Diagnostic criteria in urinary tract infections and the role of metagenomic sequencing of 16S rDNA - Beyond The Abstract

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 [2014] 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.

 

Conclusions

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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REFERENCES

 

1. Beal MA, Glenn TC, Somers CM. Whole genome sequencing for quantifying germline mutation frequency in humans and model species: cautious optimism. Mutation research 2012;750(2):96-106. 

2. Bjerklund Johansen TE, Botto H,  Cek M et al. Critical review of current definitions of urinary tract infections and proposal of an EAU/ESIU classification system. IJAA 2011;38(Suppl):64-70

3. Brubaker L, Nager CW, Richter HE, Visco A, Nygaard I, Barber MD, Schaffer J, Meikle S, Wallace D, Shibata N, Wolfe AJ. Urinary bacteria in adult women with urgency urinary incontinence. Int Urogynecol J. 2014 Sep;25(9):1179-84. doi: 10.1007/s00192-013-2325-2. Epub 2014 Feb 11. 

4. Cai T, Mazzoli S, Mondaini N, Meacci F, Nesi G, D’Elia C, Malossini G, Boddi V, Bartoletti R. The role of asymptomatic bacteriuria in young women with recurrent urinary tract infections: to treat or not to treat? Clinical Infectious Diseases 2012; 55(6):771-7

5. EAU Guidelines on Urological Infections M. Grabe (Chair), R. Bartoletti, T.E. Bjerklund Johansen, T. Cai (Guidelines Associate), M. Çek, B. Köves (Guidelines Associate), K.G. Naber, R.S. Pickard, P. Tenke, F. Wagenlehner, B. Wullt (http://uroweb.org/guideline/urological-infections/). European Association of Urology, 2015.

6. Feng H, Shuda M, Chang Y, Moore PS. Clonal integration of a polyomavirus in human Merkel cell carcinoma. Science. 2008 Feb 22;319(5866):1096-100. doi: 10.1126/science.1152586. 

7. Fouts DE, Pieper R, Szpakowski S, Pohl H, Knoblach S, Suh MJ, Huang ST, Ljungberg I, Sprague BM, Lucas SK, Torralba M, Nelson KE, Groah SL. Integrated next-generation sequencing of 16S rDNA and metaproteomics differentiate the healthy urine microbiome from asymptomatic bacteriuria in neuropathic bladder associated with spinal cord injury. J Transl Med. 2012 Aug 28;10:174. doi: 10.1186/1479-5876-10-174.

8. Foulongne V, Sauvage V, Hebert C, Dereure O, Cheval J, Gouilh MA, Pariente K, Segondy M, Burguière A, Manuguerra JC, Caro V, Eloit M. Human skin microbiota: high diversity of DNA viruses identified on the human skin by high throughput sequencing. PLoS One. 2012;7(6):e38499. doi: 10.1371/journal.pone.0038499. 

9. Hilt EE, McKinley K, Pearce MM, Rosenfeld AB, Zilliox MJ, Mueller ER, Brubaker L, Gai X, Wolfe AJ, Schreckenberger PC. Urine is not sterile: use of enhanced urine culture techniques to detect resident bacterial flora in the adult female bladder. J Clin Microbiol. 2014 Mar;52(3):871-6. doi: 10.1128/JCM.02876-13. Epub 2013 Dec 26.

10. Hooton TM, Roberts PL, Cox ME, Stapleton AE. Voided midstream urine culture and acute cystitis in premenopausal women. N Engl J Med. 2013 Nov 14;369(20):1883-91. doi: 10.1056/NEJMoa1302186.

11. Johansson H, Bzhalava D, Ekström J, Hultin E, Dillner J, Forslund O. Metagenomic sequencing of "HPV-negative" condylomas detects novel putative HPV types. Virology. 2013 May 25;440(1):1-7. doi: 10.1016/j.virol.2013.01.023.

12. Kass EH. Bacteriuria and pyelonephritis of pregnancy. Arch Intern Med, 1960. 105: p. 194-8.

13. Khasriya R, Sathiananthamoorthy S, Ismail S, Kelsey M, Wilson M, Rohn JL, Malone-Lee J. Spectrum of bacterial colonization associated with urothelial cells from patients with chronic lower urinary tract symptoms. J Clin Microbiol. 2013 Jul;51(7):2054-62. doi: 10.1128/JCM.03314-12. Epub 2013 Apr 17.

14. Kogan MI, Naboka YL, Ibishev KS, Gudima IA, Naber KG. Human urine is not sterile – shift of paradigm. Urologia Internationalis 2015;94(4):445-52.

15. Lewis DA, Brown R, Williams J, White P, Jacobson SK, Marchesi JR, Drake MJ. The human urinary microbiome; bacterial DNA in voided urine of asymptomatic adults. Front Cell Infect Microbiol. 2013 Aug 15;3:41. doi: 10.3389/fcimb.2013.00041. eCollection 2013.

16. Litwin MS, McNaughton-Collins M, Fowler FJ Jr, Nickel JC, Calhoun EA, Pontari MA, Alexander RB, Farrar JT, O'Leary MP.The National Institutes of Health chronic prostatitis symptom index: development and validation of a new outcome measure. Chronic Prostatitis Collaborative Research Network. J Urol. 1999 Aug;162(2):369-75.

17. Nienhouse V, Gao X, Dong Q, Nelson DE, Toh E, McKinley K, Schreckenberger P, Shibata N, Fok CS, Mueller ER, Brubaker L, Wolfe AJ, Radek KA. Interplay between bladder microbiota and urinary antimicrobial peptides: mechanisms for human urinary tract infection risk and symptom severity. PLoS One. 2014 Dec 8;9(12):e114185. doi: 10.1371/journal.pone.0114185. eCollection 2014.

18. Pearce MM, Hilt EE, Rosenfeld AB, Zilliox MJ, Thomas-White K, Fok C, Kliethermes S, Schreckenberger PC, Brubaker L, Gai X, Wolfe AJ. The female urinary microbiome: a comparison of women with and without urgency urinary incontinence. MBio. 2014 Jul 8;5(4):e01283-14. doi: 10.1128/mBio.01283-14.

19. Siddiqui H, Nederbragt AJ, Lagesen K, Jeansson SL, Jakobsen KS. Assessing diversity of the female urine microbiota by high throughput sequencing of 16S rDNA amplicons. BMC Microbiol. 2011 Nov 2;11:244. doi: 10.1186/1471-2180-11-244.

20. Siddiqui H, Lagesen K, Nederbragt AJ, Eri LM, Jeansson SL, Jakobsen KS. Pathogens in Urine from a Female Patient with Overactive Bladder Syndrome Detected by Culture-independent High Throughput Sequencing: A Case Report. Open Microbiol J. 2014 Dec 31;8:148-53. doi: 10.2174/1874285801408010148. eCollection 2014. 

21. Smelov V, Arroyo Mühr LS, Bzhalava D, Brown LJ, Komyakov B, Dillner J. Metagenomic sequencing of expressed prostate secretions. J Med Virol. 2014 Dec;86(12):2042-8. doi: 10.1002/jmv.23900. Epub 2014 Feb 14.

22. Towner JS, Sealy TK, Khristova ML, Albariño CG, Conlan S, Reeder SA, Quan PL, Lipkin WI, Downing R, Tappero JW, Okware S, Lutwama J, Bakamutumaho B, Kayiwa J, Comer JA, Rollin PE, Ksiazek TG, Nichol ST. Newly discovered ebola virus associated with hemorrhagic fever outbreak in Uganda. PLoS Pathog. 2008 Nov;4(11):e1000212. doi: 10.1371/journal.ppat.1000212.

23. Wagenlehner FME, Naber KG. Asymptomatic Bacteriuria - Shift of Paradigm. Editorial. Clinical Infectious Diseases 55(6):778-80 (2012)

24. Wolfe AJ, Toh E, Shibata N, Rong R, Kenton K, Fitzgerald M, Mueller ER, Schreckenberger P, Dong Q, Nelson DE, Brubaker L. Evidence of uncultivated bacteria in the adult female bladder. J Clin Microbiol. 2012 Apr;50(4):1376-83. doi: 10.1128/JCM.05852-11. Epub 2012 Jan 25.

25. Wolfe AJ, Brubaker L. “Sterile Urine” and the Presence of Bacteria. Eur Urol. 2015 Mar 12. pii: S0302-2838(15)00206-7. doi: 10.1016/j.eururo.2015.02.041. [Epub ahead of print]

26. Wullt, B., et al., Immunity, genetics and susceptibility to urinary tract infection. International Consultation on Urological Diseases (ICUD), Urogenital infections - European Association of Urology, 2010: p. 23-31.

27. Wullt B, Sundén F. Asymptomatic bacteriuria with the model strain Escherichia coli 83972 protects against symptomatic urinary tract infections. In: Naber KG, Schaeffer AJ, Heyns C, Matsumoto T, Shoskes D, Bjerklund- Johansen TE, eds. Urogenital infections. Arnhem, Netherlands: European Association of Urology–International Consultation on Urological Diseases, 2010:314–8.