OM-89 may strengthen antimicrobial defenses in bladder epithelial cells and limit the regrowth of uropathogenic Escherichia coli following antibiotic treatment by enhancing lysosomal function and intracellular antibiotic accumulation.
Researchers investigated the direct effects of OM-89, an orally administered bacterial lysate used for recurrent UTIs, using mouse and human bladder organoids and differentiated epithelial monolayers. Mouse organoids were infected with uropathogenic E coli (UPEC) and exposed to OM-89 either72 hours prior to infection followed by a 24-hour rest period, with antibiotic treatment, or continuously for 72 hours prior to infection and throughout the study. The continuous regimen was used for subsequent experiments because it reflected sustained exposure.
The primary functional assessments were bacterial clearance during antibiotic treatment and bacterial regrowth following antibiotic withdrawal. Additional tests examined intracellular bacterial burden, antibiotic accumulation, endosomal and lysosomal remodeling, lysosomal acidification and protease activity, and autophagic flux. Transcriptomic profiling was used to characterize pathways altered by OM-89, and pharmacologic inhibition tested whether the antimicrobial effect required lysosomal acidification.
OM-89 did not alter bacterial burden during the initial infection phase in mouse bladder organoids, suggesting that it did not exert detectable direct antibacterial activity under the conditions. During antibiotic treatment, bacterial burden was reduced when OM-89 was present, and postantibiotic regrowth was reduced under all 3 exposure regimens. The strongest effect occurred when OM-89 was present during antibiotic treatment, with reduced regrowth for up to 8 hours following OM-89 withdrawal.
In differentiated mouse bladder epithelial cells, OM-89 increased intracellular accumulation of fluorescently labeled ampicillin and gentamicin. At 1 times the minimum inhibitory concentration of ampicillin, OM-89 reduced bacterial burden during 3 hours of antibiotic treatment and suppressed regrowth for up to 3 hours posttreatment. Colony-forming unit measurements showed reduced intracellular bacterial burden following gentamicin treatment, whereas the additional reduction following subsequent ampicillin exposure was not statistically significant. OM-89 did not alter the minimum inhibitory concentrations of either antibiotic.
The effects extended to human bladder models. In primary human bladder epithelial cells, OM-89 increased the number and size of late endosomal and lysosomal vesicles, increased lysosomal acidification and cathepsin L activity, and enhanced intracellular uptake of fluorescently labeled ampicillin. In contrast with mouse cells, OM-89 did not restore autophagic flux during UPEC infection, indicating that some components of the response differed between species.
In human bladder organoids, OM-89 did not affect bacterial burden during the initial infection phase. Bacterial burden was not significantly reduced during ampicillin treatment at 7 hours, but OM-89 significantly reduced bacterial regrowth at 10 hours following antibiotics. Regrowth originated preferentially from the organoid wall rather than the lumen, supporting the epithelial layer as the principal site of bacterial regrowth following antibiotic withdrawal.
Transcriptomic profiling identified the lysosomal pathway as the most strongly induced gene signature during OM-89 treatment and infection. OM-89 increased lysosomal acidification and brought intracellular UPEC into closer proximity to cathepsin L–positive proteolytic compartments. During infection, mean intraorganellar pH was 6.58 with OM-89 compared with 7.5 in controls, while cathepsin L proximity enrichment was 1.25 with OM-89 vs. 0.91 with control treatment.
The requirement for lysosomal function was supported by pharmacologic inhibition. Blocking lysosomal acidification with bafilomycin A1 or chloroquine abolished the OM-89–associated reduction in postantibiotic bacterial regrowth, restoring regrowth to control levels. The researchers concluded that lysosomal acidification and protease activity were required for the enhanced control of intracellular bacteria.
The phenotype was observed across different antibiotics and UPEC strains. OM-89 reduced bacterial burden during treatment and subsequent regrowth with fosfomycin and trimethoprim-sulfamethoxazole. Enhanced clearance and reduced regrowth were observed with UTI89, J96, and 2 primary clinical isolates. OM-89 did not alter bacterial burden during the initial growth phase in these experiments, supporting an effect that emerged in conjunction with epithelial antimicrobial defenses and antibiotic treatment.
The study had several limitations. OM-89 was applied directly to bladder epithelial cultures and organoids, whereas it is administered orally in clinical practice. They also did not address how the epithelial responses interact with systemic and immune-mediated effects under physiologic administration conditions. Fluorescence-based measurements in organoids captured the overall intraorganoid bacterial population without distinguishing luminal from intracellular bacteria, potentially masking early effects within epithelial cells. Fluorescent labeling can alter antibiotic behavior, making the antibiotic-uptake tests proxies rather than direct measurements of native antibiotic activity.
“[O]ur findings support the translational relevance of OM-89 as a clinically used therapy whose mechanism of action involves direct modulation of epithelial antimicrobial pathways,” wrote lead study author Kathrin Tomasek, of the Laboratory of Microbiology and Microtechnology at the École Polytechnique Fédérale de Lausanne, and colleagues.
Full disclosures of the study authors can be found in the study.
Source: PLOS Pathogens
