177Archivio Italiano di Urologia e Andrologia 2019; 91, 3 ORIGINAL PAPER Chronic prostatic infection: Microbiological findings in two Mediterranean populations Konstantinos Stamatiou 1, Vittorio Magri 2, Gianpaolo Perletti 3, 4, Vaia Papadouli 5, Nectaria Recleiti 5, Vassiliki Mamali 5, Olympia Zarkotou 5 1 Urology Department, Tzaneion Hospital, Piraeus, Greece; 2 Urology Secondary Care Clinic, ASST-Nord, Milan, Italy; 3 Department of Biotechnology and Life Sciences, Section of Medical and Surgical Sciences, Università degli Studi dell'Insubria, Varese, Italy; 4 Faculty of Medicine and Medical Sciences, Ghent University, Ghent, Belgium; 5 Microbiology Department, Tzaneion Hospital, Piraeus, Greece. Introduction/Aim: Despite accumulated knowledge, several microbiological aspects of chronic bacterial prostatitis (CBP) remain uncertain. The aim of our study was to determine microbiological charac- teristics on our CBP population. Materials: The material of this retrospective study consisted in bacterial isolates from urine and/or prostatic secretions or sperm cultures (total ejaculate) obtained from individuals with prostatitis symptoms and from patients with febrile relapses of CBP visiting our department, from 03/2009 to 03/2015. Retrospective data from an Italian single-center database (years 2009-2015) were also collected for a tentative compari- son of pathogen prevalence between chronic bacterial prostati- tis cases assessed in Greece and Italy. Results: A total of 389 bacterial isolates obtained from eligible Greek patients constituted the material of the study. While E coli was the most frequent individual pathogen, Gram-positive species were overly more frequent than Gram-negative. Besides the high frequency of E. coli and E. faecalis isolates the most remarkable similarity between Greek and Italian databases was the wide array of different Gram-positive and Gram-nega- tive species isolated from CBP patients. Conclusions: In Greece, the incidence of CBP is possibly higher than that reported in international surveys. Similarities between Greek and Italian databases suggest geographical trends in CBP epidemiology. KEY WORDS: Chronic prostatitis; Prostate; Infection; Stamey- Meyers. Submitted 19 June 2019; Accepted 21 July 2019 Summary No conflict of interest declared. METHODS Material The material of this retrospective study consisted in bac- terial isolates from urine and/or prostatic secretions or sperm cultures (total ejaculate) obtained from individu- als with reported pelvic discomfort and genital pain, with or without lower urinary tract symptoms and sexu- al dysfunction, and from patients with febrile relapses of CBP, visiting our department from 03/2009 to 03/2015. Retrospective data from an Italian single-center database (years 2009-2015) were also collected for a tentative comparison of pathogen prevalence’s between CBP cases assessed in Greece and Italy. Patients assessment Demographic, microbiological and clinical history of each assessed patient were reviewed. Patients suffering from conditions that influence bacterial virulence or host response (eg. immunodeficiency, abnormalities of the urogenital system) and patients who received antibiotics or immunosuppressive treatment within 4 weeks of the visit were excluded from the study. Included patients were clinically evaluated and under- went the Meares-Stamey “4-glass” test. Few cases under- went the “two-glass” test, assessing the sole VB2 and VB3 specimens. Depending on medical history and specific symptoms, urethral smear cultures and total ejaculate cultures were additionally obtained from several patients. Patients presenting with febrile prostatitis were investigated by a midstream urine culture (MUC) only. Appropriate antimicrobials were administered to con- firmed cases of CBP accordingly to antibiogram for a period of 4 weeks. Follow-up included interview, physi- cal examination and the Meares-Stamey test. Microbiological evaluation The Meares-Stamey test was considered positive when: 1) bacteria grew in the culture of expressed prostatic secretion (EPS) and VB3 urine sample and did not in VB1 and VB2 sample; 2) bacterial colonies in VB3 were higher in num- DOI: 10.4081/aiua.2019.3.177 INTRODUCTION/AIM Chronic bacterial prostatitis (CBP) is an inflammatory con- dition of the prostate, characterized by pain in the geni- tal or the pelvic area, which can lead to urinary disorders and may cause sexual dysfunction. Despite the extensive knowledge accumulated over time, several microbiolog- ical aspects remain uncertain. The aim of our study was to describe the microbiological characteristics of patients referring to a single Hellenic tertiary care center with symptoms of prostatitis combined with symptoms in the genitourinary tract. Stamatiou_Stesura Seveso 30/09/19 18:23 Pagina 177 Archivio Italiano di Urologia e Andrologia 2019; 91, 3 K. Stamatiou, V. Magri, G. Perletti, V. Papadouli, N. Recleiti, V. Mamali, O. Zarkotou 178 ber compared to VB1 and VB2 samples. Given that no standard cut-off level of the number of bacteria in both urine and prostate secretion samples is defined by con- sensus for the diagnosis of chronic bacterial prostatitis, we defined no lower acceptable level for either one. Cultures, identification and semi-quantitative assay for Mycoplasma hominis and Ureaplasma urealyticum were performed using the Mycoplasma IST 2 kit (bioMerieux). Chlamydia trachomatis was detected by direct immune-fluorescence (monoclonal antibodies against lipopolysaccharide mem- brane, Kallestad). Urine samples were cultured undiluted in blood and MacConkey agar plates (Kallestad Lab., TX, USA) and subjected to centrifugation for microscopic examination of the sediment. Evaluation of culture results was performed by two spe- cialist microbiologists. Identification of traditional pathogens was performed by conventional methods and the Vitek-2 Compact (bioMerieux, France) system and sus- ceptibility testing was performed by disc diffusion and/or the Vitek-2 system. Interpretation of susceptibility results was based on Clinical and Laboratory Standards Institute (CLSI) guidelines. The local Ethical Committee approved the research pro- tocol for the present retrospective study. RESULTS A total of 548 bacterial isolates were obtained from eligi- ble Greek patients assessed in 1324 visits and recorded during a period of 6 years (2009-2015). In 114 cases the colonies counted in VB2 cultures were as many as those assessed in VB3. In 44 cases the EPS/VB3 cultures were negative despite the presence of bacteria in the speci- mens. These cases were excluded from the study. Finally, 389 bacterial isolates were recognized as CBP cases and constituted the material of our study. Demographic and microbiological data are presented in Table 1. Microbiological findings A vast variety of pathogens, including rare bacteria, was found in both mono and polymicrobial cultures from both EPS and VB3 samples. The most common were E. coli, coagulase negative staphylococci (CoNS) (mainly S. hominis and S. haemolyticus) and Enterococcus faecalis. The most common combination in polymicrobial isolates composed of two different species was that of E. coli and CoNS (21 cases) while in polymicrobial isolates composed of three different species was that of Escherichia coli, Enterococcus faecalis and Proteus mirabilis (4 cases). 29 out of 43 sperm cultures were performed comple- mentary to EPS/VB3. Thirteen of them were identical to EPS/VB3 cultures. The remaining 16 cultures allowed diagnosing bacterial infection while the EPS/VB3 cul- tures were negative. The most frequently assessed iso- lates were Enterococcus spp. (19 monomicrobial and 3 polymicrobial). Urethral discharge cultures and cultures from urethral swab samples have diagnosed 48 cases of coexisting urethral infection mainly caused by CoNS and Chlamydia trachomatis (Table 2). Follow-up visits and outcome As far as the outcomes of follow-up visits are concerned, 263 patients reported elimination of symptoms/clinical improvement, though only 149 were completely cured. Bacterial persistence occurred in 111 cases. In 17 patients pus was found in EPS and/or VB3 in the absence of symp- toms. In 6 cases EPS/VB3 cultures were negative despite the presence of bacteria in the same samples. Six patients were diagnosed with another disease during follow-up. In most non-treated cases, the pathogens found in the fol- low-up cultures were different from those isolated in the initial visit (usually Enterococcus faecalis, CoNS and E. coli). Relapses occurred in 53 patients and almost half of them were caused by microorganisms other than those causing the initial infection. The average time interval between episodes of chronic prostatitis is 13.9 months (minimum 2 and maximum 56 months). The pathogens most com- monly associated with clinical relapses were Enterococcus faecalis, CoNS and E. coli. Table 1. Patient demographic and microbiological data. Clinical sample Number Number of patients 389 Median age 34.7 Microbiological sample Cultures of prostatic secretions 92 Urine samples collected after prostate massage 343 Mid-stream urine only cultures (febrile cases) 46 Sperm cultures (total ejaculate) 43 monomicrobial infection 297 polymicrobial infection 92 Table 2. Pathogens found in monomicrobial and polymicrobial isolates. Pathogen EPS/VB3 Sperm Urethral Escherichia Coli 142 5 1 Coagulase negative staphylococcus 108 15 Enterococcus faecalis 102 16 4 Streptococcus spp. 31 Proteus spp. 28 1 Staphylococcus aureus 21 1 Klebsiella spp. 9 5 Gemella Morbilorum 4 1 Raoultella planticola 3 Haemophilus parainfluenzae 3 1 1 Enterobacter aerogenes 2 Citrobacter freundii 2 Mycoplasma hominis 1 1 1 Acinetobacter baumannii 1 Staphylococcus lugdunensis 1 Haemophilus influenzae 1 Pseudomonas aeruginosa 1 Brevundimonas diminuta/vesicularis 1 Candida non albicans 2 Candida albicans 2 Chlamydia trachomatis 13 N. gonorrhoeae 4 TOTAL 465 30 40 Stamatiou_Stesura Seveso 30/09/19 18:23 Pagina 178 DISCUSSION Epidemiological and diagnostical issues According to the general perception a progression of prostatic inflammation and its consequences from acute to chronic occur resulting on CBP pathogenesis (1). Actually, after an episode of acute bacterial prostatitis approximately 5-10% of patients progress to chronic infection (2, 3). In accordance to the above, in our study 10.52% of the patients presented with episodic or persistent relapsing urinary tract infections and all reported a previous diag- nosis of prostatitis. CBP is considered a relatively infrequent disease, since it comprises only 10% of all prostatitis cases (4). Nevertheless, from the currently available epidemiologic studies, it appears that CBP is more common since many patients may have bacterial infection despite negative urine cultures: while less than 10% of men worldwide have a proven bacterial infection of the prostate, up to 25% receive a diagnosis of prostatitis in their lifetime (5). Negative culture results occur for various reasons includ- ing, the presence of fastidious organisms, initiation of antibiotics prior to obtaining an EPS sample, high bacter- ial count cut-offs established by laboratories (e.g., 50 000 CFU), or insufficient sample volumes. Since we defined no lower acceptable level for bacterial colonies in both urine and prostate secretion samples for the diagnosis of chronic bacterial prostatitis, the bacteriologically proven incidence of CBP in this study was 29.45%. Moreover, the fact that we recognised certain Gram-positive bacteria as pathogenic may have also contributed to this difference. In fact, the literature strongly suggests that urologic diseases involving Gram-positive bacteria may be easily overlooked due to limited culture-based assays typi- cally utilized for urine in hospital micro- biology laboratories (6). Insufficient sample volumes explain the low number of assessable EPSs in this study. This fact may indicate the need of better preparing (e.g. abstain from sexual intercourse for 3 to 5 days) before the Meares-Stamey test. On the other hand, the presence of fas- tidious organisms, anaerobic pathogens or bacteria not detectable with the usual tests may explain the 44 cases whose EPS/VB3 cultures were negative despite the presence of bacteria. As a matter of fact, traditional culture procedures show lower specificity and sensitivity in detection of bacteria in prostatic speci- mens than polymerase chain reaction (PCR)-based techniques. Choi et al. found an 11.4% incidence of bacterial infection in routine EPS or VB3 cul- tures, while PCR detected bacterial infection in 40.9% of cases (7). Similarly, Krieger and Riley found a sub- stantial proportion of positive broad- spectrum PCR assays (8). Regardless of its drawbacks, the four-glass test is cur- rently considered the diagnostic standard for CBP, while PCR techniques are cumbersome and have little use in the daily clinical setting. For this reason Magri et al. introduced the 'five-glass' test (four-glass plus post-VB3 semen culture), which showed 3.6- or 6.5-fold increases in relative sensitivity and lesser reductions (-13.2% or - 14.7%) in relative specificity for traditional and unusual pathogens (Mycoplasmata and others) compared with the four-glass or two-glass test, respectively (9). The sig- nificance and diagnostic value of complementary semen culture is supported by two other studies (10. 11), though further studies are needed to determine whether a 'five-glass' test may represent a better diagnostic tool. Similarities between Greek and Italian databases may indicate geographical trends in CBP epidemiology. In fact, similar policies in antibiotic usage and common factors that influence male sexuality and sexual behav- iour may contribute to such trends. To our knowledge, a high CBP incidence (26.9%) has been also demonstrated in a previous Greek study (12) while higher incidences were found in central and southern regions of Italy (13). Microbiological and pathophysiological issues Besides the high frequency of E. coli and E. faecalis iso- lates the most remarkable similarity between Greek and Italian databases was the wide array of different species like Coag. Neg. Staphylococcus, Streptococcus spp., S. aureus, Haemophylus spp., Citrobacter Spp., Mycoplasma hominis, Haemophylus spp., P. aeruginosa and Candida spp. isolated from both Greek and Italian CBP patients (Tables 2-4). In our study Enterococcus faecalis was by far the most fre- quent bacterial isolate from sperm cultures. In the Italian cohort, this species was the third most common isolate in sperm cultures after E. coli and U. urealyticum (Tables 179Archivio Italiano di Urologia e Andrologia 2019; 91, 3 Chronic prostatic infection: Microbiological findings in two Mediterranean populations Table 3. Monomicrobial isolates in an Italian cohort of 151 consecutively assessed Italian patients (years 2009-2015). Pathogen Isolated Isolated Isolated TOTAL from EPS/VB3 from total from both only ejaculate only specimens Escherichia coli 26 13 6 45 Proteus mirabilis 2 1 / 3 Klebsiella spp. 1 2 / 3 Morganella morganii 1 5 / 6 Pseudomonas aeruginosa / / 2 2 Haemophilus parainfluenzae / 2 / 2 Citrobacter koseri / 1 / 1 Neisseria subflava / 1 / 1 Enterococcus faecalis 11 6 3 20 Staphylococcus aureus 3 / / 3 Staphylococcus coagulase-negative 1 5 1 7 Streptococcus beta-haemolyticus gr. B / / 1 1 Streptococcus agalactiae 1 / / 1 Steptococcus anginosus / 1 / 1 Kocuria kristinae / / 1 1 Chlamydia trachomatis 3 / / 3 Ureaplasma Urealyticum 4 21 2 27 Mycoplasma hominis / 2 / 2 TOTAL 53 60 16 129 Stamatiou_Stesura Seveso 30/09/19 18:23 Pagina 179 Archivio Italiano di Urologia e Andrologia 2019; 91, 3 K. Stamatiou, V. Magri, G. Perletti, V. Papadouli, N. Recleiti, V. Mamali, O. Zarkotou 180 3, 4). Several other studies provided clear evidence of a Gram-positive predomination in bacteriospermia (14, 15), while other found a very high proportion of Gram-negative microorganisms with Escherichia coli being the commonest isolate in pos- itive cultures (22.2 to 75%) (16-18). Urethral discharge cultures and cul- tures from urethral swab samples revealed coexisting CoNS and/or Chlamydia trachomatis-induced ure- thral infection. Interestingly, in the Italian cohort, whereas Mycoplasmata were found mainly in ejaculate cultures, C. tra- chomatis was isolated mainly from EPS/VB3 samples (Table 4). Since the urinary tract acts as a nest of infection for the seminal tract, these microor- ganisms are capable of causing classi- cal infections of the urogenital tract such as epididymitis and prostatitis as well as subclinical reproductive tract infections. Outcome related issues Differences in isolated microorganisms in VB3/EPS cultures between follow-up and initial visits of untreat- ed/relapsed cases reinforce the new appreciation of chronic prostatitis as a biofilm disease. Pathogen eradi- cation rates, as high as 80%, have been reported in the pastin CBP patients treated with various fluoro- quinolones (19). Our bacterial eradication rate (68.15%) is similar with that reported in a previous Greek study (64.7%) (12) and this fact may be attributed to the quinolones overuse in Greece. CONCLUSIONS In Greece, the incidence of CBP is possibly higher than that reported in international surveys. Similarities between Greek and Italian databases suggest geographi- cal trends in CBP epidemiology. The fact that Gram-positive species were the most fre- quent isolates in both Greek and Italian databases sup- port the role of Gram-positive bacteria in CBPs patho- genicity. Regardless of its drawbacks, the Meares-Stamey is the main tool for the diagnosis of CBP. The signifi- cance and diagnostic value of complementary semen cul- ture is supported by our findings, though further studies are needed to determine whether a 'five-glass' test may represent a better diagnostic tool. REFERENCES 1. Nickel JC, Olson ME, Barabas A, et al. Pathogenesis of chronic bacterial prostatitis in an animal model. Br J Urol. 1990; 66:47-54. 2. Nickel CJ. Inflammatory and pain conditions of the male geni- tourinary tract: prostatitis and related pain conditions, orchitis, and epididymitis. 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Drugs. 1999; 58 (Suppl 2):103-6. 181Archivio Italiano di Urologia e Andrologia 2019; 91, 3 Chronic prostatic infection: Microbiological findings in two Mediterranean populations Correspondence Konstantinos Stamatiou, MD stamatiouk@gmail.com Urology Dpt, Tzaneion Hospital, Piraeus (Greece) Vittorio Magri, MD Urology Secondary Care Clinic, ASST-Nord, Milan, Italy Gianpaolo Perletti, PhD Department of Biotechnology and Life Sciences, Section of Medical and Surgical Sciences Università degli Studi dell'Insubria, Varese (Italy) Vaia Papadouli, MD Nectaria Recleiti, MD Vassiliki Mamali, MD Olympia Zarkotou, MD Microbiology Dpt, Tzaneion Hospital, Piraeus (Greece) Stamatiou_Stesura Seveso 30/09/19 18:23 Pagina 181