P. aeruginosa is an opportunistic pathogen in patients with significant underlying diseases. It is one of the most common causes of hospital-acquired pneumonia, especially in mechanically ventilated patients, in whom it leads to a high mortality rate [2, 17]. Moreover, chronic airway inflammation with recurrent P. aeruginosa infections is the major cause of morbidity and mortality in patients with cystic fibrosis . High incidence, infection severity and increasing resistance characterizing P. aeruginosa infections highlight the need for new therapeutic options. In that context, different attempts have been made to use probiotic bacteria for fighting P. aeruginosa pulmonary infections . Lactobacilli are non-pathogenic bacteria closely associated with the human microbiota and commonly used as probiotics. Some of them are used because of their positive effects on the immune system, on the barrier effect of epithelia, whereas others are used for their capacity to fight pathogens colonisation either via competitive exclusion or antimicrobial molecules production. Probiotic effects are strain-specific, consequently they do not possess the same activity and they are not all recommended for the same health effects . Specific selection criteria are then needed in order to find the right probiotic harbouring the appropriated activity (inhibition of pathogen for example) within a particular ecological niche. Some Lactobacillus spp. (L. rhamnosus GG, L. plantarum 299, L. paracasei, L. casei, L. acidophilus), administered by oropharyngeal application or via orogastric or nasogastric tube, have already been tested, with different levels of success, in mechanically ventilated patients to fight P. aeruginosa pneumonia [11, 21]. To our knowledge, none of them was specifically selected according to its capacity to inhibit P. aeruginosa, nor to survive in the oral cavity or in the oropharynx. The main goal of this work was then to test the capacity of lactobacilli isolated from oral cavities of healthy volunteers and from raw milk to inhibit the production of virulence factors by P. aeruginosa PAO1 in order to look for potential probiotic bacteria capable to prevent P. aeruginosa pneumonia.
In this study, 67 isolates belonging to 9 Lactobacillus species (L. reuteri, L. fermentum, L. vaginalis, L. rhamnosus, L. zeae, L. paracasei, L. salivarius and L. plantarum), with a prevalence of L. fermentum and L. paracasei, were recovered from the oral cavities of 23 healthy volunteers. The diversity of lactobacilli isolated from the oral cavity is generally high, and these 9 species are commonly encountered in healthy persons [22–27]. Since it has been suggested that oral lactobacilli may originate from the food , 20 Lactobacillus strains (L. fermentum, L. brevis and L. parabuchneri) isolated from raw milk and whom certain species have been previously described in the oral cavity were added to increase the pool of the tested strains. Although lactobacilli do not belong to the predominant oral microbiota, in which they account for less than 1% of the cultivable fraction, they are suspected to have a considerable effect on the homeostasis of this ecosystem .
Among the 87 tested isolates, the 10 most active ones against P. aeruginosa virulence factors were identified at the species level using a polyphasic approach combining phenotypic (MALDI-TOF, API 50 CHL) and molecular (16S rRNA and rpoA genes sequencing) methods, whereas typing of L. fermentum strains was performed using PFGE.
Finally 8 strains (six L. fermentum, one L. paracasei and one L. zeae) showed a significant inhibitory effect against P. aeruginosa PAO1 biofilm formation or elastolytic activity. With the exception of L. zeae and L. paracasei that are facultative heterofermentative, all the active strains belonged to the L. fermentum species and were then obligate heterofermentative, producing both lactic and acetic acids from glucose. It has been shown that one of the major antibacterial effects of lactobacilli is mediated via lactic and acetic acids production . Indeed, lactobacilli may produce high concentrations of lactic acid and acetic acid depending on their fermentative pathways and growth conditions. We have shown that P. aeruginosa PAO1 was sensitive to pH and acetic acid with a dose-dependent effect, growth inhibition increasing in parallel with an increase in acid concentration and pH decrease. At a pH of 4 or 5, acetic acid completely inhibited the growth of P. aeruginosa at a concentration of 25 mM, whereas high concentrations (≥50 mM) of acetic acid were necessary to partially inhibit P. aeruginosa growth at pH 6. For that reason, the inhibitory activities of lactobacilli toward P. aeruginosa PAO1 biofilm formation and elastolytic activity were not tested in MRS medium that contains a high glucose concentration (20 g/L), but in BHI medium. Indeed, this medium contains a low concentration of glucose (2 g/L) that limits the growth of Lactobacillus strains and prevents a strong acidification, allowing a better differentiation between the organic acids effects from other mechanisms of action.
Elastolytic activity and biofilm formation are two majors virulence factors observed in P. aeruginosa. Among the 8 strains (6 L. fermentum, one L. paracasei and one L. zeae) significantly inhibiting elastase activity or biofilm formation, it is interesting to note that the four L. fermentum strains of milk origin (L. fermentum K.C6.3.1D, K.C6.3.1E, K.V9.3.2B and K.V9.3.2C) inhibited elastolytic activity only, whereas the ones originating from the oral cavity (L. fermentum ES.A2, ES.F.115) inhibited biofilm formation only. The two other active strains from the oral cavity, L. zeae Od.76 and L. paracasei ES.D.88 significantly inhibited elastase activity and biofilm formation respectively. Elastase has been shown to destroy respiratory epithelium tight junctions, increasing permeability disorders and interleukin-8 levels while decreasing host immune response [30, 31]. We previously showed in a murine model of P. aeruginosa pneumonia, that elastolytic activity was positively correlated to acute lung injury . It has been shown by Rumbaugh et al. that elastolytic activity and biofilm formation are under control of the quorum sensing molecules of P. aeruginosa. Different mechanisms of action may then be hypothesized, active Lactobacillus strains inhibited the quorum sensing targets, either they secreted antagonistic analogues of acyl-homoserine lactone or they inhibited regulating lasR or lasI genes factors . Moreover, the use of the BHI medium that induced a limited pH decrease, together with the low number of active strains, suggested that other mechanisms of action than organic acids production were implicated. It has been shown that surface properties, such as cell charge and hydrophobicity, implicated in the non-specific adhesive capacity of bacteria differ among Lactobacillus strains isolated from the oral cavity of healthy volunteers, with several strains (including strains of L. fermentum and L. paracasei) showing very high adhesive properties . Such a difference in surface properties between lactobacilli strains with a prevalence of high adhesive properties in lactobacilli strains isolated from the mouth might be implicated in their higher capacity to prevent biofilm formation as compared to dairy lactobacilli. Indeed surface properties are involved in adhesion properties to plastic and/or in the co-aggregation with P. aeruginosa that could be implicated in decreasing biofilm formation. However, further studies are needed to elucidate the antagonistic mechanism of action between described lactobacilli strains and P. aeruginosa.
Antagonistic activities of probiotic bacteria require a certain capacity to survive and/or to grow in the targeted ecosystem. All active strains showed a good capacity to grow in artificial saliva, suggesting that they may survive in the oral environment. However, it has been suggested that some probiotics may be implicated in the development of dental caries . The use of poorly acidifying strains such as L. fermentum K.V9.3.2B and K.V9.3.2C inhibiting elastolytic activity and L. fermentum ES.F.115 and ES.A.2 inhibiting biofilm formation may then be encouraged in their use as probiotics to fight P. aeruginosa pulmonary infection compared to the more acidifying ones. However, knowing that acid production strongly inhibits P. aeruginosa growth, the use of more acidifying strains may be also investigated and subjected to an appropriate follow-up of dental health during probiotic application. Another theoretical concern regarding the safety of probiotics is the transfer of antibiotic resistance genes toward the oral and gastrointestinal microbiota. In our study, as expected for lactobacilli that are intrinsically resistant to vancomycin, all the strains were resistant to vancomycin . No other resistance towards the recommended antibiotics was detected. On the other hand the toxic effect of putative probiotic on the epithelial cells from the oropharynx and respiratory tract will have to be investigated.