The concept of microbiome has brought revolutionary changes to the concept of environment and development of diseases, not only in the gastrointestinal, oral, and female genital tracts, but also in the urinary tract. HMP, conducted by the National Institutes of Health began in 2008 and ended in 2013. The aim of this project was to understand the human biomes, and to compare and classify the microbiota using sequences of 3,000 genomes from cultured and uncultured bacteria of 300 healthy individuals [6]. The core microbiome was collected from five different sites: nasal passages, oral cavity, skin, gastrointestinal tract, and urogenital tract [1]. HMP used multiple analytical tools, such as taxonomic profiling using 16S rRNA gene sequences and metagenomics profiling by whole-genomic shotgun sequencing [7]. A phylogenic study largely depends on gene sequencing of the 16S rRNA gene because it exists in all animals, and it is well conserved during evolution. It also differs among species of bacteria and archaea. These regions with different sequences can be used to determine phylogenetic relatedness [3].

Recent progress in the scientific understanding of the human microbiota has revealed that there are significant differences between those of the gut and bladder. The number of gut microbiota is as high as 10¹² colony forming units (CFU) per gram of feces, whereas that of urine is 10²–10⁵ CFU per ml [8910]. Moreover, the microbiome of the urinary tract is much less diverse than that of the gut, as only a dozen to dozens of species have been detected, and most urine samples are dominated by one or two bacterial families or genera [8]. The most frequently detected dominant bacteria were Lactobacillus and Gardnerella from urine samples of 60 patients with urge urinary incontinence (UUI) and 58 cohorts without UUI [8].

New approaches using 16S rRNA gene sequencing have provided new opportunities to view the traditional concept of a healthy bladder is sterile and urine is sterile if urine culture and urinalysis are negative. Traditionally, UTIs have been defined by the culture of uropathogenic organisms from mid-stream urine of >100,000 CFU per ml. However, a standard urine culture can only detect fast-growing aerobic uropathogens, such as uropathogenic Escherichia coli, and it cannot detect any slow-growing bacteria, anaerobic bacteria, or those with special nutrient requirements [9]. By contrast, DNA-based diagnostic approaches are more accurate. Nine hypervariable regions (V1–V9) of the 16S rRNA gene can be used for distinguishing various bacteria [3]. A 454 pyrosequencing study of a 26 to 90-year-old healthy population showed that 16S rRNA DNA sequencing identified 94 genera, while only 31 genera were likely to be cultivated by standard culture techniques [11]; this indicated that the remaining two-thirds of the bacteria are not routinely cultured. There are strengths and weaknesses in clinical usefulness between the two diagnostic methods. The general detection rate of bacteria is superior in metagenomics 16S rRNA gene sequencing; however, this method cannot provide quantitative information on deciding the best course of treatment. An enhanced quantitative urine culture protocol has been suggested to overcome the weaknesses of conventional urine culture. Important parts of the methods are increased urine volume, incubation in a 5% CO₂ incubator, incubation for 48 hours, and inclusion of colistin-nalidixic acid agar, in addition to blood and MacConkey agars [912]. Price et al. [12] reported a much higher detection rate compared with the traditional urine culture: the standard urine culture method could detect only 33% of all detected uropathogens via expanded-spectrum enhanced quantitative urine culture, and only 7% of those detected in the non-UTI subjects.

Healthy female urinary microbiota often include bacteria also identified in the vagina, whereas healthy male urinary microbiota resembles the gut and skin [13]. The urinary microbiome is characterized by a preponderance of Lactobacillus in women and Corynebacterium in men [14]. Dong et al. [15] compared the microbiomes of 32 paired first-catch urine samples and urethral swab specimens from adult men in a sexually transmitted disease clinic using 16S rRNA polymerase chain reaction (PCR) and deep pyrosequencing. The distribution of the microbiomes of both specimens was remarkably similar, except only the proportions of Propionibacterium spp. and Corynebacterium [15]. Neisseria, Streptococcus, Corynebacterium, and Ureaplasma spp. were frequently found in more than 5% of sexually transmitted infections (STI)-positive men compared with Lactobacillus, Sneathia, Veillonella, Corynebacterium, Provotella, Streptococcus, and Ureaplasa spp. in STI-negative men. Lactobacillus spp. were the most frequently found organisms in urine samples in both men and women [161718]. However, there is a significant inter-subject variability in the urinary microbiome and differences are evident between the groups of STI-positive and STI-negative men [1117].

Bacterial detection by 16S rDNA sequencing is changing the etiology of STI in men and women. Although Chlamydia trachomatis and Neisseria gonorrhoeae are the most common pathogenic bacteria, they are commonly not detected in patients with STIs. Even though multiplex PCR for the six most common bacteria is available, and treatment of patients with all negative results can be confusing to clinicians. Manhart et al. [19] suggested that Leptotrichia/Sneathia spp. may be associated with nongonococcal urethritis in heterosexual men who are negative for C. trachomatis, N. gonorrhoeae, Trichomonas vaginalis, Mycoplasma genitalium, and Ureaplasma urealyticum. The results from STI-positive men are compatible with BV-positive women.

Fredricks et al. [20] compared 27 subjects with BV and 46 healthy controls using a bacterium-specific PCR assay of 16S rRNA and fluorescence in situ hybridization of vaginal fluid. They reported that Atopobium, Leptotrichia, and Megasphaera were found in a high percentage of women with BV; however, Atopobium was less specific for BV. The causative relationship between these newly detected bacteria and BV in women has not been demonstrated. Although Gardnerella vaginalis is present in all specimens from women with BV, it was also found in nearly 60% of women without BV [20]. Contrastingly, Teixeira et al. [21] reported that G. vaginalis was observed in 56.7% of women with BV and 17.6% of healthy women. Lactobacillus were more frequently detected in healthy women (97.5%) than in women with BV (76.7%). Due to a high detection rate of G. vaginalis in asymptomatic women, there is a debate on whether this organism is commensal or pathogen, as well as on the needs of treatment. Although it is clear that G. vaginalis and Atopobium vaginae are present in high concentrations in grade III BV, Lactobacillus crispatus was found in lower concentration in grades II and III BV [22]. After G. vaginalis increased and lactobacilli decreased in hypoxic condition, G. vaginalis can colonize the vaginal epithelium as a biofilm [23]. G. vaginalis predominant biofilms are 5 times more tolerable for hydrogen peroxide and 4 to 8 times more tolerable for lactic acid produced from lactobacilli. In addition to a shift of predominant organisms from lactobacilli to G. vaginalis, different virulence factors of G. vaginalis can affect occurrence of BV; for example, the production of sialidase A in accordance with the subtypes of G. vaginalis. Among G. vaginalis found in 87% of healthy women, clade 4 type was found in 79.4% of healthy women, and virulence was detected in low frequency due to the lack of gene coding for sialidase [24]. In contrast, all clade 1 and clade 2, which were more commonly isolated in women with BV, have gene coding for sialidase. Castro et al. [25] also reported that G. vaginalis strains from non-BV subjects showed a less virulence expression with respect to biofilm formation, initial adhesion ability, cytotoxic effect, susceptibility to antibiotics, as well as levels of vaginolysin and sialidase. Treatment is not generally recommended for woman with asymptomatic BV due to G. vaginalis. However, benefits of preventive antibiotics treatment for recurrent BV and infection in pregnant woman, along with adjuvant therapy with probiotics, such as lactobacilli, remain to be elucidated.

The degree of diversity of urine microbiota differs in men and women; it is the largest in healthy men, followed by healthy women, and women with acute BV. Diversity was lowest after metronidazole treatment of BV [26].

Gram negative bacteria are isolated in 75% to 90% of uncomplicated UTIs. Contrastingly, Gram positive bacteria constitute 5% to 15% of all UTI bacteria, and Staphylococcus saprophyticus, Enterococcus faecalis, and Streptococcus agalactiae are frequently isolated organisms [27]. Corynebacterium, Actinobaculum, Gardnerella, and Aerococcus are uncommon Gram-positive bacteria found in the urinary tract, and UTIs caused by Gram-positive organisms usually occur in elderly patients and pregnant women [27].

Fouts et al. [14] suggested a mechanism in which the urinary microbiome in healthy subjects changes into a susceptible microbiome for UTI, comparing a healthy urinary microbiome with asymptomatic bacteriuria in patients with neuropathic bladder (NB) associated with a spinal cord injury. Their results indicated that the presence of Lactobacillus decreased over time, and Enterobacteria increased with increasing duration of NB. Finally, Enterobacteria became abundant one year after NB diagnosis, when the urinary microbiome was devoid of Lactobacillus [14]. By contrast, Klebsiella (males), Escherichia, Enterococcus (both genders), and Gardnerella (females) were significantly more enriched in urine from subjects with NB. Corynebacterium, Staphylococcus, and Streptococcus were more abundant in healthy men and women [14].

Non-bacterial microbiome, such as fungi, viruses, archaea, and protozoa, remain unknown. Mycobiome refers to the fungal microbiome, and it is an essential part of the human microbiota [28]. Although fungal infections in humans are very common, most are superficial infections, with invasive infections being rare. Because fungi are less abundant compared with bacteria, their communities are less stable and are significantly influenced by environmental conditions [28]. As a consequence, it is unlikely that a simple fungal infection, or a change in the mycobiome results in disease without alterations in the host environment that facilitate the pathology; e.g., immunosuppressed status, altered metabolism, or the tissue microenvironments [28]. Candida spp. are the most common pathogens in UTIs; a significantly greater prevalence of Candida and Saccharomyces was detected in 15.7% of standard culture-negative female patients with chronic pelvic pain syndrome in the flare status [29].

Virome is the viral microbiota. To date, very little is known about the virome in the genitourinary tract. Santiago-Rodriguez et al. [13] investigated the urinary virome in 10 patients with UTIs and 10 subjects without UTIs. They found approximately 10⁷ virus-like particles in the urine, which was lower than those detected in the human saliva. The most identifiable viruses were bacteriophages, and a low-risk herpes virus type detected in 95% of subjects. Interestingly, even though the bacterial microbiome was significantly altered by UTI, the urinary virome was not different. Moreover, the diversity of viral communities was not different between UTI+ and UTI− subjects, as bacterial diversity is higher in urine from UTI− subjects [13].

It is unclear why women with BV are more prone to developing UTI. It is widely accepted that lactobacilli are a dominant vaginal organism in healthy women, whereas women with BV have a more diverse microbiota, mainly Gram-negative anaerobes, such as Actinobacteria and G. vaginalis. Interestingly, both UTIs and BV are frequently associated with sexual activities and the use of diaphragms. Sumati and Saritha [33] reported that women with BV have an increased risk of developing UTI with an odds ratio of 13.75; pregnancy may also be an important co-risk factor. Yan et al. [34] reported that L. crispatus was cultured in 94% of healthy women and 83% of those with BV. Interestingly, a significant difference in the number of lactobacilli was observed, and the mean number of colonies of L. crispatus was 10⁶ in healthy women and 10³ in women with BV.

The genus Lactobacillus includes more than 130 species, and more than 20 species have been detected in the vagina [1835]. Lactobacillus ferment glycogen produced by vaginal epithelial cells, which produces lactic acid. They inhibit the colonization of pathogenic bacteria; however, this mechanism can be weakened to achieve sufficient inhibitory concentrations under hypoxic conditions [36]. Moreover, lactobacilli have a higher affinity for host cell receptors and can displace adherence of G. vaginalis and N. gonorrhoeae [3738]. They can also inhibit uropathogenic E. coli and E. faecalis [3940].