Methicillin-resistant Staphylococcus aureus (MRSA) is a multi-drug resistant (MDR) microorganism and one of the principal nosocomial pathogens worldwide [81]. Different strains belonging to the genus Lactobacillus (as well as Bifidobacterium) isolated from various sources have been shown to contrast the growth of S. aureus and even of clinical isolates of MRSA in vitro [82]. Their effects were mediated both by direct cell competitive exclusion and the production of short chain fatty acids or bacteriocin-like inhibitors. In addition, L. acidophilus was also reported to inhibit S. aureus biofilm formation and lipase production. In another study, L. fermentum TCUESC01, isolated from cocoa seeds, was shown to effectively inhibit S. aureus biofilm formation. The inhibition mechanism was based on the release of soluble molecules which suppressed the expression of two genes (icaA and icaR) with an important role in biofilm synthesis [83].

MDR Proteus mirabilis isolates show low antibiotic susceptibility and biofilm-forming activity that can cause serious urinary tract infections [84]. A recent study demonstrated that cultures and cell-free supernatants of L. casei DSM 20011 and L. reuteri DSM 20016 exhibited strong antimicrobial, anti-adherence, and antibiofilm formation activities against MDR P. mirabilis. In addition, supernatants of L. casei and L. reuteri significantly reduced mature biofilm formation and adherence (>60% compared to controls), indicating that these species of lactobacilli could be utilized to combat Proteus-associated urinary tract infections [85].

Dental caries has multifactorial causes and arises from an imbalance between the host and the microbiota of the mouth. For a long time, Streptococcus mutans in its biofilm form has been known to contribute to dental caries formation significantly; recently, the one pathogen –one disease approach has been deeply challenged, and the concurrent role of the entire microbiota in the health of the oral cavity tends to be more prominent [86]. The capacity of different Lactobacillus species to inhibit growth, biofilm formation, and gene expression of S. mutans has been evaluated. Susceptibility testing indicated antibacterial (pH-dependent) and antibiofilm activities of L. casei (ATCC 393), L. reuteri (ATCC 23272), L. plantarum (ATCC 14917), and L. salivarius (ATCC 11741) against S. mutans. All Lactobacillus species previously mentioned contrasted and limited the growth and virulence of S. mutans. Reduction in microcolony formation and exopolysaccharide structural changes were also highlighted by scanning electron microscopy. The highest antimicrobial activities were reported for L. casei and L. reuteri, whereas the lowest antimicrobial activities were observed with L. plantarum and L. salivarius. The highest antibiofilm and peroxide-dependent antimicrobial activities were reported for L. salivarius. Reduced expression of genes involved in exopolysaccharide production, acid tolerance, and quorum sensing were reported for all biofilm-forming cells treated with Lactobacillus spp. supernatants [87]. In a study on mixed biofilm formation by fungi and bacteria on silicone in vitro, Lactobacillus supernatant showed high efficiency against both microorganisms [88]. In the field of oral infections, the probiotic strain L. brevis CD2 was shown to inhibit the opportunistic anaerobe Prevotella melaninogenica (PM1), a well-known causative agent of periodontitis. The inhibitory effect of L. brevis CD2 on P. melaninogenica PM1 biofilms was evaluated in vitro using two different methods: the anaerobe was exposed to the supernatant of the strain in one case, or the two microorganisms were grown together to obtain single or mixed biofilms, in the second case. The inhibitory effect of CD2 on PM1 was also checked by the agar overlay method. The development of PM1 biofilm was strongly affected (56% decrease in OD₅₇₀ value) by the CD2 supernatant after 96 h—with a dose-dependent biofilm reduction using several supernatant dilutions. Confocal microscopy on the mixed biofilms revealed the ability of CD2 to prevail over PM1, greatly reducing the biofilm of the latter. The authors hypothesized that the strong adherence ability of the CD2 strain and the release of metabolites may be responsible for reducing the PM1 biofilm [89].

The use of antibiotics for the treatment of cholera is associated with side effects, such as gut dysbiosis, due to the depletion of beneficial microbiota and the risk of spreading antibiotic resistance; hence, the search for alternative therapeutic agents is extremely active. Different strains of Lactobacillus spp., screened and isolated from fecal samples of healthy children in cholera endemic area, were tested for their abilities to prevent biofilm formation and to disperse the preformed biofilms of Vibrio cholerae and V. parahaemolyticus. The results showed that the culture supernatant (CS) of seven isolates of Lactobacillus spp. used in the study inhibited the biofilm formation of V. cholerae by more than 90% [90].

A recent study showed the role of L. gasseri in contrasting the adhesion of the protozoan parasite Trichomonas vaginalis to host cells, a critical virulence aspect of this pathogen [91]. The aggregation-promoting factor-2 (APF-2) produced by L. gasseri ATCC 9857 was found to be highly inhibitory in the adhesion of T. vaginalis to human vaginal ectocervical cells. This important finding highlights that lactobacilli remain of key importance for the development of specific therapeutic strategies, even towards non-bacterial pathogens.

As a matter of fact, probiotics are active against non-bacterial biofilms as well. For example, C. albicans biofilm is associated with denture-related stomatitis and oral candidiasis, especially in elderly people. A study investigating a C. albicans biofilm on a denture base resin treated with L. rhamnosus and L. casei showed that the probiotics’ surfactant exhibited strong antifungal activity against blastoconidia and biofilm of C. albicans. Even when the C. albicans biofilm was already formed and sequentially treated with L. rhamnosus and L. casei, inhibition of the biofilm on the denture surface was reported [92]. Therefore, L. rhamnosus and L. casei probiotics could have practical applications for preventing and treating denture-related stomatitis and other Candida infections, even in neonates [93,94].

It is not uncommon to register discrepancies between the effectiveness of probiotics in vitro and in vivo. Therefore, in vitro antimicrobial activity does not necessarily assure efficacy in animal infectious models. However, cases in which the in vitro and in vivo results were congruent are also reported. As an example, L. plantarum, which showed the highest inhibition activity against S. aureus in vitro, was also very effective topically in preventing skin wound infection in S. aureus-infected mice. Bacteriocin-producing Lactobacillus sakei 2a has been shown to protect gnotobiotic mice against experimental challenge with L. monocytogenes [95]. A recent study aimed at evaluating the effects of Lactobacillus administered intranasally on a murine model of P. aeruginosa pneumonia (strain PAO1). Two probiotic combinations were selected for in vivo testing (1-L.rff for L. rhamnosus and two L. fermentum strains, and 2-L.psb for L. paracasei, L. salivarius, and L. brevis) out of 50 clinical isolates screened for the ability to decrease the synthesis of two PAO1 produced QS-dependent virulence factors (elastase and pyocyanin). Intranasal priming with both probiotic blends acted as a prophylaxis and avoided fatal complications caused by PAO1 pneumonia in mice, showing encouraging results to move towards clinical trials [96].

Among the Bifidobacteria, Bifidobacterium bifidum BGN4 is a widely used probiotic strain that has been included as a major ingredient to produce nutraceutical products for the last 20 years [97]. The various bio-functional effects and potential for industrial application of B. bifidum BGN4 have been characterized and proven in vitro (i.e., phytochemical bio-catalysis, cell adhesion, anti-carcinogenic effects on cell lines, and immunomodulatory effects on immune cells) and in vivo experiments (see below).

A study investigated the effect of Bifidobacterium spp. on the interference with the production of quorum-sensing (QS) signals and biofilm formation by enterohemorrhagic E. coli (EHEC) O157:H7. In an AI-2 bioassay, cell extracts of different Bifidobacterium reference strains (B. longum ATCC 15707, B. adolescentis ATCC 15706, and B. breve ATCC 15700) were rather effective; they resulted in a 36% reduction in biofilm formation. Cell extracts of B. longum ATCC 15707 were also able to reduce the virulence of EHEC O157:H7 in the Caenorhabditis elegans nematode in vivo model [98]. Another study highlighted how B. lactis and B. infantis, alone or in combination, have an antagonist effect on biofilms of periodontopathogens, such as Porphyromonas gingivalis and Fusobacterium nucleatum, but minimal influence on Streptococcus oralis growth in vitro [99].

Bifidobacteria strains are often used in probiotic combination with other LAB. One of these combinations, constituted of L. rhamnosus GG, L. rhamnosus LC705, B. breve 99, and P. freudenreichii JS was shown to inhibit pathogen adhesion (including Salmonella enterica, Clostridium difficile, L. monocytogenes, and S. aureus) to human intestinal mucus (in vitro). The same combination with another bifidobacterial strain (B. lactis Bb12) was less effective [100].

The studies regarding the ability of Bifidobacteria to contrast pathogenic biofilms are not so numerous as the ones on lactobacilli. Some experimental works have also highlighted a lower effectiveness compared to other LAB. As an example, Miyazaki et al. (2010) highlighted that CS of a Lactobacillus strain has a strong bactericidal effect on auto aggregative E. coli, while no effect was reported for Bifidobacteria [101]. Discrepancies among laboratory results and experiments in animal models are known for Bifidobacteria as well. For example, the S. aureus 8325-4 strain was shown to be sensitive in vitro to L. acidophilus, while B. bifidum best inhibited experimental intravaginal staphylococcosis in mice caused by the same bacteria [82]. For B. bifidum BGN4, a wide spectrum of beneficial effects in vivo (i.e., suppressed allergic responses in mouse model and anti-inflammatory bowel disease) and in clinical studies (eczema in infants and adults with irritable bowel syndrome) have been demonstrated.

Specific probiotic combinations are demonstrating day-by-day to have a marked utility in the human field, and data on antibiofilm activity on various respiratory, genito-urinary, wound, and tissue pathogens, are starting to become convincing. However, there is still a long way to go, especially in their in vivo routine usage. Herein should encourage better investigation on probiotic–biofilm interactions and how to fight biofilm infections through the so-called “good bacteria”, such as bifidobacteria and lactobacilli, highlighting that there are “useful” or “good” biofilms as well. Mechanisms of action and antibiofilm activities must be considered as strain-related; therefore, we will need to focus our research on the development of such promising strains. It is often debated whether probiotics will become broadly used drugs or medicaments in the future; it is still too early to say, but given the uncertain longevity of antibiotics, it would be recommended to explore alternative means, and so far, probiotics represent one of the most promising.