Unraveling the vaginal microbiota-based etiology of recurrent cystitis: the potential role of Lactobacillus vaginal suppositories

Abstract

Recurrent cystitis is a common problem in women that substantially impairs quality of life and contributes to increasing healthcare costs. Traditionally regarded as an ascending infection originating from the gut, recurrent cystitis is now increasingly understood as a disorder rooted in vaginal dysbiosis, with the vagina acting as a key reservoir for uropathogenic Escherichia coli. Loss of a Lactobacillus-dominant vaginal microbiota and overgrowth of enteric and anaerobic bacteria are strongly associated with recurrent cystitis, particularly in postmenopausal women. Lactobacillus species promote vaginal health by producing lactic acid, maintaining a low pH, generating antimicrobial compounds, inhibiting pathogen adhesion, and modulating mucosal immune responses. At the same time, long-term antimicrobial prophylaxis, although effective in reducing recurrences, is limited by adverse effects and the promotion of antimicrobial resistance. These concerns have driven interest in strategies that restore the protective vaginal ecosystem rather than relying on repeated antimicrobial exposure. This narrative review summarizes current concepts linking vaginal dysbiosis to recurrent cystitis, highlights the protective role of Lactobacillus, and synthesizes clinical data on Lactobacillus vaginal suppositories, particularly Lactobacillus crispatus (L. crispatus)-based preparations, as a targeted, non-antimicrobial prophylactic option. Across pilot, phase II, and randomized trials, Lactobacillus vaginal therapy has demonstrated a generally favorable safety and tolerability profile, with preliminary evidence suggesting a potential to reduce recurrent cystitis episodes while restoring a Lactobacillus-dominant vaginal microbiota. However, larger, standardized studies are still needed. Collectively, these findings provide a rationale for investigating Lactobacillus vaginal suppositories as a microbiome-informed, non-antimicrobial strategy for the long-term prevention of recurrent cystitis, particularly in postmenopausal and other high-risk women, and highlight the importance of further research to define the role of vaginal microbiota-directed therapies in future recurrent cystitis management.

Plain language summary

How vaginal bacteria may contribute to repeated bladder infections: a review of Lactobacillus vaginal suppositories to help prevent recurrence

What is this review about? This review examines how changes in vaginal bacteria may contribute to recurrent cystitis (repeated bladder infections) and whether Lactobacillus vaginal suppositories could help prevent infections from returning. Why does the vagina matter? Long-term antibiotics may cause side effects and contribute to antibiotic resistance, so non-antibiotic options are being explored. Increasing evidence suggests the vagina can act as a reservoir for bacteria such as Escherichia coli. When vaginal balance is disrupted ("vaginal dysbiosis"), protective Lactobacillus bacteria may decrease, allowing harmful bacteria to persist and ascend to the bladder. What did we do and find? We summarized studies linking vaginal microbiota patterns to recurrent cystitis and reviewed clinical research on Lactobacillus vaginal products, including Lactobacillus crispatus. The available trials are still limited in number and vary in design and population, but they suggest a generally favorable safety and tolerability profile for these suppositories, with preliminary evidence of a potential to reduce recurrences and help restore Lactobacillus-dominant vaginal communities. The overall evidence base, however, remains limited by small sample sizes and differences between studies. What do these findings mean? These findings provide a rationale for further investigation of Lactobacillus vaginal suppositories as a microbiome-informed, non-antibiotic strategy for preventing recurrent cystitis. Larger, well-standardized clinical trials are needed to confirm their benefit and determine optimal dosing and duration.

Keywords

cystitis microbiota urinary tract infection vagina

Introduction

Urinary tract infections (UTIs) are among the most common bacterial infections in women. An estimated 50%–60% of women experience at least one UTI in their lifetime, and up to 25% of those develop recurrent infections.^1,2 Recurrent cystitis (RC), characterized by repeated symptomatic UTIs over time, poses a substantial challenge for patients and healthcare systems.² The burden of RC is not limited to the direct medical costs of diagnosis and treatment but also includes substantial impairment in quality of life, with symptoms such as dysuria, frequency, urgency, and suprapubic pain disrupting work, social activities, and sexual health. Moreover, the need for repeated antibiotic courses contributes to the growing global antimicrobial resistance crisis, making effective long-term prevention strategies essential.^3,4

Most UTIs, including recurrent episodes, are caused by uropathogenic Escherichia coli (UPEC), originating from the host’s fecal flora. In postmenopausal women, declining estrogen and changes in the urogenital epithelium further elevate UTI risk.^2,5 The precise mechanisms underlying recurrence, particularly the pathways by which bacteria repeatedly access the urinary tract, remain areas of active investigation. Importantly, emerging evidence implicates the vagina as a reservoir for uropathogenic bacteria. Vaginal colonization with Escherichia coli (E. coli) and loss of protective lactobacilli create a dysbiotic vaginal microbiota that can seed the bladder.^5,6 Recognizing this vaginal-bladder axis shifts the paradigm of recurrent UTI (rUTI) pathogenesis and opens novel prevention strategies. Furthermore, understanding this dysbiosis provides a strong rationale for exploring strategies to restore or enhance the protective vaginal microbiome.⁷ Probiotics are defined as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. In the female genital tract, Lactobacillus species are the dominant commensals and represent the principal probiotic candidates for maintaining a protective vaginal environment.⁸ Among these microbiome-targeted, non-antimicrobial approaches, Lactobacillus vaginal suppositories represent a probiotic strategy that offers a direct, localized approach to replenish beneficial bacteria, potentially preventing pathogen colonization and subsequent UTIs without contributing to systemic antibiotic resistance.⁹

The aim of this narrative review is to summarize the current definitions and management of RC, to elucidate key pathogenic aspects linking vaginal microbiota to RC, and to critically appraise the use of Lactobacillus vaginal suppositories as non-antibiotic prophylaxis, including their mechanisms of action, clinical evidence, and future research directions.

Methods

This narrative review was prepared in accordance with the Scale for the Assessment of Narrative Review Articles (SANRA) guidelines.¹⁰ A structured literature search was performed using PubMed and Google Scholar, supplemented by manual reference screening, from database inception to November 2025. The following terms were searched individually and in combination: “vaginal microbiota,” “vaginal microbiome,” “recurrent cystitis,” “recurrent urinary tract infection,” “Lactobacillus,” “vaginal suppository,” “vaginal probiotic,” “probiotics,” “uropathogens,” “uropathogenic Escherichia coli,” “UPEC,” “vaginal dysbiosis,” “genitourinary microbiome,” “postmenopausal women,” “antimicrobial resistance,” and “antibiotic prophylaxis.” Articles were eligible if they were written in English and addressed the vaginal microbiota–urinary tract infection relationship, the pathophysiology of RC, or clinical evidence for Lactobacillus-based vaginal interventions. Emphasis was placed on peer-reviewed original research, randomized controlled trials, prospective cohort studies, and systematic reviews or meta-analyses. Case reports were excluded from the review. The completed SANRA checklist is provided as Supplemental Material S1.

Definition and current management of recurrent cystitis

RC is generally defined as ⩾3 symptomatic UTIs in 12 months, or ⩾2 in 6 months, with resolution of symptoms between episodes.¹¹ Throughout this review, we use the term RC to refer to recurrent symptomatic lower UTIs in women, consistent with contemporary guideline definitions of rUTI. For the diagnosis of RC, microbiological confirmation by urine culture is strongly recommended. In healthy premenopausal women, risk factors include sexual intercourse, spermicide use, and a history of UTIs at a young age. In postmenopausal women, a prior history of UTI before menopause and estrogen deficiency are predominant contributors to increased UTI susceptibility.^2,11 Moreover, randomized trials in acute uncomplicated cystitis have shown that extending antibiotic therapy from 3 to 7 days does not significantly improve microbiological cure or short-term recurrence rates, supporting the principle that longer antibiotic courses do not necessarily translate into better prevention of future episodes.¹² Behavioral modifications, such as increasing fluid intake, ensuring complete bladder emptying, voiding after sexual intercourse, and avoiding spermicidal contraceptives, are often recommended as the initial management of RC. However, the evidence supporting some of these practices is inconsistent.^13,14 For prophylaxis, guidelines recommend either continuous low-dose antibiotic prophylaxis (nitrofurantoin or trimethoprim-sulfamethoxazole for several months) or post-coital antibiotics when non-antibiotic interventions have failed. Both strategies can reduce recurrence but carry long-term risks of antimicrobial resistance and adverse effects.^2,11 Long-term antimicrobial use carries significant drawbacks, including the risk of developing antimicrobial resistance in both uropathogens and the patient’s commensal flora, as well as potential side effects such as gastrointestinal upset and vaginal candidiasis.¹⁵

Various non-antibiotic approaches have been explored as long-term strategies to prevent future RC episodes. Commonly studied alternatives include vaginal estrogen in atrophic postmenopausal women (which boosts lactobacilli and lowers recurrence), cranberries (proanthocyanidins), which have modest efficacy, and D-mannose (a bacterial adhesion inhibitor).^2,14 However, meta-analyses and guidelines note that, except for vaginal estrogen and possibly cranberry, few non-antibiotic therapies have robust evidence.^14,16 Indeed, one systematic review found no significant benefit of generic probiotics for RC prevention, mainly due to small, heterogeneous trials.¹⁴ Thus, current UTI prevention follows an algorithm of risk factor reduction, supplemental estrogen (in postmenopausal women), and prophylactic antibiotics.^2,11 Cranberry products, believed to inhibit bacterial adhesion to the uroepithelium, are a popular traditional remedy, with some studies supporting their efficacy, though results have been variable. Vaginal estrogen therapy is an established non-antibiotic option for postmenopausal women with RC and is recommended in current guidelines.^2,11,14

When compared directly, these non-antibiotic strategies differ substantially in their mechanism of action, evidence base, and patient applicability. Vaginal estrogen restores the hypoestrogenic urogenital mucosa by normalizing vaginal pH and lactobacillary flora, thereby reducing uropathogen colonization; meta-analyses of randomized controlled trials report a significant reduction in RC recurrence (relative risk 0.42; 95% CI 0.30–0.59), and the European Association of Urology assigns it a strong recommendation for postmenopausal women, though its use is inherently restricted to women with hypoestrogenism.^11,14 Cranberry proanthocyanidins inhibit E. coli adhesion to the uroepithelium through an anti-adhesive mechanism, yet clinical trial results remain heterogeneous across formulations and proanthocyanidin dosages, with major guidelines assigning only weak-to-moderate recommendations.^2,11,16 D-mannose competitively blocks FimH adhesin-mediated bacterial adhesion; however, a recent large randomized trial found no significant reduction in RC recurrence compared with placebo, and current guidelines caution that D-mannose alone may not be effective for RC prevention.^14,16

Given the limited durability and inconsistent evidence supporting many non-antibiotic interventions, attention has shifted toward modulating the host microbiome, particularly the vaginal microbiota, as a preventive strategy. This evolving paradigm highlights the protective role of a Lactobacillus-dominant vaginal environment as a natural barrier against uropathogen colonization and ascension, providing a strong biological rationale for microbiome-targeted and probiotic-based interventions.¹⁷ Subsequent sections focus on the vaginal microbiota-based pathogenesis of RC.

The vaginal reservoir of uropathogenic E. coli in recurrent cystitis

Epidemiological and molecular evidence

The majority (>75%) of acute bacterial cystitis cases are caused by E. coli, often UPEC.¹⁶ The gastrointestinal tract is the primary source of UPEC, and the infecting strains causing cystitis in the bladder often originate from the individual’s own gut flora, passing through the vaginal environment. Factors promoting this migration include the close anatomical proximity of the anus, vagina, and urethra, which can facilitate the transfer of bacteria from the periurethral area into the bladder.¹⁸ The vagina serves as a crucial intermediate reservoir before bacteria colonize the periurethral area and ascend into the bladder. Studies have consistently shown that the vaginal introitus and vagina are frequently colonized by fecal bacteria, including UPEC, in women prone to RC. This colonization is often facilitated by disruptions in the normal vaginal microbiome, particularly the loss of protective Lactobacillus species, which allows Gram-negative enteric bacteria to overgrow.^18
–20 The vaginal environment thus plays a critical role in determining the likelihood of periurethral colonization and subsequent infection.

Recent molecular studies have traced the importance of the vagina as a reservoir. Sekito et al. found that paired E. coli isolates from urine and vaginal swabs of the same RC patients were essentially identical, as evidenced by pulsed-field gel electrophoresis showing up to 100% similarity, with four of seven patients exhibiting 100% matches, and the isolates also matched in antimicrobial susceptibilities.²¹ All patients’ vaginal and urine E. coli belonged to the same clonal group (including ST131 in some cases). These data firmly establish that vaginal E. coli often mirrors bladder E. coli in RC and provide compelling evidence for the vaginal reservoir concept, particularly in vulnerable populations such as postmenopausal women, where vaginal flora changes are common. Similarly, earlier work by Gupta et al. demonstrated that women with RC were significantly more likely to have persistent vaginal E. coli colonization (35% of cases vs 11% of controls; p < 0.001).²² This vaginal colonization is rarely found in women without a UTI history, indicating that vaginal E. coli is a specific risk factor for recurrence. In practical terms, these findings suggest that uropathogens can ascend from the vagina into the urethra and bladder to trigger cystitis episodes. Another study demonstrated that the same E. coli strain can be repeatedly isolated from the urine, periurethra, and vagina of women with RC, and that certain virulence factors, such as papG and cellulose, are associated with persistence and recurrence.²³ Consistently, a recent fimbrial gene profiling study showed that the papG2 adhesin is significantly enriched in UPEC isolates from symptomatic UTIs compared with asymptomatic bacteriuria, underscoring the contribution of specific fimbrial repertoires to clinical disease expression.²⁴ Moreover, genomic analyses reveal that E. coli strains causing RC do not possess unique virulence determinants compared to those causing sporadic cystitis, but instead exhibit rapid, host-specific adaptation, including changes in metabolism and membrane transport genes.²⁵ The risk and outcome of RC are further influenced by the interplay between host susceptibility and E. coli’s diverse virulence phenotypes, rather than by a specific genetic signature.²⁶ Consistent with this concept, recent experimental work has shown that prior UPEC infection can epigenetically reprogram bladder urothelial stem cells and establish epithelial-intrinsic trained immunity that differentially shapes mucosal responses and susceptibility to subsequent infections.²⁷ Studies using advanced typing and sequencing methods have shown that while relapse with the exact E. coli clone is common, reinfection with genetically distinct strains also occurs, highlighting the genetic diversity of UPEC in RC and the need for tailored prevention strategies.²⁸

The fecal-vaginal-urinary axis

The concept of fecal-vaginal-urinary transmission has long been recognized, explaining that the host’s gut and vaginal flora often harbor the infecting E. coli strain.^20,22,29 It suggests that recurrent infections are not necessarily due to entirely new invasions from the gut each time but may instead involve the persistence of UPEC in the vaginal niche, followed by repeated ascension. This perspective can explain why simply treating the urine infection without addressing vaginal colonization often leads to relapse and highlights the importance of the vaginal environment as a target for preventive interventions.

Vaginal dysbiosis and recurrent cystitis in postmenopause

A healthy vaginal microbiota is typically dominated by Lactobacillus species, which acidify the niche and inhibit pathogens. Specifically, species such as L. crispatus, L. jensenii, L. gasseri, and L. iners are the most prevalent.^7,30,31 These commensal bacteria contribute to vaginal health by maintaining a low pH (typically 3.5–4.5) through the production of lactic acid, which inhibits the growth of many pathogenic bacteria and yeasts.^31,32 A healthy Lactobacillus-dominant flora forms a protective barrier by competing with potential pathogens for adhesion to vaginal epithelial surfaces and for nutrients, and by producing antimicrobial substances such as hydrogen peroxide and bacteriocins.^33
–36 Moreover, estrogen levels play a significant role in maintaining a healthy vaginal microbiome. Lower estrogen levels, particularly in postmenopausal women, lead to decreased glycogen production by vaginal epithelial cells. Glycogen is the primary metabolic substrate for most vaginal Lactobacillus species. Reduced glycogen availability results in decreased lactobacilli and increased vaginal pH, making the environment more susceptible to colonization by enteric bacteria.^37,38

Vaginal dysbiosis refers to a disruption of the lactobacilli-dominant vaginal microbiota. This disruption leads to overgrowth of anaerobic bacteria, such as Gardnerella, Prevotella, and Streptococcus species, as well as enteric Gram-negative rods, including E. coli.¹⁷ Lactobacillus-dominated communities maintain vaginal homeostasis by producing lactic acid, hydrogen peroxide, and bacteriocins, and by forming biofilms. In contrast, loss of these organisms permits the overgrowth of biofilm-forming pathogens and is associated with increased susceptibility to urogenital infections, including UTIs.⁸ L. crispatus-dominated community state types are considered to be most strongly associated with a stable, protective vaginal environment. In contrast, transitions to Lactobacillus-depleted, polymicrobial communities are associated with higher vaginal pH and recurrent vaginal infections that may create a permissive milieu for ascending uropathogens.⁸ This dysbiotic state is thought to compromise the natural defense mechanisms of the vaginal environment. It facilitates the colonization and ascension of uropathogenic bacteria such as E. coli, which can then trigger RC.²¹

In postmenopause, declining estrogen exacerbates the vaginal dysbiosis. Pabich et al. found that only 62% of older women had any detectable vaginal lactobacilli (vs higher rates in premenopause), whereas 39% harbored vaginal E. coli.⁶ Robust lactobacilli colonization strongly correlated with lower E. coli presence. Consequently, postmenopausal women, especially those not on estrogen therapy, have a documented depletion of protective lactobacilli and an increase in pathogenic vaginal colonizers.⁶ Moreover, Raz et al. also reported that intravaginal estriol increased vaginal lactobacilli and reduced RC episodes from 5.9 to 0.5 per patient-year (p < 0.001).³⁹ Intravaginal estrogen treatment has demonstrated efficacy in reducing RC incidence and time to recurrence in postmenopausal women.⁴⁰ Regarding local adverse effects, vaginal discomfort, burning, itching, or irritation have been reported, though these were not significantly higher than placebo.⁴¹ Despite the favorable safety profile of low-dose vaginal estrogen, its use remains limited by patient fear, misunderstanding, and concerns about side effects that may compromise long-term adherence.^42,43 Local Lactobacillus probiotics, therefore, represent a promising non-hormonal alternative capable of restoring the vaginal microbiome without hormonal exposure.

Several studies have demonstrated that the loss of Lactobacillus dominance is associated with an increased risk of RC.^5,44 They also suggest that restoring Lactobacillus levels with vaginal suppositories may shift the microbiota toward a healthier profile, reduce the abundance of pathogenic bacteria, and significantly decrease the frequency of cystitis episodes.^5,45 In postmenopausal women, RC appears to be closely linked to vaginal dysbiosis. Sekito et al.⁵ showed that postmenopausal women with RC exhibit a near-complete absence of vaginal Lactobacillus, with marked expansion of Enterobacteriaceae, forming discrete microbiome clusters distinct from those of healthy controls and uncomplicated cystitis. These findings support the view that RC is a dysbiosis-driven condition rather than solely a consequence of ascending infection. Importantly, in the same study, restoration of Lactobacillus dominance using intravaginal L. crispatus significantly increased Lactobacillus abundance, suppressed pathogenic bacterial predominance, and reduced the annual incidence of cystitis.⁵ Collectively, these data support the hypothesis that restoring vaginal Lactobacillus dominance may represent a biologically plausible strategy to reduce the risk of RC.

Taken together, vaginal dysbiosis, defined by loss of lactobacilli and overrepresentation of enteric and anaerobic species, is a key driver of postmenopausal RC. It provides a pathway for vaginal bacteria to enter the urinary tract and a diminished mucosal defense. Therefore, restoring a lactobacilli-dominated vaginal microbiome represents a rational strategy to prevent RC.

Protective and antimicrobial mechanisms of Lactobacillus

Lactobacilli exert multiple antimicrobial effects against uropathogens. Beyond their acidification of the vagina, which itself inhibits many organisms, lactobacilli produce H₂O₂ and various bacteriocins/peptides with direct bactericidal or anti-biofilm activity.^32,46 H₂O₂-producing lactobacilli have been shown to inhibit E. coli growth in vitro, and the absence of such strains is a risk factor for vaginal E. coli colonization.^22,47 Gupta et al. showed that women lacking H₂O₂-producing lactobacilli were 4–6 times more likely to be colonized by vaginal E. coli.²² Depletion of lactobacilli reduces lactic acid and H₂O₂ production, disrupting the acidic and antimicrobial environment that usually restricts uropathogen overgrowth. This dysbiotic state enables UPEC to adhere to and invade vaginal epithelial cells, forming intracellular bacterial communities within the vaginal mucosa. This intracellular vaginal reservoir allows UPEC to resist immune clearance and antimicrobials, thereby serving as a persistent infection reservoir.⁴⁸

Notably, the antimicrobial efficacy of L. crispatus in preventing RC has been demonstrated through multiple mechanisms, including the inhibition of UPEC and other pathogens. Recent molecular studies have identified specific Lactobacillus-derived factors that suppress pathogens independent of pH. Consistent with these findings, Nair et al.⁴⁹ demonstrated that tryptamine, a biogenic amine produced by probiotic lactobacilli, significantly inhibits UPEC biofilm formation, identifying a pH-independent, small-molecule mechanism by which Lactobacillus can suppress uropathogen persistence. Watanabe et al. demonstrated that the cell-free culture supernatant of L. crispatus GAI98322 markedly inhibited the growth and biofilm formation of UPEC (including ESBL-producing UPEC) and Pseudomonas aeruginosa, even when pH and acids were controlled.⁵⁰ Proteomic analysis of this supernatant revealed two key proteins, an “aggregation-promoting factor” and a “lysin,” that appear to mediate this effect. These factors can clump or lyse bacterial cells, disrupting biofilm integrity and viability. Remarkably, this killing effect occurred regardless of the pathogens’ antibiotic resistance, suggesting that L. crispatus produces broad-spectrum antimicrobials that bypass conventional resistance mechanisms.⁵⁰ In addition, lactobacilli can competitively inhibit pathogen adherence. They bind to uroepithelial cells and produce surfactant-like compounds that block pathogen attachment.^51,52 They also may modulate host immunity, for example, by inducing defensin production or dampening pro-inflammatory cascades, thereby further protecting the urinary tract.

In addition, Lactobacillus jensenii has been shown to produce (-)-Terpinen-4-ol, a compound that restores antibiotic sensitivity in drug-resistant E. coli and K. pneumoniae by inhibiting bacterial efflux pumps. This finding further underscores the diverse and potent antimicrobial strategies employed by vaginal lactobacilli.⁵³ In line with these observations, Das and Konwar recently demonstrated that vaginal isolates of L. crispatus, L. gasseri, and L. vaginalis, as well as their cell-free supernatants, potently inhibited multiple urogenital pathogens in vitro. These antimicrobial effects were retained when incorporated into encapsulated beads or sanitary materials, highlighting the prophylactic potential of Lactobacillus-based probiotic and postbiotic formulations against urogenital infections.⁵⁴ Altogether, these findings explain why restoring vaginal lactobacilli not only acidifies the niche but also actively suppresses uropathogens via secreted antimicrobials. This rich antimicrobial repertoire underlies the potential of Lactobacillus probiotics to prevent RC episodes by targeting pathogens at the vaginal reservoir.

Clinical research on and therapeutic rationale for Lactobacillus vaginal suppositories

Clinical evidence from trials

Clinical trials investigating Lactobacillus vaginal suppositories, most commonly those containing L. crispatus GAI98322, have provided preliminary clinical evidence suggesting a potential role for the prevention of RC. Key clinical trials of Lactobacillus vaginal suppositories are summarized in Table 1. Uehara et al. conducted a pioneering pilot trial in which women with RC self-administered vaginal suppositories containing L. crispatus GAI98322. The study showed a highly significant reduction in recurrence (p = 0.0007) without any adverse effects.⁵⁵ Although small, this trial provided early safety data and suggested potential efficacy, paving the way for larger investigations. A subsequent phase II trial by Sadahira et al.⁵⁶ followed 28 women who received L. crispatus GAI98322 vaginal suppository therapy for 1 year. Among the 21 who completed the study, 18 (86%) experienced a clinically meaningful reduction in UTI frequency, with mean episodes decreasing from approximately 5.6 pre-treatment to approximately 2.5 during treatment (p < 0.001). Notably, the suppressive effects persisted for at least 1 year post-treatment, suggesting a durable modulation of the vaginal microbiota. No treatment-related adverse events were reported, providing preliminary support for the safety and tolerability of this intervention.^55,56 In addition to these trials, a phase I randomized, double-blind, placebo-controlled study of L. crispatus CTV-05 vaginal suppositories in premenopausal women with a history of RC showed that a 5-day course was well tolerated, with only mild-to-moderate local symptoms and no serious adverse events, thereby further supporting the safety and feasibility of this intravaginal probiotic strategy.⁵⁷ Another double-blind randomized trial using L. crispatus suppositories (commercially known as Lactin-V) demonstrated a significant reduction in the relative risk of RC compared with placebo, especially in women who achieved sustained high-level colonization.⁴⁴ These individual trials are consistent with a recent systematic review of eight prospective studies evaluating vaginal Lactobacillus suppositories for RC prophylaxis, which concluded that such preparations are safe non-antibiotic options but that evidence for a definitive reduction in RC remains limited.⁵⁸ In that review, only two non-randomized studies of L. crispatus showed a statistically significant benefit,^55,56 and optimal dosing and therapy duration remain unknown.⁵⁸

Table 1.

Key clinical trials evaluating Lactobacillus vaginal suppositories for the prevention of recurrent cystitis.

Study	Study design	Participants (N)	Intervention	Comparator	Efficacy outcomes	Microbiome findings	Limitations
Uehara et al., 2006⁵⁵	Open-label, single-arm, pilot study	n = 9 Premenopausal women with recurrent UTI (⩾3 episodes in prior 12 months; recurrent UTI for ⩾2 years)	L. crispatus GAI 98322 vaginal suppository (1 × 10⁸ CFU), every 2 days for 12 months	None (single-arm study)	UTI frequency: 5.0 ± 1.6 to 1.3 ± 1.2 episodes/year (p = 0.0007) No adverse events reported	Strain selected for highest H₂O₂ production (32.3 ± 4.1 mM) No sequencing-based microbiome assessment performed	Small sample size No control group No microbiome sequencing Single-center; single-ethnicity population
Czaja et al., 2007⁵⁷	Phase I double-blind, placebo-controlled RCT	n = 30 Premenopausal women aged 18–35 years; recurrent UTI (⩾3 UTIs in prior year or ⩾2 UTIs in prior 6 months)	L. crispatus CTV-05 vaginal suppository (5 × 10⁸ CFU), daily for 5 days	Placebo vaginal suppository	No efficacy conclusion: phase I trial focused on safety/tolerability and was not powered for recurrence outcomes	L. crispatus CTV-05 detected by rep-PCR in 4/15 (27%) participants in the active arm No microbiome sequencing Other finding: asymptomatic pyuria in 7/15 (47%) participants in the active arm	Not powered for efficacy (Phase I) Short intervention period Low colonization rate (4/15) Limited microbiome characterization
Stapleton et al., 2011⁴⁴	Phase II double-blind, placebo-controlled RCT (NCT00305227)	n = 100 Premenopausal women aged 18–40 years; current symptomatic uncomplicated cystitis and ⩾1 prior symptomatic UTI treated within the past 12 months	Lactin-V (L. crispatus CTV-05; 1 × 10⁸ CFU), daily for 5 days, then weekly for 10 weeks	Placebo vaginal suppository	Recurrent UTI: 15% (7/48) in the Lactin-V group vs 27% (13/48) in the placebo group; RR 0.5 (95% CI 0.2–1.2) High-level colonization (>10⁶ copies/swab): 93% vs 68% (p = 0.004) Among high colonizers: UTI RR 0.07 (p < 0.01)	High-level vaginal L. crispatus colonization at 10 weeks: 93% in the Lactin-V group vs 68% in the placebo group (p = 0.004) Sustained high-level colonization was associated with reduced recurrent UTI	Primary endpoint not statistically significant Colonization assessed by qPCR only Predominantly White participants Single-strain intervention
Sadahira et al., 2021⁵⁶	Phase II single-arm, open-label trial (jRCTs061180053)	n = 28 enrolled; n = 21 completed Women aged ⩾20 years; mean age 68.3 years; recurrent cystitis (⩾2 antibiotic-treated episodes in the prior year) with current acute cystitis or acute exacerbation of chronic complicated cystitis	L. crispatus GAI 98322 vaginal suppository (1 × 10⁸ CFU), every 2 days or three times weekly for 12 months	None; historical comparator (pre-treatment frequency)	86% efficacy during administration UTI frequency: 5.62–2.52 episodes/year (p = 0.00007) Post-treatment: 1.86 episodes/year (p = 0.00054 vs baseline) No adverse events	Amplicon sequencing in four patients L. crispatus colonized the vagina during cystitis-free periods Microbiota shifts correlated with UTI prevention	Single-center; single-ethnicity cohort No control group; historical comparator only Small sample size Microbiome sequencing limited to 4 patients
Gupta et al., 2024⁵⁹	Four-arm, double-blind RCT (CTRI/2014/02/004425)	n = 174 Premenopausal women, ages 18–45 years; ⩾3 UTIs/year	G3: FloraBalance vaginal probiotic (1 × 10⁹ CFU; L. plantarum, L. salivarius, and L. brevis), 8 days per month for 4 months G4: Combined oral and vaginal probiotic regimen	G1: Placebo G2: Oral probiotic only (Visbiome)	UTI incidence at 4 months: G1 = 70.5%; G2 = 61.4%; G3 = 40.9%; G4 = 31.8% Time to first UTI: G1 = 69.3 days; G2 = 71.9 days; G3 = 123.9 days; G4 = 141.9 days (p < 0.001) Combination therapy (G4) showed numerically better outcomes than vaginal probiotic alone (G3), without a statistically significant difference	qRT-PCR: reduced vaginal E. coli, K. pneumoniae, and P. mirabilis in G3/G4 Increased lactobacilli and bifidobacteria after treatment; post-treatment lactobacilli highest in G4	Single-center; single-ethnicity cohort Modest sample size No vaginal microbiome sequencing Mixed-strain formulation limits strain-level attribution

CFU, colony-forming units; CI, confidence interval; E. coli, Escherichia coli; K. pneumoniae, Klebsiella pneumoniae; L. brevis, Levilactobacillus brevis; L. crispatus, Lactobacillus crispatus; L. plantarum, Lactiplantibacillus plantarum; L. salivarius, Ligilactobacillus salivarius; P. mirabilis, Proteus mirabilis; qPCR, quantitative polymerase chain reaction; qRT-PCR, quantitative reverse transcription polymerase chain reaction; RCT, randomized controlled trial; rep-PCR, repetitive element sequence-based polymerase chain reaction; RR, relative risk; UTI, urinary tract infection.

In addition to L. crispatus-based suppositories, other Lactobacillus vaginal formulations have also yielded promising preliminary results. In a recent randomized, double-blind, placebo-controlled trial, each probiotic vaginal tablet contained 1 × 10⁹ colony-forming units (CFU) of a blend of L. plantarum, L. salivarius, and L. brevis. In that trial, the vaginal probiotic group and the combined oral–vaginal probiotic group had substantially lower incidences of symptomatic UTI at 4 months than the placebo group (40.9% and 31.8% vs 70.4%; relative risks 0.59 and 0.45, respectively) and fewer recurrences at 12 months.⁵⁹ In addition, the mean time to the first symptomatic UTI was significantly longer in the vaginal probiotic arms than in the placebo arm (123.9 and 141.9 vs 69.3 days; p < 0.05 for both comparisons). Similarly, intravaginal estrogen, known to promote lactobacilli growth, has been shown to significantly reduce recurrence in postmenopausal women with RC.⁶⁰ Beyond trials in women with RC, microbiome-focused studies in healthy reproductive-age women also support the importance of direct vaginal delivery. In a recent randomized, placebo-controlled trial, a slow-release multi-strain L. crispatus-based vaginal synbiotic tablet achieved markedly higher rates of sustained L. crispatus-dominant colonization and conversion to an optimal community state type I than placebo, whereas an oral formulation containing the same strains failed to achieve meaningful vaginal colonization.⁶¹ Although this study did not evaluate UTI outcomes, it reinforces the concept that vaginally delivered Lactobacillus formulations are biologically more plausible than oral probiotics for reliably restoring a protective Lactobacillus-dominant vaginal microbiome.

In terms of dosing, the optimal bacterial load for intravaginal Lactobacillus is increasingly understood to fall within the range of 10⁸–10¹⁰ CFU per application. This dosing range is supported by both mechanistic studies, showing that high-density inoculation is necessary to overcome established dysbiosis and achieve mucosal adherence,^62,63 and by clinical trials, in which preparations containing approximately 10⁸–10⁹ CFU of Lactobacillus have consistently demonstrated successful vaginal colonization and reductions in RC episodes.^58,59 Taken together, current basic and clinical evidence suggests that inoculation within this range may represent a biologically and clinically appropriate dosing strategy for intravaginal Lactobacillus therapy.

Collectively, these early-phase clinical trials provide preliminary evidence for a potential role of Lactobacillus vaginal therapies as a non-antibiotic strategy for preventing RC. While some studies are randomized and placebo-controlled with objective endpoints, the overall evidence base remains limited by small sample sizes and methodological heterogeneity.⁵⁶ While effectiveness may vary by strain, formulation, regimen, and patient population, several trials suggesting reduced recurrence rates provide preliminary support for this approach as a potentially durable, non-antibiotic preventive strategy. Lactobacillus vaginal suppositories are generally well tolerated. Reported adverse events are typically mild and localized, such as transient vaginal discharge or irritation, and rarely necessitate treatment discontinuation.⁵⁷

Therapeutic advantages and practical considerations

Vaginal suppositories delivering live Lactobacillus have several theoretical and practical advantages. First, they target the intervention to the site of dysbiosis, the vaginal introitus, ensuring high local concentrations. Vaginal suppositories are designed to release their contents gradually, enabling lactobacilli to colonize the vaginal epithelium and maintain a sustained presence that provides continuous protection against pathogen colonization and ascension.^44,64 This localized, sustained delivery contrasts with oral probiotics, which may not reliably survive gastrointestinal transit or colonize the vagina.⁶⁵ Second, compared with antibiotic prophylaxis, vaginal Lactobacillus therapies represent a non-antibiotic strategy that may reduce antimicrobial exposure and resistance. They act on the microbiome to restore a protective flora rather than directly killing bacteria with antimicrobials, potentially offering broader protection.⁶⁶ Indeed, clinical trials report that repeated vaginal dosing safely “stabilizes vaginal dysbiosis” and displaces pathogens.^5,56 While other preventive strategies may focus on preventing bacterial adhesion or clearing bacteria after entry, vaginal probiotics address the ecological imbalance that facilitates initial colonization and ascension of uropathogens from the vaginal niche.⁶⁶ Third, these probiotics have an excellent safety profile, with no serious side effects reported across multiple studies.^44,55,56 Although direct resistance data for vaginal formulations are limited, a randomized, double-blind, noninferiority trial in postmenopausal women demonstrated that oral Lactobacillus prophylaxis achieved reductions in RC episodes similar to those with trimethoprim-sulfamethoxazole, without causing the marked increase in antibiotic-resistant uropathogens observed in the antibiotic group.⁶⁷ Together with probiotic trials in bacterial vaginosis showing favorable safety and minimal concern for resistance,⁶⁸ these findings support the concept that Lactobacillus-based prophylaxis, including vaginal formulations, can provide effective prevention without driving antimicrobial resistance. Indeed, women in intravaginal trials report good tolerability and often minimal local symptoms only.^44,55,56 Finally, protocols can be integrated into standard UTI management (e.g., administering lactobacilli after completing antibiotics) without causing drug interactions. Given the urgent need for non-antibiotic prophylaxis, Lactobacillus vaginal suppositories may currently be regarded as a low-risk, biologically plausible investigational option; phase II trials and a recent systematic review have described them as a “safe and promising” non-antibiotic strategy for RC prevention.^56,58,69

Future prospects and limitations of Lactobacillus vaginal suppositories

Although probiotics, particularly vaginal formulations, may represent a potentially promising adjunct in RC prevention, current clinical evidence remains heterogeneous,^16,70 and Lactobacillus vaginal therapy is not without limitations. First, consistent vaginal delivery requires patient compliance with regular suppository use, which some may find inconvenient or uncomfortable. The need for ongoing insertion, often multiple times per week, may limit long-term adherence outside a trial setting and is consistent with real-world data showing that adherence to non-antibiotic prophylactic regimens for RC is frequently suboptimal.⁷¹ Second, only a small number of Lactobacillus vaginal products with documented clinical data are currently available, and there is no broad standardization of strains, doses, or quality control.¹⁶ Clinical trials have utilized different Lactobacillus strains, formulations (e.g., number of species, CFU count, suppository base), and administration regimens (daily vs weekly, duration). This heterogeneity makes direct comparisons across studies challenging and complicates the identification of optimal strains or protocols.^44,56,58 A recent gynecologic review concluded that Lactobacillus-based probiotics may be considered part of a multimodal, non-antibiotic prevention strategy for RC, but cannot yet be recommended as standalone prophylaxis due to heterogeneity in strains and formulations, and limited product standardization.⁷² Third, while trials report no adverse effects, rare complications (e.g., local irritation and yeast overgrowth) could occur and need monitoring. Importantly, most published trials are small and short- to intermediate-term, and large-scale data on real-world effectiveness are lacking.⁵⁸ Finally, evidence of benefit is currently strongest for particular formulations (e.g., L. crispatus GAI98322), and it would be premature to generalize to all lactobacilli or probiotic mixtures. Further research is needed to fully elucidate the precise mechanisms by which specific Lactobacillus strains exert their protective effects in vivo. While in vitro studies provide clues,⁵⁰ the complex interactions within the vaginal microbiome and with the host immune system require further investigation.

To advance this field, several research avenues are critical. First, multicenter, double-blind, randomized trials of specific Lactobacillus vaginal suppositories with adequate sample sizes are needed to confirm efficacy and safety.^58,59 Such trials should compare suppositories with placebo and standard prophylaxis (e.g., low-dose antibiotics), with extended follow-up, and stratify by menopausal status. Second, head-to-head comparisons of probiotic strains and formulations are warranted. L. crispatus has shown promise, but other species and strain combinations (e.g., the blend of L. plantarum, L. salivarius, and L. brevis evaluated in a recent vaginal probiotic trial) might also be effective or synergistic.⁵⁹ Comparative trials evaluating different Lactobacillus species and strains are necessary to determine which are most effective for vaginal colonization and RC prevention.⁸ Dose-ranging studies are also needed to establish optimal bacterial counts and administration frequencies. Third, deeper microbiome analyses should accompany clinical trials. Using 16S rRNA sequencing and metagenomics, researchers can track changes in the vaginal (and perhaps bladder) microbiota over time. Whole-genome sequencing of isolates can help determine whether prophylaxis reshapes the overall community or adds a transient strain.^56,59 Studies in diverse patient populations are needed, with priority given to women of different ethnic backgrounds, because vaginal microbiome composition can vary between populations,⁷ as well as to those with specific comorbidities such as diabetes.⁷³ These studies might identify individual-specific microbial signatures or biomarkers that predict therapeutic success or failure. In postmenopausal women, recent work has proposed a predictive, preventive, and personalized medicine framework in which vaginal community state types and diversity indices are used to stratify genitourinary risk and tailor hormonal and microbiome-targeted interventions.⁷⁴ This concept could be extended to RC prophylaxis with Lactobacillus-based products. Fourth, mechanistic studies should explore the active components underlying Lactobacillus-mediated protection. Proteomic analyses, exemplified by the work of Watanabe et al., suggest that it is feasible to purify and characterize novel bacteriocins or enzymes (lysins) that inhibit uropathogens.⁵¹ Such efforts could even lead to non-living products (e.g., defined antimicrobial peptides) for prophylaxis. In parallel, innovative citizen-science initiatives have shown that women can successfully isolate their own L. crispatus strains and participate in the co-design of multi-strain, over-the-counter vaginal probiotics. These efforts underscore both the feasibility of such products and the growing demand for accessible, woman-centered Lactobacillus-based interventions.⁷⁵ These efforts remain exploratory and conceptual, but they highlight the potential for patient-driven, microbiome-based strategies to complement conventional RC prevention. Fifth, immunological studies are warranted to elucidate host responses. A recent study showed that application of an L. crispatus live biotherapeutic product can alter genital immune parameters in vivo.⁷⁶ Such studies may clarify whether restoration of lactobacilli modulates mucosal immunity in ways that contribute to UTI prevention. Furthermore, understanding the interplay between the vaginal microbiota and the uroepithelium may reveal additional preventive or therapeutic targets. Finally, health-economic analyses evaluating the cost-effectiveness of Lactobacillus vaginal suppositories relative to long-term antibiotic prophylaxis are also warranted, given the potential for reduced antibiotic prescriptions and associated downstream savings. Furthermore, population-level studies examining the impact of a broad shift toward probiotic-based prophylaxis on antimicrobial resistance patterns represent an important and timely research agenda.

Conclusion

RC is increasingly understood as a condition rooted in vaginal dysbiosis, in which depletion of protective Lactobacillus species permits uropathogens, particularly UPEC, to persist in the vaginal niche and ascend into the bladder. This vaginal-based etiology reframes the vagina, rather than the bladder alone, as a critical therapeutic target for long-term prevention. Lactobacillus vaginal suppositories, especially L. crispatus-based formulations, represent a biologically plausible and antibiotic-sparing approach that may help restore a protective vaginal microbiota and potentially reduce recurrence.

However, current evidence is still based on relatively small, heterogeneous studies, and these therapies cannot yet be considered guideline-standard care. Large, multicenter, randomized trials with standardized strains, dosing regimens, and well-defined target populations are needed to clarify efficacy, safety, and optimal integration with existing prophylactic pathways. If these gaps are addressed, vaginal Lactobacillus therapies may become a key component of microbiome-informed, antibiotic-sparing prevention strategies for RC.

Supplemental Material

sj-docx-1-tau-10.1177_17562872261448600 – Supplemental material for Unraveling the vaginal microbiota-based etiology of recurrent cystitis: the potential role of Lactobacillus vaginal suppositories

Supplemental material, sj-docx-1-tau-10.1177_17562872261448600 for Unraveling the vaginal microbiota-based etiology of recurrent cystitis: the potential role of Lactobacillus vaginal suppositories by Takanori Sekito, Hidetada Hirakawa and Takuya Sadahira in Therapeutic Advances in Urology

Footnotes

Acknowledgements

None.

Declarations

ORCID iD

Takanori Sekito

Supplemental material

Supplemental material for this article is available online.

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