Abstract
Background/Objectives: Alternative therapies for urinary tract infections (UTIs) have been explored, but their efficacy remains inconsistent. With rising antibiotic resistance, this study aimed to evaluate simplified postbiotic formulations derived from heat-killed probiotics for long-term protection against primary and recurrent UTIs in a murine model.
Methods: We compared a multi-strain (seven-strain) versus a single-strain postbiotic in preventing Escherichia coli-induced UTIs and recurrent polymicrobial UTIs, assessed protection persistence after treatment discontinuation, and established a novel sustained UTI model via intravesical co-inoculation of three uropathogens. Mice were allocated to three experimental groups: a placebo group (PBS), Postbiotic I group (a seven-strain heat-killed probiotic formulation), and Postbiotic II group (a single-strain heat-killed probiotic). After two weeks of treatment, mice were challenged with uropathogenic E. coli (UPEC) and treated for seven days. Following a 14-day washout and bacterial clearance, they were rechallenged with multidrug-resistant UPEC, Klebsiella pneumoniae, and Staphylococcus pseudintermedius.
Results: Both postbiotics significantly accelerated bacterial clearance in primary UTIs (p < 0.05). In recurrent UTIs, placebo-treated mice exhibited persistent bacteriuria, while Postbiotic I maintained a significantly higher sterile urine rate (50–80%, p < 0.01) post-treatment. Histopathological analysis confirmed reduced bladder and kidney inflammation (p < 0.05) with Postbiotic I.
Conclusions: These findings demonstrate the superior efficacy of Postbiotic I in mitigating UTIs, with sustained protection post-treatment, supporting its potential as a long-term, non-antibiotic strategy. Additionally, our reproducible chronic UTI model, achieved through the co-inoculation of three uropathogens, provides a valuable tool for future research on chronic UTI pathogenesis and treatment.
Introduction
Urinary tract infections (UTIs) are among the most prevalent bacterial infections globally, affecting individuals across all age groups and genders in both community and healthcare settings [1]. For example, a recent study reported that the global prevalence of UTIs in older adults is 23.6% (95% Confidence Interval [CI]: 19.4–28.4). The highest prevalence was observed in specific subgroups, including individuals in Africa (30%; 95% CI: 12.7–55.8), women (30%; 95% CI: 14.6–51.7), those diagnosed via urine culture (25.3%; 95% CI: 18.3–33.8), and nursing home residents (47.2%; 95% CI: 24.2–71.5) [2]. Thus, UTIs represent a significant clinical concern worldwide.
UTIs typically begin when uropathogens colonize the urethra and ascend to the bladder, forming biofilms and evading immune defenses. Progression to the kidneys can also lead to bacteremia [3,4]. The most common uropathogens include Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Enterococcus faecalis, and Staphylococcus saprophyticus [5]. Moreover, the standard treatment for UTIs involves oral antibiotics; however, prolonged use disrupts the vaginal and gastrointestinal microbiota and promotes multidrug resistance [6,7].
Moreover, although prophylactic antibiotics can reduce recurrent UTIs by up to 85%, concerns regarding antibiotic resistance and potential side effects still underscore the urgent need for alternative therapeutic approaches [8,9]. Importantly, distinguishing asymptomatic bacteriuria (ASB) from true UTI is crucial, as antibiotic treatment is often unnecessary for non-pregnant patients with ASB [4]. Collectively, non-antibiotic strategies—such as live probiotics, dietary supplements (e.g., cranberry, D-mannose), and herbal medicine—have shown promise, but their clinical efficacy remains inconsistent across studies, as further discussed below [10,11,12].
A recent randomized controlled trial found that probiotics, when used as an adjunct to conventional therapy in pregnant women with recurrent UTIs, significantly improved clinical indicators such as leukocyte and nitrite levels compared to placebo [13]. However, the small sample size (n = 60) limits the generalizability of these findings. A meta-analysis likewise found no conclusive evidence that probiotics outperform placebo or no treatment in UTI prevention [14]. While potential benefits cannot be entirely dismissed, the evidence remains limited and methodologically inconsistent. Comparisons with antibiotics also yield inconclusive results [14]. Although some studies report benefits in pediatric cases [15,16], a noninferiority trial in postmenopausal women showed that Lactobacillus rhamnosus GR-1 and L. reuteri RC-14 were less effective than trimethoprim–sulfamethoxazole, though they did not contribute to antibiotic resistance [17].
Overall, clinical evidence supporting the widespread use of probiotics for UTI prevention remains limited. Challenges such as selecting appropriate strains, determining optimal dosing, and ensuring product consistency continue to hinder broader clinical adoption [18]. These limitations highlight the need for alternative approaches. In this context, non-viable probiotic preparations (postbiotics) offer a promising strategy, with the potential to provide enhanced safety, stability, and immunomodulatory benefits—features that are explored in the present study.
Postbiotics, as defined by the International Scientific Association for Probiotics and Prebiotics (ISAPP), are “inanimate microorganisms and/or their components conferring health benefits” and offer several advantages over live probiotics, including enhanced safety and stability [12,13]. However, some reports continue to define postbiotics as “non-viable bacterial products or metabolic byproducts produced by probiotic microorganisms that exert biological activity in the host” [19]. To minimize ambiguity, in the present study, we define postbiotics as non-viable, non-replicating probiotic microorganisms, as only heat-killed probiotic cells were used.
Nevertheless, the efficacy of postbiotics (inactivated probiotic cells) in preventing and treating tract UTIs remains largely unexplored. To address this gap, we first demonstrated that 10 heat-killed lactoferrin-expressing (LF-expressing) probiotic strains exhibited superior antibacterial efficacy compared to 12 naturally occurring probiotics. Furthermore, we showed that a three-week supplementation with a viable probiotic mixture (LAB), a heat-killed probiotic mixture (HK-LAB), or a heat-killed LF-expressing probiotic mixture (HK-LAB/LF) significantly reduced daily bacteriuria by 103- to 104-fold in a primary Escherichia coli-challenged UTI mouse model. These previously defined heat-killed probiotic formulations are referred to as postbiotics in the current study to ensure consistent terminology. Additionally, these treatments improved the bacteriological cure rate (BCR) with varying degrees of efficacy [20]. To further elucidate the therapeutic mechanism of postbiotic strains, we demonstrated that five additional postbiotic strains exhibited significant in vitro biofilm inhibition and dispersal activity.
Notably, RT-qPCR analysis revealed that these inactivated probiotics downregulated genes associated with pili and biofilm formation (fimA, csgA) while upregulating genes involved in quorum sensing (luxS, qseBC, sdiA). These findings suggest that postbiotic treatment may inhibit pili and biofilm formation while promoting biofilm dispersion. Additionally, we have previously tested postbiotic composites containing 5 and 16 heat-killed probiotic strains. Both formulations significantly reduced bacterial load and morbidity in a primary UTI mouse model, with the 16-strain mixture reducing morbidity from bacteriuria by up to 30% within five days post-infection compared to placebo. However, preparing and producing formulations with higher numbers of strains involves greater complexity and labor intensity, which may hinder large-scale application. These findings provided the basis for the current study’s focus on a simplified postbiotic mixture containing either one or seven strains, selected for their potent anti-biofilm and antimicrobial activities, aiming to maintain comparable efficacy while enhancing production feasibility [15]. Furthermore, whether inactivated probiotics can effectively treat recurrent UTIs remains uncertain, particularly in cases involving severe antibiotic-resistant strains and high bacterial loads in urine.
In this study, we evaluate the efficacy of single-strain and a simplified seven-strain postbiotic formulation against both primary and recurrent UTIs. Based on our prior work with larger multi-strain heat-killed probiotic mixtures, we aim to determine whether a reduced number of selected strains can maintain comparable protective effects while improving practicality for production and application. Crucially, we also investigate the duration of protection conferred by these inactivated probiotics, assessing whether they provide sustained defense against recurrent UTIs even after a prolonged discontinuation period (two weeks). To our knowledge, this is the first study to demonstrate that distinct postbiotic formulations effectively prevent and manage both primary and recurrent severe antibiotic-resistant UTIs in a murine model, even after significant treatment cessation, highlighting their potential for long-lasting protection.
Download the full article as PDF here Oral Administration of Heat-Killed Multi-Strain Probiotics Confers Durable Protection Against Antibiotic-Resistant Primary and Recurrent Urinary Tract Infections in a Murine Model
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Chen, B.-Y.; Liu, Z.-S.; Lin, Y.-S.; Lin, H.C.; Chen, P.-W. Oral Administration of Heat-Killed Multi-Strain Probiotics Confers Durable Protection Against Antibiotic-Resistant Primary and Recurrent Urinary Tract Infections in a Murine Model. Antibiotics 2025, 14, 634. https://doi.org/10.3390/antibiotics14070634
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