الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
The current antibiotic crisis depicted as a combination of increasing rates of antibiotic resistant infections and a declining rates of antibiotic discovery (Ventola, 2015), necessitates finding new alternatives to the conventional antibiotic agents. A recent report on antibiotic resistant infections warns that the number of deaths due to antibiotic resistant infections will reach 10 million deaths annually by 2050 preceding even cancer if the crisis has not been tackled correctly (THE REVIEW ON ANTIMICROBIAL RESISTANCE, 2016). This highlighted the potential role of probiotics as anti-infective agents. Bacterial genera classified under the lactic acid bacteria group are being frequently studied in this context. This includes the genus Lactobacillus which is characterized by a long history of safe use in humans and wide distribution among different habitats (Saxelin et al., 1996; Tannock, 2004).
In this study, the antagonistic activities of the two Lactobacillus strains L. gasseri DSM 20077 and L. rhamnosus ATCC 7469 against three bacterial pathogens were investigated.
The three pathogens used in this study were Pseudomonas aeruginosa (P. aeruginosa), Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). P. aeruginosa and S. aureus were obtained as clinical isolates and E. coli is a reference strain deposited in the culture collection of
public health of England under the deposition number NCTC 10959. The three pathogens are frequently implicated in biofilm infections; which are estimated by the national institutes of health in the US to associate 80% of human infections.
The cell free supernatants (CFS), the neutralized cell free supernatants (NCFS), heat-treated and protease-treated supernatants collected from 24 h- and 48 h-cultures of the two Lactobacillus strains were tested for their antimicrobial activity against the three tested pathogens using a radial diffusion assay. The 24 h- and 48 h-CFS of both Lactobacillus strains showed an antimicrobial activity against the three pathogens. However, the highest activity was recorded against P. aeruginosa.
The antimicrobial activity of both strains against the three test pathogens was completely abolished upon neutralization. On the contrary, the heat-treated and protease-treated supernatants collected from 24 h and 48 h cultures of both tested Lactobacillus strains retained the same antimicrobial activity as their untreated counterparts (CFS). Thus it was concluded that the antimicrobial activity of the two tested Lactobacillus strains is mediated by the organic acids produced by them or probably by an acid-dependent non-bacteriocin antimicrobial compound.
The CFS of the tested Lactobacillus strains showed a superior antimicrobial activity compared to HCl of similar pH values under the same test conditions. This indicates that the antimicrobial activity observed in case of Lactobacillus CFS was specific to the organic acids produced by them and not just a pH effect.
The two Lactobacillus strains were investigated for their antibiofilm activity. In crystal violet assay, the two strains could successfully form biofilms when grown as single-species biofilms for 24 or 48 h. This contributed to the increased biomass of dual-species biofilms of the tested pathogens when co-cultured with the Lactobacillus strains compared to their single-species counterparts. This was further confirmed by viable count technique and scanning electron microscopy.
Viable count technique showed a marked decrease of the pathogens count when grown as dual species biofilms with lactobacilli compared to the counts obtained when they were grown as single-species biofilms when assessed after both 24 h and 48 h. However, the count of S. aureus grown in dual-species biofilm with L. gasseri showed a slight increase after 24 h.
SEM substantiated these findings where the scanning electron micrographs of the 48 h single- and dual-species of the test pathogens showed that they were largely replaced by the two tested lactobacilli.
The effect of L. gasseri and L. rhamnosus on some extracellular enzymes production by the test pathogens during biofilm formation was also evaluated.
Neither of the two Lactobacillus strains showed a significant effect on the production of phospholipase C by any of the three pathogens. On the other hand, they could inhibit the production of protease by the test pathogens in their mixed dual-species biofilms. This effect was most significant with S. aureus which showed the highest protease activity in its single-species biofilms. The protease activity of S. aureus showed a 4-fold decrease in its 24 h dual-species biofilm and 2-fold decrease in its 48 h dual-species biofilm with L. rhamnosus compared to their corresponding single species biofilms. The dual-species biofilms of S. aureus with L. gasseri also showed 2-fold decrease of their protease activity compared to the single-species biofilms of S. aureus at both points of time.
Finally, the immunomodulatory activity of the two tested Lactobacillus strains was assessed. The expression of interferon gamma (IFN-γ) by an in vitro culture of human peripheral blood mononuclear cells (PBMC) in response to stimulation by pathogens was investigated after pretreatment of the PBMC with different Lactobacillus preparations including viable cells, heat-killed cells and NCFS. This was compared to the expression of IFN-γ in response to pathogen stimulation by the untreated PBMC.
Both P. aeruginosa and S. aureus increased the expression of IFN-γ 16.21% and 21.59%, respectively compared to the control experiment. While a 12.42% reduction of IFN-γ expression was observed with E. coli. Although L. gasseri preparations increased IFN-γ production by ≥ 20% when added to a culture of PBMC stimulated with P. aeruginosa, these differences together with the differences obtained with other pathogens in presence of different Lactobacillus preparations were considered non-significant when statistically analyzed.
In conclusion, L. gasseri and L. rhamnosus proved to have antimicrobial, antibiofilm as well as an inhibitory effect against the proteolytic activity of P. aeruginosa, E. coli and S. aureus. Thus, the two Lactobacillus strains can be considered as promising tools for combating biofilm infections caused by these pathogens.
Further studies are recommended to investigate of the role of quorum sensing in the observed activities of the tested Lactobacillus strains and the encoding genes involved. Their immunomodulatory role should also be reassessed via monitoring the expression of cytokines other than IFN-γ in both in vitro and in vivo models. The cell structures and the extracellular products and the genes mediating the antagonistic activities displayed by the Lactobacillus strains also need to be characterized.