Relations to urinary tract infection, hormonal status, gender and age

It has become clear that the urine bladder is not sterile. It contains a microbiome. The bacterial species in this bladder microbiome often do not grow under routine urine culture conditions but can be identified under culture conditions that better resemble the bladder environment and by bacterial DNA analysis.

Identification of bacterial presence using DNA enhancing techniques holds the risk of DNA false positive identifications due to multiplication of minor contaminations that occurred during sample collection. However, analysis of matched urine samples and samples taken from potential sources for contamination strongly supports the conclusion that the bacteria identified with such techniques in a urine sample do represent a true urine microbiome.

The existence of a urine microbiome has several implications:

• It raises questions on the stability of that microbiome.
• It raises questions on how this microbiome survives over time.
• It changes the way we should look at the development of urinary tract infection.
• It opens up the potential use of urine microbiome analysis as a diagnostic tool.

Stability of the urine microbiome size

The doubling time of E coli in urine under laboratory conditions averages one hour. If we accept that a microbiome exists permanently in the bladder there must be some mechanism to balance such growth of bacterial numbers in the microbiome given the fact that bacteriuria is not the normal condition for healthy people. A simple balancing mechanism could be the normal voiding cycle in which most of the urine is excreted regularly. When a person has a residual urine volume of 10 ml, the average for normal men, containing 100 E coli/ml (a negative culture) and does not void for six hours the total E coli population size may reach 64000, after 10 hours this would be half a million. When urine volume in these periods increases to 0.5 liter this translates into 120 (stabilization) respectively 1000 E coli/ml (a ten-fold increase).

After new rounds of delayed voiding and ten-fold increases the number of E coli reaches values connected to UTI symptoms. This simple model of microbiome dynamics shows that the normal habit of emptying the bladder almost completely four to six times per day may already stabilize the urine microbiome at a level that does not cause problems. Upon delayed voiding and/ or residue after voiding, the balance moves towards growth of bacterial numbers and potential problems. This mechanism clarifies why having residual urine in the bladder after voiding increases the risk for development of UTI5 and why vesicoureteral reflux would increase the risk for renal infection.

Both clinically proven risk factors make less sense when bladder urine would be sterile. It also explains the phenomenon of low bacterial count UTI in children and explains why for adult men with normal urinary tract function an increase of residual urine volume within the normal range correlates with positive urine cultures. This mechanism stresses the importance of regular and complete voiding for prevention of UTI. In fact, frequency linked to UTI is a natural defense mechanism in this light.

Stability of the urine microbiome composition

Analysis of urine microbiome composition in males and females of different age groups shows that microbiome composition is gender specific. In women from the fertile age range the urine microbiome is dominated by Lactobacillus species. This Lactobacillus presence is low after birth, increases during puberty and decreases after menopause. In boys Lactobacillus is largely absent except for newborns. In women coming for IVF treatment microbiome composition has been determined over a 30-week interval. In women who did not become pregnant microbiome composition was stable. In women who did become pregnant the composition drastically changed.

Mechanisms for long-term survival of a urine microbiome

Next to simply having luck and staying behind in residual urine, bacteria can prolong their stay by adhesion to the bladder wall through biofilm formation, by invading urothelial cells or by being located inside crystalline material that is not excreted. Further research into how to disturb these survival techniques may lead to new therapies for prevention of UTI.

Urine microbiome as a source for urinary tract infection

When accepting that the bladder contains a microbiome, invasion of the urinary tract by a pathogen from an outside source is not the only mechanism by which urinary tract infection (UTI) may start. When the bladder microbiome contains potential causes of UTI, UTI may start when the microbiome as a whole is given the chance to multiply as outlined above. In a study on 96 children with and without a history of UTI and with or without problems of urinary tract function we found that for E coli, the most common cause of UTI, we could very accurately predict the risk of forming a new E coli UTI using the percentage of E coli in the microbiome.

The reporter operated curve (ROC) for the prediction of a new E coli UTI from the percentage of E coli in the microbiome had an area under the curve of 0·86, asymptotic significance =0·000, 95% confidence interval 0·771-0·950. It should be stressed that this prediction is based on the relative presence of E coli expressed as percentage of the total microbiome not on the absolute number (the unit for urine culture). In the light of the doubling mechanism described above, absolute numbers may be more variable than the relative presence.

Microbiome analysis as a diagnostic tool

Using microbiome analysis for prediction of UTI risk would be one application. In the study on IVF patients we found another surprising application. The urine microbiome composition determined before treatment very accurately predicts the chance that a woman will become pregnant from IVF. Our proposed explanation for this marker function is that the marker bacteria that we found prefer a urothelial environment that resembles the endometrial environment that the fertilized egg will encounter after transfer during IVF. In view of the rapid doubling times of bacteria this suggests that microbiomes might be used as sensitive, fast reacting sensors for the internal human environment.

Author:
D.J. Kok, Assistant Professor Erasmus MC Dept. of Urology, Rotterdam (NL)
Co-Author: D.M. Maghdid, Erasmus MC, Rotterdam (NL)

Declaration of interests

DJ Kok and DM Maghdid are co-owners of a patent based on microbiome analysis: New method and kit for prediction success of in vitro fertilization, PCT/ NL2011/050563.