Isolation and Physiomorphological Characterization of Escherichia coli

Bacteriophages, recovered from beef cattle environment and specifically targeting Escherichia coli O157:H7, were examined for their physiological and morphological characteristics. Degree of bacterial lysis and host range of isolated bacteriophages was determined against 55 isolates of E. coli O157:H7. Morphology of phages was examined under transmission electron microscope. Phage growth parameters, particularly rate of adsorption, rise period, latent period, and burst size were also determined. The stability of isolated phages was tested at acidic and alkaline pH, at high temperatures, and in cold storage. A total of 7 phages were isolated which showed lytic activity against 50 out of 55 isolates of E. coli O157:H7. Based on the morphology, phages were classified into Myoviridae or Siphoviridae family. Phages had a rise period between 19 and 40 min, a short latent period between 12 and 30 min, and a large burst size (89–631 virions per infected cell), indicating high lytic activity. Phages remained stable for 24 h at a wide pH (1–11) and temperature range (40–60°C) and for 90 d in cold storage. Characterization of bacteriophages, with a diverse host range of E. coli O157:H7, could aid in the development of effective biocontrol strategies for this pathogen in the food industry

Development of a competitive exclusion product to reduce the carriage of Escherichia coli O157:H7 in cattle

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 \u3c 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

LACTIC ACID BACTERIA CULTURES THAT INHIBIT FOOD-BORNE PATHOGENS: U.S. Patent No. US 7,323,166 B2

The present invention provides methods and compositions for preventing or inhibiting human food-borne pathogens in animals, and methods for increasing feed efficiency in animals by administering to the animal effective amounts of probiotic lactic acid producing bacteria. Further provided are feed compositions comprising probiotic lactic acid producing bacteria. A preferred probiotic lactic acid producing bacteria is Lactobacillus acidophilus strain ATCC accession number PTA-5249. This bacterial strain inhibits nalidixic acid-resistant Escherichia coli 0157:H7

Identification of Contamination Sources and Assessment of Risk Factors Associated with the Occurrence of Escherichia coli O157:H7 on Small-scale Cow-calf Operations in Oklahoma and Louisiana

Escherichia coli O157:H7 is a human pathogen that exists as part of the commensal microflora of cattle and is shed in animal feces. Little is known about the effect of management practices on its occurrence and transmission on small-scale cow-calf operations. Identification of risk factors associated with farm practices could help implement effective measures to control E. coli O157:H7. This study quantified the risk of E. coli O157:H7 occurrence associated with cow-calf farm practices using risk modeling. Management practices of small-scale cow-calf operations in OK and LA were assessed through survey-based research. Fecal, water, sediments and water-trough-swab samples were collected to determine the incidence of E. coli O157:H7, and potential on-farm contamination sources and risk factors identified. Association between the occurrence of pathogen and farm practices was determined using two risk assessment models (I and II). Model I determined the association of E. coli O157:H7 occurrence with water source, water container, feed, cattle breed, and herd density, while Model II determined its association with farm cleanliness. For both models, logistic regression was followed using a two-step approach, univariable and multivariable analysis. In OK and LA, E. coli O157:H7 was present in 5.8% and 8.8% fecal, 4.4% and 9.4% water, 10.3% and 9.6% sediments, and 1.5% and 10.6% water-trough-swab samples, respectively. In Model I, univariable analysis identified water container and feed, whereas multivariable analysis identified feed as a significant risk factor. In Model II, the univariable analysis found cleanliness of cattle-contact areas, such as, alleyways, water-trough, chute and equipment, to be a significant risk factor. In multivariable analysis, only the cleanliness of water-trough was identified to be a significant risk factor. Results from the study could aid in the development of on-farm best management practices for the reduction of E. coli O157:H7