In recent years, inhibition of bacterial biofilm formation has been an attractive target for therapeutic intervention . This strategy leads to the discovery and development of antibiofilm compounds for MDR bacteria including A. baumannii and E. faecalis. E. faecalis and A. baumannii are among the important pathogens found in many healthcare-related infections, and are difficult to eradicate because of their resistance to the broad spectrum antibiotics and production of biofilm . In the current study, the clinical isolates of A. baumannii and E. faecalis were resistant to the most tested antibiotics and were reported as MDR strains. Many researchers studied the antimicrobial effects of probiotics but few investigations reported the effects of synbiotics on pathogens. We observed that all tested probiotics in this study have good antimicrobial properties. Furthermore, cell-free supernatant of L. rhamnosus showed the most potent inhibitory effect against A. baumannii and E. faecalis, and L. plantarum and L. rutteri exhibited the lowest inhibitory activities. These results are in agreement with the opinion of Coconnier et al., which reported that probiotics could influence the growth and pathogenesis of Klebsiella . Production of metabolites such as acetic acid and lactic acid by probiotic bacteria can alter the pH and inhibit adhesins and, invasins of pathogenic bacteria . In a similar study, Grimoud et al. reported that Lactobacillus strains could produce the antimicrobial agents against intestinal pathogens in comparison with Bifidobacterium, and had the most antimicrobial activities . Mamianas et al. stated that L. plantarum has good antimicrobial effects against S. aureus, E. coli and Bacillus subtilis . To date, only a few carbohydrates have been defined and reported as prebiotics, including inulin, lactulose, β-galacto- oligosaccharides, and fructo-oligosaccharides . In the present study, raffinose, trehalose, riboflavin, citrate, inulin, and sorbitol was used as prebiotic. These prebiotics were used in lower concentrations (0.3%) than previous studies (0.5–5%) [34, 35].The results show the efficiency of prebiotics at lower concentration. Our results showed that cell-free supernatant of L. rhamnosus with citrate or trehalose and L. fermentum with trehalose have the best antibacterial activity against Gram-negative A. baumannii. The findings also showed, the supernatant of L. rhamnosus followed by L. fermentum with inulin have potent inhibitory effects against Gram-positive E. faecalis. In the study by Mandadzieva et al., antimicrobial activity of different strains of Lactobacillus against Enterobacter aerogenes was increased in the presence of oligosaccharides [34, 35] which was similar with our findings. The study found that the adsorption of abnormal sugars could increase the production of antimicrobial agents on specific pathways [34, 35]. The mechanism of this stimulatory activity and how the lactic acid bacteria use oligosaccharides is still unclear due to the unique characteristics of the strain. The present study indicate that prebiotics can trigger the antimicrobial properties of probiotics, by increasing the production of antimicrobial metabolites and bacteriocins. In this study, the antimicrobial effect was studied at different conditions between probiotic and prebiotic, as well as in synergism with two probiotics. Likewise, the synergism of two probiotics had less antimicrobial activity compared to the synbiotic. This observation can be due to the competitive effects of two bacteria to uptake nutrients, etc. The increased antimicrobial activity is dependent on the type of probiotic strains, the strains of the pathogenic bacteria, and the presence of one or more prebiotic. In the current study, the prebiotics improve the antimicrobial activity of probiotics against the Gram-negative compare to the Gram-positive isolates suggesting that the metabolites involved in inhibitory effects are different or act differently.
Comparison with the controls, we found that the pathogenic strains were moderate biofilm producer (p < 0.05). The inhibitory effects on biofilm formation by cell-free supernatant of probiotics and synbiotics revealed that, biofilm formation ability of A. baumannii was decreased by 75 ± 6.5% in presence of L. rhamnosus with inulin but this combination could not remove biofilm after its formation. L. plantarum in presence of riboflavin was found to be more potent than the others in inhibition of E. faecalis biofilm formation and had good antibiofilm effect (50 ± 0.86% decrease). We did not found any significant difference between antibiofilm effects of probiotics alone or in combined with prebiotics, revealed that, prebiotics had no significant effect on biofilm control. Probiotics also showed more activity in biofilm control against A. baumannii than E. faecalis. In the current study none of probiotics and synbiotics removed biofilm after formation. In another study, B. cereus displayed significant decrease in biofilm formation in the presence of L. plantarum or L. pentosus supernatants that is in parallel with our findings . Besides, other sties stated that supernatant of both of these probiotics showed good antibiofilm effect against P. aeruginosa and K. pneumonia [36, 17]. In the same way, the antibiofilm property of L. acidophilus has been exhibited only in co-culture with S. aureus but not reduction of biofilm mass of E. coli . Surface adhesion of bacteria is major factor of biofilm formation. The anti-adhesion property of probiotics is commonly associated with competitive adherence for binding sites or ability of metabolite production of synbiotics to inhibit the adhesion of pathogenic bacteria to a surface. In another study was described biosurfactants derived from probiotic lactobacilli showed both antibacterial and antifungal activities against several resistant pathogens, A. baumannii, E. coli and S. aureus [11, 5]. In addition, biosurfactants have been reported to remove stablished biofilms of Bordetella bronchiseptica and B. pumilus [38, 39]. Probably, this means that antibiofilm effects involved to preventing adhesion of pathogenic bacteria to 96-well microtiter plate surface. Another possibility could be referred to the inhibition of quorum sensing .
There is an urgent need for developing novel agents to control biofilms in medical settings. A wide range of promising treatments have been evaluated in different biofilm-related infections.