twenty - four coryneform strains were obtained from human microbiota biobank ( humb , http://biomedicum.ut.ee/armb/biobank/ ) of tartu . the strains were originally isolated from either healthy volunteers ( n=7 ) , men with inflammatory prostatitis ( n=12 ) , or otherwise healthy men who were male partners of the couples with idiopathic infertility ( n=5 ) . because the latter were without prostatitis ( including asymptomatic inflammation , etc . ) we included antibiotic use within the past 3 months as one of several exclusion criteria . briefly , the samples were cultured quantitatively to detect anaerobic , microaerophilic and aerobic bacteria within 1 h from collection . aerobic ( blood agar ) and microaerobic ( mrs agar and gardnerella vaginalis - selective agar in 10% co2 atmosphere ) cultures were incubated at 37c for 13 days , anaerobic cultures ( wilkins  chalgren media in an anaerobic glove box ) for 35 days . colonies with different morphology were gram stained and examined microscopically . the strains in humb had been characterized by -glucuronidase test or , if negative , by api coryne 2.0 biochemical identification system ( biomrieux , france ) according to manufacturer 's instructions ( the pre - sequencing identification of the strains is shown in table 1 ) . interpret with caution . a coryneform strain with identification signature that discourages suggesting a particular species . we chose two primer sets for identification of coryneform bacteria : broad - range primers ( 5-gttaag - tcccgcaagga-3 and 5-aggaggtgatccagcc-3 ) for 440 bp 16s rrna product and latter primers ( 5-cgtagggtgcgagcgttgtccg-3 and 5-cgttgcgggacttaacccaacatctc-3 ) for 516 bp 16s rrna product according to adderson et al . quantitative assessment of biofilm production was performed using crystal violet binding assay method modified from the classic christensen microwell assay ( 14 ) . briefly , strains were grown in mueller - hinton broth for 48 h in elisa plate together with negative control ; growth was verified by measuring optical density ( od ) with elisa plate reader . then , the broth was discarded , and the wells were washed and then stained with crystal violet . note that crystal violet stains the cells rather than slime . extinction at 490 nm of the inoculated wells was measured with an elisa plate reader after 48 h growth . after 48 h incubation , the broth was removed , the plates were rinsed and the wells were stained with crystal violet . after staining and drying , the wells were read again with elisa plate reader with measuring the extinction at 620 nm . arbitrarily , we considered the strains as biofilm producers if their extinction at 620 nm after staining with crystal violet was at least twice the mean of negative controls . for statistical analyses , sigmastat ( systat software , chicago , il ) and excel ( the differences between the groups were calculated with fisher exact test , t - test ( in case of normal distribution ) , and mann  whitney rank sum test ( in case of nonparametric distribution ) . twenty - four coryneform strains were obtained from human microbiota biobank ( humb , http://biomedicum.ut.ee/armb/biobank/ ) of tartu . the strains were originally isolated from either healthy volunteers ( n=7 ) , men with inflammatory prostatitis ( n=12 ) , or otherwise healthy men who were male partners of the couples with idiopathic infertility ( n=5 ) . because the latter were without prostatitis ( including asymptomatic inflammation , etc . ) we included antibiotic use within the past 3 months as one of several exclusion criteria . briefly , the samples were cultured quantitatively to detect anaerobic , microaerophilic and aerobic bacteria within 1 h from collection . aerobic ( blood agar ) and microaerobic ( mrs agar and gardnerella vaginalis - selective agar in 10% co2 atmosphere ) cultures were incubated at 37c for 13 days , anaerobic cultures ( wilkins  chalgren media in an anaerobic glove box ) for 35 days . colonies with different morphology were gram stained and examined microscopically . the strains in humb had been characterized by -glucuronidase test or , if negative , by api coryne 2.0 biochemical identification system ( biomrieux , france ) according to manufacturer 's instructions ( the pre - sequencing identification of the strains is shown in table 1 ) . interpret with caution . a coryneform strain with identification signature that discourages suggesting a particular species . we chose two primer sets for identification of coryneform bacteria : broad - range primers ( 5-gttaag - tcccgcaagga-3 and 5-aggaggtgatccagcc-3 ) for 440 bp 16s rrna product and latter primers ( 5-cgtagggtgcgagcgttgtccg-3 and 5-cgttgcgggacttaacccaacatctc-3 ) for 516 bp 16s rrna product according to adderson et al . quantitative assessment of biofilm production was performed using crystal violet binding assay method modified from the classic christensen microwell assay ( 14 ) . briefly , strains were grown in mueller - hinton broth for 48 h in elisa plate together with negative control ; growth was verified by measuring optical density ( od ) with elisa plate reader . then , the broth was discarded , and the wells were washed and then stained with crystal violet . note that crystal violet stains the cells rather than slime . extinction at 490 nm of the inoculated wells was measured with an elisa plate reader after 48 h growth . after 48 h incubation , the broth was removed , the plates were rinsed and the wells were stained with crystal violet . after staining and drying , the wells were read again with elisa plate reader with measuring the extinction at 620 nm . arbitrarily , we considered the strains as biofilm producers if their extinction at 620 nm after staining with crystal violet was at least twice the mean of negative controls . for statistical analyses , sigmastat ( systat software , chicago , il ) and excel ( microsoft , redmond , wa ) software programs were used . the differences between the groups were calculated with fisher exact test , t - test ( in case of normal distribution ) , and mann  whitney rank sum test ( in case of nonparametric distribution ) . altogether , 14 different coryneform bacteria were found from the investigated semen samples ( table 1 ) . the most common species in semen were c. seminale , or as it is synonymously called , c. glucuronolyticum ( n=5 ) , c. minutissimum ( n=4 ) , and dermabacter hominis ( n=4 ) as identified by sequencing methods . the distribution of species was heterogenous , and the sequencing method sometimes produced results that conflicted with another sequencing method ( exact match between 16s and latter primer identification was seen in 11 out of 24 strains ) , or both of them produced results that did not allow any identification at all . therefore , the identification was not conclusive for many of the strains and their identification can be considered presumptive . api results were in clear agreement with genomic methods in 6 of 18 cases ( 4 c. seminale and 2 dermabacter hominis strains ) . biofilm was observed in 7 out of 24 strains , assuming that biofilm yields od greater than two ( 0.123 ) or four ( 0.246 ) times greater than that of negative control , respectively ( table 2 ) . the biofilm - producing strains were arthrobacter cumminsii ( from prostatitis patient ) , dermabacter hominis ( two strains from patients , one from a healthy man ) , c. minutissimum ( one strain from a patient , one strain from a control ) , and actinomyces neuii ( isolated from a healthy man ) . no differences were found between the strains originating from prostatitis patients and healthy men . dermabacter hominis strains were more potent biofilm producers than c. seminale strains ( p=0.048 ) . we investigated 24 coryneform strains originating from the male genital tract and could identify 21 of them to species level with at least 96% confidence . our main interest was related to etiopathogenesis of chronic prostatitis ( cp ) , therefore , half of the strains originated from cp patients . patients with cp / chronic pelvic pain syndrome ( cpps ) ( category nih iii prostatitis ) or asymptomatic inflammatory prostatitis ( category nih iv prostatitis ) usually lack  traditional uropathogens in prostate - specific materials . to this date , no single etiopathogenetic mechanism has been proved yet , but an interrelated and multifactorial cascade has been proposed ( 15 ) where an initiating event ( infection , trauma , etc . ) may lead to immunologic stimulation , inflammation , neurogenic stimulation , neuropathic damage with afferent nerve upregulation , and ultimately , pain . whether and when the usual commensals of male genital tract may act as pathogens is a good question without a good answer . hua et al . ( 16 ) have reviewed the topic of unusual pathogens and concluded that specific microorganisms ( atypical pathogens ) explain the studies have also suggested the role of immune cross - reactivity ( 17 ) and association with urinary tract infection history ( 18 ) . former studies by our research group had revealed numerous different aerobic , anaerobic , and microaerophilic microorganisms in the seminal fluid ( 1 , 8 , 19 , 20 ) that coincided with the data of szke et al . ( 21 ) . the main difference between healthy controls and inflammatory prostatitis patients was quantitative the latter harbored significantly higher total concentration of bacteria ( mean 10 cfu / ml ) as well as a higher number of different species ( mean 5 ) in their semen than controls ( mean 10 cfu / ml and 3 species ) . instead , we found abundant polymicrobial communities that may be comparable to bacterial vaginosis in women , suggesting that prostatitis might be viewed as an unfavorable shift in the balance of genital tract microbiota . ( 22 ) that dysbacteriosis in the male genital tract may be an underlying , primary cause of cp and that the wide use of antibiotics may be an initiating risk factor for prostatitis . during our former studies ( 1 , 8 
 19 ) we found that coryneform bacteria constitute a significant part of these microbial communities , being present in three fourths of men . ( 23 ) have also shown that corynebacterium sp . were the most common bacteria in the prostatitis patients expressed prostatic secretion or urine : they were more prevalent , abundant , and represented by higher number of species in prostatitis patients than in controls . tanner et al . ( 9 ) even found some unidentified species similar to corynebacterium coyleae , corynebacterium imitans , and c. seminale that were restricted to the prostatitis patients , exclusively . in the present study , we applied molecular identification for the seminal strains collected during our previous studies and tested their biofilm formation ability as putative virulence factor . the broad , less - specific 16s primer yielded more results with a species name than the latter primer . the latter primer often failed to provide a name for a particular strain , but if it did , then the identification was usually with higher confidence than in the case of 16s primer . the most common species in semen were c. seminale , c. minutissimum , and dermabacter hominis . no statistically significant differences between the subject groups were observed , which may be partly associated with the low number of strains . as concerns specific bits of information regarding particular species , c. seminale was first isolated from the semen of a prostatitis patient ( 6 ) , although it can be found frequently in the semen of healthy men , as was confirmed by our study . c. minutissimum is a coryneform bacterium associated mainly with erythrasma , but more dangerous opportunistic infection cases also have been recorded ( 24 , 25 ) . that species has been isolated from prostate secretion as well ( 10 ) . one strain of our study gave conflicting blast results ( zimmermannella , leifsonia , agrococcus , or pseudoclavibacter ) , although all these bacteria are coryneform bacteria . brevibacterium and dermabacter species have been found from the skin of healthy adults , and seldom from various clinical samples ( 26 ) . actinomyces neuii is an atypical actinomycete ( 27 ) that has been associated with opportunistic infections . the majority of the strains were previously identified by a biochemical method , coryne api ( biomerieux ) , which had poor agreement with previously mentioned genomic methods . although in earlier studies coryne api has been lauded as a valuable species identification tool , in our study the concordance with genomic methods was not encouraging . at the same time , biochemical signatures may still give some additional information that could be related to disease ( 28 ) . corynebacterium group g ( according to api profile ) strains that were associated with prostatitis were identified as c. minutissimum , c. aurimucosum , and c. aurimucosum / c . ( they had large rather than tiny colonies , and these strains did not ferment fructose . ) corynebacterium group g is a heterogenous group and it may contain several unidentified corynebacterium species . for example , c. pilbarense was identified from this group rather recently ( 29 ) . it had been also speculated ( but not proven ) that coryneforms could grow in the prostate as a biofilm that would enhance their antibiotic resistance ( 9 ) as uropathogenic coli bacteria do ( 12 ) . the respective data regarding minimal bactericidal concentration ( mbc ) of vancomycin , moxifloxacin , or ciprofloxacin for c. urealyticum biofilm is about two or three orders of magnitude greater than the mbc of respective planctonic forms ( 30 ) . therefore , we aimed to find out whether coryneform bacteria have a proclivity to produce biofilm in vitro . according to our data , biofilm production was characteristic of 29% of the strains belonging to different species and originating from different patients groups . it was interesting to note that biofilm producers and non - producers formed two distinct populations as if there were a binomial distribution of biofilm production . some former studies have shown biofilm production ability of corynebacteria and these bacteria have been associated with catheter and prostheses infections , too ( 3134 ) . hence , we might classify coryneform bacteria as possible or probable prostate pathogens ( 15 ) , given that their initiatory , propagatory , or complicatory role remains yet unproven . microbiologically , they may be responsible for recurrence of infections that render antibiotic therapy ineffective ( 35 ) ; clinically , the initiatory role may be masked by symptom - propagating neural sensitization ( 15 ) . complicatory role of bacterial overgrowth may seem likely as well ( 22 ) . in summary , a wide variety of coryneform bacteria can be found from the male genital tract , although their exact identification is problematic due to insufficient representation in databases . nearly one third of the strains are able to form biofilm that may give them an advantage for surviving several host- and treatment - related conditions . this study was supported by the estonian ministry of education and research ( target financing no .
backgroundcoryneform bacteria constitute an important segment of male urogenital microbiota . they have been generally considered as saprophytes , although some species have been associated with prostatitis as well . at the same time , 
 biofilm infections have been suspected as a cause of prostatitis.objectiveto identify a set of coryneform bacteria isolated from semen of either healthy men or prostatitis patients applying different methods to reveal inter - assay variability and to determine their ability of adhesion and biofilm production.designcoryneform bacteria were identified by api coryne 2.0 biochemical identification system and 16s rdna sequencing using different primer sets . 
 quantitative assessment of biofilm production was performed using crystal violet binding assay method.resultsthe most common species were corynebacterium seminale , c. minutissimum , and dermabacter hominis . altogether 
 14 species and related genera were found . 
 we observed the best inter - assay agreement when identifying c. seminale . 
 biofilm was observed in 7 out of 24 strains . 
 the biofilm - producing strains belonged to arthrobacter cumminsii , dermabacter hominis , c. minutissimum , and actinomyces neuii . 
 no differences were found between the strains originating from prostatitis patients and healthy men . 
 dermabacter hominis strains were more potent biofilm producers than c. seminale strains ( p=0.048).conclusionswe can conclude that a wide variety of coryneform bacteria can be found from the male genital tract , although their exact identification is problematic due to insufficient representation in databases . 
 nearly one third of the strains are able to form biofilm that may give them an advantage for surviving several host- and treatment - related conditions .