0640) 10,000 Staph haemolyticus 10640) 1/9/93 4,000 Staph epidermichs (3000) 2/19/93 4,000 Staph haemolyticus 10440) Y8/92 CB-33 5,000 Staph epidermidis (3000) ll/l!U91 cc-53 30,000 Streptococcus morbillorum 3,400 Staph intermedius 4/1/92 5,000 Staph capitis (0040) 8/10/93 RD-43 10,000 Corynebactenum species 1,000 Staph haemolyticus (4640) 1V5/91 JF40 1,000 K pneumoniae 3/16/92 2,100 Staph. haemolyticus 104601 400 Enterococcus faecalis 200 E. coli GK4l 11/12/91 13,000 Staph. epidermidis (3040) 38.000 Enter. faecalis 50,000 Staph. haemolyticus (0440) PS-43 1V5/9 1 JS-46 12/10/91 4,500 Strep. sanguis 6,000 Staph. epidermidis (3040) TS-38 12/17/91 5/25/93 200 Staph. epidermidis (3040) 12/14/93 700 Staph. saprophyticus * Analytab Products, Inc. biotype number is in parentheses.
RB-34
1111 1/92
from prostatodynia patients. We wanted to determine if cirto the infecting organisms were culating antibodies (1s) present i n t h e sera of patients. In immunological studies using enzyme-linked immunosorbent assay with whole bacteria and bacterial extracts as sources of antigen, no serological differences were noted between patients and controls when assayed for humoral (IgG) immunity. We did not assay for secretory (IgA) immunoglobin, the dominant prostatic immunoglobin, but confined our studies to serum I&. Thus, secretory or cell mediated immunity may be operative i n this host-pathogen interaction. Effects of empirical antibiotic treatment. Results of the treatment of prostatodynia patients were provocative. Empirical treatment was individualized for each patient, and included oral and intramuscular antibiotics (chiefly nitrofurantoin, trimethopridsulfamethoxazole, fluoroquinolones, carbenicillin and gentamicin), prazosin and nonsteroidal anti-inflammatory drugs. Treatment d a t a a r e complete for 7 of t h e expressed prostatic secretion culture-positive patients. When treated empirically, 5 of the 7 patients h a d a response to antimicrobials, as indicated by followup cultures (table 2). Clinically many patients reported a response to antimicrobial treatment with decreased complaints of pain and dysuria. Of the 14 patients for whom followup d a t a a r e complete 9 reported a n improvement in symptoms: 6 expressed prostatic secretion culture-positive patients noted relief versus 3 expressed prostatic secretion culture-negative patients. Although treatments were not standardized, microbiological and symptomatic end points suggest the efficacy of antimicrobial therapy and support a role for bacteria in prostatodynia. Nitrofurantoin had a striking effect with 4 of the 5 patients responding to treatment on followup culture. We cannot explain this finding since this drug is not known to accumulate in t h e prostate.7 However, it is possible t h a t the bacteria were located i n the prostatic urethra and not the prostatic ducts, accounting for the clinical efficacy of nitrofurantoin. I t is of interest to mention that, although nitrofurantoin is known to be most active against gram-negative enterobacterial organisms, it possesses a gram-positive spectrum. In some cases of idiopathic hematuria a similar clinical response to nitrofurantoin h a s been demonstrated against gram-positive organisms.'s These 5 patients with a response to nitrofurantoin were initially given a 4 to %week course, and prolonged low dose maintenance therapy (50 to 100 mg. daily) of the drug was considered with recrudescence of
Neg. Followup Culture
0-10 0-10 0- 10 0-10 0-10
No Yes Yes
0-10 0-10
No
0-10 0-10
Yes 0-10 Lost to followup Yes Lost to followup
0-10 0-10 0-10 0-10 11-20
Yes
symptoms or positive cultures on stopping the medication. The antibiotic treatment effects were not permanent. treated expressed prostatic secretion-positive patients had at least 1 positive culture within 2 years. DISCUSSION
Urodynamic aspects of prostatodynia have been well described although a possible underlying infectious etiology has not received adequate attention. We have reported t h e isolation of bacteria from expressed prostatic secretions of prostatodynia patients, which is statistically significant compared to normal controls. Previously B a r b a l i a ~ + ~and Hellstroms e t al, and Meares6 reported dysfunction of the bladder neck and prostatic urethra. Meares suggested t h a t prostatodynia patients have a n intraprostatic reflux of urine during voiding, causing "chemical p r o ~ t a t i t i s . " Although ~ these studies are descriptive of prostatodynia and its symp tomatic treatment, they offer no definitive etiology for thh acquired functional disorder. We propose t h a t prostatodynia may be caused by a n ascending subclinical infection of the prostate by bacteria, including recognized uropathogens and commensals. Of the 9 expressed prostatic secretion culture-positive patients 8 had low concentrations of inflammatory cells associated with prostatic secretions (0to 10 white blood cells per high power field, table 1). Thus, for a subset of patients prostatodynia may be a n infectious, minimally inflammatory disorder of the prostate. The somatic and functional complaints of prostatodynia may derive from intraprostatic host-cell interactions with elaborated bacterial products, such as cell wall components, toxins and degradation products. I t is also possible t h a t bacteria t h a t are difficult to culture (cell wall defective and/or nutritionally deficient) may contribute to this chronia condition. Furthermore, prostatodynia may be mediated by complex processes between host cell products and host immunity. In our study 68% of the prostatic bacterial isolates were coagulase-negative staphylococci. The most common isolate, Staph. epidermidis, h a s been implicated by Nickel and Costerton,'" and Wedren17 in the etiology of chronic bacterid prostatitis. Another coagulase-negative staphylococci speciee isolated in our study (Staph. haemolyticus) has been reported by Gunn and Davis,'* and Sanchis-Bayarri e t all'' to cause urinary tract infections in men. In fact, these congulasenegative staphylococci species possess virulent properti-
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BACTERIAL INFECTION IN PROSTATODYNLA
that may be operative in prostatodynia patients.14 These bacteria can adhere to urothelial cells20and cellular proteins, including laminin, fibronectin, vitronectin and collagen,21 which may allow ascending infection and colonization of the prostate. Furthermore, these bacteria produce an extracellular slime substance with anti-phagocytic and antichemotactic PI-OpertieS affecting neutrophils as well as anti-proliferativeproperties that affect lymphocytes.14 Thus, extracellular slime substance can impair host defenses. Extracellular slime substance also has cytoprotective properties, which can conceal bacteria from otherwise bactericidal levels of antibiotics and lead to recrudescent infections resistant to therapy.16 It seems likely that the antimicrobial treatment failures of patients R. B. and R. D. could have resulted from the virulent properties of coagulase-negative staphylococci (table 2). Our patients were diagnosed with prostatodynia clinically and microscopically. They had noninflammatory and minimally inflammatory intraprostatic disease, as indicated by the concentration of white blood cells in expressed prostatic secretions. Thus, prostatodynia may involve a type of host immune deficiency. Wedrkn et al reported on several nonbacterial prostatitis patients in whom leukocytes had decreased phagocytie activity against autologous bacterial isolates, suggesting that there exists a selective process involving prostatic pathogens and specific host defenses.22 Similarly, Sanchts-Bayarri et a1 concluded that coagulase-negative staphylococci, particularly Staph. haemolyticus, can function as an opportunistic pathogen in immune depressed hosts. Thus, when Blacklocks and Millers reported associations of stress, anxiety and tension with prostatodynia, they may have described a clinical situation predisposing to infection. Circulating glucocorticoid levels become elevated with psychological and autonomic stimulation, causing anti-inflammatory effects that may diminish host immunity.= These suggestions of host immune defects may explain why 3 patients (C.C., J. F. and T. s.)were reinfected with different organisms afbr an initial response to antimicrobial treatment (table 2). These factors may also explain why no expressed prostatic secretion culture-positive patients had negative cultures for longer than 2 years. Glucocorticoids are also known to cause local and systemic release of catecholamines,23 which may stimulate the sympathetic noradrenergic nerve plexus innervating the bladder neck and preprostatic sphincters.6.8 Formal study of the role of glucocorticoids and catecholamines in prostatodynia may be warranted. CONCLUSIONS
In some prostatodynia patients bacteria may have an etiological role in concert with diminished host immunity. That a subset of patients responds to antimicrobial therapy adds weight to this postulated role for bacteria, particularly those species with known virulent properties. Further research is indicated to evaluate operative host-pathogen interactions and the role for antimicrobial therapy in prostatodynia. It would be worthwhile to use molecular techniques, including the polymerase chain reaction, to determine if bacterial nucleic acids persist in prostatic secretions from culturenegative specimens. Dolise Nelson provided laboratory support. REFERENCES
1. B m n e r , H., Weidner, W.and Schiefer, H. G.: Studies on the role of Ureaplasma urealyticum and Mycoplasma hominis in pro%
tatitis. J. Infect. Dis., 141: 807,1983.
2. Drach, G. W.,Meares, E. M., Fair, W. R. and Stamey, T. A: Classificationofbenign disease associated with prostatic pain: prostatitis or prostatodynia?Letter to the Editor. J. Urol., 120: 266,1978. 3. Barbalias, G.A., Meares, E. M., Jr. and Sant, G. R.: Prostatedynia: clinical and urodynamic characteristics. J. Urol., 1sO. 514,1983. 4. Barbalias, G.A.: ProstaWynia or painful male urethral syndrome? Urology, 36: 146,1990. 5. Hellstrom, W. J. G., Schmidt, R. A., Lue, T. F. and Tanagho, E. A: N e u r o m d a r dysfunction in nonbacterial prostatitis. Urology, 30: 183,1987. 6. Meares, E.M., Jr.: F’rostaWynia: clinical findingsand rationale for treatment. In: Therapy of Prostatitis. Edited by W. Weidner, H. Brunner, W. Krause and C. F. Rothauge. San Francisco: W. Zuckschwerdt Verlag, vol. 11, pp. 207-212, 1986. 7. Meares, E. M., Jr.: Prostatitis and related disorders. In. Campbell’s Urology, 6th ed. Edited by P. C. Walsh, A. B. Retik, T. A. Stamey and E.D. Vaughan, Jr. Philadelphia: W.B. Saunders Co., vol. 1,chapt. 18,pp. 807-822, 1992. 8. Blacklock,N. J.: Unxiynamic and psychometric observations and their implication in the management of prostatodynia. In: Therapy of Prostatitis. Edited by W. Weidner, H. Brunner, W. Krause and C. F. Rothauge. San Francisco:W.Zuckeehwerdt Verlag, vol. 11, pp. 201-206.1986. 9. Miller, H. C.:Stress prostatitis. Urology, 3 2 507,1988. 10. Meares, E. M.and Stamey, T. A.: Bacteriologiclocalization p a t terns in bacterial prostatitis and urethritis. Invest.Urol.,5: 492,1968. 11. Bhaduri, S.:Rapid disruption of bacteria for protein extraction. Current protucob in molecular biology. Red Book Bull., suppl., winter 1993. 12. Shortliffe, L. M. D., Sellers, R. G. and Sehachter, J.: The characterization of nonbacterial prostatitk search for an etiology. J. Urol., 148: 1461, 1992. 13. Henriksen, A Z.,Maeland, J. A. and Brakstad, 0. G.: Monoclonal antibodies against three ditrerent enterobacterial outer membrane proteins. Characterization, Cmse-reactivity, and binding to bacteria. Acta Path. Microbiol. Immunol. Scand., BI: 559,1989. 14. Kloos, W.E. and Bannerman, T. L.: Update on clinical signiiim c e of wdase-negative Btaohvlococci. Ch. Microbiol. Rev., ?: 117,iSS4. 15. Dominaue, G. J., Thomas, R., Walters, F., Serrano. A. and Heidi;;er; P. M.,. Jr.: Cell wall deficient bacteria aa a cause of idiopathic hematuria. J. Urol., 1M):483,1993. 16. Nickel, J. C.and Costerton, J. W.: Coagulasenegative staphylococcus in chronic prostatitis. J. Urol., 141:398,1992. 17. W a n , H.:On chronic prostatitis with special studies of Staphylococcus epidermidis. Scand. J. Urol. Nephrol., suppl., 18s:1, 1989. 18. Gunn,B. A. and Davis, C. E., Jr.: Staphylococcus haemolyticue urinary tract infection in a male patient. J. Clin. Microbiol., 28: 1055,1988. 19. Sanchfs-Bayarri Vaillant, V., Sdnchez sbnehez, R., M d d a Benito, G. and Sanchh-Bayarri Bemal, V.: A Staphylococcus haemolyticue study in urinary infections. An analysis of 8 cases.Rev. Clin. Esp., 190: 443,1992. 20. Mgrdh, P.-A., Colleen, S. and Hovelius, B.: Attachment of bacteria to exfoliated cells from the urogenitaltract. Invest. Urol., 1 6 322,1979. 21. Paulsson, M., Ljungh, A. and Wadstriim, T.: Rapid identification of fibronectin, vitronectin, laminin, and collagen cell surface binding proteins on coagulase-negative staphylococci by particle agglutination assays. J. Clin Microbiol., 30:2006,1992. 22. W a n , H., Holm, S. E. and Bergman,B.: Can decreased phagocytosis and killing of autologous gram-positive bacteria explain the finding of gram-pitive bacteria in ‘non-bacterial proetatitis”? Acta Path. Microbiol. Immunol.Scand., aection B, 96 75,1987. 23. Irwin,M.: Strewinduced immune s u p p d o n . Role of the autonomic nervous system.Ann. New York Acad. Sci.. gsI:203, 1993.
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Vol. 154, 1378-1381, October 1995 Printed in U . S A
THE JOURNAL OF UROWGY Copyright 0 1995 by
h l E R l C A N UROLAXICALASSOCIATION, I N C
BACTERIAL INFECTION IN PROSTATODYNIA JOSHUA E. LOWENTRITT, KAZUYA KAWAHARA,* LISET G. HUMAN, WAYNE J. G. HELLSTROM AND GERALD J. DOMINGUE From the Departments of Urology and Microbiology and Immunology, Tulane University School of Medicine, New Orleans, Louisiana
ABSTRACT
Purpose: We investigated a possible bacterial etiology for prostatodynia. Materials and Methods: We evaluated segmented urine specimens from 22 patients and 16 controls by bacteriological localization studies. Immunological studies were performed on patient and control sera. Results: Nine patients had positive cultures from prostatic secretions. When compared to controls, this novel finding was statistically significant (p <0.025). Coagulase-negative staphylococci were the most common isolates (68%).No humoral ( 1 6 )immune differences were found between patients and controls. Conclusions: In a subset of prostatodynia patients bacteria may have a n etiological role. Antibiotic treatment demonstrated clinical efficacy. KEYWORDS:prostate, infection, prostatitis, Staphylococcus
Prostatodynia is a commonly diagnosed and poorly treated urological syndrome. In a large study of 597 prostatitis patients nearly a third were diagnosed with prostatodynia, which is a significant fraction of the urological patient population.' Clinically prostatodynia frustrates the patient and physician due to its chronicity and resistance to therapy. The syndrome is usually characterized by persistent perineal pain, and functional and somatic urological complaints, including abnormal urine flow, frequency, urgency and dysuria. The diagnosis is differentiated from other common prostatitis syndromes by a lack of prostatic inflammation and no Significant leukocytes or bacteria in the expressed prostatic secretions.2 However, the etiology of prostatodynia is unknown despite these distinct diagnostic criteria. Urodynamic research by Barbalias3.4 and Hellstrom~e t al, and Meares6 established that prostatodynia patients have an acquired functional disorder of the bladder neck and prostatic urethra, causing an intraprostatic reflux of urine and resulting in "chemical prostatitis."7 Blacklocks and Millerg suggested that prostatodynia is causally associated with psychological factors, including anxiety states and excessive tension or stress. However, no definitive pathophysiological agent or mechanism has been clearly established. To evaluate a possible infectious etiology of prostatodynia we studied segmented urine specimens and sera from a series of patients. In this prospective nonrandomized bacteriological, clinical and immunological study we compared nonblinded patient findings to those of a normal control group. We did not standardize empirical antibiotic treatment regimens in this study although we analyzed treatment data, and performed microbiological and symptomological evaluations for therapeutic efficacy. MATERIALS AND METHODS
Patient populations. During 3 years 26 symptomatic patients with a clinical diagnosis of prostatodynia were referred to our institution for study. Most patients had been treated by many urologists and had completed multiple courses of different antibiotics before being examined a t our clinic. We Accepted for publication March 31. 1995. Supported in part by a fellowship from the American Foundation for Urologic Diseases, Inc. and instltutional grants from Tulane University School of Medicine. * Current address: Kagoshima IJniversity School of Medicine, Kagoshima, 890, Japan.
later confirmed each clinical diagnosis by microscopic examination of prostatic secretions, particularly the concentration of white blood cells per high power field. Investigators who study prostatodynia disagree regarding the precise concentration delimiting normal from excessive white blood cells per high power field in the expressed prostatic ~ e c r e t i o n . ~ . ~ We used the level of 20 white blood cells per high power field in expressed prostatic secretions as an exclusionary upper limit to separate patients with a noninflammatory prostatitis syndrome (prostatodynia) from inflammatory disorders, chiefly nonbacterial prostatitis and chronic bacterial prostatitis. Four patients referred with symptoms suggestive of prostatodynia who had greater than 20 white blood cells per high power field in expressed prostatic secretions were excluded from the study and further analysis. Thus, 22 patients with the clinical diagnosis of prostatodynia confirmed by microscopy were included. Controls included 16 asymptomatic men with no recent genitourinary complaints. Clinical evaluations. Patients and controls were evaluated by the same physician for routine bacteriological localization studies, as described by Meares and Stamey.10 The urethral urine, midstream bladder urine, expressed prostatic secretion by prostate massage and post-massage urine were collected, examined microscopically and cultured bacteriologically. Venous blood specimens for immunological studies were allowed to clot and were centrifuged. Serum was stored a t -70C in small aliquots. Serum specimens were thawed no more than twice before immunological study. Bacteriological methods. Quantitative routine cultures were performed on urine, and centrifuged urine sediments were inoculated into tryptose broth to detect colony counts fewer than 100 colony-forming units per ml. Standard microscopy was accompanied by fluorescent acridine orange staining for bacterial, leukocytic and cellular nucleic acids. All cultured bacteria were identified by biotyping. Extractions of bacterial proteins. To determine possible immune responses to solubilized bacterial antigens, a protocol was adapted from Bhaduri." Bacteria isolated from patients were cultured in 1,600 ml. tryptose broth overnight, collected by centrifugation, washed twice in phosphate b d ered saline and resuspended in 25 ml. cold acetone. After 1 hour of incubation with agitation at OC cells were collected and excess acetone w a s removed over a nitrogen stream. Cells were resuspended in 10 ml. I(.; sodium dodecyl sulfate a t room temperature for 15 minutes. The mixture was cen-
1378
1379
BACTERIAL INFECTION IN PROSTATODYNIA
trifuged, the supernatant was removed and proteinase inhibitors (0.5 mg./ml. phenylmethylsulfonylfluoride and 1.0 mgJ ml.knzamidine) were added. The supernatant was fdtered by ultrafiltration over a nitrogen stream, dialyzed with 3 changes of 0.01 M. phosphate buffered saline (pH 7.2)for 72 hours at 4C and stored at 4C. Protein concentration was determined by a modified Lowry assay. Indirect enzyme-linked immunosorbent assay. Our indirect enzymelinked immunosorbent assay method was adapted from that of ShortWe et a1.12 Binding and diluting buffers were identical: 0.01 M. phosphate buffered saline (pH 7.2) with 0.05% polysorbate 20, 1% bovine serum albumin and 0.05% sodium azide. The washing buffer was 0.01 M. phosphate buffered saline (pH 7.2)with 0.5% polysorbate 20 and 0.01% sodium azide. Briefly, in each well of a U-bottom microtiter enzyme-linked immunosorbent assay plate 50 pg. solubilized bacterial antigen were suspended in the binding buffer and incubated for 1 hour at 37C, and wells were washed 3 times with washing buffer. Serum (100pl.) from a patient or control was serially diluted in diluting buffer in each well and incubated for 2 hours at 37C and wells were washed as noted previously. Goat anti-human IgG horseradish peroxidase (100pl.) was diluted in diluting buffer to 15 units per ml. and incubated for 2 hours at 37C, and wells were washed 5 times as noted previously. "hen 100 p,l. substrate solution were added to each well and allowed to develop for 1 hour in a dark chamber at room temperature. Absorbance was read at 410 nm. and the titer was read as the reciprocal of the highest dilution producing an absorbance greater than 0.100. Whole cell enzyme-linked immunosorbent assay. Whole cell enzyme-linked immunosorbent assay protocols were adapted from those of Henricksen et al.13 Using bacteria cultured from patients, and sera from patients and controls assays were performed for humoral (IgG) immune responses to the organisms isolated. Briefly, bacteria were cultured in Luria broth overnight. Cells were collectedby centrhgation, washed in phosphate buffered saline with divalent cations (phosphate buffered saline, calcium and magnesium) and adjusted to standard optical density. A solution of los cells per ml. (900 4.)and 100 4. serially diluted patient or control serum with 5% bovine serum albumin was inoculated in microcentrifuge tubes for 1 hour at 37C.Cells were washed twice with phosphate buffered saline, resuspended in 1 ml. phosphate buffered saline with 0.25% bovine serum albumin containing a 1:1,000dilution of goat anti-human IgG horseradish peroxidase conjugate and absorbed for 1 hour at 37C. Cells were washed 3 times, resuspended in 50 pl. phosphate buffered saline, transferred to a modified flabbottom micm titer enzyme-linked immunoaorbent assay plate and 100 pl. substrate were added. After developing in a dark chamber for 1 hour at mom temperature absorbance was read at 410 nm. Titers were defined as the reciprocal of the highest dilution with a minimum absorbance of 0.200.
TABLE1. Comparison of patient and contrul subject ages, presenting complaints, micmscopy and culture of segmented urine specimens with localization studies characteristic PLe. Controls 22
No. subjects No. specimens
Age: Mean
f SD
h P No. plun complaint 1%)
46
16 16
40.1 2 10.6 31.4 f 10.7 21-62 20-60 20 (89)
No. umdynamic complaint (8) No. expressed pmtatic -tiom with 0 to 10 white blood cells/high power field (%): Cdtllre-p.*
cultureneg. No. expressed prostatic secretion with 11 to 20
white blood cells/hiph power field (46): Culture-p. CUltllre-neK.
No. dtllre-p&. expressed pmntatic secretion (%)t * chi-squareanalysie, p <0.001.
9 (41)
1 (6)
blood cells per high power field) of which 7 were expressed prostatic secretion culture-negative (table 1). In addition, of these 8 patients 3 had culture-negative results in all 4 urine segments; the diagnosis could have been uonbacterial prostatitis or prostatodynia depending on the level of white blood cells per high power field used as an exclueionary limit between inilammatory and noninflammatory prostatic seamtions. Thus, our study confirms that of Meares that the diagnostic distinction between culture-negative p r o s t a h dynia and nonbacterial prostatitis is not obvious in some patients.6 B m ~ When ~ routine . bacterial localization criteria were used on the bacterial counts from segmented urine cultures, 9 of the 22 patients had culture-positive expressed prostatic secretions (less than lo6 colony-forming units per ml.).l0 In 15 specimens from these 9 patients 22 bacterial isolates were localized to the prostatic aecretions. As shown in table 2, eoagulase-negative staphylococci predominated (68%), particularly Staphylococcus epidermidis and Staph. haemolyticus. A known uropathogen, Staph. saprophyticus, was also isolated. Other isolates included the gram-negative rods, Escherichia coli and Klebsiella pneumoniae, and grampositive organisms, streptucmci, enterococci and diphtheroids. Mean number of bacteria isolated per expressed prostatic secretion culture-positive specimen was 13,500colony-forming units per ml. (range 200 to 50,000, table 2). These generally low numbers are consistent with other studies suggesting that coagulase-negative staphyloeocei species grow more slowly in urine than other uropathogens, such as the gramnegative rods.14 It follows that low colony counts of these organisms may represent significant infection rather than commensal growth or contamination. Unfortunately most routine clinical microbiology laboratories would report these RESULTS results as commensals, attributing no significance to their Patient population. Summary patient and control data are isolation. We do not support this interpretation in this shown in table 1. The patient population, mean age and unique patient population. range, and presenting complaints are consistent with previPatient group prostatic bacteriology becomes significant OUS studies on prostatodynia.6 The patients were generally when compared to that of the control group (table 1). Young to middle-aged men. The most common complaint was Whereas 41% of the patients had positive expressed prostatic an intermittent, often severe pain localized to 1 or more areas secretion cultures despite multiple previous courses of anti(penis, testis, perineum, suprapubic area and/or lower back). biotics, only 6% of control cultures were positive. The 1 conMore than half had 1 or more nonspecific d y n a m i c com- trol subject with a positive expressed prostatic secretion culPlaints (frequency, urgency, nacturia and/or dysuria). ture was totally asymptomatic and had no history of Meares demonstrated that a subset of patients diagnosed genitourinary disease.This difference in frequency is statisW h prostatodynia (25%) had moderately elevated (11to 15 tically Signiscant (p <0.025),supporting the premise of a role white blood cells per high power field) inflammatory cells in for bacteria in wme prostatodynia patients. Immunological studies. We assayed patient and control the expressed prostatic secretion.6 In our study 8 cases (36%) had moderately elevated inflammatory cells (11ta 20 white sera with bacterial antigens derived from organisms cultured
1380
BACTERIAL INFECTION IN PROSTATODYNIA
TABLE2. Quantitative exoressed orostatic secretion bacterial cultures and concentration of inflammatory cells fronl specimens of expressed prostatic secretion cn culture-positiue patients F't-Age
Date
White Blood Cellsl Hieh Power Field
-
Bactena Isolated*
3,700 Staph epidermidis (30001 1,000 Staph haemolyticus (0640) 10,000 Staph haemolyticus 10640) 1/9/93 4,000 Staph epidermichs (3000) 2/19/93 4,000 Staph haemolyticus 10440) Y8/92 CB-33 5,000 Staph epidermidis (3000) ll/l!U91 cc-53 30,000 Streptococcus morbillorum 3,400 Staph intermedius 4/1/92 5,000 Staph capitis (0040) 8/10/93 RD-43 10,000 Corynebactenum species 1,000 Staph haemolyticus (4640) 1V5/91 JF40 1,000 K pneumoniae 3/16/92 2,100 Staph. haemolyticus 104601 400 Enterococcus faecalis 200 E. coli GK4l 11/12/91 13,000 Staph. epidermidis (3040) 38.000 Enter. faecalis 50,000 Staph. haemolyticus (0440) PS-43 1V5/9 1 JS-46 12/10/91 4,500 Strep. sanguis 6,000 Staph. epidermidis (3040) TS-38 12/17/91 5/25/93 200 Staph. epidermidis (3040) 12/14/93 700 Staph. saprophyticus * Analytab Products, Inc. biotype number is in parentheses.
RB-34
1111 1/92
from prostatodynia patients. We wanted to determine if cirto the infecting organisms were culating antibodies (1s) present i n t h e sera of patients. In immunological studies using enzyme-linked immunosorbent assay with whole bacteria and bacterial extracts as sources of antigen, no serological differences were noted between patients and controls when assayed for humoral (IgG) immunity. We did not assay for secretory (IgA) immunoglobin, the dominant prostatic immunoglobin, but confined our studies to serum I&. Thus, secretory or cell mediated immunity may be operative i n this host-pathogen interaction. Effects of empirical antibiotic treatment. Results of the treatment of prostatodynia patients were provocative. Empirical treatment was individualized for each patient, and included oral and intramuscular antibiotics (chiefly nitrofurantoin, trimethopridsulfamethoxazole, fluoroquinolones, carbenicillin and gentamicin), prazosin and nonsteroidal anti-inflammatory drugs. Treatment d a t a a r e complete for 7 of t h e expressed prostatic secretion culture-positive patients. When treated empirically, 5 of the 7 patients h a d a response to antimicrobials, as indicated by followup cultures (table 2). Clinically many patients reported a response to antimicrobial treatment with decreased complaints of pain and dysuria. Of the 14 patients for whom followup d a t a a r e complete 9 reported a n improvement in symptoms: 6 expressed prostatic secretion culture-positive patients noted relief versus 3 expressed prostatic secretion culture-negative patients. Although treatments were not standardized, microbiological and symptomatic end points suggest the efficacy of antimicrobial therapy and support a role for bacteria in prostatodynia. Nitrofurantoin had a striking effect with 4 of the 5 patients responding to treatment on followup culture. We cannot explain this finding since this drug is not known to accumulate in t h e prostate.7 However, it is possible t h a t the bacteria were located i n the prostatic urethra and not the prostatic ducts, accounting for the clinical efficacy of nitrofurantoin. I t is of interest to mention that, although nitrofurantoin is known to be most active against gram-negative enterobacterial organisms, it possesses a gram-positive spectrum. In some cases of idiopathic hematuria a similar clinical response to nitrofurantoin h a s been demonstrated against gram-positive organisms.'s These 5 patients with a response to nitrofurantoin were initially given a 4 to %week course, and prolonged low dose maintenance therapy (50 to 100 mg. daily) of the drug was considered with recrudescence of
Neg. Followup Culture
0-10 0-10 0- 10 0-10 0-10
No Yes Yes
0-10 0-10
No
0-10 0-10
Yes 0-10 Lost to followup Yes Lost to followup
0-10 0-10 0-10 0-10 11-20
Yes
symptoms or positive cultures on stopping the medication. The antibiotic treatment effects were not permanent. treated expressed prostatic secretion-positive patients had at least 1 positive culture within 2 years. DISCUSSION
Urodynamic aspects of prostatodynia have been well described although a possible underlying infectious etiology has not received adequate attention. We have reported t h e isolation of bacteria from expressed prostatic secretions of prostatodynia patients, which is statistically significant compared to normal controls. Previously B a r b a l i a ~ + ~and Hellstroms e t al, and Meares6 reported dysfunction of the bladder neck and prostatic urethra. Meares suggested t h a t prostatodynia patients have a n intraprostatic reflux of urine during voiding, causing "chemical p r o ~ t a t i t i s . " Although ~ these studies are descriptive of prostatodynia and its symp tomatic treatment, they offer no definitive etiology for thh acquired functional disorder. We propose t h a t prostatodynia may be caused by a n ascending subclinical infection of the prostate by bacteria, including recognized uropathogens and commensals. Of the 9 expressed prostatic secretion culture-positive patients 8 had low concentrations of inflammatory cells associated with prostatic secretions (0to 10 white blood cells per high power field, table 1). Thus, for a subset of patients prostatodynia may be a n infectious, minimally inflammatory disorder of the prostate. The somatic and functional complaints of prostatodynia may derive from intraprostatic host-cell interactions with elaborated bacterial products, such as cell wall components, toxins and degradation products. I t is also possible t h a t bacteria t h a t are difficult to culture (cell wall defective and/or nutritionally deficient) may contribute to this chronia condition. Furthermore, prostatodynia may be mediated by complex processes between host cell products and host immunity. In our study 68% of the prostatic bacterial isolates were coagulase-negative staphylococci. The most common isolate, Staph. epidermidis, h a s been implicated by Nickel and Costerton,'" and Wedren17 in the etiology of chronic bacterid prostatitis. Another coagulase-negative staphylococci speciee isolated in our study (Staph. haemolyticus) has been reported by Gunn and Davis,'* and Sanchis-Bayarri e t all'' to cause urinary tract infections in men. In fact, these congulasenegative staphylococci species possess virulent properti-
1381
BACTERIAL INFECTION IN PROSTATODYNLA
that may be operative in prostatodynia patients.14 These bacteria can adhere to urothelial cells20and cellular proteins, including laminin, fibronectin, vitronectin and collagen,21 which may allow ascending infection and colonization of the prostate. Furthermore, these bacteria produce an extracellular slime substance with anti-phagocytic and antichemotactic PI-OpertieS affecting neutrophils as well as anti-proliferativeproperties that affect lymphocytes.14 Thus, extracellular slime substance can impair host defenses. Extracellular slime substance also has cytoprotective properties, which can conceal bacteria from otherwise bactericidal levels of antibiotics and lead to recrudescent infections resistant to therapy.16 It seems likely that the antimicrobial treatment failures of patients R. B. and R. D. could have resulted from the virulent properties of coagulase-negative staphylococci (table 2). Our patients were diagnosed with prostatodynia clinically and microscopically. They had noninflammatory and minimally inflammatory intraprostatic disease, as indicated by the concentration of white blood cells in expressed prostatic secretions. Thus, prostatodynia may involve a type of host immune deficiency. Wedrkn et al reported on several nonbacterial prostatitis patients in whom leukocytes had decreased phagocytie activity against autologous bacterial isolates, suggesting that there exists a selective process involving prostatic pathogens and specific host defenses.22 Similarly, Sanchts-Bayarri et a1 concluded that coagulase-negative staphylococci, particularly Staph. haemolyticus, can function as an opportunistic pathogen in immune depressed hosts. Thus, when Blacklocks and Millers reported associations of stress, anxiety and tension with prostatodynia, they may have described a clinical situation predisposing to infection. Circulating glucocorticoid levels become elevated with psychological and autonomic stimulation, causing anti-inflammatory effects that may diminish host immunity.= These suggestions of host immune defects may explain why 3 patients (C.C., J. F. and T. s.)were reinfected with different organisms afbr an initial response to antimicrobial treatment (table 2). These factors may also explain why no expressed prostatic secretion culture-positive patients had negative cultures for longer than 2 years. Glucocorticoids are also known to cause local and systemic release of catecholamines,23 which may stimulate the sympathetic noradrenergic nerve plexus innervating the bladder neck and preprostatic sphincters.6.8 Formal study of the role of glucocorticoids and catecholamines in prostatodynia may be warranted. CONCLUSIONS
In some prostatodynia patients bacteria may have an etiological role in concert with diminished host immunity. That a subset of patients responds to antimicrobial therapy adds weight to this postulated role for bacteria, particularly those species with known virulent properties. Further research is indicated to evaluate operative host-pathogen interactions and the role for antimicrobial therapy in prostatodynia. It would be worthwhile to use molecular techniques, including the polymerase chain reaction, to determine if bacterial nucleic acids persist in prostatic secretions from culturenegative specimens. Dolise Nelson provided laboratory support. REFERENCES
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tatitis. J. Infect. Dis., 141: 807,1983.
2. Drach, G. W.,Meares, E. M., Fair, W. R. and Stamey, T. A: Classificationofbenign disease associated with prostatic pain: prostatitis or prostatodynia?Letter to the Editor. J. Urol., 120: 266,1978. 3. Barbalias, G.A., Meares, E. M., Jr. and Sant, G. R.: Prostatedynia: clinical and urodynamic characteristics. J. Urol., 1sO. 514,1983. 4. Barbalias, G.A.: ProstaWynia or painful male urethral syndrome? Urology, 36: 146,1990. 5. Hellstrom, W. J. G., Schmidt, R. A., Lue, T. F. and Tanagho, E. A: N e u r o m d a r dysfunction in nonbacterial prostatitis. Urology, 30: 183,1987. 6. Meares, E.M., Jr.: F’rostaWynia: clinical findingsand rationale for treatment. In: Therapy of Prostatitis. Edited by W. Weidner, H. Brunner, W. Krause and C. F. Rothauge. San Francisco: W. Zuckschwerdt Verlag, vol. 11, pp. 207-212, 1986. 7. Meares, E. M., Jr.: Prostatitis and related disorders. In. Campbell’s Urology, 6th ed. Edited by P. C. Walsh, A. B. Retik, T. A. Stamey and E.D. Vaughan, Jr. Philadelphia: W.B. Saunders Co., vol. 1,chapt. 18,pp. 807-822, 1992. 8. Blacklock,N. J.: Unxiynamic and psychometric observations and their implication in the management of prostatodynia. In: Therapy of Prostatitis. Edited by W. Weidner, H. Brunner, W. Krause and C. F. Rothauge. San Francisco:W.Zuckeehwerdt Verlag, vol. 11, pp. 201-206.1986. 9. Miller, H. C.:Stress prostatitis. Urology, 3 2 507,1988. 10. Meares, E. M.and Stamey, T. A.: Bacteriologiclocalization p a t terns in bacterial prostatitis and urethritis. Invest.Urol.,5: 492,1968. 11. Bhaduri, S.:Rapid disruption of bacteria for protein extraction. Current protucob in molecular biology. Red Book Bull., suppl., winter 1993. 12. Shortliffe, L. M. D., Sellers, R. G. and Sehachter, J.: The characterization of nonbacterial prostatitk search for an etiology. J. Urol., 148: 1461, 1992. 13. Henriksen, A Z.,Maeland, J. A. and Brakstad, 0. G.: Monoclonal antibodies against three ditrerent enterobacterial outer membrane proteins. Characterization, Cmse-reactivity, and binding to bacteria. Acta Path. Microbiol. Immunol. Scand., BI: 559,1989. 14. Kloos, W.E. and Bannerman, T. L.: Update on clinical signiiim c e of wdase-negative Btaohvlococci. Ch. Microbiol. Rev., ?: 117,iSS4. 15. Dominaue, G. J., Thomas, R., Walters, F., Serrano. A. and Heidi;;er; P. M.,. Jr.: Cell wall deficient bacteria aa a cause of idiopathic hematuria. J. Urol., 1M):483,1993. 16. Nickel, J. C.and Costerton, J. W.: Coagulasenegative staphylococcus in chronic prostatitis. J. Urol., 141:398,1992. 17. W a n , H.:On chronic prostatitis with special studies of Staphylococcus epidermidis. Scand. J. Urol. Nephrol., suppl., 18s:1, 1989. 18. Gunn,B. A. and Davis, C. E., Jr.: Staphylococcus haemolyticue urinary tract infection in a male patient. J. Clin. Microbiol., 28: 1055,1988. 19. Sanchfs-Bayarri Vaillant, V., Sdnchez sbnehez, R., M d d a Benito, G. and Sanchh-Bayarri Bemal, V.: A Staphylococcus haemolyticue study in urinary infections. An analysis of 8 cases.Rev. Clin. Esp., 190: 443,1992. 20. Mgrdh, P.-A., Colleen, S. and Hovelius, B.: Attachment of bacteria to exfoliated cells from the urogenitaltract. Invest. Urol., 1 6 322,1979. 21. Paulsson, M., Ljungh, A. and Wadstriim, T.: Rapid identification of fibronectin, vitronectin, laminin, and collagen cell surface binding proteins on coagulase-negative staphylococci by particle agglutination assays. J. Clin Microbiol., 30:2006,1992. 22. W a n , H., Holm, S. E. and Bergman,B.: Can decreased phagocytosis and killing of autologous gram-positive bacteria explain the finding of gram-pitive bacteria in ‘non-bacterial proetatitis”? Acta Path. Microbiol. Immunol.Scand., aection B, 96 75,1987. 23. Irwin,M.: Strewinduced immune s u p p d o n . Role of the autonomic nervous system.Ann. New York Acad. Sci.. gsI:203, 1993.
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