الفهرس 
LITERATURE REVIEWOnly 14 pages are availabe for public view
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Abstract
There is a growing frequency in using probiotics as a cure and prevention of number of medical disorders and pathogenic infections. C. difficile infection (CDI) is causing health problems range from mild diarrhea to fulminant colitis, toxic megacolon and death. The alarming increase of multidrug resistant strains of C. difficile claimed the necessity to find non-antibiotic alternative strategy to control the CDI. Many research studies have suggested the possible use of probiotics for the avoidance of C. difficile associated disease (CDAD) as a consequence to long antibiotic treatment periods which cause the imbalance of the gut microbiota. This imbalance gives the chance of enteric pathogens for colonization in the intestinal epithelial cells and secreting their toxins. In the present study, we aimed at isolation of new probiotic candidates from human origin capable of inhibiting the pathogenic C. difficile and controlling its colonization within the human gut.
Fecal samples from twenty breast-fed infants were used as a good source of probiotics. The samples were manipulated in PBS buffer and serial dilutions of them were incubated on MRS agar plates at 37ºC. Twenty Gram positive and catalase negative isolates have been screened for their in vitro ability to inhibit the C. difficile using broth culture inhibition assay by means of their cell-free supernatant. Three isolates out of the twenty showed inhibition ability more than 55% and were investigated in more details. Neutralization the supernatant of these three isolates showed decrease of the inhibition to 39.00, 31.98, and 36.11% which confirmed that the inhibition action could be due to combination of organic acids and bacteriocins that produced by the isolates. The three isolates were analyzed for their ability for auto-aggregation and co-aggregation against C. difficile, the results revealed significant aggregation ability for them ranged from 44 –76%. This property would ensure their action against C. difficile because auto-aggregation ability is enabling the adherence and promotes colonization in the human gut, while the co-aggregation ability against the C. difficile would support the displacement of this pathogen and so would protect from infection. The three isolates also showed a significant inhibition to C. difficile when tested in co-culture with C. difficile as they suppressed the growth of the pathogen and even the C. difficile toxin A and B genes were effectively inhibited within the genome extracted from this co-culture. Molecular identification of these isolates have been achieved by amplification and sequencing their 16S rRNA genes thus, they were named as E. faecalis NM815, E. faecalis NM915, and E. faecium NM1015 and registered at the GenBank under accession numbers KU365166, KU365167, and KU365168 respectively. Moreover, they met all the required properties of probiotics. They tolerated the harsh conditions of human GIT as they showed significant survival at low pH 1.5, 2.0 % w/v bile salts, and 1.9 mg/ml pancreatic enzyme. In addition, they exhibited significant adhesion to Caco-2 cell line and showed antibacterial activities against some Gram positive and Gram negative bacteria. Since they are belonging to Enterococcus therefore the safety was determined in respect to the antibiotic resistance, common enterococcal virulence genes and hemolytic activity. The results confirmed their safety profile. These three strains when administrated to mice groups and followed by C. difficile challenge showed good inhibition to the pathogenic effects of the CDI which confirmed by histological analysis of the intestines and liver compared to the mice group which administrated PBS buffer without probiotic. In addition, the fecal samples from mice group treated with probiotics were effectively inhibited from toxin A and B when tested by q PCR using specific primers, compared to the fecal samples from mice group untreated with probiotics. Moreover the serum analysis of the mice groups showed the ability of the probiotic strains to activate the immune system.
Accordingly to the in vitro and in vivo experiments done along this study, these strains are highly recommended as potential probiotics to control the C. difficile infection.