This study presents a novel experimental design to distinguish potential pathogens from commensal bacteria in healthy human bladder. It is hypothesized that the bladder immune system is constantly at work to maintain immune homeostasis and suppress the activity of pathogens from causing an overt infection. The aim is to identify the presence of these potential pathogens by isolating bacteria associated with white blood cells in urine samples of healthy volunteers.

Six healthy female volunteers (Samples A to F) were recruited and informed consent was obtained before participating in the study. They answered a 39-point questionnaire on lower urinary tract symptoms (LUTS) grouped into storage, stress incontinence, voiding and pain. The questions were close-ended with a yes or no response. Urine samples were then collected and separated into sediment and supernatant by centrifugation. These were designated as “neat urine”. The cells in the sediment were labelled with CD45 microbeads (Miltenyi Biotec, Germany) and sorted with magnetic-activated cell sorting (autoMACS® Pro Separator, Miltenyi Biotec, Germany) into CD45-positive “white blood cell (WBC)” and CD45-negative “non-WBC” fractions. Individual aliquots of the supernatant and sediment of neat urine, WBC and non-WBC fractions were cultured on chromogenic agars (chromID® CPS® Elite). After 24-hour incubation at 37oC, colony growth was observed and recorded. The cells in the sediments were also stained with DAPI for cell nuclei and bacteria, wheat germ agglutinin (WGA) for cell membrane, anti-lipoteichoic acid antibody for Gram-positive bacteria and anti-lipid A antibody for Gram-negative bacteria. The fluorescently-labelled cells were examined using a TCS SP8 deconvolution laser scanning confocal microscope (Leica Microsystems, Germany).

The volunteers (mean age 25 ± SD 5 years) were all healthy with only two respondents scoring one point each on the questionnaire. Culture of supernatant fractions provided information on planktonic bacteria present in the urine while sediment fractions showed bacteria associated with white blood cells (WBC) in the WBC fraction and epithelial cells in the non-WBC fraction. The bacterial colonies were presumptively identified based on the manufacturer’s documentation. Possible organisms were Enterococcus sp. (turquoise), Proteus sp. (brown), Escherichia coli (red to burgundy) and Klebsiella, Enterobacter, Serratia, Citrobacter (KESC group) (blue to blue-green). However, the diversity of colours that were observed in this study could not be grouped entirely into these four classes and will, therefore, be described solely based on colour when mentioned. Samples A and B did not show any colony growth from all cultured fractions. In Sample C (Figure 1), five types of colonies were observed in the sediment of neat urine and WBC fraction. They were E. coli (burgundy), Enterococcus sp. (turquoise), KESC group (blue), Proteus sp. (brown) and white colonies. The turquoise, blue and brown colonies were similarly present in the neat urine supernatant while the white colonies were present in the non-WBC sediment. Supernatant of non-WBC and WBC fractions did not show any growth. In Sample D, Proteus sp. (brown) and yellow colonies were observed in the sediment of neat urine. The same yellow colony was also present in the sediment of WBC fraction, with additional green-white and white colonies. Non-WBC fraction as well as the supernatant of neat urine and WBC fraction did not show any growth. In Sample E, three types of colonies were present in all fractions. They were Enterococcus sp. (turquoise), white and blue-coloured lawn. In Sample F, six types of colonies were observed in the sediment of neat urine – E. coli (burgundy), KESC group (blue, dark blue and green), yellow and white. All were also present in the supernatant of neat urine and non-WBC fraction except for the white. In the non-WBC fraction, all were present except for E. coli in the sediment. In the WBC fraction, all were present except for the green KESC group in the sediment while the supernatant showed the blue KESC group, yellow and white colonies. White blood cells and both Gram-positive and Gram-negative bacteria could be observed in the sediment of WBC fractions on confocal microscopy (Figure 2).

It has been shown, in recent years, that the urine is not sterile [1] and bacteria are ubiquitously present in health and disease. The emerging field of the urobiome has complicated the diagnosis of urinary tract infection, particularly in respect to standard culture methods. White blood cells that are shed into the urine provide a useful means to study bacteria implicated in immune system activation, subsequently aiding in distinguishing pathogens from commensals. Even though this study examined healthy pre-menopausal females, differential bacterial growth could be observed in three out of six urine samples, suggesting transient subclinical infections which are being successfully subdued by the host immune response. Asymptomatic bacteriuria occurs in healthy individuals and is thought to be a beneficial colonization of commensals which can persist in the bladder by modifying host gene expression [2]. However, the observation in this study shed new light on this clinical presentation where the lack of symptoms could be due to an active engagement of the immune system in preventing the infection from manifesting.

This study demonstrates a workflow for isolating bacteria that are being targeted by the immune system. The presence of bacteria that is unique to the WBC sediment fraction indirectly distinguishes pathogens from commensals in the bladder. The same approach can be applied to urine samples from patients with urinary tract infection to single out aetiological pathogens and potentially characterize deficiencies in their immune response that lead to symptomatic infections. More samples from healthy and diseased individuals are planned for future work to compare between the groups and accurately identify pathogenic microorganisms of the urinary tract.

Hilt, E. E., McKinley, K., Pearce, M. M., Rosenfeld, A. B., Zilliox, M. J., Mueller, E. R., Brubaker, L., Gai, X., Wolfe, A. J., & Schreckenberger, P. C. (2014). Urine is not sterile: use of enhanced urine culture techniques to detect resident bacterial flora in the adult female bladder. Journal of Clinical Microbiology, 52(3), 871–876. https://doi.org/10.1128/JCM.02876-13
Lutay, N., Ambite, I., Grönberg Hernandez, J., Rydström, G., Ragnarsdóttir, B., Puthia, M., Nadeem, A., Zhang, J., Storm, P., Dobrindt, U., Wullt, B., & Svanborg, C. (2013). Bacterial control of host gene expression through RNA polymerase II. The Journal of Clinical Investigation, 123(6), 2366–2379. https://doi.org/10.1172/JCI66451

Continence 2S2 (2022) 100350
DOI: 10.1016/j.cont.2022.100350