Decreased shedding of Escherichia coli O157:H7 by cattle following vaccination with type III secreted proteins

Cattle are an important reservoir of Escherichia coli O157:H7 leading to contamination of food and water, and subsequent human disease. This pathogen colonizes its hosts by producing several proteins such as Tir and EspA that are secreted by a type III secretion system. These proteins play a role in colonization of the intestine, suggesting that they might be useful targets for the development of a vaccine to reduce levels of this organism in cattle. Vaccination of cattle with proteins secreted by E. coli O157:H7 significantly reduced the numbers of bacteria shed in feces, the numbers of animals that shed, and the duration of shedding in an experimental challenge model. Vaccination of cattle also significantly (P=0.04) reduced the prevalence of E. coli O157:H7 in a clinical trial conducted in a typical feedlot setting. This strategy suggests it is possible to vaccinate cattle to decrease the level of E. coli O157:H7 shedding for the purpose of reducing the risk of human disease.     

Efficacy of a vaccine and a direct-fed microbial against fecal shedding of Escherichia coli O157:H7 in a randomized pen-level field trial of commercial feedlot cattle

Our primary objective was to determine the efficacy of a siderophore receptor and porin proteins-based vaccine (VAC) and a Lactobacillus acidophilus-based direct-fed microbial (DFM) against fecal shedding of Escherichia coli O157:H7 in commercial feedlot cattle fed a corn grain-based diet with 25% distiller's grains. Cattle projected to be on a finishing diet during the summer were randomly allocated into 40 study pens within ten blocks based on allocation dates. Blocks were complete; each of the four pens within a block was randomly assigned one treatment: control, VAC, DFM, or VAC+DFM. The DFM was fed (10(6)CFU/animal/day of Lactobacillus) throughout the study periods (84-88 days) and cattle were vaccinated at enrollment and again three weeks later. Fresh fecal samples (30/pen) from pen floors were collected weekly for four consecutive weeks (study days 52-77). Two concurrent culture procedures were used to enable estimates of E. coli O157:H7 shedding prevalence and prevalence of high shedders. From 4800 total samples, 1522 (31.7%) were positive for E. coli O157:H7 and 169 (3.5%) were considered high shedders. Pen-level linear mixed models were used for data analyses. There were no significant interactions among treatments and time of sampling. However, vaccinated pens had lower (P<0.01) overall prevalence of E. coli O157:H7 (model-adjusted mean ±SEM=17.4±3.95%) and lower (P<0.01) prevalence of high shedders (0.95±0.26%) than unvaccinated pens (37.0±6.32% and 4.19±0.81%, respectively). There was no evidence of a DFM effect on either measure of E. coli O157:H7 shedding. Results indicate that a two-dose regimen of the vaccine significantly reduces fecal prevalence of E. coli O157:H7 (vaccine efficacy of 53.0%) and prevalence of E. coli O157:H7 high shedders (vaccine efficacy of 77.3%) in commercial feedlot cattle reared in the summer on a finishing diet with 25% distiller's grains.     

肠出血性大肠埃希菌O157:H7疫苗的研究进展

O157:H7是肠出血性大肠埃希菌(EHEC)最常见的血清型.目前国内外尚无EHEC O157:H7疫苗问世,因此疫苗研究对防治EHEC O157:H7有着重大意义.外膜蛋白A(OmpA)作为抗EHEC O157:H7基础疫苗为疫苗研究提供了新的思路,此文对EHEC O157:H7疫苗研究的有关进展作了综述.     

肠出血型大肠埃希菌O157∶H7基因工程疫苗研究进展

肠出血型大肠埃希菌O157∶H7(EHEC O157∶H7)是重要的新发传染病,多次在世界范围内暴发流行。EHEC O157∶H7可引起出血性肠炎、溶血性尿毒综合症、血栓性血小板减少性紫癜等,严重者可导致死亡。研制安全、有效的疫苗是预防其感染最简单、经济、快捷的手段,但尚未有可用于人的疫苗。EHEC O157∶H7的毒力因子主要有T3SS相关蛋白、志贺毒素和表面菌毛及非菌毛黏附物质等,目前研究的疫苗种类包括单个毒力因子或者多个毒力因子融合的亚单位疫苗、DNA疫苗、载体活疫苗及BG疫苗等。本文通过阐述EHEC O157∶H7主要保护性抗原,就近年来EHEC O157∶H7基因工程疫苗研究进展进行综述,为其疫苗的研制提供新思路。     

Prevalence of Escherichia coli O157:H7 in range beef calves at weaning

This study was designed to determine the prevalence of Escherichia coli O157:H7 infection of beef calves at weaning, prior to arrival at the feedlot or mixing with cattle from other sources. Fifteen range cow-calf herds, which weaned calves in October and November, were sampled in Kansas, Missouri, Montana, Nebraska and South Dakota. Faecal culture for E. coli O157:H7 was performed and anti-O157 serum antibody titres were determined by blocking ELISA. Thirteen of the 15 herds (87%) were found to have at least one positive isolation of E. coli O157:H7 in faecal samples. Within positive herds, prevalence ranged from 1.7-20.0%, with an average of 7.4+/-6.2% S.D. of individual animals shedding E. coli O157:H7 in faeces. All herds had high prevalence of anti-O157 antibodies, ranging 63-100% of individuals within herds seropositive. This study indicates that E. coli O157:H7 infection before weaning, prior to entry into feedlots, is widespread. Furthermore, serologic evidence suggests that most calves (83%) and all herds (100%) have been exposed to E. coli O157.     

Antimicrobial activity of commercial citrus-based natural extracts against Escherichia coli O157:H7 isolates and mutant strains

Due to increasing concerns about the development of antimicrobial resistance amongst pathogenic bacteria, alternative strategies have been sought that do not use antibiotics to reduce pathogenic bacteria from foods and patients. A natural compound that has potent antimicrobial properties is citrus peel, which contains a variety of essential oils that inhibit the growth of or kill pathogenic bacteria. In the present study, seven citrus-based natural antimicrobials were evaluated for their ability to inhibit the growth of the pathogen Escherichia coli O157:H7. Zones of inhibition of E. coli O157:H7 by the citrus-derived fraction (10 microL/6 mm disk) were determined by a disk-diffusion assay on Sorbitol-MacConkey agar. Inhibition zones were observed after 48 h lawn growth of E. coli O157:H7 cells at 37 degrees C. Two citrus-based fractions, orange CP VAL terpeneless FAB 968611 and Limonene 1x Dist FAB 955430, inhibited E. coli O157:H7 with inhibition zones of approx. 11-24 mm dia. The remaining other five citrus-derived extracts (orange oil FL VAL 1121 ARR 974760, Orange 5x Conc VAL 4121 ARR 968374, orange terpenes ESS 1120 ARR 986259, orange terpenes CP 1100 ARR 986255, and orange terpenes OEO HP 1100 ARR 986257) were noninhibitory to E. coli O157:H7, yielding no clear inhibition zones. These studies show that citrus-derived natural compounds differ in their inhibitory activity against E. coli O157:H7 and some have potential applications as inhibitory agents against E. coli O157:H7 in various pathogen reduction strategies.     

Strategies to reduce person-to-person transmission during widespread Escherichia coli O157:H7 outbreak

During the Escherichia coli O157:H7 outbreak in 2006 in the United States, the primary strategy to prevent illness was to advise consumers not to eat spinach. No widespread warnings were issued about preventing person-to-person (secondary) transmission. A disease transmission model, fitted to the current data, was used to investigate likely reductions in illnesses that could result from interventions to prevent secondary transmission. The model indicates that exposure to contaminated spinach occurred early in the outbreak and that secondary transmission was similar to that in previous E. coli outbreaks ( 12%). The model also suggests that even a modestly effective strategy to interrupt secondary transmission (prevention of only 2%-3% of secondary illnesses) could result in a reduction of 5%-11% of symptomatic cases. This analysis supports the use of widespread public health messages during outbreaks of E. coli O157:H7 with specific advice on how to interrupt secondary transmission.     

Escherichia coli O157:H7 outbreak associated with consumption of ground beef, June-July 2002

OBJECTIVE: A case-control and environmental study tested the hypothesis that purchasing and eating ground beef from a specific source was the cause of a cluster of cases of hemolytic uremic syndrome (HUS) and Escherichia coli (E. coli) O157:H7 gastroenteritis. METHODS: A case-control study comparing risk factors was conducted over the telephone on nine case-patients with 23 selected controls. An environmental investigation was conducted that consisted of reviewing beef handling practices at a specific local supermarket and obtaining ground beef samples from the store and two households with case-patients. RESULTS: The analysis of the case-control study showed that eight case-patients (89%) purchased ground beef at Grocery Chain A compared with four controls who did not develop illness (17%) (matched odds ratio=undefined; 95% confidence interval 2.8, infinity; p=0.006). The environmental investigation showed that Grocery Chain A received meat from Meatpacker A. Laboratory analysis of meat samples from Meatpacker A and Grocery Chain A and stool samples from some patients recovered an identical strain of E. coli O157:H7 according to pulse-field gel electrophoresis. CONCLUSIONS: Both the case-control and environmental studies showed that purchasing ground beef at Grocery Chain A, which received ground beef from Meatpacker A, was the major risk factor for illness in eight case-patients; the ninth case-patient was found to be unrelated to the outbreak. Furthermore, meat from Meatpacker A was associated with a nationwide outbreak of E. coli O157:H7 illness that resulted in the second largest recall of beef in U.S. history at the time.     

Community-wide outbreak of Escherichia coli O157:H7 associated with consumption of frozen beef burgers

On 24-25 October 2005 a cluster of five haemolytic uraemic syndrome (HUS) cases was reported in southwest France. An investigation was undertaken to identify the outbreak source and implement control measures. Cases were defined as individuals with HUS or diarrhoea with isolation of Escherichia coli O157:H7 in stools or a positive antibody response to E. coli O157 lipopolysaccharide, resident in southwest France with symptom onset after 19 September 2005. Sixty-nine identified patients had symptom onset between 5 October and 3 November 2005, including 17 cases of HUS. One brand of frozen beef burgers produced on 22 August 2005 was consumed by all patients in the week before symptom onset. E. coli O157:H7 strains from patients, patients' burgers and the manufacturing plant were genetically related. This is the largest community-wide outbreak of E. coli O157:H7 in France to date and the first associated with consumption of contaminated frozen beef burgers.     

Bacteriophage isolated from feedlot cattle can reduce Escherichia coli O157:H7 populations in ruminant gastrointestinal tracts

Escherichia coli O157:H7 can live undetected in the gut of food animals and be spread to humans directly and indirectly. Bacteriophages are viruses that prey on bacteria, offering a natural, nonantibiotic method to reduce pathogens from the food supply. Here we show that a cocktail of phages isolated from commercial cattle feces reduced E. coli O157:H7 populations in the gut of experimentally inoculated sheep. A cocktail of phages was used in order to prevent the development of resistance to the phages. In our first in vivo study we found that our cocktail of phages reduced E. coli O157:H7 populations in the feces of sheep (p < 0.05) by 24 hours after phage treatment. Upon necropsy, populations of inoculated E. coli O157:H7 were reduced by phage treatment in both the cecum (p < 0.05) and rectum (p < 0.1). In our second in vivo study, several ratios of phage plaque-forming units (PFU) to E. coli O157:H7 colony-forming units (CFU) were used (0:1, 1:1, 10:1, and 100:1 PFU/CFU) to determine the most efficacious phage dose. A 1:1 ratio of phage to bacteria was found to be more effective (p < 0.05) than either of the higher ratios used (10:1 or 100:1). Ruminal levels of E. coli O157:H7 were not significantly reduced (p > 0.10) in any of the studies due to relatively low inoculated E. coli O157:H7 ruminal populations. Our results demonstrate that phage can be used as a preharvest intervention as part of an integrated pathogen reduction scheme.     

Antibodies among healthy population of developing countries against enterohaemorrhagic Escherichia coli O157:H7

In Thailand, no reports are available on Escherichia coli serotype O157:H7, a causative agent of severe bloody diarrhoea, sometimes associated with haemolytic-uraemic syndrome and thrombotic thrombocytopenic purpura. The reason for the non-identification of infection due to E. coli O157 in this country and in other developing countries has not been rigorously discussed. The aim of this study was to determine the humoral response against the infectious organism. The IgM and IgG antibody responses against E. coli O157 lipopolysaccharide were studied using indirect enzyme-linked immunosorbent assay. Three hundred and thirty-two serum samples obtained from healthy blood donors and patients with diseases unrelated to diarrhoea were investigated. With a cut-off value of mean +2 SDs for each age-group, the frequency of the IgM and IgG responses to O157 lipopolysaccharide was 11.74% (39 of 332 samples) and 22.59% (75 of 332 samples) respectively. Furthermore, agglutination test of 173 subjects revealed titres ranging from 10 to 40 in all the samples. The results suggest possible exposure of the Thai population to cross-reacting antigens from other intestinal organisms in addition to infection due to E. coli O157:H7.     

Escherichia coli O157:H7 infection in cows and calves in a beef cattle herd in Alberta,Canada

Escherichia coli O157:H7 infection of cows and calves in a naturally-infected beef cattle herd in Alberta, Canada, was investigated over 2 years, encompassing two calf production cycles. In both years of the study, E. coli O157:H7 was isolated from the faeces of cows shortly after but not before parturition in late winter: 6/38 (16%) in 1996 and 13/50 (26%) in 1997. At < 1 week post-partum, 13/52 (25%) calves born in 1997 were shedding the organism. Faecal shedding of E. coli O157:H7 by cows and calves continued over the 7 weeks that they were in the calving pens, with the organism being isolated from the faeces of 2-18% of cows and 23-26% of calves during this period. Five weeks after they were moved onto a native grass pasture, all the calves and all but one cow in 1997 had ceased shedding the organism. When the calves were weaned in the fall, E. coli O157:H7 was isolated from the faeces of 0-1.5% of the calves 1 week prior to weaning and from 6-14% of the calves within 2 weeks after weaning. Parturition, calving pens and weaning appear to be important factors in maintaining E. coli O157:H7 infections in this beef cattle herd. Isolates from cows and calves during the immediate post-partum period were mostly of the same pulsed-field gel electrophoresis (PFGE) type of E. coli O157:H7. Similarly, at weaning a common PFGE type of E. coli O157:H7, which differed slightly from the post-partum PFGE type, was isolated from the calves. These typing data suggest a common source of infection for the animals as well as demonstrate clonal turnover of resident populations of this pathogen.     

The prevalence of Escherichia coli O157.H7 in dairy and beef cattle in Washington State.

Escherichia coli O157.H7 was found in 10 of 3570 (0.28%) faecal samples from dairy cattle in 5 of 60 herds (8.3%). Several tentative associations with manure handling and feeding management practices on dairy farms were identified. Faecal/urine slurry samples, bulk milk samples, and milk filters from dairy herds were negative for E. coli O157.H7. E. coli O157.H7 was also isolated from 10 of 1412 (0.71%) faecal samples from pastured beef cattle in 4 of 25 (16%) herds. The prevalence of E. coli O157.H7 excretion in feedlot beef cattle was 2 of 600 (0.33%). The identification of cattle management practices associated with colonization of cattle by E. coli O157.H7 suggests the possibility that human E. coli O157.H7 exposure may be reduced by cattle management procedures.     

Natural and synthetic plant compounds as anti-biofilm agents against Escherichia coli O157:H7 biofilm

Escherichia coli is a common gram-negative bacterium found in the gut and intestinal tract of warm-blooded animals including humans. An evolved seropathotype E. coli O157:H7 (STEC) came into existence in 1982, since then it has been evolved as a stronger and more robust drug-resistant pathotype of E. coli. This drug resistance is due to horizontal gene transfer, natural gene evolution for survival, and most of the cases due to the ability of STEC to switch to the biofilm growth mode from planktonic lifestyle. During the growth in biofilm mode, Escherichia coli O157:H7 opts more robust ability to grow in adverse environments i.e., in presence of antibiotics and other antimicrobial chemicals. Due to the biofilm matrix, the microbial community acquires drug resistance. This makes the treatment of diseases caused by E. coli O157:H7 a complex challenge. To address the illnesses caused by this biofilm-forming pathogen, there are several possible strategies such as antibiotic therapies, synthetic antimicrobial chemicals, adjunct therapy of synergistic effect of multiple drugs, and more importantly plant originated compounds as a new anti-biofilm candidate. The present review summarizes various phytochemicals and their derivatives reported in the last decade mostly to eliminate the biofilm of STEC. The review will progressively reveal the antibiofilm mechanism of the phytochemicals against STEC and to be a potential candidate for the development of the future antibacterial drugs to STEC induced infections.     

Escherichia coli O157:H7 infection of calves: infectious dose and direct contact transmission.

Cattle are considered to be a reservoir host of Escherichia coli O157:H7 and contaminated foods of bovine origin are important vehicles of human infection. In this study, the susceptibility of calves to experimental E. coli O157:H7 infection following low oral exposures was determined. Two of 17 calves exposed to very low (< 300 c.f.u.) doses, and 3 of 4 calves exposed to low (< 10,000 c.f.u.) doses, subsequently excreted the challenge strains in their faeces. All calves (n = 12) sharing isolation rooms with calves that excreted the challenge strain in their faeces similarly began faecal excretion of the same strains within 21 days or less. The identity between the challenge strains and the strains excreted in calf faeces was confirmed by restriction digestion electrophoretic patterns using pulsed field gel electrophoresis. Calves shed E. coli O157:H7 in their faeces after very low dose exposures at concentrations ranging from < 30 to > 10(7) c.f.u./g, and for durations similar to the values previously reported for calves challenged by larger doses. The susceptibility of calves to infection following very low exposures or direct contact with infected calves has important implications for programmes for pre-harvest control of this agent.     

肠出血性大肠埃希菌O157∶H7的快速检测方法比较

目的比较肠出血性大肠埃希菌O157∶H7不同快速检测方法的现场检测效果,以寻找一种可以用于野外现场检测的简便方法。方法对商品销售的3种代表性试剂盒进行检测灵敏度、特异性及操作简便性的比较。结果试剂A及C检测大肠埃希菌O157∶H7的灵敏度分别为约8.64×105 CFU/ml和约8.64×104 CFU/ml,且操作简便,检测过程<30min;试剂B约为4.32×101 CFU/ml,但阳性时间为72h。结论试剂A、试剂C可用于大肠埃希菌O157∶H7急性腹泻的现场检测;试剂B的检测周期长,不适合现场检测,对急性腹泻的快速检测需要研究更加敏感、易于操作的检测方法。     

Thermal tolerance of acid-adapted and non-adapted Escherichia coli O157:H7 and Salmonella in ground beef during storage

Thermal tolerance of acid-adapted Escherichia coli O157:H7 or Salmonella in ground beef was evaluated during storage at 4 degrees C or -20 degrees C. Both pathogens were adapted to acidic conditions (pH approximately 4.6) by growing in tryptic soy broth supplemented with 1% glucose. A five-strain cocktail of E. coli O157:H7 or Salmonella was grown separately in TSB (pH approximately 6.6) and TSB + 1% glucose for 24 h at 37 degrees C to provide cells with or without acid adaptation. Irradiated ground beef was inoculated with either acid-adapted or non-adapted E. coli O157:H7 or Salmonella; the samples stored at 4 degrees C were subjected to heat treatment at 62 degrees C or 65 degrees C on days 1, 7, 14, 21, and 28, and the samples stored at -20 degrees C were subjected to heat treatment at 62 degrees C or 65 degrees C on days 1, 30, 60, 90, and 120. Decimal reduction time (D values) of the pathogens was determined as an indicator of thermal tolerance. Significantly higher D(62) values were observed on days 21 and 28 for non-adapted E. coli O157:H7 stored at 4 degrees C and on days 90 and 120 for non-adapted E. coli O157:H7 stored at -20 degrees C (P < 0.05). Higher D(62) values were observed on days 21 and 28 among non-adapted Salmonella strains stored at 4 degrees C and on day 28 for acid-adapted strains of Salmonella stored at 4 degrees C (P < 0.05). Higher D(62) values for acid-adapted strains of Salmonella stored at -20 degrees C were observed on days 30, 60, and 90 (P < 0.05), when while no differences were observed in the D(65) values of acid-adapted and non-adapted strains of E. coli O157:H7 and Salmonella throughout storage at both temperatures (P > 0.05). This suggests that acid adaptation of foodborne pathogens provides a certain level of protection against heat treatment at lower cooking temperatures, while at higher temperatures there were no observed differences between the sensitivity of acid-adapted and non-adapted strains in an actual food system over an extended period of refrigerated and frozen storage.     

Detection of Escherichia coli O157:H7 and Listeria monocytogenes in beef products by real-time polymerase chain reaction

Rapid methods for the detection of Escherichia coli O157:H7 and Listeria monocytogenes in food products are important to the food industry and for public health. Conventional microbiological methods and newly developed molecular-based techniques such as polymerase chain reaction (PCR)-based methods are time consuming. In this study, a faster method based on utilization of a hybridization probe with real-time PCR, was developed and applied for detection of E. coli O157:H7 and L. monocytogenes from artificially contaminated raw ground beef and fully cooked beef hotdogs. Target genes for E. coli O157:H7 and L. monocytogenes were rfbE and hylA, respectively. An analysis of 169 bacterial strains showed that the chosen primers and probes were specific for detection of E. coli O157:H7 and L. monocytogenes by real-time PCR. The assay was positive for nine of 10. E. coli O157:H7 strains, and all L. monocytogenes (7/7) strains evaluated. Bacterial strains lacking these genes were not detected by these assays. Detection limits of real-time PCR assays ranged from 10(3) to 10(8) colony forming units (CFU)/ml for E. coli O157:H7 in modified tryptic soy broth and 10(4) to 10(8) CFU/mL for L. monocytogenes in Fraser Broth. Detection sensitivity ranged from 10(3) to 10(4) CFU/g of raw ground beef or hotdog without enrichment for E. coli and L. monocytogenes. Approximately 1.4-2.2 CFU/g of E. coli O157:H7 in raw ground beef were detected following an enrichment step of 4 h. Approximately 1.2-6.0 CFU/g of L. monocytogenes in beef hotdogs were detected following an enrichment step of 30 h. The real-time PCR assays for detection of E. coli O157:H7 and L. monocytogenes in raw ground beef and beef hotdogs were specific, sensitive and rapid.     

Development of a dual vaccine for prevention of Brucella abortus infection and Escherichia coli O157:H7 intestinal colonization

Zoonoses that affect human and animal health have an important economic impact. In the study now presented, a bivalent vaccine has been developed that has the potential for preventing the transmission from cattle to humans of two bacterial pathogens: Brucella abortus and Shiga toxin-producing Escherichia coli (STEC). A 66 kDa chimeric antigen, composed by EspA, Intimin, Tir, and H7 flagellin (EITH₇) from STEC, was constructed and expressed in B. abortus Δpgm vaccine strain (BabΔpgm). Mice orally immunized with BabΔpgm(EITH₇) elicited an immune response with the induction of anti-EITH₇ antibodies (IgA) that clears an intestinal infection of E. coli O157:H7 three times faster (t = 4 days) than mice immunized with BabΔpgm carrier strain (t = 12 days). As expected, mice immunized with BabΔpgm(EITH₇) strain also elicited a protective immune response against B. abortus infection. A Brucella-based vaccine platform is described capable of eliciting a combined protective immune response against two bacterial pathogens with diverse lifestyles—the intracellular pathogen B. abortus and the intestinal extracellular pathogen STEC.     

Molecular typing methods to investigate transmission of Escherichia coli O157:H7 from cattle to humans.

The utility of phage typing, pulsed-field gel electrophoresis (PFGE), and plasmid profile analysis was compared, to differentiate between Canadian Escherichia coli O157:H7 strains of human (n = 27) and cattle (n = 24) origin. The diversity indices for phage typing, plasmid analysis and PFGE were 0.85, 0.69 and 0.93, respectively. PFGE and phage typing were also applied to study the role of direct transmission of E. coli O157:H7 from cattle to humans on isolates collected from two separate farm outbreaks. PFGE showed that more than one E. coli O157:H7 strain with varying PFGE DNA subtype profiles, may be responsible for an outbreak, and that more than one E. coli O157:H7 subtype may be circulating on a particular farm at any one time. To our knowledge, this is one of the first reports where PFGE typing was used to verify the direct transmission of E. coli O157:H7 from cattle to humans.