International data showed that only 5–10 % of prostatitis patients have a microbiologically demonstrated bacterial infection [17]. A correct microbiological analysis should include the use of adequate biological material that has to be homogeneous, quantitatively sufficient, and representative of the site infection organ [18, 19]. Collection and sampling have to be simple, noninvasive, and not bothersome for patients, have a good compliance, and have to be not contaminated by a saprophytic microflora. A correct microbiological analysis should also include identification of a low number of bacteria in expressed prostatic secretion or post-massage urines, as this can be the causative agent in chronic bacterial prostatitis [20]. Critical points of microbiological sampling are immediate/rapid transport to suitable conditions for biological materials, immediate/rapid culture after collection, and the application of bacteriological techniques able to quantify a small number of pathogens [21].

The most common isolated pathogens from prostatitis patients are those involved in urinary tract infections (uropathogens), which include: Gram-negative organisms, most commonly Escherichia coli, Proteus spp., Klebsiella spp. and Pseudomonas spp., enterococci, Staphylococcus aureus, and rarely anaerobic organisms such as Bacteroides spp. [17] (Fig. 2.1).

In a retrospective study Mazzoli S. evaluated 1,686 isolated strains from chronic prostatitis patients [17]. The microbiological findings were as follows: 371 strains of Gram-negatives (22.00 %) and 1,112 Gram-positives (65.9 %), 14 yeast strains (0.83 %), and 189 mycoplasmata (11.2 %) [17]. It is worth considering that Gram-positives represented the majority of isolates with strain Enterococcus faecalis being the most common with a prevalence of 42.7 % [17]. Similar results have been confirmed by Cai et al. who enrolled more than 15,000 consecutive outpatients with chronic bacterial prostatitis attending a single sexually transmitted disease center from January 1997 and December 2008 [22]. All patients underwent microbiological cultures of first-void early morning urine, midstream urine, expressed prostatic secretion, and post-prostate massage urine. The prevalence of different bacterial strains was stratified in four different periods: 1997–1999, 2000–2002, 2003–2005, and 2006–2008 [22]. In this large cohort of patients, it demonstrated an increasing prevalence of E. faecalis strains reaching a peak in the 2006–2008 period [20], highlighting the emerging role of Gram-positive strains [22]. These microbial data follows the widespread use of large-spectrum antibiotics in outpatients.

On the other hand, some cases of prostatitis are caused by atypical pathogens, such as Chlamydia trachomatis or mycoplasmata [23, 24]. In fact, a large prospective study of men with chronic prostatitis found that 74 % had an infectious etiology: the most common isolates were Chlamydia trachomatis (37 % of cases) and Trichomonas vaginalis (11 %), whereas 5 % of patients had infection due to Ureaplasma urealyticum [25]. Classical bacterial uropathogens were found in 20 % of patients, and more patients with these pathogens had prostatic specimens with leukocytes as compared with patients with nonbacterial pathogens [25]. Other reports showed that possible prostatitis pathogens include: Mycoplasma genitalium, Neisseria gonorrhoeae, Mycobacterium tuberculosis, various fungi, and several viruses [23, 24]. However, future microbiological studies should be performed in order to clarify the role of atypical bacteria.

The role of biofilm-producing bacteria in the development of urinary tract infections and in acute and chronic prostatitis particularly has been elucidated in recent studies [26, 27]. Bacteria living in a biofilm usually have significantly different properties compared with free-floating bacteria (planktonic) of the same species, as the dense and protected environment of the biofilm allows them to interact in various ways [28]. In this environment bacteria exhibit an increased resistance to antibiotics, as the dense extracellular matrix and the outer layer of cells protect the interior of the community [28]. This aspect is particularly important for the management of chronic bacterial prostatitis patients. Recently, Bartoletti et al. evaluated the role of biofilm-producing bacteria in the clinical response to antibiotic therapy among patients affected by chronic bacterial prostatitis, showing that biofilm-producing bacteria were commonly found and had a significant negative impact on the clinical response to antibiotic therapy [27]. As also highlighted by Tenke et al., this aspect has an important impact on the diagnostic approach. In fact, the diagnosis of chronic bacterial prostatitis can be difficult because colonized bacteria in biofilms will not enter the prostatic secretion or urine sample [29].

In 2006, Kanamaru demonstrated in an “in vitro study” a clear association between acute bacterial prostatitis and biofilm formation [30]. It has been suggested that if bacteria persist after an acute or, more likely, clinically subacute prostate inflammation, they can form small, sporadic bacterial microcolonies or biofilms within the ductal system adherent to the epithelium [31]. There is a quiescent period, a sort of “hibernation,” when the environment becomes adverse to and difficult for bacterial existence. The presence of focal sites of bacterial persistence can be postulated by areas of inflammation with subsequent lymphocyte invasion and infiltration of plasma cells and macrophages, which has been demonstrated in both nonbacterial and bacterial prostatitis [32, 33]. It appears that the persistence of bacteria in the prostate gland in these focal biofilms leads to persistent immunologic stimulation and subsequent chronic inflammation. It is suspected that the creation of a chemically and immunologically distinct microenvironment may induce microorganisms to crystallize, calcify, and form calculi [28, 31]. Recently, Mazzoli aimed to isolate potential biofilm-producing bacteria from chronic bacterial prostatitis patients and to evaluate their ability to produce in vitro biofilms [28]. The author evaluated 150 bacterial strains isolated from chronic prostatitis NIH-II patients: 50 Enterococcus faecalis, 50 Staphylococcus spp., 30 Escherichia coli, and 20 Gram-negative miscellanea. Quantitative assay of biofilm production and adhesion was performed according to the classic Christensen microwell assay. Isolates were classified as nonproducers or weak, moderate, or strong producers. The majority of E. coli, Gram-negative bacteria, staphylococci, and enterococci strains were strong or medium producers: 63–30 %, 75–15 %, 46–36 %, and 58–14 %, respectively. In this sense, the author highlighted the association between bacteria that were strong producers of biofilm and chronic prostatitis [28]. In line with these “in vitro” observations, Sfanosa et al. demonstrated that acute inflammatory proteins constitute the organic matrix of prostatic corpora amylacea and calculi in men with prostate cancer and presented a definitive analysis of the protein composition of prostatic corpora amylacea and calculi. Moreover, they suggested that acute inflammation has a role in calculi biogenesis: in fact, proteins identified in calcifications, including calprotectin, myeloperoxidase, and a-defensins, are contained in neutrophilic granules [34]. Immunohistochemistry suggested the source of lactoferrin to be prostate-infiltrating neutrophils as well as inflamed prostate epithelium and suggested prostate-infiltrating neutrophils as a major source of protein for calprotectin, because this protein was absent from other prostate compartments. These data confirm the inflammatory genesis and components of prostatic calcifications: inflammation, which resembles the “primum movens” of the calcifications, may now also be the starting point for an additional cascade, amplifying and perpetuating the inflammation itself and the tissue damage. Inflammatory cytokines, especially interleukin 8, were also proved to be present in men with prostatitis as a surrogate marker of prostatitis in general as well as in well-specified Chlamydia trachomatis prostatitis [35, 36].