Among virulence factors for pathogens, biofilms are associated with a broad array of topical infections, including periodontal diseases, chronic wounds, and vaginosis (1). The burden of biofilm-related disease contributes to patient morbidity and increased mortality rates, thus representing a major public health issue in developed countries, which adds over $1 billion to US hospitalization costs annually (2, 3). Biofilm is a structured community of microorganisms that irreversibly adhere to an abiotic surface or biological tissue and represents a phenotypically distinct state from the planktonic counterpart, particularly in terms of growth rate and gene expression (1, 4).

Biofilms are morphologically distinguishable by the presence of a self-produced extracellular polymeric matrix that encloses microbial cells, offering great protection against chemical disinfectants, antimicrobials, and human immune response. The host immune system is not only ineffective in fully eradicating mature biofilm, but the presence of antibodies in the surrounding environment can contribute to tissue damage (5). Moreover, sessile microbial biofilm cells are up to 1,000-fold more resistant to antibiotics than planktonic cells, mainly due to the difficulty of high-molecular-weight antibiotics to penetrate the viscous matrix and to reach the deeper layers of biofilm (6, 7). The antimicrobial tolerance can be also ascribed to the presence in the biofilm core of a subpopulation of persistent cells, which in virtue of their dormant nonproliferative phenotype are not susceptible to mechanisms of action of common antibiotics (6, 8). Other factors, such as the slow growth rate, the presence of multidrug efflux pumps, and stress response regulation, and the peculiar biofilm microenvironment characterized by differences in pH, pCO₂, and pO₂, contribute to reducing the susceptibility of biofilm to antimicrobials (9). Furthermore, the higher cell density in biofilms favors the horizontal gene transfer, which in turn, increases the probability of emergence of strains with resistance or altered virulence profiles (10, 11). Although antimicrobial treatment can alleviate the symptoms of infection by killing the free-floating microorganisms disseminated from the adherent population (12), it usually fails to completely eradicate pathogens in the biofilm. As a result, when antibiotic therapy stops, the persistent cells can revert to their phenotype (13), causing a relapse of the condition and potentially leading to recurrent or chronic infections (5). Given the importance of clearance of mature biofilm for the successful resolution of such infections, alternative strategies to the use of antibiotics are highly desirable. These may include the use of plant-derived compounds, antimicrobial peptides from various sources and probiotics (14). Traditionally, probiotic microorganisms, mainly lactic acid bacteria (LAB) and bifidobacteria, have been orally administered to treat or prevent several gastrointestinal disorders, including diarrhea, gastritis, Crohn's disease, colitis, allergies, food intolerances, and obesity (15). In the last years, there has been an increased interest in the topical administration of probiotic cells to overcome the biofilm-related problem of antibiotic resistance. Probiotics are defined as “Live microorganisms which, when administered in adequate amounts, confer a health benefit to the host” (16). Among LAB bacteria, Lactobacillus is the largest genus and comprises the most widely used probiotic species (i.e., L. rhamnosus, L. acidophilus, L. casei, L. plantarum, L. reuteri, L. delbrueckii). Lactobacilli are gram-positive, nonsporing, nonrespiring rods, and they are considered as Generally Recognized As Safe (GRAS) (17). In the present mini-review, we explored the possibility of using lactobacilli, intended both as living cells and as derivatives, in the treatment of biofilm-mediated oral infections.

Oral diseases (i.e., periodontitis, gingivitis, and dental caries) are an excellent example of infection associated with the formation of a highly pathogenic biofilm (18, 19). Periodontitis is one of the most prevalent diseases worldwide and is caused by gram-negative bacteria, such as Porphyromonas gingivalis and Treponema denticola (20), able to form polymicrobial dental plaque biofilms and to produce virulence factors responsible for an exacerbated inflammatory response, which leads to the destruction of the supporting periodontal tissue and eventually teeth loss (19). Severe forms of periodontitis are also associated with the colonization of subgingival sites by Aggregatibacter actinomycetemcomitans and the concomitant reduction of commensal bacteria (21).

The treatment of periodontitis includes hygiene practices, mechanical debridement, and eventually the use of antimicrobials. However, these interventions are not enough to achieve long-term stability, especially in more severe cases, and regular maintenance care based on biofilm control is essential to preserve the equilibrium of the oral microbiome (22). This aspect is also pivotal in the management of dental caries. Members of the Streptococcus genus, such as S. gordonii, S. oralis, S. sanguinis, and S. salivarius, promote the initial colonization on dental pellicle and thus allow for subsequent binding of S. mutans (23). S. mutans is the main etiological agent of dental caries owing to its ability to produce exopolysaccharides (EPS) such as glucan and to rapidly form a mature biofilm on the tooth surface (22). Besides bacteria, the fungal pathogen C. albicans can be present in mixed-species biofilms in dental plaque together with S. mutans (24) and is a frequent cause of stomatitis in children, elderly, and immunocompromised patients (25). In oral candidiasis, the switch of Candida from budding yeast to filamentous hyphae allows for covalent attachment to the oral mucosal surface, followed by biofilm formation, invasion, and tissue damage.

The use of probiotics to face up to biofilm-mediated oral diseases has been studied during the last 20 years (26). Examples of lactobacilli exerting anti-biofilm activity against pathogens causing oral diseases are reported in Table 1. The oral cavity is densely colonized by approximately 1,000 bacterial species. The current understanding is that the overgrowth of cariogenic microorganisms in dental caries is driven by a shift in the oral homeostasis toward less diversity (37), favored by acidic production from sugars that create optimal microenvironmental conditions to support the proliferation and biofilm formation of acid-tolerant S. mutans. Although in the oral cavity, certain strains of lactobacilli can be cariogenic due to their acidogenic and aciduric features, several studies have reported the therapeutic potential of other beneficial Lactobacillus strains (38–40). It has been proposed that lactobacilli may hamper oral pathogens in different ways, even if the exact mechanisms are still poorly understood and could vary among different strains (41). However, lactobacilli can interfere with harmful bacteria and fungi through competition for nutrients, co-aggregation, production of antimicrobials (i.e., bacteriocin, hydrogen peroxide, and organic acids), and modulation of the immune system (42). Moreover, there are few studies showing that lactobacilli are able to integrate into the target biofilm and transiently colonize the oral cavity, thus, competing with pathogens for adhesion sites (43). As an example, Romani et al. demonstrated that L. reuteri PTA5289 can be temporarily incorporated in oral microbiota during a 6-weeks intervention, causing a delay in the regrowth of S. mutants after full-mouth disinfection with chlorhexidine (44). Some authors reported that commercially available Lactobacillus strains (L. rhamnosus GG, L. plantarum 299v, L. reuteri PTA5289 and SD2112, L. paracasei DSM16671) reduced biofilm formation of S. mutans clinical isolates on both smooth glass surface (27) and saliva-coated hydroxyapatite (28). The persistent presence of a Lactobacillus strain potentiates its interference with pathogens. For this reason, Wu et al. evaluated the anti-biofilm activity of Lactobacillus salivarius, a species frequently found in human saliva and tooth surface of healthy subjects (45). They observed that the co-cultivation of S. mutans with a panel of 64 strains of L. salivarius resulted in a reduction of biofilm formation up to 69%, which was higher than that obtained with the reference strain L. rhamnosus GG. Moreover, L. salivarius reduced the expression of genes (gtfB, gtfC, and gtfD) encoding for three glucosyltransferase (Gtfs) involved in the synthesis of exopolysaccharide matrix, and consequently crucial for S. mutans biofilm formation (29). Similarly, Lee at al. demonstrated that L. rhamnosus GG exerted an anti-biofilm activity by decreasing the expression of gtfs in S. mutans but it was not able to integrate into the oral biofilm model. Contrariwise, L. acidophilus ATCC4356 and L. reuteri ATCC334 integrated into S. mutants biofilm without affecting glucan production (30). S. mutans produces proteins anchored in the cell walls to facilitate binding to C. albicans, which in turn, supports streptococcal colonization and caries progression of the formed biofilm (46, 47). Krzyściak et al. showed that L. salivarius (HM6 Paradens) not only reduced the biomass of mono-species biofilms of S. mutans and C. albicans clinical strains, but also the multispecies biofilm. Interestingly, SEM images revealed that the addiction to viable probiotics inhibited the formation of hyphae and germ tubes in C. albicans, consequently hindering the fungal pathogenesis (32). Other authors supported this observation. In particular, three Lactobacillus strains isolated from the saliva of caries-free subjects were able to reduce biofilm development when co-incubated with two oral strains of C. albicans (35). The anti-biofilm activity seemed to be related to the downregulation of Candida biofilm-specific genes, including HWP1 (a glycoprotein located in the hyphal surface), ALS3 (a protein similar to alpha-agglutinin that is essential for the adhesion of Candida), and CPH1 (a regulator of morphogenesis implicated in the maintenance of cell wall organization, pseudohyphal formation in response to oxidative stress, and biofilm development) (39, 40). Viela et al. demonstrated that L. acidophilus ATCC4356, a strain widely employed as food supplements, inhibited the formation of C. albicans ATCC18804 biofilm (57%) and filamentation in vitro, with the best results achieved after 24 h of incubation; the reduction in the number of fungal hyphae produced after probiotic treatment was also observed in Galleria mellonella infection model (48). James et al. have found that a multistrain probiotic combination (Lactobacillus helveticus CBS N116411, L. plantarum SD57870, S. salivarius DSM14685) was effective at both preventing the formation of (>67%) and removing preformed (>63%) C. albicans biofilms (36). Moreover, the combination of two lactobacilli supernatant significantly reduced the expression of several C. albicans genes involved in the yeast–hyphae transition, such as ALS3, EFG1 (hyphae-specific gene activator), SAP5 (secreted protease), and HWP1, in agreement with the results of Rossoni et al. (35).

Regarding periodontal disease therapy, in a study carried out by Jaffar et al., different species of lactobacilli were sought for their ability to eradicate preformed biofilm of three strains of A. actinomycetemcomitans, with promising results. In particular, biofilms of A. actinomycetemcomitans Y4 (serotype b) and A. actinomycetemcomitans OMZ 534 (serotype e) were effectively dispersed after the addiction of lactobacilli, with reduction rates up to 90% and, interestingly, the authors identified three probiotic enzymes, namely protease, lipase, and amylase, that may be responsible for the anti-biofilm activity (34).

Beneficial outcomes of lactobacilli can be obtained not only by living cells, but their byproducts (cell components or metabolites) may be also able to trigger probiotic effects (49). The terms “postbiotics” and “paraprobiotics” have emerged recently and have gained increasing interest since they can exert a wide range of positive effects on the host, such as immunomodulatory, antitumor, and antimicrobial activities, as well as preservation of intestinal barrier (50). Postbiotics are defined as soluble products or metabolites secreted by probiotics capable of providing physiological benefits through direct or indirect mechanisms. They include metabolic byproducts of live probiotic cells, such as cell-free supernatant (CFS), secreted proteins, bacteriocins, organic acids, and secreted biosurfactants (BS) (51). The term paraprobiotics is used to indicate nonviable probiotics (inactivated or dead intact cells), or their cell structural components that can be recovered after cell rupture. The latter comprise peptidoglycans, teichoic acid, cell wall polysaccharides, surface proteins, and cell wall-bound BS (52). Concerns about the administration of living probiotics have been described in experimental models, clinical trials, and case reports (50, 53). Postbiotics/paraprobiotics display prolonged shelf life and good stability (54) and, in this regard, can be potentially employed in oral therapy as a safer alternative to the use of viable cells. Lactobacilli postbiotics/paraprobiotics with anti-biofilm properties toward oral pathogens are listed in Table 2 and briefly described in the following sections.

Therapies for periodontal diseases are costly and may not be sufficiently effective. Findings explored in the present mini-review highlighted that lactobacilli can behave as anti-biofilm agents against a variety of microorganisms responsible for oral diseases, although the activity strongly varies among different Lactobacillus strains. Notably, not only living cells but also Lactobacillus derivatives may exert antipathogenic effects. Thus, the employment of postbiotics, paraprobiotics, or a mixture of both can represent a novel bio-therapeutic approach to face biofilm-related oral diseases, favoring the formulation stability and safety. It is also a step forward in the search for alternative solutions to the use of antibiotics, in the perspective of containing the spread of antibiotic resistance.

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

This study was supported by the Italian Ministry for University and Research.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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