Since the importance of microbiota was established, numerous studies have addressed the impact of microbiota and the balance between host and resident microbiota on the human body [11, 25]. In addition to gut microbiota, urine microbiota also impacts urinary diseases, but is poorly understood. Furthermore, the noninvasive voided urine sample is easier to collect. In this study, we describe the urinary microbiota in males with and without BPH, and establish the association between abundance of species and clinical characteristics of participants, using 16S rDNA amplicon sequencing. To our knowledge, few studies have discussed the detailed impact of urinary microbiota in BPH patients. It was incorrectly considered that urine is sterile, primarily on the basis of culture-dependent methods. However, microbes exist in urine of adult men and women even without clinical infection [26, 27].
In a study by Lewis et al. [9], 16S rRNA sequencing on mid-stream voided urine samples revealed that the number and diversity of microbiota change with age in healthy, asymptomatic individuals of both genders. It is assumed that these changes may be associated with the occurrence of BPH with aging, in men [28]. Although both BPH and LUTS are highly prevalent in men and increase in prevalence with age, only 25%-50% of patients with BPH suffer from significant LUTS, while only half to two-thirds of patients with LUTS showed bladder outlet obstruction (BOO) on urodynamic testing [29]. In addition, the difference in individual response suggests that the pathophysiology of BPH is heterogeneous and poorly understood. The causes of BPH/LUTS are multifactorial, and inflammation is one of the important factors that was proposed on observation of chronic inflammation coexisting with histologic changes in resected prostate specimen in BPH [30]. Prostate inflammation may be triggered by bacterial infection, followed by the secretion of cytokines, chemokines, and growth factors including CD4, CD8, CD45, CD68, C-reactive protein, tumor necrosis factor, interleukin-6, and others [31-33]. Due to the above hypothesis, a link between BPH and microbiota was considered and a target for its diagnosis and treatment was set.
LUTS is composed of voiding and storage symptoms, which were mainly evaluated by IPSS. Clinically, we can stratify mild to severe symptoms with a symptom score. Bajic et al. [34] observed a distinct presence of microbiota in severe symptomatic patients who needed surgical therapy, and in minimally symptomatic controls. Compared to the study by Bajic et al., our results detected statistically significant differences in the relative abundance of specific microbes in the urine samples of BPH patients compared to the samples of the control group. Further evaluation of the urine microbiota elucidates association of some microbes with degrees of LUTS as well as with the voiding and storage symptoms. Significant differences in microbes were also present in this subset of symptoms. Nevertheless, it is hypothesized that dysbiosis of the urine microbiota may play an important role in the progression of BPH and LUTS. Within pathogens that over-represented in urine samples of BPH patients, Haemophilus, Staphylococcus, Dolosigranulum, Listeria, Phascolarctobacterium, Enhydrobacter, Bacillus, [Ruminococcus]torques, Faecalibacterium, and Finegoldia are associated with more severe LUTS.
Haemophilus spp. is reported as a rare urinary tract pathogen but needs to be investigated in patients with urinary tract abnormalities such as urolithiasis [35]. A previous study demonstrated that Haemophilus influenzae isolated from urine specimens may induce prostatitis and epididymitis, and may have been ignored and underestimated as a uropathogen [36]. Staphylococcus saprophyticus, a Gram-positive uropathogen, causes urinary tract infections, especially in elderly and vulnerable patients, and may be related to prostatitis mentioned in the previous studies [37-38]. The presence of Listeriamonocytogenes in urine can be considered as the cause of urinary tract infection, mostly in prostatitis [39]. Extracellular vesicles induced by Enhydrobacter aerosaccus may interfere with communication between the host and the bacteria. Samra M et al. analyzed that bacteria-derived extracellular vesicles may cause allergic airway diseases in children [40]. Faecalibacterium was more abundant in patients with diabetes mellitus (DM) and hyperlipidemia, compared to patients with only DM, as seen in the study by Fengping Liu et al. They also demonstrated that Faecalibacterium is associated with incidence of UUI. In addition, Bacillus is also common in patients with DM, hypertension and hyperlipidemia [41]. After culture-independent 16S rDNA gene sequencing, Finegoldia was identified in urine samples from patients with severe preoperative urinary symptoms, undergoing surgery for pelvic organ prolapse and stress incontinence [42]. The presence of Lactobacillus was positively associated with HbA1c and glucose levels in our study as well as in the study by Jiawei Chen et al. Their results suggested that changes in the microbial community, specifically in Lactobacillus, may be an impact factor in LUTS [43]. According to previous studies, special microbes can be identified in culture-negative urine. Through DNA sequencing, we have opportunities to investigate the role of urine microbiota in the development of diseases.
In order to decrease the possibility of urine contamination, we collected mid-stream voided urine samples with sterile containers. Furthermore, voided urine is less easily contaminated with microbes in males than in females. Although collecting urine by sterile catheterization was an alternative method for our study, it was considered invasive, uncomfortable and not clinically feasible. Different urine collection methods show different urine microbiota. This may be due to differences between the microbiome of the urethra and the bladder [44]. According to this hypothesis, results of the urine microbiota in our study may represent a mixture of bacteria from the urethra and the bladder. Because antibiotic treatment can impact the composition of urine microbiota and the influence may persist for at least two months [45], we excluded participants who had taken antibiotics within the last two months.
Our study has some limitations. First, it is a cross-sectional study in which the cause-effect relationship is difficult to define between clinical characteristics and bioinformatics indicators. Therefore, further animal experimentation is needed to clarify the role of specific microbes in the development of BPH and LUTS. Second, we only analyzed urine microbiota from voided urine; further work would include catheterized urine collection to identify the different microbes residing in the bladder and urethra. Finally, we did not analyze the relationship between the medications for BPH and urine microbiota. Thus, prospective studies need to clarify if different strategies will disturb the environment of the microbiota. The strength of our study is that we demonstrated the statistically significant differences in urine microbiota in samples of patients with BPH/LUTS as well as in the subset of symptoms.