Although new diagnostic approaches have facilitated a qualitative and quantitative identification of individual bacteria species with very high accuracy, there are some significant concerns due to the limitations of the methodology, the low biomass of the urinary microbiota, and its immediacy to other bacterial niches with higher microbial biomass ^[12][14][12,14]. The techniques that have been used to obtain urine samples to investigate the urinary microbiota include the midstream clean-catch urine, transurethral catheterization, or suprapubic aspiration ^[14][15][14,15]. The choice of a specific method of a urine sample collection may affect the obtained results [14] with difficulties in distinguishing bacteria from the bladder from microbial contamination from the skin, vulvovaginal and perineal flora ^[12][14][15][12,14,15]. Additionally, urethral or bladder tissue samples, obtained by urethral swabs, bladder biopsy, or scraping, might be used in urobiome research [16]. Pohl et al. showed that the urobiome of healthy men and women differs according to sampling method (voiding versus catheterized samples). In addition, their results indicate that the microbiome of the urethra and bladder are different. In the urethral samples, the most abundant genera were Veillonella, Staphylococcus, and Neisseria, while in bladder samples Lactobacillus, Streptococcus, and Gardnerella [17]. According to Wolfe et al., suprapubic aspiration sampling minimizes contamination from non-urinary sites and provides the best view of the bladder bacteria [18]. The recommended sampling method to use in studies investigating the microbiome of the urinary tract is transurethral catheterization ^[18][19][18,19]. However, the use of invasive techniques to collect urine for urobiome research in the pediatric population raises an ethical concern. It is worth underlining that urine cultures of clean-catch urine samples have good efficacy in diagnosing urinary tract infections in children ^[20][21][20,21], and the contamination rate of urine collected via urethral catheterization is lower but not significantly different from that of clean-catch [21]. Interestingly, Bundgaard-Nielsen et al., who investigated the impact of the collection method on the urobiome composition, did not report any interpersonal daily or day-to-day deviations in microbiota composition in women, girls, or boys [22].

Another methodological challenge is the inability of the sequencing technique to differentiate DNA from living versus dead bacteria ^[11][12][11,12]. To assess the viability of identified microorganisms, modified clinical cultivation procedures were implemented, and an enhanced quantitative urine culture (EQUC) protocol, which uses multiple culture media and various incubation conditions to cultivate bacteria that do not grow under standard conditions, was developed ^[15][18][19][15,18,19]. Considering a non-invasive urine collection method for the urobiome studies, Ozer et al. using first voided or midstream urine samples showed no significant difference regarding 16s ribosomal RNA analysis [23].

Furthermore, sequencing of 16S ribosomal RNA gene cannot provide information below the species level, in particular data about the strain level or presence of virulence factors. These details should be taken into consideration, as not all strains belonging to the same species exhibit pathogenic behavior [24]. For instance, several virulence genes are characteristic of uropathogenic Escherichia coli (E. coli) and are not present in other E. coli strains. These genes encode virulence factors essential in UTI development, such as adhesins (fimH gene encoding type 1 fimbrial tip adhesin), siderophores, and toxins (hlyA gene encoding α-hemolysin, cnf1 gene encoding cytotoxic necrotizing factor) ^[25][26][25,26].

3. The Urobiome Origin and Composition

3.1. The Origin of Urinary Microbiota

The origin of urinary microbiota is still not entirely clear. A urinary microbiome was identified even in neonates [27]. The fetus is considered sterile during normal pregnancy and acquires bacteria through transmission from the mother at delivery [28]. The maternal microbiome has a strong influence on the neonatal skin, oral mucosa, and nasopharyngeal microbiome development, and might similarly influence newborns’ urobiome [29]. The composition and function of the early infant microbiota are primarily determined by birth mode, maternal microbiota, exposure to antibiotics, and feeding practices in early life [30]. It is hypothesized that the main origins of urinary microbiota are microbial communities of the gastrointestinal tract and vagina, majorly because of the anatomical proximity. Evidence suggests that the infant microbiota assembles and stabilizes 2–3 years after birth ^[28][30][28,30]. In adolescents, pubertal hormonal changes may influence the maturation of the microbiome of the urinary tract [11]. However, the effect of age on the urobiome of healthy children has been scarcely analyzed.

3.2. Urobiome Composition Depending on the Child’s Age

Kinneman et al. aimed to examine the urinary microbiome of eighty-five children younger than 48 months who presented to the Emergency Department mainly because of fever. Urine samples were collected via transurethral catheterization. The urinary microbiome was identified in every child. The most abundant families detected in urine samples were tissierellaceae, prevotellaeae, veillonellaceae, enterobacteriaceae, and comamonadaceae, while the five most abundant genera were Prevotella, Peptoniphilus, Escherichia, Veillonella, and Finegoldia. There were no significant differences according to gender, probably because urine samples were collected via catheterization, which minimized periurethral and perineal contamination. Nine children were diagnosed with urinary tract infection, in this group decreased urobiome diversity was observed [27].

Two studies investigated the urobiome in toilet trained children ^[31][32][31,32]. Fredsgaard et al. described the voided urinary microbiota in a group of thirty asymptomatic prepubertal children aged 6 to 10 years old. Bacterial DNA was extracted from all “clean-catch” midstream urine samples. The voided urinary microbiota differed significantly according to gender. The urine of girls was dominated by Prevotella (18.2%), Porphyromonas (12.9%), Ezakiella (8.1%), Prevotella (7.4%), and Dialister (7.0%). In boys, the most abundant genus was Porphyromonas (22.4%), followed by Ezakiella (12.0%), Campylobacter (11.6%), Prevotella (8.6%), and Dialister (3.7%). The authors suggest that the discrepancy in the urobiome composition between girls and boys could be due to gender-specific anatomy and differences in sex hormones levels in the prepubertal period [31].

Another study conducted by Kassiri et al. focused on prepubertal boys aged from 3 months to 8 years. The catheterized urine samples did not reveal a clear predominance of a particular bacterial genus. The majority of urine samples showed the presence of Staphylococcus and Varibaculum species, and to a lesser extent Peptoniphilus and Actinobaculum. The authors support the opinion that the development of the urinary microbiota starts and evolves in early life and becomes more stable in adulthood, similarly to the microbiota of the gastrointestinal tract. However, the study group consisted of patients who required elective urologic procedures, therefore the composition of urobiome may not adequately represent the bacteria present in the urinary tract of children without urological pathologies [32].

From puberty, the shift in the composition of the microbiota of the urinary tract may reflect physiological changes. Presumably, the urobiome of adolescents begins to resemble the urobiome of healthy adults. However, no studies were performed on this topic, and further investigations regarding alterations of the urinary microbiota in this age group are needed.

4. Dysbiosis and Urinary Tract Infection

An imbalance in urinary microbiota may cause an overgrowth of pathogenic bacteria. Changes in urobiome composition may impact disease susceptibility and pathophysiology [3]. Dysbiosis of the urobiome has been related to an increased risk of urinary tract infection, nephrolithiasis, and dysfunction of the lower urinary tract ^[9][10][33][9,10,33]. Although most studies focused on adults, the number of investigations evaluating the relationship between the urobiome, dysbiosis, and urinary tract diseases in children increases ^{[11][24][34][35]}[11,24,34,35].

The findings of recent studies in this field may have significant clinical implications. Strategies of manipulating and reshaping disease-prone microbiomes may serve as an alternative or supportive option in the management of pediatric urological diseases. The modifications of the urobiome include microbial supplements (probiotics or synbiotics), foods or substrates (diet or prebiotics), microbial suppression or elimination (antibiotics) strategies [3].

Urinary tract infection is one of the most common bacterial infections in children [36]. The predominant pathogen is E. coli which accounts for 90% of primary UTIs in girls and 80% of primary UTIs in boys ^{[36][37][38][39]}[36,37,38,39]. Other bacterial pathogens include gram-negative Klebsiella, Enterobacter, Proteus and Citrobacter ^[37][38][37,38], and gram-positive Enterococcus and Staphylococcus saprophyticus [38]. The clinical manifestation of UTI has a broad spectrum and may present as infection of the lower or upper urinary tract. According to patient comorbidities, UTIs can be divided into uncomplicated and complicated. In children, complicated UTIs are mainly associated with congenital anomalies of kidneys and urinary tract. A serious problem is the recurrence of urinary tract infections, defined as three or more infections per year [40]. Noteworthy, different microbial communities are associated with UTIs in specific patient groups (Table 1).

Table 1. UTIs classification according to complicating factors ^[40][41].

UTIs classification according to complicating factors [40,41].

UTI	Characteristic	Related Microorganisms
uncomplicated	occurs in immunocompetent patients with anatomically and functionally normal urinary tract	uropathogenic E. coli (causative factor in 80%)
complicated	occurs in patients with anatomical or neurological abnormalities of urinary tract (i.e., hydronephrosis, vesicoureteral reflux), in patients with compromised immunity, or if foreign bodies are present in patient’s urinary tract (i.e., calculi, catheters)	E. coli (often presenting combination of many virulence factors, or multi-drug resistant profile), non-	E. coli	infection (i.e., Klebsiella spp., Enterococcus spp., Pseudomonas aeruginosa)

Most UTIs result from ascending infection, and periurethral colonization with uropathogenic bacteria is the first step in the development of UTI [38]. Among several host defense mechanisms, a diverse urobiome has been associated with a protective role. According to Kinneman et al., children with UTIs had a significantly decreased alpha diversity, and the composition of the microbiome clustered separately compared with children without UTIs [27]. There is no consensus regarding the taxonomy of healthy urobiota. Additionally, the proportion of species forming the urobiome is dynamic. Commensal bacteria of the urinary tract are believed to serve as a barrier for uropathogens by blocking access to urothelium, producing antimicrobial compounds, or out-competing pathogenic bacteria for common resources [42]. It is suggested that probably most bacteria causing UTIs are part of the resident urinary tract bacteria and reveal their uropathogenicity due to an imbalance in normal urobiome composition [43]. UTIs develop as a result of several host-microbial interactions, and host susceptibility and bacterial virulence factors are crucial elements of that interplay. The interaction between the uroplakin receptors expressed at superficial urothelial cells and bacterial type 1 fimbrial adhesin fimH may serve as an example [43]. The process of E. coli invasion and formation of an intercellular population of bacteria starts after uropathogenic E. coli fimbriae connect with uroplakin Ia/Ib [44].

The reservoirs of uropathogens are the gastrointestinal tract and vagina ^[45][46][45,46]. Paalanne et al. conducted a case-control study that assessed differences in the gut microbiome between children with UTI and healthy controls. The biodiversity of the intestinal microbiome of children was similar in both groups, but there were differences at the family and genus levels. The genus Enterobacter was more abundant in the UTI patients, and the family peptostreptococcaceae was more abundant in controls. These findings suggest that the intestinal environment and its microbial community are associated with the risk of UTI in the pediatric population [47].

Similarly, the vaginal microbiota might impact host susceptibility to UTI and can either protect against or increase the risk of UTIs in girls. Gorbachinsky et al. demonstrated that vulvovaginitis may cause UTIs by changing the perineal microbiome and increased colonization of uropathogens [48]. Previous studies indicated that the vaginal microbiome alters with age. In prepubertal age, a variety of anaerobes, diphtheroids, coagulase-negative staphylococci, and E. coli dominate, while the postmenarcheal vaginal microbiome is dominated by Lactobacillus spp. ^[11][29][11,29]. In contrast, a prospective longitudinal study among perimenarcheal girls conducted by Hickey et al. demonstrated that bacteria producing lactic acid were dominant in the vaginal microbiota of most girls well before the onset of menarche [49]. A protective effect of Lactobacillus spp. is associated with the following mechanisms: maintaining the vagina’s characteristic low pH (mainly due to lactic acid production), releasing antimicrobial compounds such as hydrogen peroxide and bacteriocins, out-competing pathogenic bacteria, preventing the adhesion of pathogens to epithelial cells, and modulation of the host immune system by activation of the Toll-like receptor pathway and interleukins production ^{[11][29][50][51]}[11,29,50,51].