Vitamin K Analogs Influence the Growth and Virulence Potential of Enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) causes serious foodborne disease worldwide. It produces the very potent Shiga toxin 2 (Stx2). The Stx2-encoding genes are located on a prophage, and production of the toxin is linked to the synthesis of Stx phages. There is, currently, no good treatment for EHEC infections, as antibiotics may trigger lytic cycle activation of the phages and increased Stx production. This study addresses how four analogs of vitamin K, phylloquinone (K1), menaquinone (K2), menadione (K3), and menadione sodium bisulfite (MSB), influence growth, Stx2-converting phage synthesis, and Stx2 production by the EHEC O157:H7 strain EDL933. Menadione and MSB conferred a concentration-dependent negative effect on bacterial growth, while phylloquinone or menaquinone had little and no effect on bacterial growth, respectively. All four vitamin K analogs affected Stx2 phage production negatively in uninduced cultures and in cultures induced with either hydrogen peroxide (H2O2), ciprofloxacin, or mitomycin C. Menadione and MSB reduced Stx2 production in cultures induced with either H2O2 or ciprofloxacin. MSB also had a negative effect on Stx2 production in two other EHEC isolates tested. Phylloquinone and menaquinone had, on the other hand, variable and concentration-dependent effects on Stx2 production. MSB, which conferred the strongest inhibitory effect on both Stx2 phage and Stx2 production, improved the growth of EHEC in the presence of H2O2 and ciprofloxacin, which could be explained by the reduced uptake of ciprofloxacin into the bacterial cell. Together, the data suggest that vitamin K analogs have a growth- and potential virulence-reducing effect on EHEC, which could be of therapeutic interest.IMPORTANCE Enterohemorrhagic E. coli (EHEC) can cause serious illness and deaths in humans by producing toxins that can severely damage our intestines and kidneys. There is currently no optimal treatment for EHEC infections, as antibiotics can worsen disease development. Consequently, the need for new treatment options is urgent. Environmental factors in our intestines can affect the virulence of EHEC and help our bodies fight EHEC infections. The ruminant intestine, the main reservoir for EHEC, contains high levels of vitamin K, but the levels are variable in humans. This study shows that vitamin K analogs can inhibit the growth of EHEC and/or production of its main virulence factor, the Shiga toxin. They may also inhibit the spreading of the Shiga toxin encoding bacteriophage. Our findings indicate that vitamin K analogs have the potential to suppress the development of serious disease caused by EHEC.

Changes in the faecal bile acid profile in dogs fed dry food vs high content of beef: a pilot study.

BACKGROUND: Dogs are fed various diets, which also include components of animal origin. In humans, a high-fat/low-fibre diet is associated with higher faecal levels of bile acids, which can influence intestinal health. It is unknown how an animal-based diet high in fat and low in fibre influences the faecal bile acid levels and intestinal health in dogs. This study investigated the effects of high intake of minced beef on the faecal bile acid profile in healthy, adult, client-owned dogs (n = 8) in a 7-week trial. Dogs were initially adapted to the same commercial dry food. Thereafter, incremental substitution of the dry food by boiled minced beef over 3 weeks resulted in a diet in which 75% of each dog's total energy requirement was provided as minced beef during week 5. Dogs were subsequently reintroduced to the dry food for the last 2 weeks of the study. The total taurine and glycine-conjugated bile acids, the primary bile acids chenodeoxycholic acid and cholic acid, and the secondary bile acids lithocholic acid, deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA) were analysed, using liquid chromatography-tandem mass spectrometry. RESULTS: The faecal quantities of DCA were significantly higher in dogs fed the high minced beef diet. These levels reversed when dogs were reintroduced to the dry food diet. The faecal levels of UDCA and taurine-conjugated bile acids had also increased in response to the beef diet, but this was only significant when compared to the last dry food period. CONCLUSIONS: These results suggest that an animal-based diet with high-fat/low-fibre content can influence the faecal bile acids levels. The consequences of this for canine colonic health will require further investigation.

Detection of PPCPs in marine organisms from contaminated coastal waters of the Saudi Red Sea.

The occurrence of PPCPs in macroalgae, barnacle and fish samples from contaminated coastal waters of the Saudi Red Sea is reported. Solvent extraction followed by solid phase extraction was applied to isolate the compounds, and their quantification was carried out by high performance liquid chromatography-tandem mass spectrometry. Atenolol, ranitidine, chlorpheniramine, DEET, and atrazine were detected in one or more macroalgae at

Photolysis of pharmaceuticals and personal care products in the marine environment under simulated sunlight conditions: irradiation and identification.

The photochemical fate of 16 pharmaceuticals and personal care products (PPCPs) found in the environment has been studied under controlled laboratory conditions applying a sunlight simulator. Aqueous samples containing PPCPs at environmentally relevant concentrations were extracted by solid-phase extraction (SPE) after irradiation. The exposed extracts were subsequently analysed by liquid chromatography combined with triple quadrupole mass spectrometry (HPLC-MS/MS) for studying the kinetics of photolytic transformations. Almost all exposed PPCPs appeared to react with a half-life time (τ 1/2) of less than 30 min. For ranitidine, sulfamethoxazole, diclofenac, warfarin, sulfamethoxazole and ciprofloxacin, τ1/2 was found to be even less than 5 min. The structures of major photolysis products were determined using quadrupole-time-of-flight mass spectrometry (QToF) and spectroscopic data reported in the literature. For diclofenac, the transformation products carbazol-1-yl-acidic acid and 8-chloro-9H-carbazol-1-yl-acetic acid were identified based on the mass/charge ratio of protonated ions and their fragmentation pattern in negative electrospray ionization (ESI--QTOF). Irradiation of carbamazepine resulted in three known products: acridine, carbamazepine-10,11-epoxide, and 10,11-dihydro-10,11-dihydroxy-carbamazepine, whereas acetaminophen was photolytically transformed to 1-(2-amino-5 hydroxyphenyl) ethenone. These photochemical products were subsequently identified in seawater or fish samples collected at sites exposed to wastewater effluents on the Saudi Arabian coast of the Red Sea.

Cereulide production by Bacillus weihenstephanensis strains during growth at different pH values and temperatures.

Besides Bacillus cereus, some strains of the psychrotolerant, potentially foodborne pathogen Bacillus weihenstephanensis can produce the emetic toxine (cereulide). This toxin is a heat- and acid-stable cyclic dodecadepsipeptide that causes food intoxication with vomiting. However, some severe clinical cases with lethal outcomes have been described. If cereulide can be produced during refrigerated storage, it will not be inactivated by reheating food, representing an important risk of food intoxication for consumers. In this paper, we determined the capacity of the B. weihenstephanensis strains BtB2-4 and MC67 to grow and produce cereulide on agar media at temperatures from 8 °C to 25 °C and at a pH from 5.4 to 7.0. At 8 °C, strain BtB2-4 produced quantifiable amounts of cereulide, whereas the limit of detection was reached for strain MC67. For BtB2-4, cereulide production increased 5-fold between 8 °C and 10-15 °C and by more than 100-fold between 15 °C and 25 °C. At temperatures of 10 °C and higher, cereulide concentrations were within the range of those reported by previous works in foods implicated in emetic poisoning. At 25 °C, decreasing the pH to 5.4 reduced cereulide production by strain BtB2-4 by at least 20-fold.

Occurrence of pharmaceuticals and personal care products in effluent-dominated Saudi Arabian coastal waters of the Red Sea.

The occurrence of selected pharmaceuticals and personal care products (PPCPs) and the pesticide atrazine were investigated in seawater samples collected from stations located at effluent dominated sites in the Saudi Arabian coastal waters of the Red Sea. PPCPs were analysed using solid phase extraction (SPE) followed by high performance liquid chromatography - tandem mass spectrometry (HPLC-MS/MS). A multi component method for the ultra-trace level quantification of 13 target PPCPs in Seawater was developed and validated for the here performed study. The method procedure is described in detail in the supplementary material section. 26 samples from 7 distinct locations (2 directly influenced by continuous sewage release) were chosen for the sampling of surface seawater. Based upon local sales information, 25 target substances (20 PPCPs, 4 pesticides and 1 stimulant) were chosen for the here reported method development. Thirteen PPCPs were detected and quantified in a total of 26 seawater samples. Metformin, diclofenac, acetaminophen, and caffeine were identified as the most abundant PPCPs, detected in maximum concentration higher than 3 µg/L (upper quantification limit for the here developed method). Concentrations were in the range of 7- >3000 (metformin), 3000 ng/L (caffeine). The contribution of direct sewage release on the PPCP levels detected was obvious, the target PPCPs were detected in the Al-Arbaeen and Al-Shabab coastal lagoons in high concentrations due to the low water exchange with the open sea and still ongoing sewage releases in the lagoons. Also, substantial amounts of antibiotics were detected in all samples. Levels and distribution profile of the detected PPCPs revealed high level release rates and give raise to concern on potential environmental risks associated with the here document long term exposure on the fragile coastal marine environment of the region but particularly in the nearby protected coral reef environment outside the harbour region of Jeddah.

Identification and quantification of lichenysin - a possible source of food poisoning.

Lichenysin produced by 53 different Bacillus licheniformis strains has been structurally examined with a qualitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using quadrupole-time-of-flight mass spectrometry. The same lichenysin isoforms are produced from all strains, indicating that the growth conditions have a stronger influence on the lipopeptide production than the genotype. A rapid method for the quantification of lichenysin from bacterial cell cultures with LC-MS/MS after a simple methanol extraction has been refined. For the first time commercially available lichenysin has been used as calibrant, making quantification more accurate. The trueness for C15-lichenysin has been improved to 94% using matrix-matched calibration with lichenysin compared with 30% using solvent calibration with surfactin. The quantitative method was fully validated based on Commission Decision 2002/657/EC. The LOD of the method was below 1 µg g(-1) and the repeatability ranged from 10% to 16%.

Determination and quantification of the emetic toxin cereulide from Bacillus cereus in pasta, rice and cream with liquid chromatography-tandem mass spectrometry.

A rapid and sensitive method has been developed for determination and quantification of cereulide in cream, rice and pasta. Samples are homogenised after addition of amylase to cooked rice and pasta, and cereulide is extracted with methanol. After the removal of water with methyl-tert butyl ether/hexane and evaporation until dryness, no further purification was required before analysis with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Recently, both cereulide and (13)C6-cereulide has become commercially available at high purities; hence, this method offers a more reliable quantification of positive samples than previous methods using valinomycin or in-house produced and purified cereulide as calibration standard. The introduction of amylase in the sample preparation improves both the extraction yield of cereulide from positive samples of starch-rich matrices such as pasta and rice, and the within-laboratory reproducibility of the analytical method. The LoQ of the method is 1.1 ng/g cereulide with RSDs ranging from 2.6% to 10%. The method is fully validated based on Commission Decision 2002/657/EC, suitable for routine analysis, and has been used to analyse samples from a cereulide food poisoning outbreak in a kindergarten in Norway. Cereulide production in different rice and pasta samples was investigated, showing that cereulide was unexpectedly produced by emetic Bacillus cereus in all eight pasta and rice samples.

Toxin production and growth of pathogens subjected to temperature fluctuations simulating consumer handling of cold cuts.

It is crucial for the quality and safety of ready-to-eat (RTE) foods to maintain the cold chain from production to consumption. The effect of temperature abuse related to daily meals and elevated refrigerator temperatures on the growth and toxin production of Bacillus cereus, Bacillus weihenstephanensis and Staphylococcus aureus and the growth of Listeria monocytogenes and Yersinia enterocolitica was studied. A case study with temperature loggings in the domestic environment during Easter and Christmas holidays was performed to select relevant time and temperature courses. A model for bacterial surface growth on food using nutrient agar plates exposed to variations in temperatures was used to simulate food stored at different temperatures and exposed to room temperature for short periods of time. The results were compared with predicted growth using the modeling tool ComBase Predictor. The consumers exposed their cold cuts to room temperatures as high as 26.5°C with an average duration of meals was 47 min daily for breakfast/brunch during the vacations. Short (≤ 2 h) daily intervals at 25°C nearly halved the time the different pathogens needed to reach levels corresponding to the levels associated with human infection or intoxication, compared with the controls continuously stored at refrigerator temperature. Although the temperature fluctuations affected growth of both B. weihenstephanensis and S. aureus, toxin production was only detected at much higher cell concentrations than what has been associated with human intoxications. Therefore, growth of L. monocytogenes and Y. enterocolitica was found to be the limiting factor for safety. In combination with data on temperature abuse in the domestic environment, modeling programs such as ComBase Predictor can be efficient tools to predict growth of some pathogens but will not predict toxin production.

Liquid chromatography and mass spectrometry in food allergen detection.

Food allergy is an important issue in the field of food safety because of the hazards for affected persons and the hygiene requirements and legal regulations imposed on the food industry. Consumer protection and law enforcement require suitable analytical techniques for the detection of allergens in foods. Immunological methods are currently preferred; however, confirmatory alternatives are needed. The determination of allergenic proteins by liquid chromatography and mass spectrometry has greatly advanced in recent years, and gel-free allergenomics is becoming a routinely used approach for the identification and quantitation of food allergens. The present review provides a brief overview of the principles of proteomic procedures, various chromatographic set ups, and mass spectrometry instrumentation used in allergenomics. A compendium of published liquid chromatography methods, proteomic analyses, typical marker peptides, and quantitative assays for 14 main allergy-causing foods is also included.

Determination of chloramphenicol residues in meat, seafood, egg, honey, milk, plasma and urine with liquid chromatography-tandem mass spectrometry, and the validation of the method based on 2002/657/EC.

A simple and rapid method for the determination and confirmation of chloramphenicol in several food matrices with LC-MS/MS was developed. Following addition of d5-chloramphenicol as internal standard, meat, seafood, egg, honey and milk samples were extracted with acetonitrile. Chloroform was then added to remove water. After evaporation, the residues were reconstituted in methanol/water (3+4) before injection. The urine and plasma samples were after addition of internal standard applied to a Chem Elut extraction cartridge, eluted with ethyl acetate, and hexane washed. Also these samples were reconstituted in methanol/water (3+4) after evaporation. By using an MRM acquisition method in negative ionization mode, the transitions 321-->152, 321-->194 and 326-->157 were used for quantification, confirmation and internal standard, respectively. Quantification of chloramphenicol positive samples regardless of matrix could be achieved with a common water based calibration curve. The validation of the method was based on EU-decision 2002/657 and different ways of calculating CCalpha and CCbeta were evaluated. The common CCalpha and CCbeta for all matrices were 0.02 and 0.04 microg/kg for the 321-->152 ion transition, and 0.02 and 0.03 microg/kg for the 321-->194 ion transition. At fortification level 0.1 microg/kg the within-laboratory reproducibility is below 25%.