In a recent paper in the Open Microbiology Journal Siddiqui et al report 16S rDNA findings in the urine of a woman diagnosed with overactive bladder disease (OAB). A complex bacterial profile, with fastidious and anaerobic bacteria, was observed in contrast to the findings in routine urine culture. Thus the authors question the role of microorganisms in disease classification (Siddiqui et al, 2014). Their question is relevant as some diseases of the urinary tract are defined by the presence of microorganisms while others are defined by their absence.
Diagnoses based on the presence of micororganisms
Urinary tract infections (UTI) is a big group of disorders usually classified as uncomplicated and complicated, but more recently also by risk factors and severity grading depending on the clinical presentation (Bjerklund Johansen et al, 2011). The diagnosis requires clinical symptoms and evidence of living bacteria in the urine, usually quantified by colony forming units per milliliter (CFU/ml). Leucocytes and blood in urine are signs of the host response. Leucocytes infiltrate the mucosa and release immunoactive proteins. Urothelial cells burst and are shed into urine; sometimes bleeding occurs (Wullt et al., 2010).
Culture tests and urine microscopy have been the gold standard for diagnosing UTI. When sexually transmitted pathogens are suspected an amplification system is also used [Litwin et al, 1999; EAU guidelines, 2015]. In order to differentiate between infection and contamination the concept of significant bacteriuria was introduced by Kass. The concept was based on monoculture of a dominant pathogen [Kass, 1960]. However, no fixed bacterial count has been considered indicative for significant bacteriuria in all kinds of UTIs and under all circumstances [Hooton et al, 2013]. Currently, the counts of uropathogens in a midstream sample of urine (MSU) should exceed 104 CFU/ml in men or vary from ≥103 CFU/ml in acute uncomplicated cystitis till ≥105 CFU/ml in women with complicated UTIs. The lower the colony counts in MSU the higher the likelihood of contamination. In a suprapubic bladder puncture specimen, any count of bacteria is considered diagnostic [EAU Guidelines, 2015]. The currently routine urine culture method is limited to detect easily culturable aerobic bacteria only and not fastidious and anaerobic bacteria. The underlying idea has always been that urine from healthy subjects is sterile and a negative or positive urine culture has usually been taken as discriminative for an infection to be absent or present, respectively.
Diagnoses based on the absence of microorganisms
Several urological disorders with symptoms that resemble infections are regarded as separate entitites based on the exclusion of bacterial growth with conventional techniques. Examples are overactive neurogenic bladder, female urethral syndrome, bladder pain syndrome/interstitial cystitits and chronic pelvic pain syndrome/chronic inflammatory prostatitis. Interestingly, the differentiation of chronic abacterial prostatitis in inflammatory and non inflammatory depends on the presence of white celles in expressed prostatic secretion which may be a sign of a host reaction to infection. Biopsy has a role in the diagnostic work-up in interstitial cystitis to detect mast cells in the mucosa, a specific host reaction.
Change of paradigmata
During the recent years two pretended paradigmata related to the role of bacteria in urological disease classification have changed completely (Wagenlehner and Naber 2012; Kogan et al., 2014) :
i) We have learned that asymptomatic bacteriuria, although “significant” according to the bacterial load, should not be considered an infection anymore. It rather represents colonization or at best a risk factor under certain circumstances, and treatment is generally not necessary and sometimes even harmful (Cai et al., 2012¸EAU guidelines, 2015). Moreover, it has been shown that bladder instillation with certain E. coli strains can even prevent recurrent infections by bacterial interference (Wullt et al., 2010). Hence, bacteriuria does not neccesarily mean an infection.
ii) There is increasing evidence that if extended sets of culture media for identification of fastidious and anaerobic bacteria or culture-independent metagenomic sequencing (MGS) is applied a broad range of even non-culturable bacteria has been detected in the ”sterile” bladder urine in healthy individuals [Kogan et al. 2014; Hilt et al 2014; Siddiqui et al, 2011; Fouts et al, 2012; Wolfe et al, 2012; Lewis et al, 2013; Hilt et al, 2014; Pearce et al, 2014] and in female patients with different urological disorders [Khasriya et al, 2013; Brubaker et al, 2014; Nienhouse et al, 2014; Pearce et al, 2014; Siddiqui et al, 2014] or expressed prostatic secretions (EPS) in men [Smelov et al, 2014]. The terms urinary microbiota and urinary microbiome have been adopted to define the microorganisms that normally exist within the bladder [Wolfe and Brubaker, 2015]..
Thus, sterile urine seems to be a myth and the recent MGS-based findings on the urine microbiome encourage a discussion to redefine the criteria for UTIs and non-infectious urological disorders with similar symptoms.
The role of MGS in diagnosing urological disorders
In their case report Siddiqui and colleagues  presented a female patient with a 10 years history of overactive bladder (OAB) syndrome diagnosed after exclusion of other urological disorders, e.g. interstitial cystitis and bladder cancer. The patient had been treated with various anticholinergic agents with temporary relief only. When urine culture revealed a “significant” bacteriuria of >105 cfu/ml with α-hemolytic streptococci (Streptococcus viridans group) she was treated for one week with trimethoprim 160 mg b.i.d. About a month later the routine culture was negative, but the urinary symptoms persisted.
By comparison of midstream and catheter urine samples Hooton et al (2014) demonstrated, that streptococci are rarely uropathogens but rather contaminants. Nevertheless he also demonstrated that very low counts of E. coli could be relevant, whereas similar conclusions could not be drawn for other Gram-negative rods or S. saprophyticus. Although trimethoprim is not recommended for treatment of streptococcal infections, it would most likely be an adequate treatment for a low count UTI caused by E. coli. Therefore, according to the classical approach the OAB diagnosis made by Siddiqui et.al. can still be regarded as confirmed, especially as an intermittent electric stimulation treatment was satisfactory, which the patient wanted to continue.
On two occasions, when the streptococcal bacteriuria was registered and one year later, respectively, the authors analyzed the patient’s urine with a culture-independent 16S ribosomal DNA pyrosequencing analysis. Findings in the 1st urine sample were consistent with the routine culture of α-hemolytic streptococci, but also for several fastidious bacteria, such as Atropium, Prevotella, and Ureaplasma spp.. The latter were also confirmed in the routine culture negative 2nd urine sample one year later. The authors now speculate whether or not these bacteria, especially the Ureaplasma sp., may be the infectious agents causing the patient`s chronic symptoms. Unfortunately, no adequate antibiotic therapy for Ureaplasma sp. with macrolides or tetracyclines was administered and without the results of adequate treatment no conclusions can be drawn as to the role of these “difficult-to-culture” bacteria for the patient`s symptoms. Thus, despite highly sophisticated investigations the gain of knowledge from this case report is rather limited.
Extended sets of culture media and culture-independent metagenomic sequencing (MGS) enable us to detect a wide range of fastidious and anaerobic and even non-culturable bacteria in the ”sterile” bladder urine in healthy individuals and in patients with different urological disorders. We must of course take advantage of these new technologies and it is now high noon to redefine UTIs and various urological disorders based on findings by routine culture. Other positive examples are the metacron etiological relationship between microorganisms and malignant tumors of the urinary tract, like Herpes virus and Schistosomiasis in bladder cancer. It is expected that the application of novel MGS technologies will further improve findings, as the significance of sequencing data is based on the number of sequencing reads [Beal et al, 2012]. For cost reasons it will take time before novel assays become clinically applicable, but while the MGS methods remain expensive, the sequencing related costs have decreased rapidly. Further MGS studies will extend our knowledge on the microbiome of the urogenital tract in both men and women, and the method opens intriguing new perspectives on the role of microorganisms in diseases of the urinary tract.
However, as we recently learned from the case of asymptomatic bacteriuria, the pure detection of bacteria in the urine by itself does not prove an infectious etiology of a specific disorder. Well designed clinical studies in which extended sets of culture media and MGS are integrated, are needed to clarify the pathogenesis of urological disorders where bacteria may play a role. Having said this, it is important to avoid that results of new technologies lead to unnecessary antibiotic consumption with unwanted collateral damage and adverse events.
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