Local administration of commensal probiotics as a sort of “bacteria-therapy”, able to interfere with disease-associated disease is gaining increasing interest, finding applications in many fields .
Probiotics own different mechanisms that interfere with the activity of pathogenic bacteria, including: the production of antagonistic substances (e.g., bacteriocins, fatty acids, hydrogen peroxide and lactic acid), generation of environment conditions unfavorable for pathogens (e.g., competition for nutrients or pH alteration) and the competitive adhesion to human tissues preventing the colonization by harmful bacteria. Among bacterial species recently recognize as probiotic, S. salivarius 24SMB and S. oralis 89a isolated from the rhinopharynx of healthy children are suggestive strains of a healthy flora. S. salivarius 24SMB and S. oralis 89a are administered in a ratio of 98:2 by means of a nasal spray are present, with the aim of preventing ear, nose and throat diseases. The abundance of S. salivarius in the solution is due to its primary and predominant presence in the upper respiratory tract surfaces of humans and because its non-pathogenic behavior in healthy individuals . In particular, the strain 24SMB was isolated in 2012 and selected as a promising probiotic due to the absence of virulence traits and antibiotic resistance genes and its ability to inhibit S. pneumoniae growth . Santagati et al. demonstrated its safety and tolerability, and its capability to colonize the rhinopharynx when administrated as a nasal spray .
Conversely, S. oralis 89a was isolated from a recalcitrant healthy child during a tonsillitis outbreak and it was found to be able to inhibit the growth of group A streptococci in vitro . Since then, this strain has been used in in vitro and in vivo studies to evaluate its clinical effects on streptococcal tonsillitis and otitis media [19, 20, 30,31,32]. The whole genome of this strain was recently sequenced, identifying the gene encoding for the bacteriocin Colicin V and for tolerance to Colicin E2 .
As a preliminary step, the reciprocal interaction between S. salivarius 24SMB and S. oralis 89a was assessed. Using the transwell device, the two strains were physically separated by a membrane that permitted only the passage of diffusible molecules form one compartment to the other. In both cases, a significant increase in biofilm formation was observed, suggesting a possible positive synergistic effect between the two strains. Furthermore, when S. salivarius 24SMB and S. oralis 89a were simultaneously cultured in direct contact, the resulting biofilm appeared to be like the sum of the two strains grown individually. Unfortunately, the contribution of a single strains in mixed biofilms was not evaluated, being the used staining technique not species-specific, representing a limit of the study.
Then, the anti-biofilm activity resulting from the combination of S. salivarius 24SMB and S. oralis 89a against pathogens of the upper airways was investigated. In particular, the anti-biofilm activity was tested against S. pneumoniae, S. pyogenes and M. catarrhalis being the most common bacterial pathogens causing acute otitis media and bacterial pharyngotonsillitis [33, 34]. Moreover, the anti-biofilm effect was assessed on S. aureus, involved in 50% of recalcitrant chronic rhinosinusitis , and coagulase negative staphylococci and anaerobes, including P. acnes .
Biofilms are comprised of microorganisms enclosed in a hydrated self- produced polymeric matrix attached to a solid surface. They represent an important cause of chronic infectious diseases of the upper airways, including recurrent middle ear diseases, chronic rhinosinusitis and recurrent pharyngotonsillitis , but also teeth or in implant-associated infections. Biofilm-related infections are often resistant to antibiotic therapy, posing serious concerns about the infection control. Frequent antibiotic intakes may have deleterious effects due to the depletion of the commensal microbiome and the subsequent colonization by microorganisms that are less susceptible to the prescribed antibiotics . In this context, the use of probiotics able to disperse pathogens biofilm may be therapeutically beneficial and may offer a valid alternative or a coadjuvant treatment to conventional antimicrobials.
With the aim to test the aforementioned hypothesis, in the present study, co-culture experiments by means of the direct or indirect culture of the probiotic strains with the respiratory tract pathogens was performed. The combination of probiotics was able to both inhibit the biofilm development and to disperse the already established biofilms of all the tested pathogens with the exception of S. pyogenes. This behavior is not surprising; the inhibitory activity of S. salivarius 24SMB against S. pyogenes depends on the choice of the growth media . Indeed, Santagati and co-workers described how S. salivarius was not able to inhibit S. pyogenes in Todd Hewitt broth supplemented with blood, while an increase in the inhibitory activity was observed on Columbia blood agar. In our experimental setting, the lack of activity against S. pyogenes could also be explained by the lack of supplementation of yeast extract, glucose or calcium salts in the growth medium, which are necessary supplement for an optimal production of bacteriocins [38, 39].
A more detailed investigation was carried out by CLSM analysis, which considered additional outcome variables as live/dead cells ratio and percentage of substratum coverage. Specifically, the total biomass volume was significantly lower when the tested pathogens were incubated with probiotics compared to that of controls. Differently to what observed in the spectrophotometric assay, the higher sensitivity of the CLSM analysis allowed to appreciate a significant biomass reduction also for S. pyogenes. Indeed, while the spectrophotometric assay allows the semi-quantitative measurement of the air-dried biofilm biomass, CLSM gives the possibility to collect three-dimensional images of hydrated biological structures without fixation . This non-destructive technique has radically transformed optical imaging in biology and microbiology, providing a useful tool for the examination of the structure of biofilms.
Concerning the live to dead cells ratio, no differences between treated and untreated samples were found, indicating a prevalent inhibitory effect rather than a potential bactericidal activity of the probiotic strains. On the contrary, substratum coverage was significantly lower in treated biofilms, with particular regard to that of S. epidermidis and S. aureus, which appeared more scattered than controls.
Recent studies shown that chemical interactions through secretion of molecules by different microbial species may affect spatial biofilm structure, regulating both its formation and dispersion [41,42,43]. Recently, Santagati et al. described the presence of a blpU-like bacteriocine cassette in the genome of S. salivarius 24SMB, which has been shown to mediate intra- and interspecies competition with inhibitory activity against S. pyogenes and S. pneumoniae and also to provide competitive advantage in colonization in vivo [15, 44]. However, no other genes responsible for production of bacteriocine (i.e. salivaricins, commonly produced by other S. salivarius strains) were identified throughout the genome . Similarly, S. oralis 89a possess a locus for the production of a Colicin V, a proteolitically processed peptide antibiotic, which kills sensitive cells by disrupting membrane potential . Interestingly, S. oralis 89a is characterized by the presence of luxS gene, responsible for the production of the quorum-sensing molecule AI-2, involved in cell-to-cell communication and able to influence the expression of virulence factors, motility and biofilm formation . In particular, controlled concentrations of AI-2 are able to promote mutualistic biofilm formation and to influence structure and composition of other commensal streptococcal species biofilm [47, 48]. Unluckily, the genome sequence of S. salivarius 24SMB is not available in public databases and no information on the presence of quorum-sensing clusters involved in biofilm formation and regulation (e.g., Rgg transcriptional regulators family) are available.
Nonetheless, signaling is restricted only to those species with appropriate receptors, suggesting that other kind of unspecific interactions may play an important role in determining biofilm spatial structure . For example, metabolic end products like lactic acid and hydrogen peroxide produced by streptococci,
can act with a broader spectrum by cause acid and oxidative stress, respectively. As expected, the presence of S. salivarius 24SMB and S. oralis 89a in the co-cultures slightly lowered the pH in all the cases except for S. pyogenes and S. pneumoniae (data shown in Additional file 1). Since the inhibitory activity was observed also for the two streptococci, it can be supposed that alteration in pH is not the only mechanism of action, but other specific interactions might occur.
Finally, a high reduction of biofilm biomass in transwell co-cultures was observed. This event might suggest that the anti-biofilm activity of the probiotic mixture is mediated by diffusible molecules secreted by the probiotic strains, rather than depending on a mechanism requiring physical contact. This was confirmed by supplementation of the cell-free extract to the medium, displaying an effect comparable to that observed in the transwell co-culture. In the attempt to elucidate the nature of the inhibition, the cell-free extracts were neutralized to a pH of 7.0, to evaluate the contribution of the acidic environment resulting from the streptococcal fermentation or were heated, to eliminate all the thermolabile secreted molecules. Even though, both the treatments did not completely impair the inhibitory effect of the cell-free extract, indicating a likely multifactorial and strain-specific strategy. Furthermore, the contribution to biofilm formation between pathogenic and probiotic strains in mixed species biofilms was not discriminated. Indeed, mixed biofilms were not analyzed by confocal microscopy, because of the lack of species-specific dyes able to differentiate the presence of different microbes. Stains able to discriminate among different bacteria should be used in future studies in order to investigate the role of probiotic strains in the biofilm production by pathogenic bacteria.
As the goal of the probiotics tested in this study is to create a barrier against pathogens, an interesting issue would be to investigate if pathogens are able to invade and establish within pre-existing probiotic biofilms.