Differences in Growth of Salmonella enterica and Escherichia coli O157:H7 on Alfalfa Sprouts

Sprout producers have recently been faced with several Salmonella enterica and Escherichia coli O157:H7 outbreaks. Many of the outbreaks have been traced to sprout seeds contaminated with low levels of human pathogens. Alfalfa seeds were inoculated with S. enterica and E. coli O157:H7 strains isolated from alfalfa seeds or other environmental sources and sprouted to examine growth of these human pathogens in association with sprouting seeds. S. enterica strains grew an average of 3.7 log₁₀ on sprouting seeds over 2 days, while E. coli O157:H7 strains grew significantly less, an average of 2.3 log₁₀. The initial S. enterica or E. coli O157:H7 inoculum dose and seed-sprouting temperature significantly affected the levels of both S. enterica and E. coli O157:H7 on the sprouts and in the irrigation water, while the frequency of irrigation water replacement affected only the levels of E. coli O157:H7. Colonization of sprouting alfalfa seeds by S. enterica serovar Newport and E. coli O157:H7 strains transformed with a plasmid encoding the green fluorescent protein was examined with fluorescence microscopy. Salmonella serovar Newport colonized both seed coats and sprout roots as aggregates, while E. coli O157:H7 colonized only sprout roots.     

Reduction of Escherichia coli O157:H7 on radish seeds by sequential application of aqueous chlorine dioxide and dry‐heat treatment

Aims: To assess the effectiveness of sequential treatments of radish seeds with aqueous chlorine dioxide (ClO₂) and dry heat in reducing the number of Escherichia coli O157:H7. Methods and Results: Radish seeds containing E. coli O157:H7 at 5·5 log CFU g^?1 were treated with 500 μg ml^?1 ClO₂ for 5 min and subsequently heated at 60°C and 23% relative humidity for up to 48 h. Escherichia coli O157:H7 decreased by more than 4·8 log CFU g^?1 after 12 h dry‐heat treatment. The pathogen was inactivated after 48 h dry‐heat treatment, but the germination rate of treated seeds was substantially reduced from 91·2 ± 5·0% to 68·7 ± 12·3%. Conclusions: Escherichia coli O157:H7 on radish seeds can be effectively reduced by sequential treatments with ClO₂ and dry heat. To eliminate E. coli O157:H7 on radish seeds without decreasing the germination rate, partial drying of seeds at ambient temperature before dry‐heat treatment should be investigated, and conditions for drying and dry‐heat treatment should be optimized. Significance and Impact of the study: This study showed that sequential treatment with ClO₂ and dry‐heat was effective in inactivating large numbers of E. coli O157:H7 on radish seeds. These findings will be useful when developing sanitizing strategies for seeds without compromising germination rates.     

Differences in Attachment of Salmonella enterica Serovars and Escherichia coli O157:H7 to Alfalfa Sprouts

Numerous Salmonella enterica and Escherichia coli O157:H7 outbreaks have been associated with contaminated sprouts. We examined how S. enterica serovars, E. coli serotypes, and nonpathogenic bacteria isolated from alfalfa sprouts grow on and adhere to alfalfa sprouts. Growth on and adherence to sprouts were not significantly different among different serovars of S. enterica, but all S. enterica serovars grew on and adhered to alfalfa sprouts significantly better than E. coli O157:H7. E. coli O157:H7 was essentially rinsed from alfalfa sprouts with repeated washing steps, while 1 to 2 log CFU of S. enterica remained attached per sprout. S. enterica Newport adhered to 3-day-old sprouts as well as Pantoea agglomerans and 10-fold more than Pseudomonas putida and Rahnella aquatilis, whereas the growth rates of all four strains throughout seed sprouting were similar. S. enterica Newport and plant-associated bacteria adhered 10- to 1,000-fold more than E. coli O157:H7; however, three of four other E. coli serotypes, isolated from cabbage roots exposed to sewage water following a spill, adhered to sprouts better than E. coli O157:H7 and as well as the Pseudomonas and Rahnella strains. Therefore, attachment to alfalfa sprouts among E. coli serotypes is variable, and nonpathogenic strains of E. coli to be used as surrogates for the study of pathogenic E. coli may be difficult to identify and should be selected carefully, with knowledge of the biology being examined.     

An Improved Enrichment Broth for Isolation of Escherichia coli O157, with Specific Reference to Starved Cells, from Radish Sprouts

An enrichment broth was developed for the efficient isolation of Escherichia coli O157 from radish sprouts. The broth was buffered peptone water containing 0.5% sodium thioglycolate (STG-BPW), which was designed to allow growth of E. coli O157 in starved and unstarved states. However, this medium suppressed the growth of non-carbohydrate-fermenting obligate aerobes whose colonial appearance on sorbitol MacConkey agar containing cefixime and tellurite (CT-SMAC) resembled that of E. coli O157. Both starved and unstarved cells of E. coli O157 experimentally inoculated into radish sprouts were successfully recovered with STG-BPW enrichment in all cases, most of which showed marked disappearance of E. coli O157-like colonies on CT-SMAC.     

Selective Enrichment with a Resuscitation Step for Isolation of Freeze-Injured Escherichia coli O157:H7 from Foods

We studied injury of Escherichia coli O157:H7 cells in 11 food items during freeze storage and methods of isolating freeze-injured E. coli O157:H7 cells from foods. Food samples inoculated with E. coli O157:H7 were stored for 16 weeks at −20°C in a freezer. Noninjured and injured cells were counted by using tryptic soy agar and sorbitol MacConkey agar supplemented with cefixime and potassium tellurite. Large populations of E. coli O157:H7 cells were injured in salted cabbage, grated radish, seaweed, and tomato samples. In an experiment to detect E. coli O157:H7 in food samples artificially contaminated with freeze-injured E. coli O157:H7 cells, the organism was recovered most efficiently after the samples were incubated in modified E. coli broth without bile salts at 25°C for 2 h and then selectively enriched at 42°C for 18 h by adding bile salts and novobiocin. Our enrichment method was further evaluated by isolating E. coli O157:H7 from frozen foods inoculated with the organism prior to freezing. Two hours of resuscitation at 25°C in nonselective broth improved recovery of E. coli O157:H7 from frozen grated radishes and strawberries, demonstrating that the resuscitation step is very effective for isolating E. coli O157:H7 from frozen foods contaminated with injured E. coli O157:H7 cells.     

Transmission of Escherichia coli O157:H7 from Contaminated Manure and Irrigation Water to Lettuce Plant Tissue and Its Subsequent Internalization

The transmission of Escherichia coli O157:H7 from manure-contaminated soil and irrigation water to lettuce plants was demonstrated using laser scanning confocal microscopy, epifluorescence microscopy, and recovery of viable cells from the inner tissues of plants. E. coli O157:H7 migrated to internal locations in plant tissue and was thus protected from the action of sanitizing agents by virtue of its inaccessibility. Experiments demonstrate that E. coli O157:H7 can enter the lettuce plant through the root system and migrate throughout the edible portion of the plant.     

Potential Uptake of Escherichia coli O157:H7 from Organic Manure into Crisphead Lettuce

To investigate the potential transfer of Escherichia coli O157:H7 from contaminated manure to fresh produce, lettuce seedlings were transplanted into soil fertilized with bovine manure which had been inoculated with approximately 10⁴ CFU g⁻¹ E. coli O157:H7. The lettuce was grown for approximately 50 days in beds in climate-controlled rooms in a greenhouse. As the bacterium was not detected in the edible parts of the lettuce, the outer leaves of the lettuce, or the lettuce roots at harvest it was concluded that transmission of E. coli O157:H7 from contaminated soil to lettuce did not occur. The pathogen persisted in the soil for at least 8 weeks after fertilizing but was not detected after 12 weeks. Indigenous E. coli was detected only sporadically on the lettuce at harvest, and enterococci were not detected at all. The numbers of enterococci declined more rapidly than those of E. coli in the soil. Pseudomonas fluorescens, which inhibited growth of E. coli O157:H7 in vitro, was isolated from the rhizosphere.     

Survival of Escherichia coli O157:H7 in Soils from Jiangsu Province,China

Escherichia coli O157:H7 (E. coli O157:H7) is recognized as a hazardous microorganism in the environment and for public health. The E. coli O157:H7 survival dynamics were investigated in 12 representative soils from Jiangsu Province, where the largest E. coli O157:H7 infection in China occurred. It was observed that E. coli O157:H7 declined rapidly in acidic soils (pH, 4.57 – 5.14) but slowly in neutral soils (pH, 6.51 – 7.39). The survival dynamics were well described by the Weibull model, with the calculated t_d value (survival time of the culturable E. coli O157:H7 needed to reach the detection limit of 100 CFU g⁻¹) from 4.57 days in an acidic soil (pH, 4.57) to 34.34 days in a neutral soil (pH, 6.77). Stepwise multiple regression analysis indicated that soil pH and soil organic carbon favored E. coli O157:H7 survival, while a high initial ratio of Gram-negative bacteria phospholipid fatty acids (PLFAs) to Gram-positive bacteria PLFAs, and high content of exchangeable potassium inhibited E. coli O157:H7 survival. Principal component analysis clearly showed that the survival profiles in soils with high pH were different from those with low pH.     

Effects of Acid Adaptation, Product pH, and Heating on Survival of Escherichia coli O157:H7 in Pepperoni

The thermotolerance of E. coli O157:H7 cells (strain 380-94) heated in pepperoni is reported. Information on the pattern of thermal inactivation of E. coli O157:H7 in pepperoni was applied in the development of heating processes designed to reduce E. coli O157:H7 numbers therein by 5 log₁₀ units.     

Plant Lesions Promote the Rapid Multiplication of Escherichia coli O157:H7 on Postharvest Lettuce

Several outbreaks of Escherichia coli O157:H7 infections have been associated with minimally processed leafy vegetables in the United States. Harvesting and processing cause plant tissue damage. In order to assess the role of plant tissue damage in the contamination of leafy greens with E. coli O157:H7, the effect of mechanical, physiological, and plant disease-induced lesions on the growth of this pathogen on postharvest romaine lettuce was investigated. Within only 4 h after inoculation, the population sizes of E. coli O157:H7 increased 4.0-, 4.5-, and 11.0-fold on lettuce leaves that were mechanically bruised, cut into large pieces, and shredded into multiple pieces, respectively. During the same time, E. coli O157:H7 population sizes increased only twofold on leaves that were left intact after harvest. Also, the population size of E. coli O157:H7 was 27 times greater on young leaves affected by soft rot due to infection by Erwinia chrysanthemi than on healthy middle-aged leaves. Confocal microscopy revealed that leaf tip burn lesions, which are caused by a common physiological disorder of lettuce, harbored dense populations of E. coli O157:H7 cells both internally and externally. Investigation of the colonization of cut lettuce stems by E. coli O157:H7 showed that the pathogen grew 11-fold over 4 h of incubation after its inoculation onto the stems, from which large amounts of latex were released. The results of this study indicate that plant tissue damage of various types can promote significant multiplication of E. coli O157:H7 over a short time and suggest that harvesting and processing are critical control points in the prevention or reduction of E. coli O157:H7 contamination of lettuce.     

Prevalence of Escherichia coli O157:H7 in Gallbladders of Beef Cattle

Gallbladders and rectal contents were collected from cattle (n = 933) at slaughter to determine whether the gallbladder harbors Escherichia coli O157:H7. Both gallbladder mucosal swabs and homogenized mucosal tissues were used for isolation. Only five gallbladders (0.54%) were positive for E. coli O157:H7. Fecal prevalence averaged 7.1%; however, none of the cattle that had E. coli O157:H7 in the gallbladder was positive for E. coli O157:H7 in feces. Therefore, the gallbladder does not appear to be a common site of colonization for E. coli O157:H7 in beef cattle.     

Multiplex Fluorogenic Real-Time PCR for Detection and Quantification of Escherichia coli O157:H7 in Dairy Wastewater Wetlands

Surface water and groundwater are continuously used as sources of drinking water in many metropolitan areas of the United States. The quality of water from these sources may be reduced due to increases in contaminants such as Escherichia coli from urban and agricultural runoffs. In this study, a multiplex fluorogenic PCR assay was used to quantify E. coli O157:H7 in soil, manure, cow and calf feces, and dairy wastewater in an artificial wetland. Primers and probes were designed to amplify and quantify the Shiga-like toxin 1 (stx1) and 2 (stx2) genes and the intimin (eae) gene of E. coli O157:H7 in a single reaction. Primer specificity was confirmed with DNA from 33 E. coli O157:H7 and related strains with and without the three genes. A direct correlation was determined between the fluorescence threshold cycle (C_T) and the starting quantity of E. coli O157:H7 DNA. A similar correlation was observed between the C_T and number of CFU per milliliter used in the PCR assay. A detection limit of 7.9 × 10⁻⁵ pg of E. coli O157:H7 DNA ml⁻¹ equivalent to approximately 6.4 × 10³ CFU of E. coli O157:H7 ml⁻¹ based on plate counts was determined. Quantification of E. coli O157:H7 in soil, manure, feces, and wastewater was possible when cell numbers were ≥3.5 × 10⁴ CFU g⁻¹. E. coli O157:H7 levels detected in wetland samples decreased by about 2 logs between wetland influents and effluents. The detection limit of the assay in soil was improved to less than 10 CFU g⁻¹ with a 16-h enrichment. These results indicate that the developed PCR assay is suitable for quantitative determination of E. coli O157:H7 in environmental samples and represents a considerable advancement in pathogen quantification in different ecosystems.     

Rectoanal Junction Colonization of Feedlot Cattle by Escherichia coli O157:H7 and Its Association with Supershedders and Excretion Dynamics

Feedlot cattle were observed for fecal excretion of and rectoanal junction (RAJ) colonization with Escherichia coli O157:H7 to identify potential “supershedders.” RAJ colonization and fecal excretion prevalences were correlated, and E. coli O157:H7 prevalences and counts were significantly greater for RAJ samples. Based on a comparison of RAJ and fecal ratios of E. coli O157:H7/E. coli counts, the RAJ appears to be preferentially colonized by the O157:H7 serotype. Five supershedders were identified based on persistent colonization with high concentrations of E. coli O157:H7. Cattle copenned with supershedders had significantly greater mean pen E. coli O157:H7 RAJ and fecal prevalences than noncopenned cattle. Cumulative fecal E. coli O157:H7 excretion was also significantly higher for pens housing a supershedder. E. coli O157:H7/E. coli count ratios were higher for supershedders than for other cattle, indicating greater proportional colonization. Pulsed-field gel electrophoresis analysis demonstrated that isolates from supershedders and copenned cattle were highly related. Cattle that remained negative for E. coli O157:H7 throughout sampling were five times more likely to have been in a pen that did not house a supershedder. The data from this study support an association between levels of fecal excretion of E. coli O157:H7 and RAJ colonization in pens of feedlot cattle and suggest that the presence of supershedders influences group-level excretion parameters. An improved understanding of individual and population transmission dynamics of E. coli O157:H7 can be used to develop preslaughter- and slaughter-level interventions that reduce contamination of the food chain.     

Modification of sorbitol MacConkey medium containing cefixime and tellurite for isolation of Escherichia coli O157:H7 from radish sprouts

A modified version of sorbitol MacConkey medium containing cefixime and tellurite (CT-SMAC medium) was produced by adding salicin and 4-methylumbelliferyl-beta-D-galactopyranoside to CT-SMAC medium; this medium was designated CT-SSMAC medium and was used to isolate Escherichia coli O157:H7 from radish sprouts. Of 101 non-E. coli bacteria isolated from radish sprouts that produced colorless colonies similar to colonies of E. coli O157:H7 grown on CT-SMAC medium, 92 (91%) formed colonies that were red to pink or were beta-galactosidase negative and colorless on CT-SSMAC medium. On the other hand, colonies of E. coli O157:H7 strains were colorless and beta-galactosidase positive on CT-SSMAC medium. Our results suggest that CT-SSMAC medium is more selective than CT-SMAC medium for isolating E. coli O157:H7.     

Isolation of Potentially Pathogenic Escherichia coli O157:H7 from the Ganges River

Escherichia coli serotype O157:H7 was detected among bacteria collected from the Ganges River. O157:H7 isolates tested positive for stx₁, stx₂, and eae gene sequences. Identification of potentially pathogenic isolates from extensively used source water indicates that O157:H7 may be a significant but as yet underacknowledged public health concern in India.     

Extractable Organic Components and Nutrients in Wastewater from Dairy Lagoons Influence the Growth and Survival of Escherichia coli O157:H7

The influence of nutrients in wastewater from dairy lagoons on the survival of Escherichia coli O157:H7 was monitored. Initially, the survival of E. coli O157:H7 in wastewater from which the competing native organisms had been removed by filter sterilization or autoclaving was compared with that in wastewater from which competing organisms had not been removed. Numbers of E. coli O157:H7 or E. coli ONT (O-nontypeable):H32 cells declined rapidly in filter-sterilized water and exhibited a slower decline in nonsterile water, while the organisms proliferated in autoclaved water. Subsequently, the growth of E. coli O157:H7 strains was monitored in 300 μl of Luria-Bertani (LB) broth supplemented with incremental proportions of filter-sterilized wastewater. E. coli O157:H7 and E. coli ONT:H32 strains failed to grow in filter-sterilized wastewater, and their growth was reduced incrementally with wastewater supplementation of LB broth. Consequently, the influence of organic extracts of wastewater on the growth of E. coli O157:H7 and E. coli ONT:H32 in reduced-strength LB was monitored, followed by scale-up tests in wastewater. Acidic and basic extracts inhibited growth of both strains, while the neutral aqueous extract improved growth. However, a scale-up with a threefold increase in the acidic components supplementing the wastewater did not result in any additional decline in numbers of E. coli O157:H7 cells. When protected inside a 300-kDa dialysis tube and exposed to diffusible components, E. coli O157:H7 survived longer, with a decimal reduction time of 18.1 days, compared to 3.5 days when inoculated directly into wastewater. Although wastewater can potentially provide nutrients to naturally occurring human pathogens, the chemical components, protozoa, and coliphages in wastewater can inhibit the growth of freshly introduced pathogens from manure.     

Coevolution of Bacteriophage PP01 and Escherichia coli O157:H7 in Continuous Culture

The interaction between Escherichia coli O157:H7 and its specific bacteriophage PP01 was investigated in chemostat continuous culture. Following the addition of bacteriophage PP01, E. coli O157:H7 cell lysis was observed by over 4 orders of magnitude at a dilution rate of 0.876 h⁻¹ and by 3 orders of magnitude at a lower dilution rate (0.327 h⁻¹). However, the appearance of a series of phage-resistant E. coli isolates, which showed a low efficiency of plating against bacteriophage PP01, led to an increase in the cell concentration in the culture. The colony shape, outer membrane protein expression, and lipopolysaccharide production of each escape mutant were compared. Cessation of major outer membrane protein OmpC production and alteration of lipopolysaccharide composition enabled E. coli O157:H7 to escape PP01 infection. One of the escape mutants of E. coli O157:H7 which formed a mucoid colony (Mu) on Luria-Bertani agar appeared 56 h postincubation at a dilution rate of 0.867 h⁻¹ and persisted until the end of the experiment (~200 h). Mu mutant cells could coexist with bacteriophage PP01 in batch culture. Concentrations of the Mu cells and bacteriophage PP01 increased together. The appearance of mutant phage, which showed a different host range among the O157:H7 escape mutants than wild-type PP01, was also detected in the chemostat culture. Thus, coevolution of phage and E. coli O157:H7 proceeded as a mutual arms race in chemostat continuous culture.     

Experimental and Field Studies of Escherichia coli O157:H7 in White-Tailed Deer

Studies were conducted to evaluate fecal shedding of Escherichia coli O157:H7 in a small group of inoculated deer, determine the prevalence of the bacterium in free-ranging white-tailed deer, and elucidate relationships between E. coli O157:H7 in wild deer and domestic cattle at the same site. Six young, white-tailed deer were orally administered 10⁸ CFU of E. coli O157:H7. Inoculated deer were shedding E. coli O157:H7 by 1 day postinoculation (DPI) and continued to shed decreasing numbers of the bacteria throughout the 26-day trial. Horizontal transmission to an uninoculated deer was demonstrated. Although E. coli O157:H7 bacteria were recovered from the gastrointestinal tracts of deer necropsied from 4 to 26 DPI, attaching and effacing lesions were not apparent in any deer. Results are similar to those of inoculation studies in calves and sheep. In field studies, E. coli O157 was not detected in 310 fresh deer fecal samples collected from the ground. It was detected in feces, but not in meat, from 3 of 469 free-ranging deer in 1997. In 1998, E. coli O157 was not detected in 140 deer at the single positive site found in 1997; however, it was recovered from 13 of 305 dairy and beef cattle at the same location. Isolates of E. coli O157:H7 from deer and cattle at this site differed with respect to pulsed-field gel electrophoresis patterns and genes encoding Shiga toxins. The low overall prevalence of E. coli O157:H7 and the identification of only one site with positive deer suggest that wild deer are not a major reservoir of E. coli O157:H7 in the southeastern United States. However, there may be individual locations where deer sporadically harbor the bacterium, and venison should be handled with the same precautions recommended for beef, pork, and poultry.     

Effects of Common Forage Phenolic Acids on Escherichia coli O157:H7 Viability in Bovine Feces

Ruminant animals are carriers of Escherichia coli O157:H7, and the transmission of E. coli O157:H7 from cattle to the environment and to humans is a concern. It is unclear if diet can influence the survivability of E. coli O157:H7 in the gastrointestinal system or in feces in the environment. Feces from cattle fed bromegrass hay or corn silage diets were inoculated with E. coli O157:H7, and the survival of this pathogen was analyzed. When animals consumed bromegrass hay for <1 month, viable E. coli O157:H7 was not recovered after 28 days postinoculation, but when animals consumed the diet for >1 month, E. coli O157:H7 cells were recovered for >120 days. Viable E. coli O157:H7 cells in feces from animals fed corn silage were detected until day 45 and differed little with the time on the diet. To determine if forage phenolic acids affected the viability of E. coli O157:H7, feces from animals fed corn silage or cracked corn were amended with common forage phenolic acids. When 0.5% trans-cinnamic acid or 0.5% para-coumaric acid was added to feces from silage-fed animals, the E. coli O157:H7 death rate was increased significantly (17-fold and 23-fold, respectively) compared to that with no addition. In feces from animals fed cracked corn, E. coli O157:H7 death rates were increased significantly with the addition of 0.1% and 0.5% trans-cinnamic acid (7- and 13-fold), 0.1% and 0.5% p-coumaric acid (3- and 8-fold), and 0.5% ferulic acid (3-fold). These data suggest that phenolic acids common to forage plants can decrease viable counts of E. coli O157:H7 shed in feces.     

Characterization of Shiga Toxin-Producing Escherichia coli Isolates Associated with Two Multistate Food-Borne Outbreaks That Occurred in 2006

Shiga toxin-producing Escherichia coli isolates from two 2006 outbreaks were compared to other O157:H7 isolates for virulence genotype, biofilm formation, and stress responses. Spinach- and lettuce-related-outbreak strains had similar pulsed-field gel electrophoresis patterns, and all carried both stx₂ and stx_2c variant genes. Cooperative biofilm formation involving an E. coli O157:H7 strain and a non-O157:H7 strain was also demonstrated.