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The aim of the present study was to investigate the most effective probiotic combinations to prevent death and necrotizing enterocolitis (NEC) in a premature rat model.One hundred fifty-eight premature Sprague-Dawley premature rats were enrolled. Probiotic strains Bifidobacterium bifidum, B longum, Lactobacillus acidophilus, L plantarum, and B breve were fed as a single strain or mixture with 2 or 3 strains for a total of 9 study groups; control groups received no exogenous probiotic supplement. Fecal samples were collected for 72 hours to detect probiotic strains and pathologic strains by real-time polymerase chain reaction. Colony counts of probiotic strains Escherichia coli and Klebsiella were compared between groups before and after 36 hours of the study period. The incidence of death and NEC were compared via Fisher exact test between groups.The results demonstrated that L plantarum alone (P = 0.0026) and B bifidum with B longum together (P = 0.0017) were more effective in reducing NEC as compared with the control group. All of the study groups except B breve and B bifidum with B breve definitely prevented death compared with controls. B bifidum and B longum together had significantly lower mortality than the control group (P < 0.0001). Colony counts of E coli and Klebsiella in stool samples were significantly decreased in the B bifidum, B longum, and L plantarum group compared with the other study and control groups after 36 hours.Administration of a mixture of probiotic strains with B bifidum and B longum was most effective in preventing death and NEC in this animal model, and these observations provide an evidence-based strategy for designing further neonatal clinical trials.