| Several experiments were conducted to select lactic acid bacteria (LAB) for a competitive exclusion product (CEP) that would potentially inhibit E. coli O157:H7 in the intestinal tract of live cattle. Fecal samples from cattle that were culture negative for E. coli O157:H7 were collected every three weeks over a period of 12 weeks. Lactic acid bacteria were isolated from cattle feces by repeated plating over de Man-Rogosa-Sharpe (MRS)/Lactobacillus Selection (LBS) agar. Six hundred eighty-six pure colonies were isolated and each isolate was tested for inhibition of a four-strain mixture of E. coli O157:H7 using agar spot test. Three hundred fifty-five isolates (52%) showed significant inhibition. Seventy-five isolates showing maximum inhibition were screened for bile tolerance by monitoring growth in MRS broth with 0, 0.05, 0.15 and 0.3% oxgall over a 24 h period. Most isolates were tolerant to bile, and were subsequently identified using the Analytical Profile Index (API) system. The following strains of LAB were most commonly identified, Lactobacillus acidophilus, L. fermentum, L. delbreukii, L. salivarius, L. brevis, L. cellobiosus, Leuconostoc spp., and Pediococcus acidilactici. Well-identified strains were further tested for antibiotic resistance and inhibition towards E. coli O157:H7 in manure and rumen fluid. Four of the 19 strains showed susceptibility to all the antibiotics. Seven of the 19 strains (37%) significantly reduced E. coli counts in manure and 13 of the 19 strains (68%) significantly reduced E. coli counts in rumen fluid (P < 0.05). Two of the isolates, M35 and L411, that were found closely related to each other by ribotyping analysis, were finally selected for CEP and further tested for acid tolerance and resistance to vancomycin, cephalosporin, and polymixin B. For acid tolerance, growth was monitored at pH 2, 4, 5, and 7 in MRS broth. Both isolates showed acid tolerance and were susceptible to the three antibiotics tested for. The 16S rRNA sequence analysis of M35 revealed its close homology to L. crispatus. The developed CEP will be further used in cattle feeding trials. |
