Construction and Characterization of Escherichia coli O157:H7 Strains Expressing Firefly Luciferase and Green Fluorescent Protein and Their Use in Survival Studies

Manure samples were collected from 16 organic (ORG) and 9 low-input conventional (LIC) Dutch dairy farms during August and September 2004 to determine the prevalence of the STEC virulence genes stx 1 (encoding Shiga toxin 1), stx 2 (encoding Shiga toxin 2), and eaeA (encoding intimin), as well as the rfbE gene, which is specific for Escherichia coli O157. The rfbE gene was present at 52% of the farms. The prevalence of rfbE was higher at ORG farms (61%) than at LIC farms (36%), but this was not significant. Relatively more LIC farms were positive for all Shiga toxin-producing E. coli (STEC) virulence genes eaeA, stx 1 , and stx 2 , which form a potentially highly virulent combination. Species richness of Enterobacteriaceae, as determined by DGGE, was significantly lower in manure positive for rfbE. Survival of a green fluorescent protein-expressing E. coli O157:H7 strain was studied in the manure from all farms from which samples were obtained and was modeled by a biphasic decline model. The time needed to reach the detection limit was predominantly determined by the level of native coliforms and the pH (both negative relationships). Initial decline was faster for ORG manure but leveled off earlier, resulting in longer survival than in LIC manure. Although the nonlinear decline curve could theoretically be explained as the cumulative distribution of an underlying distribution of decline kinetics, it is proposed that the observed nonlinear biphasic pattern of the survival curve is the result of changing nutrient status of the manure over time (and thereby changing competition pressure), instead of the presence of subpopulations differing in the level of resistance.Shiga-toxin producing Escherichia coli (STEC) is an important group of zoonotic human pathogens, with E. coli O157 being the best known and most studied serotype (10). STEC strains are generally carried asymptomatically by cattle and shed in their feces, which in turn may serve as a means of maintenance and spread of these pathogens among cattle herds (47). Since the reported increase in food-borne disease associated with the consumption of fresh vegetables (51, 54), the potential contamination of vegetable crops that are grown in fields enriched with manure has raised increasing concern (18, 29, 52). Understanding on-farm survival and spread of human pathogens is of utmost importance in preventing the spread of this organism to the environment, groundwater, food, and crops and subsequently back to cattle.A considerable body of literature exists on the prevalence of STEC on cattle farms and statistically associated management factors which could be used for potential intervention strategies to reduce shedding of these pathogens (2, 53, 57). However, these studies are not unambiguous and have resulted in the identification of only a very few risk factors, like season and cattle age, which are ubiquitous. The direction of influence of the majority of the management practices like feeding regimen and housing conditions remains unclear and is under debate.D...

Section: Methodsmentioning

confidence: 85%

Prevalence of Shiga Toxin-Producing Escherichia coli stx ₁ , stx ₂ , eaeA , and rfbE Genes and Survival of E. coli O157:H7 in Manure from Organic and Low-Input Conventional Dairy Farms

Franz

Klerks

Vos

et al. 2007

“…All cores were inoculated with a rifamycin-resistant derivative of E. coli O157:H7 strain B6914 containing plasmid pGFP with genes for green fluorescent protein and ampicillin resistance (8). B6914 was grown to the mid-log phase in Luria-Bertani broth (Gibco BRL, Gaithersburg, Md.)…”

Section: Methodsmentioning

confidence: 99%

Leaching of Escherichia coli O157:H7 in Diverse Soils under Various Agricultural Management Practices

Gagliardi

Karns

2000

273

Application of animal manures to soil as crop fertilizers is an important means for recycling the nitrogen and phosphorus which the manures contain. Animal manures also contain bacteria, including many types of pathogens. Manure pathogen levels depend on the source animal, the animal's state of health, and how the manure was stored or treated before use. Rainfall may result in pathogen spread into soil by runoff from stored or unincorporated manure or by leaching through the soil profile. Steady rainfall consisting of 16.5 mm h ؊1 was applied to 100-mm disturbed soil cores that were treated with manure and inoculated with Escherichia coli O157:H7 strain B6914. The level of B6914 in leachate was near the inoculum level each hour for 8 h, as was the level of B6914 at several soil depths after 24 h, indicating that there was a high rate of growth. Bacterial movement through three different types of soil was then compared by using disturbed (tilled) and intact (no-till) soil cores and less intense rainfall consisting of 25.4 mm on 4 consecutive days and then four more times over a 17-day period. Total B6914 levels exceeded the inoculum levels for all treatments except intact clay loam cores. B6914 levels in daily leachate samples decreased sharply with time, although the levels were more constant when intact sandy loam cores were used. The presence of manure often increased total B6914 leachate and soil levels in intact cores but had the opposite effect on disturbed soil cores. Ammonia and nitrate levels correlated with B6914 and total coliform levels in leachate. We concluded that tillage practice, soil type, and method of pathogen delivery affect but do not prevent vertical E. coli O157:H7 and coliform transport in soil and that soluble nitrogen may enhance transport.

“…This strain does not produce Shiga-like toxins I and II (Stx1 Ϫ Stx2 Ϫ ) but contains the pGFP cDNA vector (Clontech Laboratories, Inc., Palo Alto, CA) expressing green fluorescent protein (GFP) and ampicillin resistance. The survival characteristics of the GFP-labeled strain were indistinguishable from those of the wild-type strain (13). In addition, Kudva et al (26) reported no differences in survival in bovine manure and manure slurry between toxin-positive (Stx1 ϩ Stx2 ϩ ) and toxin-negative (Stx1 Ϫ Stx2 Ϫ ) E. coli O157:H7.…”

Section: Methodsmentioning

confidence: 95%

“…Escherichia coli O157:H7 strain B6-914 gfp-91 was kindly provided by Pina Fratamico (13). This strain does not produce Shiga-like toxins I and II (Stx1 Ϫ Stx2 Ϫ ) but contains the pGFP cDNA vector (Clontech Laboratories, Inc., Palo Alto, CA) expressing green fluorescent protein (GFP) and ampicillin resistance.…”

Section: Methodsmentioning

confidence: 99%

Effects of Cattle Feeding Regimen and Soil Management Type on the Fate of Escherichia coli O157:H7 and Salmonella enterica Serovar Typhimurium in Manure, Manure-Amended Soil, and Lettuce

Franz

Diepeningen

Vos

et al. 2005

175

155

Survival of the green fluorescent protein-transformed human pathogens Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium was studied in a laboratory-simulated lettuce production chain. Dairy cows were fed three different roughage types: high-digestible grass silage plus maize silage (6:4), low-digestible grass silage, and straw. Each was adjusted with supplemental concentrates to high and low crude protein levels. The pathogens were added to manure, which was subsequently mixed (after 56 and 28 days for E. coli O157:H7 and Salmonella serovar Typhimurium, respectively) with two pairs of organically and conventionally managed loamy and sandy soil. After another 14 days, iceberg lettuce seedlings were planted and then checked for pathogens after 21 days of growth. Survival data were fitted to a logistic decline function (exponential for E. coli O157:H7 in soil). Roughage type significantly influenced the rate of decline of E. coli O157:H7 in manure, with the fastest decline in manure from the pure straw diet and the slowest in manure from the diet of grass silage plus maize silage. Roughage type showed no effect on the rate of decline of Salmonella serovar Typhimurium, although decline was significantly faster in the manure derived from straw than in the manure from the diet of grass silage plus maize silage. The pH and fiber content of the manure were significant explanatory factors and were positively correlated with the rate of decline. With E. coli O157:H7 there was a trend of faster decline in organic than in conventional soils. No pathogens were detected in the edible lettuce parts. The results indicate that cattle diet and soil management are important factors with respect to the survival of human pathogens in the environment.