Volatile compounds from Escherichia coli O157:H7 and their absorption by strawberry fruit

Volatile compounds emitted by cultures of two strains of the pathogenic bacterium Escherichia coliO157:H7 and a nonpathogenic strain of E. coli were trapped on Super-Q porous polymer and identified by GC-MS. The predominant compound produced by all three strains was indole with lesser amounts of other components including methyl ketones, 2-heptanone, 2-nonanone, 2-undecanone, and 2-tridecanone. The vapor-phase profiles of these strains were similar for most chemicals identified but differed with regard to ketones. Strawberry fruit was shown to be a suitable host for E. coli O157:H7 with the population of the bacterium either increasing or remaining stable after 3 days depending on inoculation level. Headspace analysis of the volatile compounds from inoculated fruit yielded no detectable quantity of indole. Strawberry fruit readily absorbed indole and other volatile compounds produced by the bacteria and in some cases metabolized the compounds to new volatile products. Thus, headspace "marker" compounds indicating possible bacterial contamination of fruit were largely removed from the vapor phase by the strawberries.     

Fate of chlorate present in cattle wastes and its impact on Salmonella Typhimurium and Escherichia coli O157:H7

Chlorate salts are being developed as a feed additive to reduce the numbers of pathogens in feedlot cattle. A series of studies was conducted to determine whether chlorate, at concentrations expected to be excreted in urine of dosed cattle, would also reduce the populations of pathogens in cattle wastes (a mixture of urine and feces) and to determine the fate of chlorate in cattle wastes. Chlorate salts present in a urine-manure-soil mixture at 0, 17, 33, and 67 ppm had no significant effect on the rates of Escherichia coli O157:H7 or Salmonella Typhimurium inactivation from batch cultures. Chlorate was rapidly degraded when incubated at 20 and 30 degrees C with half-lives of 0.1 to 4 days. Chlorate degradation in batch cultures was slowest at 5 degrees C with half-lives of 2.9 to 30 days. The half-life of 100 ppm chlorate in an artificial lagoon system charged with slurry from a feedlot lagoon was 88 h. From an environmental standpoint, chlorate use in feedlot cattle would likely have minimal impacts because any chlorate that escaped degradation on the feedlot floor would be degraded in lagoon systems. Collectively, these results suggest that chlorate administered to cattle and excreted in wastes would have no significant secondary effects on pathogens present in mixed wastes on pen floors. Lack of chlorate efficacy was likely due to low chlorate concentrations in mixed wastes relative to chlorate levels shown to be active in live animals, and the rapid degradation of chlorate to chloride at temperatures of 20 degrees C and above.     

Antibacterial activities of plant essential oils and their components against Escherichia coli O157:H7 and Salmonella enterica in apple juice

We evaluated 17 plant essential oils and nine oil compounds for antibacterial activity against the foodborne pathogens Escherichia coli O157:H7 and Salmonella enterica in apple juices in a bactericidal assay in terms of % of the sample that resulted in a 50% decrease in the number of bacteria (BA(50)). The 10 compounds most active against E. coli (60 min BA(50) range in clear juice, 0.018-0.093%) were carvacrol, oregano oil, geraniol, eugenol, cinnamon leaf oil, citral, clove bud oil, lemongrass oil, cinnamon bark oil, and lemon oil. The corresponding compounds against S. enterica (BA(50) range, 0.0044-0.011%) were Melissa oil, carvacrol, oregano oil, terpeineol, geraniol, lemon oil, citral, lemongrass oil, cinnamon leaf oil, and linalool. The activity (i) was greater for S. enterica than for E. coli, (ii) increased with incubation temperature and storage time, and (iii) was not affected by the acidity of the juices. The antibacterial agents could be divided into two classes: fast-acting and slow-acting. High-performance liquid chromatography analysis showed that the bactericidal results are related to the composition of the oils. These studies provide information about new ways to protect apple juice and other foods against human pathogens.     

Storage stability and antibacterial activity against Escherichia coli O157:H7 of carvacrol in edible apple films made by two different casting methods

The antimicrobial activities against Escherichia coli O157:H7 as well as the stability of carvacrol, the main constituent of oregano oil, were evaluated during the preparation and storage of apple-based edible films made by two different casting methods, continuous casting and batch casting. Antimicrobial assays of films and high-performance liquid chromatography (HPLC) analysis of film extracts following storage up to 49 days at 5 and 25 degrees C revealed that (a) optimum antimicrobial effects were apparent with carvacrol levels of approximately 1.0% added to the purees prior to film preparation, (b) carvacrol in the films and film weights remained unchanged over the storage period of up to 7 weeks, and (c) casting methods affected carvacrol concentration, bactericidal activity, physicochemical properties, and colors of the apple films. Carvacrol addition to the purees used to prepare the films reduced water vapor and oxygen permeability of apple films. The results indicate that carvacrol has a dual benefit. It can be used to both impart antimicrobial activities and enhance barrier properties of edible films. The cited observations facilitate relating compositional and physicochemical properties of apple puree films containing volatile plant antimicrobials to their use in foods.     

Effect of Molasses on Regrowth of E. coli O157:H7 And Salmonella in Compost Teas

Compost water extracts (compost teas) are gaining popularity among organic growers, largely because of their disease suppressive activity when applied to foliage or soil. Production methods often include addition of supplemental constituents, particularly molasses, to stimulate plant-beneficial microbial populations. We have found that molasses amendments also favor regrowth of human pathogenic bacteria, raising public health concerns about potential contamination of treated crops, particularly produce intended for fresh consumption. Using disease outbreak strains marked with green fluorescent protein (GFP) and spontaneous antibiotic-resistance, we found that regrowth of Salmonella enterica serovar Thompson and Escherichia coli O157:H7 was positively correlated with molasses concentration. For Salmonella, regrowth was also dependent on the type of starter compost material used. Salmonella populations increased from 1 at time 0 to over 1000 CFU ml^?1 in dairy manure compost tea with 1% molasses, and from 1 at time 0 to over 350,000 CFU ml^?1 in chicken manure compost tea by 72 h. E. coli populations increased from 1 at time 0 to approximately 1000 CFU ml^?1 in both types of tea by 72 h. Pathogen regrowth did not occur when molasses was eliminated or kept to 0.2%.     

A rapid multiplexed chemiluminescent immunoassay for the detection of Escherichia coli O157:H7, Yersinia enterocolitica, Salmonella typhimurium, and Listeria monocytogenes pathogen bacteria

A simple and rapid multiplexed sandwich chemiluminescent enzyme immunoassay has been developed for the simultaneous detection of Escherichia coli O157:H7, Yersinia enterocolitica, Salmonella typhimurium, and Listeria monocytogenes. To achieve the multiplexed detection of the four pathogens, a new polystyrene 96 well microtiter plate format has been designed, in which each main well contains four subwells in the bottom. The monoclonal antibodies specific for each bacteria were separately immobilized in each subwell. When the samples were added to the main wells, the bacteria able to specifically bind to the corresponding monoclonal antibody were captured in one of the four subwells. Subsequently, a mixture of peroxidase-labeled polyclonal antibodies against the four bacteria was added and the peroxidase activity of the bound polyclonal labeled antibodies in each well was measured by an enhanced luminol-based chemiluminescent cocktail using a low-light charge-coupled imaging device. The assay was simple and fast, and the limit of quantification was in the order of 104-105 CFU/mL for all bacterial species. The accuracy of the method, evaluated by comparison of the results with a conventional culturing methodology, was satisfactory, with recovery values ranging from 90 to 120%. This method can be used as a screening test to evaluate the presence of these pathogen bacteria in different foodstuffs.     

Plant extracts, spices, and essential oils inactivate Escherichia coli O157:H7 and reduce formation of potentially carcinogenic heterocyclic amines in cooked beef patties

Meats need to be heated to inactivate foodborne pathogens such as Escherichia coli O157:H7. High-temperature treatment used to prepare well-done meats increases the formation of carcinogenic heterocyclic amines (HCAs). We evaluated the ability of plant extracts, spices, and essential oils to simultaneously inactivate E. coli O157:H7 and suppress HCA formation in heated hamburger patties. Ground beef with added antimicrobials was inoculated with E. coli O157:H7 (10(7) CFU/g). Patties were cooked to reach 45 °C at the geometric center, flipped, and cooked for 5 min. Samples were then taken for microbiological and mass spectrometry analysis of HCAs. Some compounds were inhibitory only against E. coli or HCA formation, while some others inhibited both. Addition of 5% olive or apple skin extracts reduced E. coli O157:H7 populations to below the detection limit and by 1.6 log CFU/g, respectively. Similarly, 1% lemongrass oil reduced E. coli O157:H7 to below detection limits, while clove bud oil reduced the pathogen by 1.6 log CFU/g. The major heterocyclic amines 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) were concurrently reduced with the addition of olive extract by 79.5% and 84.3% and with apple extract by 76.1% and 82.1%, respectively. Similar results were observed with clove bud oil: MeIQx and PhIP were reduced by 35% and 52.1%, respectively. Addition of onion powder decreased formation of PhIP by 94.3%. These results suggest that edible natural plant compounds have the potential to prevent foodborne infections as well as carcinogenesis in humans consuming heat-processed meat products.     

Survival of <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 in various soil particles: importance of the attached bacterial phenotype

The risk of enteropathogens to food and water is highly dependent on their survival in soil environments. Here, the effects of soil type, particle size, the presence of natural organic matter (NOM) or Fe/Al (hydro)oxides on pathogenic Escherichia coli O157:H7 survival in sterilized soil particles were assessed through survival, attachment, metabolic activity, and qRT-PCR analyses. The abundance of inoculated E. coli O157:H7 in Brown soil (Alfisol) particles increased 0.6–1.4 log₁₀ CFU/g within 3 days (except for NOM-stripped clay), while that in Red soil (Ultisol) particles decreased rapidly in 8 days post-inoculation. Additionally, survival of bacteria was significantly enhanced when Fe/Al (hydro)oxides had been removed from Red soil particles. For the two soils, E. coli O157:H7 survived the longest in NOM-present clays and the bacterial adenosine 5′-triphosphate (ATP) levels were 0.7–2.0 times greater in clays than in sands and silts on day 8. Moreover, clays were more effective than silts and sands in binding cells and changing the expressions of acetate pathway-associated genes (pta and ackA). For silts and sands, E. coli O157:H7 decayed more rapidly in the presence of NOM and similar trends of bacterial ATP levels were observed between NOM-stripped and NOM-present soil particles, indicating that the primary role of NOM was not as a nutrient supply. These findings indicate that soil particles function mainly through attachment to change the metabolic pathway of E. coli O157:H7 and ultimately impact the survival of bacterial pathogens in soils.     

苏云金芽胞杆菌类ABC transporter基因zwa-FEG能提高对双效菌素的抗性

双效菌素（Zwittermicin A,ZwA）是一种氨基多元醇类新型广谱抗生素,最初是从蜡状芽胞杆菌(Bacillus cereus) UW85中发现的,zmaR为它的抗性基因。在本实验室已测得的苏云金芽胞杆菌（Bacillus thuringiensis）YBT-1520菌株全基因组序列中有ZwA合成基因簇完整序列,分析发现在其末端存在一个类似ABC transporter的序列（命名为zwa-FEG）。将zwa-EFG在大肠杆菌（Escherichia coli）中表达,大肠杆菌能获得对ZwA的抗性;将zwa-EFG转移到产ZwA的菌株UW85中,能使其ZwA产量显著提高。推测zwa-EFG是一种ZwA专一性的ABC transporter,能将ZwA分泌到胞外,从而增强ZwA的合成和对ZwA的抗性。     

大肠杆菌猪水肿毒素B亚单位基因的克隆与鉴定

根据已知大肠杆菌志贺氏菌样毒素Ⅱ型变异体Ｂ亚单位（ＳＬＴ－ＩＩｖＢ）基因序列,设计并合成３条２对引物,以大肠杆菌Ｏ１３８基因组为模板,通过聚合酶链式反应（ＰＣＲ）分别扩增出２０４ ｂｐ的基因序列及包含一段信号肽的２６１ ｂｐ基因序列。将这两段ＤＮＡ分别克隆进表达性载体质粒ＰＹＡ３３３４的ＥｃｏＲⅠ和ＢａｍＨⅠ位点之间,经地高辛标记探针进行Ｓｏｕｔｈｅｒｎ杂交和酶切分析鉴定,并对两条扩增片段进行序列