Overactive bladder (OAB) is characterized as “urgency, with or without urgency urinary incontinence (UUI), usually with increased daytime frequency and nocturia” defined by the International Continence Society (ICS; Abrams et al., 2003). The pioneering works of Brading suggested that abnormal electrical couping of smooth muscle cells resulting in detrusor contraction while other researches suggesting sensation of urgency may be provoked by urothelial/suburothelial dysfunction (Drake et al., 2001; Lee et al., 2010; Chen et al., 2017). It has been proposed that metabolic syndrome, affective disorders, sex hormone deficiency, urinary microbiota, gastrointestinal dysfunction and subclinical autonomic nervous system dysfunction are potential pathophysiological cofactors of OAB (Peyronnet et al., 2019). However, there are still a number of patients failed to respond to the medications, including antimuscarinics and the β3-adrenoreceptor agonist, requiring invasive interventions (Wadie, 2014; De Wachter et al., 2019). Therefore, the multifactorial nature of OAB and the effective pharmacotherapy of refractory cases compose the two main obstacles in basic science research and clinical management.

Emerging evidence indicated that alteration to urinary microbiome is closely related to the pathogenesis of OAB using 16S rRNA gene sequencing and expanded quantitative urine culture (EQUC) (Price et al., 2016; Gasiorek et al., 2019; Lundy et al., 2021). Wolfe et al. identified urinary bacteria community with 16S rRNA sequencing in female patients undergoing urogynecologic surgery with negative standard urine cultures, and developed its linkage with diverse urinary diseases (Wolfe et al., 2012). Pearce et al. (2014) used EQUC and 16S rRNA sequencing to compare the urinary microbiome of women with and without wet OAB (UUI) and their data suggest a potentially important difference in the urinary microbiome of women in the two groups. Combined, with the in-depth research on the urinary microbiome, from sterile to fertile, a growing number of scholars realize that the current research results are just the tip of the iceberg (Hilt et al., 2014; Price et al., 2016; Brubaker et al., 2021).

The urinary microbiome, however, contains not just bacteria, but also viruses, fungi, parasites, and other species (Lloyd-Price et al., 2016). Incidentally, excluding the bacteria, studies on other microorganisms, particularly viruses and their association with OAB are limited (Brubaker et al., 2021). Among them, most frequently detected viruses in urinary tract are human polyomavirus type 1 (BK Virus) and 2 (JC Virus), both of which are mainly studied in immunocompromised patients, such as kidney and bone marrow transplant recipients (Jhang et al., 2018). Meanwhile, both JC and BK are double-stranded DNA viruses and belong to polyomavirus (Salabura et al., 2021). Detection of JC Virus is reported in recent studies of urinary diseases such as lower urinary tract symptoms (LUTS) and OAB, but its relationships with urinary bacteria and OAB symptoms require further research (Garretto et al., 2018; Thomas et al., 2021). Additionally, human papillomavirus, adenovirus, and cytomegalovirus infections have been associated with LUTS (Paduch, 2007; Brubaker et al., 2021). Although urinary tract infections (UTIs) are mainly caused by bacteria such as Escherichia coli and viral infections are rare pathogens, viral infections of the urinary tract are linked to considerable morbidity and distress, including increased mortality in immunocompromised patients (Khonsari et al., 2021; Salabura et al., 2021). However, there is an important link between bacteria and viruses, such as increased antibiotic resistance in UTI patients, which leads to an increase in the global medical burden, and bacteriophages, as the most abundant group of human viruses, can be used as a feasible treatment for UTI (Behzadi et al., 2020; Ahmadi, 2021). It is important to note that, especially in female patients, misdiagnosis of UTI and genital tract infections (GTIs) may lead to imbalances in bacterial and viral populations and worsen disease (Behzadi et al., 2019).

Next-generation sequencing (NGS) of bacterial 16S rRNA is widely applied in clinical pathogen detection including urology (Derosa et al., 2020; Salabura et al., 2021). One such application is the unbiased next-generation metagenomic sequencing (mNGS), which overcomes the limitations of current diagnostic tests and allows untargeted, culture-independent pathogen detection directly from clinical samples (Neugent et al., 2020). At the same time, the development of bioinformatics for the analysis of sequencing data can help reduce possible biases in the results to obtain accurate and reliable analysis results (Behzadi and Ranjbar, 2018). Here, we seek to evaluate the role of urinary viruses in the onset of OAB using a mNGS approach, and attempt to reveal the potential relationship between viruses, bacteria, and OAB patients.

The correlations between alteration of urine microbiome and occurrence of OAB have not been revealed until recent years (Wolfe et al., 2012; Pearce et al., 2014; Wu et al., 2017; Brubaker et al., 2021). We have previously demonstrated that at the genus level, the abundance of Aerococcus and Staphylococcus increased, while Prevotella and Lactobacillus decreased in OAB patients (Wu et al., 2017). By comparing the urinary microbiome of women with UUI and asymptomatic controls, Pearce et al. found that the microbiome from UUI showed a decreased in Lactobacillus and an increased in Grdnerella abundance from the UUI cohort by EQUC (Pearce et al., 2014). Additionally, Karstens et al. (2016) determined that an increased severity of UUI symptoms was associated with decreased microbial diversity in female UUI patients. However, most studies on urinary microbiome were measured by 16S rRNA gene sequencing, and investigations were limited to the bacteria. In the present study, we took the lead in unveiling the effects of urinary viral infection on the presence of OAB, and providing evidence on the connections between modified microbial features and degree of OAB symptoms.

Consistent with our previous report, the analysis of Chao1 and Shannon index indicated that the OAB group had lower diversity than the control group, which was consistent from both 16S rRNA gene sequencing and mNGS results (Wu et al., 2017). Since most studies on the urinary microbiome used 16S rRNA amplicon sequencing and focused on the genus level analysis, few studies have similar results at the species level. Recently, Wolfe et al. have provided new insights into taxonomic schemes for species-level bacterial annotations in 16S amplicon data (Hoffman et al., 2021). In the results obtained by mNGS, at species level, the OAB-VI group demonstrated significantly decreased Lactobacillus iners, but increased coagulase-negative Staphylococci (CoNS), including Staphylococcus epidermidis, Staphylococcus hominis and Staphylococcus warner. Previous studies of Staphylococcus warneri, Staphylococcus epidermidis and Staphylococcus hominis are mainly elaborated in human skin microbiome (Byrd et al., 2018). Staphylococcus epidermidis may act both as an opportunistic pathogen and a probiotics, which prevents bacterial biofilm formation via expression of a serine type protease, termed Esp (Otto, 2009; Sugimoto et al., 2013). On the contrary, known as the most sensitive microbiome marker in urinary tract diseases, a decreased Lactobacillus in the urine was associated with the presence of OAB/UUI (Wu et al., 2017; Peyronnet et al., 2019). In a study, whole-genome phylogenetic analysis of bacterial strains isolated from the vagina and bladder of the same female identified highly similar Lactobacillus iners and Lactobacillus crispatus, both of these Lactobacillus have been linked to health in the vagina, as well as the bladder in asymptomatic women (Thomas-White, 2018). These conclusions may indicate its potential role in prevention and treatment of OAB as a probiotics.

Viral genomes, especially JC Virus and BK Virus, were detected predominantly more frequent in patients with OAB. Similarly, Garretto et al. (2018) found that urine samples from OAB patients contain complete genomes of JC Virus while no viral genes were detected in the control group. In reality, JC Virus may infect healthy persons latently, resulting in asymptomatic replication and sporadic detection of the virus in the urine in up to 30% of the general population (White and Khalili, 2011). Previous studies have determined the subtype of JC Virus and global distribution (Forni et al., 2020). The main subtype of JC Virus strains detected in urine of OAB patients were subtype 7B (n = 8) and subtype 7A (n = 7) and these two subtypes of JC Viruses are mainly prevalent in Asia, which was obviously inconsistent with the subtype measured by Wolfe et al., since their samples were mainly from Caucasians and African Americans (Garretto et al., 2018). Interactions between virus and bacteria are widely reported in gastrointestinal tract, mostly focusing on the detrimental effects of bacteriophage on bacteria and end up with reduce in specific bacteria (Santiago-Rodriguez and Hollister, 2019). The bacterial microbiota is required for many enteric viruses during the infection of eukaryotic cells (Monedero et al., 2018). The effects of bacteria on eucaryotic viruses include direct binding with virions to enhance stability and indirect effects led by immune activation through the attachment of host cells (Rowe et al., 2019). Nevertheless, the evidence on whether the virus interacts directly or indirectly with urinary tract bacteria remains to be discovered.

In a cross-sectional study, women with unhealthy habit of holding urine suffer from more frequent LUTS, and this feature was also found in our clinical practice (Reynolds et al., 2020). Here, we found that history of pelvic surgery, holding urine habit and UTI history are risk factors for viral infection, which may make the symptoms of OAB worse. However, GTIs was not taken into account in this study. The association between history of GTIs and OAB and viral infection in women deserves further exploration (Behzadi et al., 2019, 2020). Estrogen usage history whose protective impact of vaginal estrogen therapy has been reported on postmenopausal OAB patients associated with increase of Lactobacillus is found to be a protective factor of viral infection (Thomas-White et al., 2020). Patients in the virus infected group present more severe symptoms and received higher OABSS and OAB-V8 score, which could be explained by the bladder epithelium damage caused by JC or BK viral replication (Li et al., 2013). Given the multifactorial nature of OAB, further research and clinical observations to identify the triple relationship among OAB, virus, bacteria as well as the importance of viral detection and targeted treatment in OAB patients would greatly benefit clinical practice.

In conclusion, we focused at the bacterial and viral composition of urine from OAB and healthy individuals, and found that more severe symptoms of OAB patients seems to be related to viral infection. This is the first published research on the function of human urine bacteria and viruses in OAB female patients that we are aware of. It is our hope that this work lays the foundation for future research on female urine microbiota and OAB disease.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

PW, JZ, and QSo: study concept and design. PW, HZ, YG, BW, YQ, and ZZ: investigation. PW, HZ, and BW: supervision. HZ, QSu, LL, and YW: analysis and interpretation of data. PW, QSo, QSu, and LL: writing – original draft. PW, JZ, QSo, and YW: writing – review and editing. PW: funding acquisition. All authors contributed to the article and approvedthe submitted version.

This current study was supported by funding from the National Natural Science Foundation of China (grant nos. 81870522 and 82173304) and the Natural Science Foundation of Guangdong Province (grant nos. 2018A030313148 and 2021A1515012262).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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