In this study, we found that there was no significant difference in urine microbiota results between the Gardnerella-positive NC group and Gardnerella-positive RC group. The Gardnerella-positive group could be divided into three urotypes: 1) Escherichia-dominant group, 2) Gardnerella-dominant group, and 3) Lactobacillus-dominant group, respectively. All Escherichia-dominant groups were associated with RC. In the Gardnerella-dominant and Lactobacillus-dominant groups, the NC and RC groups were mixed. In particular, bacterial vaginosis-associated strains such as Atopobium, Megasphaera, and Ureaplasma were detected only in the RC group.
Our research group has completed two papers related to the urine microbiome. The core of the first paper was that E. coli was the most causative strain in acute and recurrent cystitis, but the base from which E. coli grew, that is, the bladder condition (commensal or pathogenic organism) was completely different.13 That is, the first paper is a study on the E. coli dominant urotype. In this paper, the second study, we found out that Gardnerella affects the dominant urotype of recurrent cystitis in the process of newly elucidating the pathophysiology of recurrent cystitis.
A recent UTI guideline is based on antibiotic treatment based on urine culture for RC.25 In particular, continuous low-dose antimicrobial prophylaxis is recommended if RC persists after behavioral interventions have failed.25 In reality, 75% of the patients of clinical practice with RC are taking empirical antibiotics without undergoing tests.26 Consequently, antibiotic resistance has increased while RC prevalence has not decreased.27
Recently, new bacterial technology of 16s RNA sequencing and EQUC revealed that various bladder microbiomes play an important role in addition to the past classical uropathogens in RC.6,13 According to a previous classical urine culture-based study, the most common causative bacteria of uncomplicated UTI were E. coli (58%), mixed flora (13.4%), and K. pneumoniae (6.5%).28 However, the pattern was different in our group's previous pilot study.13 This study based on NGS additionally discovered Sphingomonas, Staphylococcus, Streptococcus, and Rothia spp., which were not found in the existing culture, especially for the RC group.
With the accumulation of the knowledge regarding this microbiome, the importance of the gut-vagina-bladder axis for RC has been increasingly emphasized. Clinical evidence of an association between the vagina and bladder is as follows. First, bacterial vaginosis is a risk factor for UTI;29 second, UTI decreases when hormone treatment such as vaginal estrogen is administered;30,31 and lastly, there are many patients who complain of frequent UTI after sex.32 On the microbiological basis, it has been reported that about two-thirds of bladder microbiota overlap with gut microbiota, and about one-third of bladder microbiota exists only in the vagina.33
The first finding of our study was that there was no difference in the detection rate of Gardnerella between the NC and RC groups. Similar with our result, a previous study reported that Gardnerella was detected in 27% of the normal population, and this ratio was not significantly different from patients with urinary symptoms.9
Second, our study suggested three patterns of Gardnerella -positive patients. First, in the E. coli dominant group, this is the first study of humans showing that Gardnerella can act as a covert pathogen that activates E. coli. It has already been shown that the vagina acts as a reservoir for uropathogens such as E. coli. Moreover, even short exposure of the bladder to Gardnerella caused bladder cell damage such as urothelial exfoliation and urothelial apoptosis in an animal study.16 The second group (Gardnerella dominant group) provides clues that increased amounts of Gardnerella itself may be associated with RC. Although rare, it has been reported that Gardnerella acts as a causative agent of UTIs.29 Considering the low culture positive rate of Gardnerella, the clinical significance is likely to be higher in practice. In the case of the third Lactobacillus-dominant group, since the protectivity effect of Lactobacillus is different depending on the type of Lactobacillus strain, cystitis can occur even with a small percentage of Gardnerella in Lactobacillus strains with poor protectivity. However, in this study, the Lactobacillus strain was not analyzed, and thus further follow-up studies are required to investigate the difference between normal and RC strains.
Considering that both NC and RC exist in group 2 (Gardnerella dominant) and group 3 (Lactobacillus-dominant), it can be inferred as follows. Asymptomatic Gardnerella infection is present in the vagina, and some are self-treated.34 Likewise, the presence of Gardnerella does not necessarily cause RC, just as the presence of Gardnerella does not necessarily cause bacterial vaginosis. Although Gardnerella itself does not have high virulence, it is affected by other risk factors for causing symptoms. Risk factors such as host immunity, Gardnerella proliferation, microbiome environment, and residence environment seem to influence the development of the phenotype (asymptomatic or RC). For example, it seems that cystitis always occurs when Gardnerella and other bacterial vaginosis strains are accompanied which can be interpreted as the influence of the microbiome environment. In addition, the fact that the proportion of Gardnerella was higher in cystitis patients could be the evidence that the amount of Gardnerella proliferation had an effect on phenotype expression. Overall, our results provided clues for a new pathophysiology of RC. The usage of antibiotics based on traditional uropathogens in group 2 or group 3 has weak clinical effects and may cause side effects of antibiotics-resistance to occur.
Our study has several advantages. First, we demonstrated the clinical importance of gut-vagina-bladder axis in humans focusing on Gardnerella for the first time. The gut-vagina-bladder axis has been explained to some extent through microbiological and animal experiments, but studies on humans are still lacking.7,16,33 Second, in our study, the specimen was collected by transurethral catheterization, so the bladder microbiome is well reflected without contamination. Finally, our study suggested several types and novel mechanisms of RC that had not been previously elucidated.
However, our study also has several limitations. First, since it is not a prospective study, it cannot be free from selection bias, especially regarding the NC group. Also, although not statistically significant, the NC group was younger than the RC group and had a lower rate of menopause, which may have affected the results. Second, our study revealed the importance of Gardnerella infection as a covert pathogen, but did not provide a cut-off for the required amount of infection for clinical symptoms to appear. Third, the difference between the Lactobacillus strains in the NC group and the RC group could not be suggested. This requires follow-up studies and is believed to provide clues about Lactobacillus prophylaxis in patients with RC in the future. Fourth, this study did not suggest whether treating Gardnerella could actually improve the clinical symptoms of RC. In general, antibiotics used for bacterial vaginosis and those used for RC are different.35 The choice of antibiotics for UTIs can also affect the vaginal microbiome. For example, the use of beta-lactam antibiotics is less effective against vaginal colonized E. coli, and recurrent UTIs caused by vaginal E. coli easily occur with such beta-lactam antibiotics.36,37 Furthermore, Gardnerella is difficult to treat due to biofilm formation.38 Metronidazole or tobramycin, which were previously recommended as therapeutic agents for Gardnerella, can prevent the formation of a new biofilm, but are known to have less effect on previously formed biofilm.38 Therefore, further studies are needed to determine which antibiotic is most suitable for RC caused by Gardnerella.
In summary, if conventional uropathogens are not detected in patients with RC, Gardnerella infection should be considered. Asymptomatic urine Gardnerella (asymptomatic bacteriuria) does not require treatment, but urine Gardnerella in symptomatic patients is considered to be the causative agent of cystitis, so treatment is necessary. Also, even for asymptomatic cases, treatment should be considered if E. coli or the causative agent of bacterial vaginosis is detected in addition to Gardnerella.