In this study we describe the healthy urine microbiome in a heterogeneous population of men and women with and without NB, using both 16S rDNA sequencing and metaproteomic analysis. Based on other reports
[50–53] and including our data, this is further confirmation that the commonly held clinical belief that healthy urine should be sterile is false. Specifically, our data indicate that (1) when collected by the routine method of “clean, midstream catch”, healthy urine is not aseptic; (2) the healthy and NB urine microbiomes differs by gender; (3) the asymptomatic bacteriuria urine microbiome of people with NB differs from that of healthy controls; and (4) the asymptomatic bacteriuria urine microbiome of people with NB differs depending on duration of exposure to and type of urinary catheter.
This is the first report comparing the healthy urine microbiome in male and female subjects. Historically, and based on cultivation results, clinicians have assumed urine to be ‘sterile’. However, Wolfe et al. recently used 16S rDNA sequencing to identify uncultivated bacteria in the urine of healthy women
. Our data confirm these results in women, and further show that uncultivated bacteria are also present in the urine of healthy men. Moreover, we demonstrate that the healthy urine microbiome of males and females differs, with men having significantly more relative abundance of Corynebacterium, a common component of the superficial skin flora, and women having significantly greater relative abundance of Lactobacillus. Critical to this discussion is an understanding that because all samples from healthy subjects and those of subjects with NB who voided were collected by midstream clean catch, it is not possible to distinguish whether the microbes identified originated in the bladder, urethra, or both. Therefore, the possibility exists that the identified urine microbiomes are populated by species that exist in the bladder, urethra, or both.
Our finding of predominance of Corynebacterium in healthy males suggests that this microbe may contribute to the healthy urine microbiome. Not only was Corynebacterium identified to a significantly greater degree in healthy males compared to those with NB, males with NB who voided and were sampled by clean catch had the lowest abundance of this species. Dong et al. compared clean catch urine and distal urethral swabs in healthy volunteers and similarly found a predominance of Corynebacterium in both types of sampling but in significantly greater amounts in the clean catch urine samples
. Taken together, the data suggest that Corynebacterium may reside in the proximal urethra and/or bladder in addition to the distal urethra, and may play a role in the healthy urine microbiome.
While it is well established that in most healthy women of childbearing age the vaginal tract is colonized by Lactobacillus species
[54–56], this has not been investigated in women with NB. We too found a clear preponderance of Lactobacillus in healthy control females; however, in addition to the greatest relative abundance of Lactobacillus in healthy control females, there was a progressive reduction in abundance of Lactobacillus in females with NB who void (clean catch sample) or who use intermittent catheterization, and females with NB who use indwelling (Foley) catheters (Figure
3 inset). This may suggest that either increasing exposure to a urinary catheter and/or increasing severity of NB can influence the ability of Lactobacillus to colonize the urinary tract. Alternatively, Lactobacillus may merely be a contaminant of the external urethra that arises from proximity to the vaginal flora. However, this appears less likely since Wolfe et al. showed the presence of Lactobacillus in urine collected by transurethral catheters and suprapubic aspirate, which samples the bladder directly
. Because lactic acid-producing Lactobacillus species contribute to controlling the growth of more virulent bacteria that cannot survive in a more acidic environment, the presence of Lactobacillus within the urethra and/or bladder may be protective. This has been shown to be the case in infants
 and may also be true for males as Lactobacillus has been shown to be present in clean catch urine samples of healthy males by Dong et al.
 and in our study. Our findings suggest that Lactobacillus may be a commensal organism present during states of health, more in females than in males, and that the microbiome of at-risk populations may be characterized by a distinct lack of Lactobacillus, which perhaps creates a better environment for the growth of pathogenic microorganisms. Together, these findings suggest that the clinical objective of ‘sterile’, microbe-free urine may not be optimal for the patient.
This is the first report of 16S rDNA sequencing of urine in people with NB, providing much more detail about the ABU state than has previously been available through cultivation-based evidence. Standard cultivation diagnostics show populations vulnerable to bacteriuria include nursing home residents utilizing long-term indwelling catheterization
, institutionalized Veterans utilizing intermittent catheterization
, and patients with SCI who utilize urinary catheters
[60–62]. Our analysis not only confirms the cultivation-based evidence, but also shows that a significantly different microbiome was present in the NB group, and that 16S rDNA sequencing specifically identifies microorganisms, such as Enterobacteriales, as potential major contributors to a pathogenic microbiome. These results supplement those from Bank et al., where urine specimens of 142 consecutive patients with varied genitourinary pathology (kidney stones, indwelling catheters, or suspected UTI) were analyzed with standard cultivation and screened for Actinobaculum schaalii using PCR
. Those authors found that the most heavily colonized patients were those who were older and who utilized indwelling urinary catheters, while the younger patients who typically use intermittent catheterization (and may have utilized urinary catheters for a shorter amount of time) were colonized with bacteria to a lesser degree. The Enterobacteriaceae was most commonly isolated in the catheterized specimens in that study. Significant variance in medical comorbidities, underlying genitourinary pathology, and method of urine collection limit any further comparisons to that study. Moreover, because our NB population was reportedly asymptomatic (i.e. infection free), our findings demonstrate that in the catheterized population, the microbiome is intrinsically different than in controls, even in the absence of illness. This distinction is important, since in the clinical setting ABU is often inappropriately treated with antibiotics, which may further disrupt the NB microbiome.
The present data also indicate that the urine microbiome of healthy subjects with NB became increasingly abundant with Enterobacteriales with increasing duration of NB, whereas Lactobacillus decreased over time, both in men and women. While the urinary microbiome of men and women with NB remained similar to that of healthy controls during the first several months after NB diagnosis, by one year the urine microbiome was nearly devoid of Lactobacillus and dominated by Enterococcus. This further suggests a change in the microbiome with duration of NB that may place patients at increased risk of UTI.
Fundamental to these discoveries is the diverse sample population and our novel analytic approach of utilizing a combination of 16S rDNA sequencing in all subjects and metaproteomics in a subsample. Clinical gold standard diagnostic testing when a patient presents with signs and/or symptoms of UTI includes (1) urinalysis to confirm urinary tract inflammation and (2) urine culture to identify, quantify and predict antimicrobial resistance to a given pathogen(s). We believe that 16S rDNA sequencing has the potential for translation to the clinic, offering significant clinical advancement over diagnostic urine culture because it provides a greater depth of understanding and sensitivity pertaining to the composition of commensal and potentially pathogenic microbes present in urine. Furthermore, prospective assessments during varying periods of health and disease may allow personalization of care that has not been possible to date with our current diagnostic methods. Urinary metaproteomic profiles in parallel may contribute to the identification of a host inflammatory response utilizing urinary biomarkers with greater sensitivity and specificity for UTI than traditional measures of urinary leukocyte esterase production or white blood cell count detected by urinalysis. We hypothesize that protein profiles with distinct abundance ratios of immunomodulatory versus pro-inflammatory and microbiocidal molecules, are indicative of either UTI or reflect asymptomatic colonization. For instance, the presence of lactotransferrin in urine has been used to support the notion of a “battle for iron” being waged between the host and the pathogen, involving E. coli, particularly in the case of UTI by E. coli given the abundance of its triad of iron acquisition receptors
. While metaproteomics holds promise as a diagnostic method to discriminate between symptomatic UTI and ABU, more in-depth fractionation of samples is needed to evaluate whether this method can reach the sensitivity of 16S rDNA profiling methods. Larger patient cohorts, including those diagnosed with UTI symptoms, are required to assess if such ‘omics methods’ more accurately differentiate UTI from ABU than current diagnostic standards. In addition, they suggest new considerations that may impact preventive and treatment options for people at-risk for UTI.
Several limitations of this study are to be considered when interpreting the results. The major limiting factor is that some subjects (healthy controls and NB subjects who voided spontaneously) had urine collection via midstream clean catch sampling while the subjects with NB who managed their bladder with intermittent catheterization or indwelling Foley (urethral) catheters were sampled directly from the catheter. Therefore, microbes identified in the former groups could be representatives from the bladder, urethra, skin, or a combination of these microbiomes, whereas urine from subjects in the latter groups represents bladder microbiota. This distinction raises other questions, such as to what degree do differing microbiomes in the urethra and bladder influence each other, and does a changing urethral microbiome represent a potential antecedent UTI state. Further, given that clinical urine sampling is unlikely to become more invasive (via direct sampling from the bladder), how do clinicians interpret the presence of bacteria in clean catch midstream urine samples that could potentially originate from multiple anatomic locations? Lastly, future studies will employ a larger sample size as our results indicate that the urine microbiome differs by a number of clinical factors requiring multiple stratification points.