The Urinary Microbiome: A Pediatric Urological Perspective

1) Benign urological diseases and UM

Several studies in benign urological diseases have investigated the UM in relation to UTI, urinary stones, UUI, OAB, neurogenic bladder (NB), lower urinary tract symptoms, STI, IC/BPS, and CP/CPPS in adults. However, literature reviews have revealed that there are very few studies on UM in pediatric urological benign diseases.

(1) UTI: Since the establishment of the standard urine culture in the 1950s until approximately 10 years ago, the UT had been considered sterile under normal conditions [7-11]. This perspective is now altered with the discovery of the genitourinary microbiome and the demonstration that most urinary bacteria identified by 16S sequencing can be cultured under expanded conditions [10,12]. As symptomatic UTI is the outcome of an altered UM makeup, some researchers have suggested that the terms “UTI” and “asymptomatic bacteriuria” be replaced by the term “dysbiosis” [14].

The relationship between UM and UTI is obvious and relatively well-studied. Several studies suggest that the microbial composition and diversity of the UM are related to the development of a UTI, with an association between decreased microbial diversity and the incidence of UTI [10,15-17]. Studies analyzing the UM reported that the UT is inhabited by a diverse bacterial community, with the predominant genera being Lactobacillus, Prevotella, and Gardnerella [4]. However, the role of these commensal bacteria and their importance in the emergence or prevention of UTIs remains to be fully elucidated.

A study of patients with indwelling urinary catheters suggested that microbial diversity plays a protective role in the development of UTIs, which are caused by dysbiosis of the commensals [16]. However, the benefits of the HU2117 E. coli strain have not been seen in patients with chronic indwelling catheters despite a similar rate of colonization with probiotics [16,18].

Yoo et al. [19] conducted a human bladder microbiome study to investigate the effect of Gardnerella on recurrent UTI (rUTI). The Gardnerella-dominant or Lactobacillus-dominant groups expressed rUTI with symptoms in the presence of risk factors such as the degree of Gardnerella proliferation or causative agents of bacterial vaginosis. Depending on other risk factors, the presence of Gardnerella in the urine is considered to be related to rUTI.

The majority of UM studies have focused on adult subjects, and pediatric reports are scarce. Mapping the UM in the pediatric population has just begun [12]. Kinneman et al. [20] assessed the UM in 85 children younger than 48 months, with and without UTI. They demonstrated that UM was identified in every child, and altered microbiome diversity and composition were observed in subjects with a standard culture-positive UTI. They also observed that antibiotic administration was effective in the UM only for a short time. Kassiri et al. [21] examined the UM in 20 prepubertal boys (aged 3 months-8 years; median age 15 months) with and without prior antibiotic exposure. The majority of patients presented with Staphylococcus and Varibaculum species and, to a lesser extent, Peptoniphilus and Actinobaculum. Several of the detected genera have been previously identified in the urine of adult men. However, urinary microbial communities profiled in children differed from those described in adults. This study also presented that the composition of the UM in children may begin to develop early in life and evolve over time, achieving stability in adulthood.

(2) UUI and OAB: OAB is a poorly understood disorder, but is thought to be multifactorial and is believed to originate from abnormal neuromuscular signaling and functioning of the detrusor muscle [10,22]. In the pediatric population, OAB not only burdens the child’s development but also negatively impacts the family [12]. OAB is a condition where frequent urination is required, which often leads to UUI. The mechanisms of OAB are not fully elucidated, but there are potential links to the involvement of the microbiome. Since anticholinergics or beta-3 agonists that inhibit detrusor contraction and promote bladder relaxation are ineffective in many patients, this indicates other etiologies besides neuromuscular dysfunction, including UM [22,23].

Several studies of the UM in adults have been conducted in patients with UUI and OAB, and have reported significant differences in bacterial urine compositions between adult female UUI and healthy controls. These differences are reported to affect the symptom severity and treatment responses [15,23-25]. Compared to controls, patients with UUI exhibit higher Gardnerella and fewer Lactobacillus sequence profiles. Additionally, culture tests using expanded quantitative urine culture (EQUC) showed nine genera, including Actinobaculum, Actinomyces, Aerococcus, Arthro-bacter, Corynebacterium, Gardnerella, Oligella, Staphylococcus, and Streptococcus, were commonly found in UUI patients [10,15]. The more frequently cultured organisms were Lactobacillus gasseri from UUI urine and Lactobacillus crispatus from control urine [10,15,23,24]. In another study, Pearce et al. [15] compared the baseline UM of women with and without UUI. They identified statistical associations between UUI and several bacterial species, including Actinomyces neuii, Actinotignum schaalii, Aerococcus urinae, Cory-nebacteria coylae, Corynebacteria riegelii, Oligella ure-thralis, and Streptococcus anginosus. Several of these species are considered uropathogens; however, most are not detected in standard urine cultures. This study also presented that while L. crispatus was associated with the non-symp-tomatic controls, L. gasseri was associated with UUI. Recently, Karstens et al. [23] found that the relative abundance of 14 bacteria significantly differed between healthy and UUI patients. They further suggested that persistent low-grade infections by bacteria that are not commonly detected by routine cultures could potentially be responsible for the irritative symptoms of UUI. This could justify the potential for the therapeutic use of Lactobacillus probiotics in UUI and OAB.

Other studies have investigated the clinical associations between UM and different UUI treatments. In 40% of UUI women treated with anticholinergics, there was minimal or no response [26]. The diversity of UM has been associated with the response to solifenacin for the treatment of UUI. The presence and number of cultivatable bacteria are directly correlated with the presence and severity of UUI [24,27]. Thomas-White et al. [24] found that a lower diversity of cultivatable bacteria is associated with a positive response to a low dose of solifenacin.

In summary, the studies published to date have demonstrated a clear role of the UM in adults with UUI/OAB, and in the response to UUI/OAB treatment. However, there is uncertainty about the possible associations between UM and OAB symptoms in the pediatric population due to a lack of reports concerning UM in pediatric UUI/OAB.

(3) Urinary stone: The role of the microbiome in urolithiasis is relatively well-established. Recently, numerous culture-independent UM studies have been published in an attempt to elucidate whether the UM contributes to urinary stone formation [28]. More recently, several studies have suggested that UM may play an important role in urinary stone formation [10]. Stern et al. [29] presented that compared to 6 controls, 23 patients with kidney stones had higher levels of Bacteroides and less Prevotella. Tang et al. [30] recently analyzed the characteristics of gut microbiomes in 13 patients with multiple kidney stones. They found an abundance of proinflammatory bacteria and fewer anti-inflammatory bacteria compared to 13 matched healthy controls.

Up to 32% of calcium oxalate stones demonstrate bacterial growth when cultured. However, the cultured bacteria may be unique to the stone. Calcium oxalate stones have been shown to harbor a microbiome that is independent of the UM in children with urolithiasis [10]. Barr-Beare et al. [31] demonstrated significant bacterial diversity in children with calcium oxalate stones, including Enterobacteriaceae, Gardnerella, and Lactobacillus. They postulated that calcium oxalate stones harbor a microbiome, and the bacteria within the stone’s microbiome contribute to the stone formation by altering urine supersaturation.

Several studies revealed an inverse relationship between intestinal colonization with Oxalobacter formigenes and the development of calcium oxalate stones [32-34]. Kaufman et al. [34] reported that colonization with O. formigenes was associated with a 70% reduction in urolithiasis risk in adult patients with recurrent calcium oxalate stones. Barr-Beare et al. [31] also demonstrated that kidney stones are associated with the Enterobacteriaceae species, such as E. coli.

However, there are still uncertainties about differences obtained in the results between studies due to experimental factors (such as sample collection, storage, DNA extraction, sequencing, or data analysis) or biological factors (such as geography, ethnicity, stone phenotype, or some other regional factors) [28].

Taken together, several studies proposed that bacteria present in calcium oxalate deposits contribute to calcium oxalate renal disease. Furthermore, the UM may play an important role in urinary stone formation in both children and adults.

(4) NB: It is well known that patients, especially children with NB, have a high risk of recurrent UTIs. UM variations were observed using 16S rRNA sequencing analysis for patients with NB and normal bladder function. Urine in the healthy control bladders was significantly predominant in Lactobacillus and Corynebacterium genera, whereas other bacterial genera, including Klebsiella, Enterococcus, and Escherichia, were more frequently found in the NB urine [35].

Groah et al. [36] demonstrated that the UM in patients with NB comprised uropathogenic bacteria, including E. coli, Enterococcus faecalis, Pseudomonas aeruginosa, and Klebsiella pneumonia. They also suggested possible causes for changes in the NB UM, which showed an altered composition of perineal bacteria due to fecal incontinence and/or the use of bowel care regimens or changes in gut microbiota that alter the ability of certain bacteria to colonize the UT. Bossa et al. [18] recently demonstrated a significant variation in the bacterial composition of the microbiome in patients with NB. They further showed a return to baseline bacterial composition after the resolution of a UTI or discontinuation of probiotics. Forster et al. [37] presented that Enterobacteriaceae are the most predominant bacteria along with Staphylococcus, Streptococcus, and Enterococcus in the UM of 34 children with NB. They also observed that the route of catheterization might affect the composition of the UM. Children who catheterize their urethra have a higher proportion of Staphylococcus, while the UM of patients who catheterize through a Mitrofanoff was composed of Enterobacteriaceae.

2) Urological cancers and UM

Dysbiosis of the microbiome contributes to carcinogenesis through several mechanisms, including DNA damage, production of carcinogenic metabolites, and stimulation of inflammation [38]. Numerous studies have focused on the relationship between the host microbiome and cancer susceptibility in systems other than the UT, such as gastric, colorectal, or hepatobiliary cancer [9,39]. However, the role of UM in UT cancers has yet to be elucidated. According to the reports of a few studies on urological cancers in adults thus far, UM is mainly associated with prostate cancer, bladder cancer, and renal cancer.

(1) Prostate cancer: Many pathogenic microorganisms including endogenous Enterobacteriaceae, such as E. coli and Pseudomonas spp., are known to infect the prostate [6]. Inflammation in the prostate plays a vital role in the generation of prostate cancer, and cytokines such as interleukin (IL)-6 and IL-8 have been associated with the propagation of prostate cancer [40]. Compared to healthy controls, the level of Bacteroides massiliensis was elevated, whereas Faecalibacterium prausnitzii and Eubacterium rectale were reduced in the gut microbiota of adult patients with prostate cancer [41]. Cavarretta et al. [42] also reported that a high abundance of Propionibacterium spp., predominantly Propionibacterium acnes, is consistent with the pro-inflammatory role of P. acnes, thus supporting reports of its association with prostate cancer. According to the Feng et al. study [43], Escherichia, Propionibacterium, Acinetobacter, and Pseudomonas were the most abundant genera in radical prostatectomy tissue of adult patients with prostate cancer. Besides, Yu et al. [44] also reported that adults with prostate cancer had a lower number of E. coli in urine samples, and no significant differences were observed in Enterococcus numbers for urine and prostatic secretion samples.

However, unlike studies in adults, to date there are no reports on the relationship between UM and any prostate disease (including benign diseases and malignant tumors such as sarcoma) in children.

(2) Bladder cancer: Some studies have demonstrated that the UM is associated with bladder cancer transformation and metastatic progression. It has further been suggested that dysbiosis of the UM produces a chronically inflammatory urothelial microenvironment which leads to bladder cancer [45]. However, it remains unknown whether the UM affects the development or progression of bladder cancer, or whether bladder cancer is responsible for the alternation of the UM [6]. Some studies indicate that the bladder microbiome may change the extracellular matrix (ECM) to promote or inhibit urothelial carcinogenesis. The ECM regulates tissue homeostasis and maintains the onset and progression of cancer, including bladder cancer [6]. Conversely, Hayatsu and Hayatsu [46] reported that the Lactobacillus casei present in the UM prevents the production of carcinogens and mutagens by intestinal bacteria and the excretion of mutagens in urine. Some studies also demonstrated that L. casei reduces superficial bladder cancer recurrence in adults [47,48]. Wu et al. [25] analyzed midstream urine from 31 adult patients with bladder cancer and 18 healthy controls. They found an abundant presence of Acinetobacter, Anaerococcus, and Sphingobacterium species in bladder cancer. Herbaspirillum, Porphyrobacter, and Bacteroides species were detected in bladder cancers at a high risk of recurrence and progression. More recently, Ahn et al. [49] demonstrated that among the six genera of Cutibacterium, Peptoniphilus, Sphingomonas, Staphylococcus, Micrococcus, and Moraxella, there was a significant increase in Micrococcus sp. In bladder cancer compared to non-cancer.

There is a well-documented association between chronic schistosomiasis and bladder squamous cell carcinoma [6,50,51]. Adebayo et al. [50] demonstrated that certain UM, such as Fusobacterium, Sphingobacterium, and Enteroc-ccus sp., distinguishes the patients with urogenital schistosomiasis from healthy individuals.

In terms of urothelial cell carcinoma (UCC), Xu et al. [52] studied the changes in the UM community of UCC patients in comparison to healthy individuals. They presented an enrichment of Streptococcus in urine in a preliminary study of the UM involving a small number of patients with UCC.

Unfortunately, unlike studies in adults, there has been no research in children to study the relationship between UM and bladder cancer or UCC.

(3) Renal cancer: Unlike UM studies in prostate cancer and bladder cancer in adults, very few studies have been conducted for renal cancer. In a recent study about the microbiome in 5 benign renal and malignant renal tissues in adults, Heidler et al. [53] are the first to demonstrate a plethora of microorganisms with significant differences between the two groups. They isolated 3 domains, 15 phyla, 16 classes, 19 orders, 27 families, 28 genera, and 30 species of microorganisms. Trachelomonas volvocinopsis, Pseudoalteromonas haloplanktis, Halomicrobium mukohataei, Aeromonas salmonicida, and Mycoplasma mycoides were abundantly found in renal cell carcinoma tissue. Microorganisms that appeared solely in renal cancer tissue were Cyanophora paradoxa, Spirosoma navajo, Phaeocystis antarctica, Euglena mutabilis, and Mycoplasma vulturii. More recently, Ahn et al. [49] performed a metagenomic analysis of urinary DNA in 12 patients with renal cancer. They demonstrated that among the six genera of Cutibacterium, Peptoniphilus, Sphingomonas, Staphylococcus, Micrococcus, and Moraxella, the Micrococcus sp., Cutibacterium acnes, Cutibacterium granulosum, Peptoniphilus lacydonensis, and Staphylococcus epidermidis were significantly increased in renal cancer compared to non-cancer.

Unfortunately, there are no reports on the association between UM and renal cancer, especially Wilms’ tumor, in children.

(4) Other genitourinary cancers: There have been no studies associating UM and other genitourinary cancers, such as testis cancer, penile cancer, urethral cancer, etc., in both adults and children. Therefore, there needs to be an escalated interest in the UM study of these genitourinary cancers.

3) Role of probiotics and diet in pediatric urological diseases

There have been recent attempts for the therapeutic application of probiotic microorganisms as a treatment of disease in clinical practice. Probiotics have also been applied to modify the intestinal microbiome. Various clinical trials, including fecal microbiota transplantation, have been performed to study the role of certain beneficial strains in UTI, bladder cancer, and urinary stone formation [9].

To date, antibacterial therapy is the primary basic management for UTIs in children. However, it is inevitable that the UM undergoes alterations during UTI and antibiotic therapy. Long-term use of broad-spectrum antibiotics negatively affects the beneficial bacterial flora in the host, with the consequential selective overgrowth of pathogenic bacteria, eventually resulting in bacterial resistance [54]. Therefore, probiotics such as Lactobacillus, cranberries, and D-Mannos have been used as an alternative or adjuvant therapy for the prevention and treatment of UTIs in both children and adults [9].

Recently, the relationship between dysbiosis and UTI suggests that altering the UM through the use of probiotics may impart therapeutic effects. Probiotics using commensal bacteria could serve as either alternatives or adjuvant options to antibiotics for preventing antibiotic resistance [10]. Trials of bacterial interference with probiotics of the HU2117 E. coli strain have shown protection from symptomatic UTI in NB [10].

According to recent cancer studies in adults, probiotics such as the oral L. casei strain Shirota could be effective for the prevention and treatment of non-invasive bladder tumors [47,55]. Ohashi et al. [56] reported that habitual intake of lactic acid bacteria reduces the risk of bladder cancer in adult patients. However, to date, there have been no reports that probiotics are efficacious for the preventing and treating pediatric malignant tumors.

Siener et al. [32] demonstrated that O. formigenes reduces urinary oxalate by decreasing intestinal absorption. Given the relationship between colonization with O. formigenes and stone formation, researchers have explored the efficacy of probiotics in reducing stone formation [10]. Some studies described O. formigenes as a probiotic with the potential to treat hyperoxaluria. However, there is conflicting data on the effect of Oxalobacter probiotics on the prevention of urinary oxalate stones [10].

Dietary factors such as cranberry, D-Mannos, and fermented milk products are also known to reduce the risk or incidence of recurrent UTIs by altering the properties of the genitourinary bacterial flora [57-60]. Therefore, dietary habits that may change the UM may be important factors associated with urological disorders, especially UTIs in children.