Candida species are ubiquitous fungal commensals of the human mucosa that can become opportunistic pathogens, leading to a wide range of infections from superficial mucocutaneous overgrowth to life-threatening disseminated candidiasis (Kojic and Darouiche, 2004; Nett and Andes, 2020). A virulence trait contributing to many of the difficult-to-treat clinical infections caused by Candida is its ability to form biofilms, i.e., structured microbial communities that are attached to biotic or abiotic surfaces and embedded in an extracellular polymeric matrix (Ramage et al., 2005; Gulati and Nobile, 2016). Biofilm formation is particularly clinically relevant as it often forms on indwelling medical devices (e.g., vascular catheters, urinary catheters, prosthetic valves, endotracheal tubes) as well as on mucosal surfaces and surgical sites. Device-associated candidemia is associated with significant morbidity and mortality (Kojic and Darouiche, 2004; Nobile and Johnson, 2015). Biofilm cells exhibit collective phenotypes that differ from planktonic cells including altered metabolism, increased stress response, altered cell surface properties, and the secretion of a protective extracellular matrix (ECM) creating a niche environment, together all these factors contribute to their enhanced tolerance to antifungal therapy and immune clearance (Fanning and Mitchell, 2012; Cavalheiro and Teixeira, 2018; Nett and Andes, 2020; Atriwal et al., 2021).

The multifactorial antifungal tolerance of Candida biofilms is attributable to several contributions. The biofilm ECM contains polysaccharides, such as mannan-glucan complexes, extracellular DNA, proteins, and extracellular vesicle cargo, that together impede fungal penetration and may sequester antifungals, leading to reduced effective concentration at the cell surface (Roy and Gow, 2023; Nett and Andes, 2020). In addition, cells in mature biofilms exhibit altered gene expression patterns, with up-regulation of drug efflux pumps and stress-response pathways (heat-shock proteins, calcineurin signaling), metabolic shifts resulting in micro-niches (areas with hypoxia or nutrient limitation) that diminish activity from fungicidal drugs (Ramage et al., 2005; Robbins et al., 2011; Fanning and Mitchell, 2012). Adaptation at the community level, rather than individual resistance mutations, continues to explain why biofilm-associated infections do not respond to standard therapies, often requiring device removal or a combination of therapies for cure (Nobile and Johnson, 2015; Cavalheiro and Teixeira, 2018).

An additional clinically relevant phenomenon that occurs in biofilms, in some conditions, is the presence of persister cells. Persisters are phenotypic variants of an isogenic population that can tolerate, for a limited period, exposures to very high concentrations of antimicrobial agents, including antifungals, without developing inheritable resistance mutations. The earliest definitive experimental evidence of antifungal persisters in C. albicans biofilms was presented by LaFleur and colleagues, who demonstrated biphasic killing kinetics in biofilms exposed to amphotericin B or chlorhexidine. They reported on a small subpopulation that survived lethal exposures and then reseeded growth in exposed biofilms (LaFleur et al., 2006). The concept of lower viability subsequently continued to expand and become more nuanced. In many bacterial biofilms, persisters are considered a potential reservoir for relapse and the eventual emergence of genetic resistance. The same holds for fungal infections, particularly in relation to the potential for repopulation in biofilms depleted of an antifungal agent (Lewis, 2010). Persister cell biology in Candida is complex and is not universal. Some studies suggest that C. albicans biofilms rarely contain cells that resist the above products, indicating the formation of persistors is complex, strain-specific, and reliant on experimental conditions (Denega et al., 2019). Together, these data indicate that while persister cells likely contribute to treatment failure in some settings, they are one of several interacting mechanisms, alongside matrix sequestration, efflux activity, and metabolic heterogeneity, that produce the high tolerance of Candida biofilms.

Considering the limited antifungal classes available (azoles, polyenes, echinocandins) and the clinical challenges of eliminating biofilm-associated candidiasis, alternative or complementary treatment strategies are becoming of interest. Probiotic-derived metabolites (often termed “postbiotics” or “cell-free supernatants from probiotics”) are a class of interest. Bacterial probiotics (Lactobacillus/Lactiplantibacillus spp.) and probiotic yeasts (Saccharomyces boulardii) are capable of secreting organic acids (lactic acid, acetic acid, a variety of short-chain fatty acids), fatty acids (medium-chain fatty acids, like capric acid), and bacteriocin-like peptides and other small molecules that can suppress adhesion, hyphal transition, biofilm formation, and planktonic growth of Candida in vitro (Murzyn et al., 2010; García-Gamboa et al., 2024; Noverr and Huffnagle, 2004). Mechanistic studies indicate that some metabolites from probiotics downregulate hypha- and adhesion-associated genes (HWP1), disrupt membrane integrity, acidify the microenvironment, and interfere with quorum sensing and matrix production (Jiang et al., 2016; Ribeiro et al., 2017). Systematic reviews of postbiotics and other mechanistic studies extend the notion of characterized microbial metabolites as adjuncts that are stable and safe, avoiding some of the potential risks of live-microbe therapy, especially in immunocompromised patients (Malagón-Rojas et al., 2020; Zdybel et al., 2025; Prajapati et al., 2023; Aguilar-Toalá et al., 2021).

Recent epidemiological studies demonstrate substantial species-specific and geographic variability in antifungal resistance across different forms of candidiasis, as summarized in Table 1. Data from national surveillance programs and multicenter studies indicate that non-albicans Candida species, particularly Candida glabrata, C. tropicalis, C. parapsilosis, and C. auris, exhibit higher and more variable resistance rates compared with C. albicans, especially in cases of candidemia and other invasive infections. Resistance patterns also differ markedly by antifungal class and region; for example, echinocandin resistance in C. glabrata bloodstream isolates remains relatively low in the United States but shows an increasing trend, underscoring emerging therapeutic challenges. Collectively, these findings highlight the dynamic and region-dependent nature of antifungal resistance in Candida species and emphasize the need for continuous surveillance and alternative or adjunctive treatment strategies to manage resistant infections (Arendrup & Patterson, 2017; Pfaller et al., 2019; Nguyen, and Ren, 2025; Kaur et al., 2016). In parallel with these resistance trends, the global population of immunocompromised individuals, including patients with malignancies, transplant recipients, individuals with HIV infection, and those receiving immunosuppressive therapies, is steadily increasing, further amplifying the clinical burden and therapeutic complexity of resistant candidiasis (Sahu et al., 2026a; Sahu et al., 2025; Mallick et al., 2025; Das et al., 2026; Sahu et al., 2026b). Consequently, resistance rates are disproportionately higher among non-albicans Candida species in these vulnerable patient populations.

Importantly, treatment efficacy against Candida biofilms is highly strain- and species-dependent (Gulati and Nobile, 2016; Nett and Andes, 2020). Clinical and laboratory studies have demonstrated substantial variability among C. albicans isolates, as well as between C. albicans and non-albicans Candida species, in terms of biofilm architecture, extracellular matrix composition, efflux pump expression, stress-response activation, and persister cell formation (Gulati and Nobile, 2016; Denega et al., 2019; Nett and Andes, 2020). These differences directly influence susceptibility to conventional antifungals and adjunctive strategies, including probiotic-derived metabolites (Kaur and Nobile, 2023). As a result, therapeutic responses observed in one strain or species cannot be universally extrapolated, underscoring the need for strain-inclusive evaluation when assessing anti-biofilm and anti-persister interventions (Denega et al., 2019; Kaur and Nobile, 2023).

The aim of this review is to summarize recent mechanistic and clinical evidence about how the combination of Candida biofilms and persister cells leads to antifungal tolerance, and the emerging literature on probiotic-derived metabolites as anti-biofilm and anti-persister therapy, emphasizing the mechanistic data, advantages and limitations of existing in vitro and in vivo studies, and potential future “postbiotic” therapies for adjunctive treatment.

Candida biofilms are characterized by the presence of an ECM, a dynamic, self-formed structure of macromolecules that encases sessile cells and alters the microenvironmental parameters of the biofilm community. Biochemical and compositional analyses of multiple Candida species indicate that the ECM has a high composition of polysaccharides, notably α-mannans and branched α-1,6/α-1,2 mannan and some β-glucan species, notably β-1,6 glucan, extracellular DNA (eDNA), and a diverse protein repertoire (enzymes and structural proteins) as well as some lipids and extracellular vesicle cargo (Pierce et al., 2017; Nett and Andes, 2020). The components of the ECM will vary in stoichiometry during different stages of biofilm development and differ among species; however, their relative presence creates a hydrated, viscoelastic gel that serves to stabilize intercellular connections, promote adhesion to surfaces, and sequester factors from both the host and/or other microbes. Recent reviews and experimental studies suggest that the ECM is not simply a passive scaffold: polysaccharides of the matrix and matrix-associated proteins actively bind small molecules and drugs, and matrix-derived extracellular vesicles provide additional enzymatic and structural factors that stabilize the integrity of the matrix (Roy and Gow, 2023; Pierce et al., 2017). Because of these properties, the matrix both physically slows the diffusion of antifungal agents and chemically interacts with them (for example, binding azoles or polyenes), thereby reducing the effective antifungal exposure to cells embedded deep in the biofilm (Nett and Andes, 2020; Roy and Gow, 2023).

Efflux pumps create an additional, complementary layer of tolerance associated with the existence of biofilms. Gene profiling and activity studies indicate that members of two major families of transporters, ATP-binding cassette (ABC) transporters (CDR1, CDR2) and the major facilitator superfamily (MFS, e.g. MDR1), are upregulated in the early biofilm community. However, persistence can occur in the mature biofilm (Atriwal et al., 2021). Upregulation may occur prior to any antifungal exposure, emphasizing that the activation of efflux is an inherent biological feature of the biofilm lifestyle, rather than a strictly downstream response to drugs. Inhibition of efflux, an experimental paradigm, has demonstrated reduced biofilm tolerance in in vitro models. Recent reviews suggest that efflux-mediated drug export is an actionable target to sensitize biofilms to conventional agents (Ren et al., 2024; Kaur and Nobile, 2023). It is essential to acknowledge that efflux contributes differently to tolerance, depending on both the drug class (efflux being particularly informative for azoles) and the fungal species, based on their transporter repertoire and expression time course.

Cellular stress-response pathways represent another key mechanism of survival for biofilm cells challenged with antifungal drugs. Biofilm growth is linked to the activation of highly conserved stress-response signaling cascades, primarily the heat shock protein (Hsp) network (Hsp90/Hsp70 and their co-chaperones), the calcineurin signaling pathway, and downstream gene expression responses, all of which help maintain protein folding, cell wall integrity, and ionic homeostasis under drug- and host-imposed stressors (Gong et al., 2017; Cowen, 2009; Roy and Tamuli, 2022; Ren et al., 2024). Hsp90, in particular, serves to stabilize client proteins, including calcineurin, important for tolerating azole, echinocandin, and polyene fungicidal activity; either pharmacologic or genetically induced destabilization of Hsp90-calcineurin signaling enhances fungicidal activity against biofilms (Cowen and Lindquist, 2005; LaFayette et al., 2010; Singh et al., 2009; Gong et al., 2017; LeBlanc et al., 2020). Recent mechanistic work also demonstrates that stress-response activation is mechanistically linked to biofilm-specific signaling phenotypes, including matrix biogenesis, metabolic reprogramming, and oxidative-stress resistance and persistence formation, which shows that stress pathways protect both individual cells and function to support community-level responses (Ren et al., 2024; Wang et al., 2025; Robbins et al., 2011; Uppuluri et al., 2018). Together, these signaling systems represent central regulators of drug tolerance in Candida biofilms and are key targets for antifungal-sensitizing strategies.

Modified membrane and cell physiology, specifically modulation of sterol biosynthesis and membrane composition, further reduces susceptibility to drugs in biofilms. Upregulation or modulation of genes involved in the ergosterol biosynthetic pathway (ERG family), as well as modifications to sterol content, may alter the binding or activity of polyenes, and in the case of azoles, change the abundance of the target enzyme (lanosterol 14α-demethylase), which affects its activity. Biofilm cells also remodel the components of their plasma membrane lipid composition and cell wall architecture, which contribute to physical and biochemical changes that reduce drug uptake or increase tolerance to membrane-directed stress (Atriwal et al., 2021; Kaur and Nobile, 2023). These changes to membranes are often integrated alongside efflux and stress-response pathways, producing layered irregularities that contribute to tolerance.

Metabolic variation in biofilms, including areas of nutrient limitation, oxygen gradients, and slow-growing or metabolically quiescent subpopulations, plays a crucial role in determining the efficacy of drugs. Many antifungals exert activity during the specific metabolic state of active growth and/or divided cells; therefore, the cells in micro-niches with low metabolic activity or hypoxia can tolerate exposures to the agent, while planktonic cells proliferating in their exponential phase can be killed (Pierce et al., 2017; Wall et al., 2019). This metabolic downregulation is also important when generating and/or maintaining persister cells, which are transiently dormant, drug-tolerant phenotypes that can reseed biofilms when they are no longer under drug pressure. Together, the interactions in time and space of matrix-mediated sequestration, efflux, stress-response buffering, membrane remodeling, and metabolic quiescence produce the antifungal tolerance seen in clinical biofilms. This complexity explains why single-target therapies often fail and why a multi-modal or adjunctive approach is indicated to eradicate device-associated and mucosal biofilm infections. Table 2 summarizes the central cellular, biochemical, and structural mechanisms contributing to antifungal tolerance in Candida biofilms.

Candida biofilms are composed of a unique population of phenotypic variants that survive the use of fungicidal agents but do not pass on their traits through hereditary means. They are considered a non-heritable group of persister cells because they do not contain stable mutations, unlike the resistant mutant forms of fungi, which result in an increased minimal inhibitory concentration (MIC). The persisters survive drug exposure by being transiently tolerant, metabolically altered, or dormant until removed from the drug treatment. Only in this state can persister cells maintain their population density during the course of treatment and rebuild their population numbers once treatment ceases (Lewis, 2010; LaFleur et al., 2006). The persistent state occurs in fungal biofilms and has been defined in terms of its clinical significance and application (Denega et al., 2019; Uppuluri et al., 2018; Delarze and Sanglard, 2015).

Experimentally, the presence of persister cells in Candida biofilms is most often inferred from biphasic killing curves: when mature biofilms are challenged with high concentrations of a fungicidal agent, a rapid initial kill reduces the majority of the population but is followed by a plateau or much slower kill rate in which a small surviving fraction persists despite prolonged exposure (Lewis, 2010; LaFleur et al., 2006). LaFleur et al.’s classic study demonstrated this biphasic response in C. albicans biofilms exposed to amphotericin B and chlorhexidine and identified surviving cells that could reseed biofilm growth after drug removal, the operational signature of persisters (LaFleur et al., 2006). Subsequent studies across Candida species and clinical isolates have reproduced biphasic kinetics in many, but not all, settings, and the size of the persister fraction is highly variable, depending on the strain, substrate, and growth conditions (Denega et al., 2019).

Instead of single-gene resistance mechanisms, molecular investigations of persister cells indicate coordinated transcriptional and metabolic reprogramming (Lewis, 2012). Differential regulation of genes involved in sterol biosynthesis (ERG1, ERG25) and cell-wall/matrix polysaccharide pathways (including β-1,6-glucan-associated genes like SKN1 and KRE1) has been reported in comparative expression studies and targeted analyses of biofilm subpopulations. These findings are consistent with altered membrane and cell-wall physiology in persister-enriched cells (Attavar, 2010). Additionally, persisters exhibit gluconeogenic flow and energy storage pathways (trehalose and glycogen buildup), which are metabolic indicators of a transition to a quiescent, stress-protected state (Kuczyńska-Wiśnik et al., 2015; Wuyts et al., 2018). These changes plausibly reduce the efficacy of drugs that rely on active metabolism or target ergosterol biosynthesis, while simultaneously helping persisters tolerate membrane- or ROS-mediated damage.

Persister survival is mainly dependent on stress-response mechanisms. Persisters are frequently shielded from drug-induced oxidative stress and apoptosis-like pathways by molecular chaperones (most notably Hsp90 and small heat-shock proteins), the calcineurin signaling pathway, and enhanced antioxidant defenses (superoxide dismutases and other ROS-detoxifying enzymes) (O’Meara et al., 2017; Gong et al., 2017; Li et al., 2021). In particular, Hsp90 stabilizes important signaling clients, such as calcineurin, allowing cells to mount efficient cell-wall and membrane stress responses; in several models, disruption of Hsp90 or calcineurin makes biofilm cells more susceptible to antifungal agents (O’Meara et al., 2017; Gong et al., 2017; Li et al., 2021). Furthermore, persisters frequently display upregulation of the ROS-detoxification machinery and stress-protective storage molecules (trehalose, glycogen), which together blunt the lethal effects of fungicide-induced oxidative damage and support recovery once the drug pressure subsides (da Silva et al., 2021; Kuczyńska-Wiśnik et al., 2015; Wuyts et al., 2018).

Despite strong evidence for persisters in numerous experimental systems, significant debates and methodological limitations persist in the field. Some of the earlier findings may represent heterogeneous drug penetration, transient microenvironmental drug gradients, or assay artifacts rather than actual phenotypic persistence because several meticulously controlled reports have failed to find a stable persister subpopulation in some C. albicans strains or under particular experimental protocols (Denega et al., 2019). Whether biphasic killing is observed depends on several factors, including inoculum, substrate (plastic vs. mucosal tissue), biofilm age, drug exposure schedules, and detection sensitivity. As a result, apparent disagreements in the literature frequently reflect experimental context rather than a single biological phenomenon (Wuyts et al., 2018). Consensus reviews emphasize that persister formation in Candida is highly context- and strain-dependent, and that rigorous assay standardization and single-cell analyses are needed to resolve outstanding discrepancies.

Clinically, persister cells are invoked as a possible explanation for relapsing device-associated infections, when removal of the biofilm source is often necessary for treatment, and recurring mucosal infections (such as recurrent vulvovaginal candidiasis). Although direct in-patient persister identification is still technically challenging, in vivo and ex vivo biofilm models (rodent catheter and denture models, tissue explants) replicate biofilm architecture and have been used to show treatment failure and regrowth consistent with persister-mediated relapse (Li et al., 2015; LaFleur et al., 2006). To prevent relapse, strategies targeting metabolic dormancy, stress-response buffering (Hsp90/calcineurin), ROS detoxification, or matrix-associated protection are being actively investigated as supplements to standard antifungal regimens (Wuyts et al., 2018; Robbins et al., 2011). The major physiological and molecular features of Candida persister cells are summarized in Table 3.

In in vitro studies and increasingly in preclinical studies, numerous types of metabolites (secretory or chemically diverse) secreted by yeasts and probiotics include short- and medium-chain fatty acids, organic acids (e.g., lactic acid and acetic acid), biosurfactants, and small-sized aromatic compounds. Demonstrate antifungal and anti-biofilm effectiveness against the increasing prevalence of Candida spp. In early research and well-referenced studies, Saccharomyces boulardii culture filtrate (containing medium-chain fatty acids, with capric acid (C10:0) as the main fatty acid) has demonstrated the following: inhibiting hyphae formation, promoting hyphal transition, decreasing C. albicans’ adherence, and lowering biofilm formation by inhibiting virulence-related genes (HWP1 and CSH1) (Suchodolski et al., 2021). Multiple, later studies from all five genera of probiotic bacteria have confirmed and expanded on these findings, including culture filtration (“post-biotic”) of Lactobacillus spp. (including L. rhamnosus, L. casei, L. acidophilus, and L. plantarum) demonstrated that these products were capable of inhibiting the initial phases of C. albicans biofilm formation and to impart a reduction in adhesion to non-living surfaces and to inhibit the yeast-to-hypha formation of Candida, a critical morphologic switch for the development of a well-structured biofilm Architecture. These inhibitory effects are observed both when probiotics are present as live cells and when only their secreted metabolites (postbiotics) are applied, indicating that soluble molecules alone can mediate a significant portion of the anti-Candida activity (Matsubara et al., 2016; García-Gamboa et al., 2024).

The degree to which probiotics affect various pathogens will depend on the type of probiotic strain, the developmental stage of the pathogens, and whether the strains have been previously exposed to their target pathogens. Laboratory-made products using L. rhamnosus and L. casei supernatants were effective in decreasing the amount of biofilm formed during the initial stages of Candida infection and preventing the growth of filamentous fungi (Matsubara et al., 2016; Song and Lee, 2017). For Candida infections caused by non-albicans species, both L. gasseri and L. rhamnosus disrupted the established biofilms (after 24 hours of growth) by decreasing the number of viable cells present in those biofilms (Tan et al., 2018). Certain types of probiotic products (e.g., defined L. plantarum postbiotic products) also show a similar level of efficacy against candidiasis infection in laboratory-developed (artificially created) mycotic (fungus) populations, and to some degree in mixed populations of Candida spp., suggesting that their effectiveness might be enhanced within larger mixed populations of varying microorganisms (García-Gamboa et al., 2024). Pediococcus-derived strains (e.g., Pediococcus acidilactici HW01) have also been shown to exhibit similar anti-Candida activities by disrupting biofilm growth and formation. However, the factors responsible for this inhibition are primarily due to the production of organic acids, rather than traditional peptide-type bacteriocins (Kim and Kang, 2019). Together, these studies indicate that the potential of commercially available probiotic secretomes to inhibit biofilms formed by multiple species of microorganisms is quite wide (there are likely many different strains in the family of probiotic bacteria that have inherent properties to inhibit the formation of biofilms within the same family). A detailed summary of key probiotic-derived metabolites, their mechanisms, and anti-Candida effects is presented in Table 4.

Lactobacilli produce organic acids (lactic, acetic and short-chain fatty acids) that help lower the local environment’s pH, thus making it difficult for filamentation and biofilm formation to occur. In addition, these organic acids may help “uncouple” the cellular metabolic activities of certain types of fungi from their ability to respire normally, resulting in a hypersensitivity to other stress agents (Paniágua et al., 2021; García-Gamboa et al., 2024). Medium-length chain fatty acids (for example, capric acid) can intercalate into fungal cell membranes, disrupting their normal hydrophobic properties and interfering with their ability to adhere to one another through their biophysical attachment sites. Furthermore, certain biosurfactant-producing probiotic microorganisms can lower the surface tension of liquid food products, thereby inhibiting the formation of stable membranes upon contact with non-biological surfaces (Murzyn et al., 2010). These observations have been confirmed by numerous studies, which have demonstrated that exposure to probiotics leads to the downregulation of genes associated with virulence and biofilm development in the fungi being exposed (Murzyn et al., 2010; Poon and Hui, 2023).

Additionally, multiple studies have demonstrated that the metabolites produced by probiotics can interact with those produced by other naturally occurring products or traditional antifungal medications. Several reported instances have demonstrated that combining honey and probiotic filtrates or low-dose antifungals with probiotic postbiotics leads to decreased biofilm biomass and reduced viability, as measured in vitro. The synergistic effect of probiotic postbiotics weakening certain defenses (adhesion, matrix, morphology) of multi-species biofilms would ultimately restore access to antifungals as well as potency against the pathogens (García-Gamboa et al., 2022). Mixed species biofilms, which typically contain both Candida and bacteria on either a mucosal surface or a device surface, are particularly effective in responding to the actions of probiotic filtrates. Data has shown that treatment with Lactobacilli supernatants or combinations of other probiotics results in lower polymicrobial biofilm thickness and lower matrix density than does treatment with a non-probiotic agent (Song and Lee, 2017; García-Gamboa et al., 2024).

Despite promising in vitro and ex vivo findings, significant limitations and gaps persist between research and clinical practice. The effectiveness of postbiotics is variable based on strain and method of preparation: Only some Lactobacillus strains or methods of preparing postbiotics produce antifungal activity that is comparable to that produced by other strains or methods of preparing postbiotics; and the concentrations that are effective in vitro are often greater than those that can be achieved in vivo without delivering the product directly to the desired site of action (via a targeted delivery system). The stability, standardization, and safety of concentrated postbiotics require assessment, particularly for individuals who are immunocompromised. The use of concentrated postbiotics in immunocompromised patients may be contraindicated, particularly when using probiotic forms of Lactobacillus. Coatings for devices or topical formulations that contain defined postbiotics are two methods of providing high concentrations at the desired site of action while also minimizing the risk of exposure to postbiotics in other locations (García-Gamboa et al., 2024; Murzyn et al., 2010). Most of the evidence regarding the ability of postbiotics to prevent or treat biofilm-associated candidiasis remains preclinical. Very few randomized clinical trials have evaluated the effect of postbiotics on the prevention/treatment of biofilm-associated candidiasis; however, the available data from both mechanistic and animal-model studies provide strong justification for developing these products for clinical use.

Available evidence indicates that probiotic-derived metabolites display both concentration-dependent and time-dependent antifungal activity against Candida species. Several studies demonstrate that increasing metabolite concentration enhances immediate inhibition of fungal growth, filamentation, and biofilm formation, whereas prolonged exposure, particularly at sub-inhibitory levels, progressively disrupts biofilm maturation, extracellular matrix integrity, and fungal viability. These pharmacodynamic patterns have been consistently observed across organic acids, fatty acids, and complex postbiotic preparations, highlighting that exposure duration is a critical determinant of anti-biofilm efficacy in addition to dose. These findings support the need for sustained delivery strategies when translating probiotic-derived metabolites into clinical or device-associated applications (Murzyn et al., 2010; Matsubara et al., 2016; Paniágua et al., 2021; García-Gamboa et al., 2024).

To accelerate the identification of postbiotics that can effectively target biofilm tolerance, a high-throughput discovery of metabolites and secretomes that act as anti-persisters will be necessary. There is growing evidence that a persister-based screen is possible, considering the development of a persister-based assay that can scale, as demonstrated by Petersen et al (Petersen et al., 2024). This really underscored the possibility of developing a drug-susceptibility assay that screens explicitly for compounds that are active against persister cells. They demonstrated this in the context of bacterial persisters, while addressing experimental parameters (i.e., starvation, exposure regimen) that enriched for persistent states, based on the design of their screen, which enabled the testing of large libraries of chemical compounds. In principle, similar persister-aware high-throughput screens could be adapted to investigate probiotic secretomes (i.e., cell-free supernatants and fractionated metabolomes) to systematically identify molecular motifs capable of killing or sensitizing fungal persisters, rather than solely inhibiting growth. Such screening would follow or combine automated fractionation (LC-MS guided), reliable miniaturized persister assays, and orthogonal readouts (viability, metabolic reporters, single cell imaging) to limit false positives that inhibit or kill only growing cells (Petersen et al., 2024).

Research in probiotic synthetic biology and metabolic engineering opens another promising avenue: it is possible to rationally engineer probiotics to overproduce specific antifungal metabolites, biosurfactants, or even secrete modified enzymes capable of degrading elements found within fungal matrices. Recent reviews and empirical studies have demonstrated that CRISPR/Cas, recombineering, and heterologous-expression toolkits enable the precise engineering of lactic acid bacteria and other probiotic chassis (Mugwanda et al., 2023; Ma et al., 2022). Engineered strains could establish localized factories (e.g., a vaginal gel or denture coating containing an engineered Lactobacillus capable of excreting the stable anti-biofilm peptide), or the secretome of the engineered strain could be purified and developed as a ready-defined postbiotic while mitigating the outset risks of using live microbes with the highest-risk patients (Yang et al., 2025; Ling et al., 2023).

Ultimately, screening and mechanistic investigations must occur in physiologically relevant models. Although simple microtiter-plate biofilm assays are ideal for initial triaging, they do not recapitulate the host factors (flow, shear, and adsorbed host proteins), polymicrobial interactions, immune effectors, or device materials. Achieving the translational goal will require the careful standardized use of ex vivo and in vivo models (catheter and denture rodent models, organoids and mucosal explants, and microfluidic “organ-on-chip” systems) that will permit controlled gradients and polymicrobial communities to be evaluated for both efficacy and host interactions with Candidate functional postbiotics and engineered probiotics (Nett et al., 2010; Carvalho et al., 2020; Andes et al., 2004; Řičicová et al., 2010). These models will further test whether Candidate metabolites penetrate the matrix, reach persister niches, or remain active in complex biological fluids.

Candida biofilms continue to pose a considerable therapeutic challenge due to their complex combination of biofilm-mediated resistance mechanisms and surviving persister cells that cause antifungal resistance and infection relapse. Recently, metabolites derived from probiotics have emerged as novel adjunctive agents that can inhibit adherence, reduce hyphal development, disrupt biofilm structure, decrease virulence, and potentially impact persister-like subpopulations. Although results are encouraging in vitro, challenges related to metabolite stability, barriers to delivery, as well as strain variability and safety issues, need to be addressed before considering clinical use. Continued mechanistic studies, improved biofilm and persister models, refined metabolite formulations, and carefully controlled clinical trials are necessary to fully realize the benefits of postbiotics in combination with integrated approaches for treating persistent Candida infections.