Bacterial outer membrane vesicles provide an alternative pathway for trafficking of Escherichia coli O157 type III secreted effectors to epithelial cells.

IMPORTANCE: Bacteria can package protein cargo into nanosized membrane blebs that are shed from the bacterial membrane and released into the environment. Here, we report that a type of pathogenic bacteria called enterohemorrhagic Escherichia coli O157 (EHEC) uses their membrane blebs (outer membrane vesicles) to package components of their type 3 secretion system and send them into host cells, where they can manipulate host signaling pathways including those involved in infection response, such as immunity. Usually, EHEC use a needle-like apparatus to inject these components into host cells, but packaging them into membrane blebs that get taken up by host cells is another way of delivery that can bypass the need for a functioning injection system.

Transcriptomic analysis reveals the antibacterial mechanism of phenolic compounds from kefir fermented soy whey against Escherichia coli 0157:H7 and Listeria monocytogenes.

Transcriptomic analysis was used to investigate the antibacterial mechanism of phenolic compounds from kefir fermented soy whey (FSP) against Escherichia coli 0157:H7 and Listeria monocytogenes. The kefir fermentation increased the concentration of several phenolic aglycones with proven antibacterial efficacy in the FSP. The time-kill curve showed that 2× MICs of the FSP killed >99.9 % of the strains within 2 h of exposure. The checkerboard fractional inhibition concentration (FIC) assay proved that phenolics were the sole antibacterial agent in the FSP. The transmission electron microscope (TEM) photomicrograph corroborated the propidium iodide (PI) uptake, protein, and nucleic acid leakage assays. They demonstrated that the phenolics permeated the cell membrane, disrupted the cytoplasm, and caused cell lysis in the treated cells leading to protein and nucleic acid leakage. The transcriptome analysis revealed that exposure of the cells to MICs of the phenolics induced molecular responses leading to differential expression of 1850 genes in E. coli 0157:H7 and 2090 in L. monocytogenes. The phenolics suppressed the expression of genes crucial for carbohydrate utilization, transmembrane glucose transport, tricarboxylic acid (TCA), and ATP synthesis. The phenolic-induced stress also downregulated the expression of quorum sensing and virulence-related genes, peptidoglycan and phospholipid synthases, and ABC transporters. The cells initiated a resistance response by stimulating the two-component signal transduction systems to trigger the over-expression of a cascade of genes involved in stress resistance, gluconeogenesis, ATPase activity and proton transmembrane transport. Nonetheless, the data indicated that the phenolics suppressed the expression of translational proteins that would have facilitated the resistance and repair of the cell damage caused by the phenolics. The study provides discrete data evidence that FSP could be used to control the pathogenicity and the proliferation of E. coli 0157:H7 and L. monocytogenes in our foods and food systems.

A Novel Nano-Antimicrobial Polymer Engineered with Chitosan Nanoparticles and Bioactive Peptides as Promising Food Biopreservative Effective against Foodborne Pathogen E. coli O157-Caused Epithelial Barrier Dysfunction and Inflammatory Responses.

For food quality and safety issues, the emergence of foodborne pathogenic bacteria has further accelerated the spread of antibiotic residues and drug resistance genes. To alleviate the harm caused by bacterial infections, it is necessary to seek novel antimicrobial agents as biopreservatives to prevent microbial spoilage. Nanoantimicrobials have been widely used in the direct treatment of bacterial infections. CNMs, formed by chitosan nanoparticles and peptides, are promising antibiotic alternatives for use as excellent new antibacterial drugs against pathogenic bacteria. Herein, the current study evaluated the function of CNMs in the protection of foodborne pathogen Escherichia coli (E. coli) O157 infection using an intestinal epithelial cell model. Antibacterial activity assays indicated that CNMs exerted excellent bactericidal activity against E. coli O157. Assessment of the cytotoxicity risks toward cells demonstrated that 0.0125-0.02% of CNMs did not cause toxicity, but 0.4% of CNMs caused cytotoxicity. Additionally, CNMs did not induced genotoxicity either. CNMs protected against E. coli O157-induced barrier dysfunction by increasing transepithelial electrical resistance, decreasing lactate dehydrogenase and promoting the protein expression of occludin. CNMs were further found to ameliorate inflammation via modulation of tumor factor α, toll-like receptor 4 and nuclear factor κB (NF-κB) expression via inhibition of mitogen-activated protein kinase and NF-κB activation and improved antioxidant activity. Taken together, CNMs could protect the host against E. coli O157-induced intestinal barrier damage and inflammation, showing that CNMs have great advantages and potential application as novel antimicrobial polymers in the food industry as food biopreservatives, bringing new hope for the treatment of bacterial infections.

Synergistic inactivation of bacteria based on a combination of low frequency, low-intensity ultrasound and a food grade antioxidant.

This study evaluated a synergistic antimicrobial treatment using a combination of low frequency and a low-intensity ultrasound (LFU) and a food-grade antioxidant, propyl gallate (PG), against a model gram-positive (Listeria innocua) and the gram-negative bacteria (Escherichia coli O157:H7). Bacterial inactivation kinetic measurements were complemented by characterization of biophysical changes in liposomes, changes in bacterial membrane permeability, morphological changes in bacterial cells, and intracellular oxidative stress upon treatment with PG, LFU, and a combination of PG + LFU. Combination of PG + LFU significantly (>4 log CFU/mL, P < 0.05) enhanced the inactivation of both L. innocua and E. coli O157:H7 compared to PG or LFU treatment. As expected, L. innocua had a significantly higher resistance to inactivation compared to E. coli using a combination of PG + LFU. Biophysical measurements in liposomes, bacterial permeability measurements, and scanning electron microscope (SEM)-based morphological measurements show rapid interactions of PG with membranes. Upon extended treatment of cells with PG + LFU, a significant increase in membrane damage was observed compared to PG or LFU alone. A lack of change in the intracellular thiol content following the combined treatment and limited effectiveness of exogenously added antioxidants in attenuating the synergistic antimicrobial action demonstrated that oxidative stress was not a leading mechanism responsible for the synergistic inactivation by PG + LFU. Overall, the study illustrates synergistic inactivation of bacteria using a combination of PG + LFU based on enhanced membrane damage and its potential for applications in the food and environmental systems.

The Impact of Plasma Membrane Lipid Composition on Flagellum-Mediated Adhesion of Enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a major cause of foodborne gastrointestinal illness. The adhesion of EHEC to host tissues is the first step enabling bacterial colonization. Adhesins such as fimbriae and flagella mediate this process. Here, we studied the interaction of the bacterial flagellum with the host cell's plasma membrane using giant unilamellar vesicles (GUVs) as a biologically relevant model. Cultured cell lines contain many different molecular components, including proteins and glycoproteins. In contrast, with GUVs, we can characterize the bacterial mode of interaction solely with a defined lipid part of the cell membrane. Bacterial adhesion on GUVs was dependent on the presence of the flagellar filament and its motility. By testing different phospholipid head groups, the nature of the fatty acid chains, or the liposome curvature, we found that lipid packing is a key parameter to enable bacterial adhesion. Using HT-29 cells grown in the presence of polyunsaturated fatty acid (α-linolenic acid) or saturated fatty acid (palmitic acid), we found that α-linolenic acid reduced adhesion of wild-type EHEC but not of a nonflagellated mutant. Finally, our results reveal that the presence of flagella is advantageous for the bacteria to bind to lipid rafts. We speculate that polyunsaturated fatty acids prevent flagellar adhesion on membrane bilayers and play a clear role for optimal host colonization. Flagellum-mediated adhesion to plasma membranes has broad implications for host-pathogen interactions.IMPORTANCE Bacterial adhesion is a crucial step to allow bacteria to colonize their hosts, invade tissues, and form biofilm. Enterohemorrhagic Escherichia coli O157:H7 is a human pathogen and the causative agent of diarrhea and hemorrhagic colitis. Here, we use biomimetic membrane models and cell lines to decipher the impact of lipid content of the plasma membrane on enterohemorrhagic E. coli flagellum-mediated adhesion. Our findings provide evidence that polyunsaturated fatty acid (α-linolenic acid) inhibits E. coli flagellar adhesion to the plasma membrane in a mechanism separate from its antimicrobial and anti-inflammatory functions. In addition, we confirm that cholesterol-enriched lipid microdomains, often called lipid rafts, are important in bacterial adhesion. These findings demonstrate that plasma membrane adhesion via bacterial flagella play a significant role for an important human pathogen. This mechanism represents a promising target for the development of novel antiadhesion therapies.

PchE Regulation of Escherichia coli O157:H7 Flagella, Controlling the Transition to Host Cell Attachment.

Shiga toxins and intimate adhesion controlled by the locus of enterocyte effacement are major enterohemorrhagic Escherichia coli (EHEC) virulence factors. Curli fimbriae also contribute to cell adhesion and are essential biofilm components. The transcriptional regulator PchE represses the expression of curli and their adhesion to HEp-2 cells. Past studies indicate that pchE also represses additional adhesins that contribute to HEp-2 cell attachment. In this study, we tested for pchE regulation of several tissue adhesins and their regulators. Three adhesin-encoding genes (eae, lpfA1, fliC) and four master regulators (csgD, stpA, ler, flhDC) were controlled by pchE. pchE over-expression strongly up-regulated fliC but the marked flagella induction reduced the attachment of O157:H7 clinical isolate PA20 to HEp-2 cells, indicating that flagella were blocking cell attachments rather than functioning as an adhesin. Chemotaxis, motor, structural, and regulatory genes in the flagellar operons were all increased by pchE expression, as was PA20 motility. This study identifies new members in the pchE regulon and shows that pchE stimulates flagellar motility while repressing cell adhesion, likely to support EHEC movement to the intestinal surface early in infection. However, induced or inappropriate pchE-dependent flagellar expression could block cell attachments later during disease progression.

Chemical-free and synergistic interaction of ultrasound combined with plasma-activated water (PAW) to enhance microbial inactivation in chicken meat and skin.

In general, the poultry industry uses 0.5-1 ppm chlorine solution in the meat sanitization process. However, chlorine can react with organic material and produce halogenated organic compounds, notably chloroform, which causes bladder and rectal cancer in humans. For this reason, many industries try to avoid chlorine. This study investigated the efficacy of ultrasound and plasma-activated water (PAW) on the inactivation of Escherichia coli and Staphylococcus aureus in chicken muscle, rough skin, and smooth skin. Samples inoculated with bacteria suspension were treated by ultrasound alone and PAW-ultrasound. The Taguchi method and desirability function approach were used for the experimental design and optimization. Combined ultrasound and PAW inactivated up to 1.33 log CFU/ml of E. coli K12 and 0.83 log CFU/ml of S. aureus at a sample thickness of 4 mm, at 40 °C for 60 min, while PAW alone only reduced E. coli K12 by 0.46 log CFU/ml and S. aureus by 0.33 log CFU/ml under the same condition. The muscle topography showed a porous structure, which facilitated the penetration of PAW. The color measurements of muscle treated with ultrasound and PAW-ultrasound were dramatically different from the untreated sample, as also perceived by the sensory evaluation panel. Therefore, the synergistic interaction of combined PAW-ultrasound could be used to enhance microbial inactivation in meat.

Lactoferrin translocates to the nucleus of bovine rectal epithelial cells in the presence of Escherichia coli O157:H7.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a foodborne pathogen which causes illness in humans. Ruminants are the main reservoirs and EHEC predominantly colonizes the epithelium of the recto-anal junction of cattle. Immunosuppression by EHEC promotes re-infection of cattle. However, bovine lactoferrin (bLF) apparently can overrule the immunosuppression by inducing EHEC-specific IgA responses at the mucosal site. The IgA responses are significantly correlated with reduced EHEC shedding and the absence of colonization at the rectal mucosa following re-infection. Therefore, to examine the interaction between bLF and bovine rectal epithelial cells, we first developed a method to establish a primary cell culture of epithelial cells of the rectum of cattle. Furthermore, we used LC-MS/MS to demonstrate the presence of secreted lactoferrin in bovine milk and the absence of a "delta" isoform which is known to translocate to the nucleus of cells. Nevertheless, lactoferrin derived from bovine milk was internalized by rectal epithelial cells and translocated to the nuclei. Moreover, nuclear translocation of bLF was significantly enhanced when the epithelial cells were inoculated with EHEC, as demonstrated by confocal fluorescence microscopy and confirmed by Raman microscopy and 3D imaging.

Inactivation mechanism of E. coli O157:H7 under ultrasonic sterilization.

Ultrasonic sterilization (US), as a promising non-thermal sterilization method, exhibits unique superiorities than traditional sterilization methods. In this study, the inactivation mechanism of E. coli O157:H7 under US was investigated in cucumber and bitter gourd vegetable juices. Results revealed that the US treatment showed good antibacterial ability in countering E. coli O157:H7. Through determinations of conductivity and ß-galactosidase activity, significant augmentation in membrane permeability of the bacteria was confirmed after the US treatment. The morphologies of the US treated E. coli O157:H7 demonstrated that the integrity of the cell membrane was disrupted by US treatment. SDS-PAGE and LSCM data further proved the disruptive action of US, leading to the leakage of proteins and DNA through the breakage on cell membrane. The decrease of metabolic-related enzyme activity was verified through investigation of bacterial metabolism. The antibacterial mechanism analysis indicated that the US can generate free radicals which resulted in the rise of intracellular oxidative stress, attenuation of energy metabolism and inhibition of hexose monophosphate pathway. As the application verification, the US treatment can cause the deprivation of E. coli O157:H7 cell viability in vegetable juices without obvious impact on the sensory quality.

Cathelicidin- derived PR39 protects enterohemorrhagic Escherichia coli O157:H7 challenged mice by improving epithelial function and balancing the microbiota in the intestine.

The zoonotic enterohaemorrhagic Escherichia coli (EHEC) O157:H7 can disrupt intestinal epithelial barrier function and in turn leading to serious intestinal and systemic disease. PR39 could effectively inhibit the growth of Gram-negative bacteria, but there is little knowledge of its effects on intestinal barrier function and the microbiota in E. coli-challenged mice. In this study, an intestinal disease caused by EHEC O157:H7 was established, to analyze the effect of PR39 on EHEC O157:H7 induced intestinal epithelial barrier injury and disorder. Interestingly, PR39 attenuated EHEC O157:H7-induced systemic symptoms and significantly decreased mortality and the degree of E. coli shedding in faeces. Furthermore, the infiltration index of macrophages and neutrophils in intestine of the PR39 treatment group were obviously attenuated, along with the level of apoptosis. PR39 treatment group had distinctly improved tight junction associated proteins' expression after EHEC O157:H7 caused injury. Additionally, the sequencing analysis of cecum microbiota showed that PR39 altered the abnormal increase in Bacteroides caused by EHEC O157:H7 and promoted the growth of probiotics such as Lactobacillus. In conclusion, cathelicidin-derived PR39 could effectively improve EHEC O157:H7-induced epithelial barrier injury, and dysfunction of immune and microbiota homeostasis in the intestinal tract, indicating that PR39 could be an excellent potential drug for zoonotic EHEC O157:H7-related intestinal disease.

Effects of Sublethal Thymol, Carvacrol, and trans-Cinnamaldehyde Adaptation on Virulence Properties of Escherichia coli O157:H7.

Essential oils (EOs) have demonstrated wide-spectrum antimicrobial activities and have been actively studied for their application in foods as alternative natural preservatives. However, information regarding microbial adaptive responses and changes in virulence properties following sublethal EO exposure is still scarce. The present study investigated the effect of sublethal thymol (Thy), carvacrol (Car), or trans-cinnamaldehyde (TC) adaptation on virulence gene expression and virulence properties of Escherichia coli O157:H7. The results demonstrated that E. coli O157:H7 grown to the early stationary phase in the presence of sublethal EO showed significantly (P < 0.05) reduced motility (reversible after stress removal), biofilm-forming ability, and efflux pump activity, with no induction of antibiotic resistance and no significant changes to its adhesion and invasion ability on a human colon adenocarcinoma (Caco-2) cell line. Reverse transcription-quantitative PCR revealed reduced expression of relevant virulence genes, including those encoding flagellar biosynthesis and function, biofilm formation regulators, multidrug efflux pumps, and type III secretion system components. This study demonstrated that Thy, Car, and TC at sublethal concentrations did not potentiate virulence in adapted E. coli O157:H7, which could benefit to their application in the food industry.IMPORTANCE The present study was conducted to evaluate changes in virulence properties in Escherichia coli O157:H7 adapted to sublethal essential oils (EOs). The results demonstrated reduced motility, biofilm-forming ability, and efflux pump activities in EO-adapted E. coli O157:H7, with no induction of antibiotic resistance or infection (adhesion and invasion) on Caco-2 cells. Reverse transcription-quantitative PCR results revealed changes in the expression of related virulence genes. Thus, the present study provides new insights into microbial virulence behavior following EO adaptation and suggests that Thy, Car, and TC sublethal exposure did not constitute a significant risk in inducing microbial virulence.

Survival of E. coli O157:H7, Salmonella Typhimurium, HAdV2 and MNV-1 in river water under dark conditions and varying storage temperatures.

The ability of Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium, Human adenovirus serotype 2 (HAdV2) and Murine Norovirus 1 (MNV-1) to survive in river water at -20, 4, room temperature (~24 °C) and 37 °C, were evaluated under dark conditions. The tested surface water was obtained from the main Nile River in the Dokki area, Giza and sterilized by autoclaving. The pathogens were inoculated separately in the autoclaved river water. Each microcosm was sampled and the test microorganisms counted after zero (immediately following inoculation), 1, 7, 15, 30, 60, 90 and 120 days. Physicochemical parameters including pH, turbidity, electrical conductivity, dissolved oxygen, total dissolved solids, total alkalinity, biological oxygen demand, chemical oxygen demand, nitrates and nitrites, and sulphate, were also measured. For HAdV2, the highest decay rates were observed at 37 °C and room temperature compared to 4 and -20 °C. A similar trend was found for the MNV-1, although unlike the HAdV2, the decay rate was higher at -20 than at 4 °C. Also, 4 °C was the best temperature for the survival of MNV-1 (T90 = 76.9 days), E. coli O157:H7 (T90 = 103 days) and Salmonella Typhimurium (T90 = 105 days). The least survival of the pathogens, except MNV-1, was recorded at 37 °C. These results indicate that under dark conditions and low temperatures, enteric pathogens could be stable for extended periods. No significant statistical correlation was observed between the experimental temperatures and the infectivity of the viral particles. This study provided useful information about the stability of these pathogens in the Nile River water and could serve as an early warning when considering the water of the river for agricultural irrigation or household use in areas with limited or no access to potable water.

Persistencia, internalización y translocación de Escherichia coli O157:H7, O157:H16 y O105ab en plantas y frutos de tomate (Solanum lycopersicum L.) / Persistence, internalization and translocation of Escherichia coli O157:H7, O157:H16 and O105ab in plants and tomato fruits (Solanum lycopersicum L.)

La presencia de bacterias patógenas, como Escherichia coli, afecta la calidad e inocuidad de las hortalizas que se consumen en fresco y se relaciona con graves problemas de salud. El objetivo de este trabajo fue determinar si 3 cepas diferentes de E. coli tienen la capacidad de penetrar y permanecer en plantas y frutos de tomate. Se siguió un diseño experimental completamente al azar, para lo cual se estableció un cultivo de tomate (variedad «Cid¼) en condiciones de invernadero y se evaluaron 3 tratamientos, T1 (E. coli O157: H7), T2 (E. coli de cultivo de tomate -#91;EcT-#93; O157: H16), T3 (E. coli de cultivo de espinaca -#91;EcH-#93; EcH O105ab) y un testigo T4, con 100 plantas cada uno y 4 formas de inoculación: en el sustrato, en el tallo, en el pecíolo y en el pedúnculo. Se realizaron muestreos en etapa vegetativa, floración, fructificación y madurez fisiológica para cuantificar en placa las UFC/g y saber si las bacterias lograban moverse y recuperarse en la raíz, el tallo, la flor y el fruto. Los grupos filogenéticos a los que correspondieron las bacterias recuperadas fueron confirmados mediante pruebas bioquímicas, serotipificación y PCR. A los 120 días la recuperación de bacterias en la planta fue del 23% (E. coli O157: H7), 28% (EcT O157: H16) y 55% (EcH O105ab) con la inoculación al sustrato, mientras que con la inoculación por punción la recuperación fue (en igual orden) del 5%, 3% y 4% a los 30 días; del 37%, 35% y 30% a los 90 días; y del 42%, 39% y 13% a los 65 días. Las cepas utilizadas mostraron la capacidad de entrar en la planta de tomate y de permanecer en ella y transportarse hasta llegar al fruto, sin producir síntomas que indiquen su presencia.

The presence of pathogenic bacteria, such as Escherichia coli affects the quality and safety of vegetables that are consumed fresh and is associated with serious health problems. The objective of this study was to determine if three different strains of E. coli can penetrate and remain in plants and tomato fruits. A completely randomized experimental design was followed for which a tomato crop ("Cid" variety) was established under greenhouse conditions and three treatments were evaluated, T1 (E. coli O157: H7), T2 (E. coli from tomato cultivation -#91;EcT-#93; O157: H16), T3 (E. coli from spinach cultivation -#91;EcH-#93; O105ab) and a T4 control, with 100 plants each and four forms of inoculation: in the substrate, steam, petiole and the peduncle. Samples were carried out in vegetative stage, flowering, fruiting and physiological maturity to quantify in petri dish CFU/g and know if the bacteria managed to move around and recover in root, stem, flower and fruit. The phylogenetic groups that corresponded to the bacteria recovered were confirmed by biochemical tests, serotyping and PCR. At 120 days the recovery of bacteria in the plant was 23% (E. coli O157: H7), 28% (EcT O157: H16) and 55% (EcH O105ab) whit inoculation to the substrate while the inoculation by puncture the recovery was (in the same order) of 5%, 3%, and 4% at 30 days; 37%, 35% and 30% at 90 days; and 42%, 39% and 13% at 65 days. The strains submit the ability to enter the tomato plant and to stay in it and transported to the fruit, without producing that indicate their presence.

Plasma membrane profiling during enterohemorrhagic E. coli infection reveals that the metalloprotease StcE cleaves CD55 from host epithelial surfaces.

Enterohemorrhagic Escherichia coli (EHEC) is one of several E. coli pathotypes that infect the intestinal tract and cause disease. Formation of the characteristic attaching and effacing lesion on the surface of infected cells causes significant remodeling of the host cell surface; however, limited information is available about changes at the protein level. Here we employed plasma membrane profiling, a quantitative cell-surface proteomics technique, to identify host proteins whose cell-surface levels are altered during infection. Using this method, we quantified more than 1100 proteins, 280 of which showed altered cell-surface levels after exposure to EHEC. 22 host proteins were significantly reduced on the surface of infected epithelial cells. These included both known and unknown targets of EHEC infection. The complement decay-accelerating factor cluster of differentiation 55 (CD55) exhibited the greatest reduction in cell-surface levels during infection. We showed by flow cytometry and Western blot analysis that CD55 is cleaved from the cell surface by the EHEC-specific protease StcE and found that StcE-mediated CD55 cleavage results in increased neutrophil adhesion to the apical surface of intestinal epithelial cells. This suggests that StcE alters host epithelial surfaces to depress neutrophil transepithelial migration during infection. This work is the first report of the global manipulation of the epithelial cell surface by a bacterial pathogen and illustrates the power of quantitative cell-surface proteomics in uncovering critical aspects of bacterial infection biology.

Fiber optic surface plasmon resonance sensor for detection of E. coli O157:H7 based on antimicrobial peptides and AgNPs-rGO.

A fiber optic surface plasmon resonance (FOSPR) sensor was developed for detection of Escherichia coli O157:H7 (E. coli O157:H7) in water and juice, based on antimicrobial peptides (AMP), Magainin I, as recognition elements and silver nanoparticles-reduced graphene oxide (AgNPs-rGO) nanocomposites assisted signal amplification. The uniform AgNPs-rGO was fixed on the surface of optical fiber and covered with gold film. Not only was the SPR response greatly enhanced, but also the AgNPs was prevented from being oxidized. The FOSPR showed a sensitivity of about 1.5 times higher than that fabricated only with gold film. In the assay, Magainin I, immobilized on the surface of gold film, could specifically capture E. coli O157:H7, resulting in the wavelength shift of the SPR absorption peak. Under the optimized conditions, the SPR resonance wavelength exhibited a good linear relationship with natural logarithm of the target bacteria concentration in the range of 1.0 × 103 to 5.0 × 107 cfu/mL with the detection limit of 5.0 × 102 cfu/mL (S/N = 3). The FOSPR sensor showed good specificity for E. coli O157:H7 detection compared to other bacteria similar to the target bacterial species. Furthermore, the FOSPR sensor was successfully applied to the detection of E. coli O157:H7 in water, fruit and vegetable juice with the satisfactory recoveries of 88-110%. This assay for E. coli O157:H7 detection possesses high sensitivity, good selectivity, reproducibility and stability. In addition, the AMP based SPR biosensing methodology could be extended to detect a wide variety of foodborne pathogens. Therefore, the versatile method might become a potential alternative tool in food analysis and early clinical diagnosis.

A microfluidic device for studying chemotaxis mechanism of bacterial cancer targeting.

Bacterial cancer targeting may become an efficacious cancer therapy, but the mechanisms underlying bacterial specificity for cancer cells need to be explored prior to adopting it as a new clinical application. To characterize the mechanism of bacterial chemotactic preference towards cancer cells, we developed a microfluidic device for in vitro study. The device consists of a cell culture chamber on both sides of a central bacteria channel, with micro-channels used as barriers between them. The device, when used as model for lung cancer, was able to provide simultaneous three-dimensional co-culture of multiple cell lines in separate culture chambers, and when used as model for bacterial chemotaxis, established constant concentration gradients of biochemical compounds in a central channel by diffusion through micro-channels. Fluorescence intensity of green fluorescence protein (GFP)-encoding bacteria was used to measure bacterial taxis behavior due to established chemotactic gradients. Using this platform, we found that Escherichia coli (E. coli) clearly illustrated the preference for lung cancer cells (NCI-H460) which was attributed to biochemical factors secreted by carcinoma cells. Furthermore, by secretome analysis and validation experiments, clusterin (CLU) was found as a key regulator for the chemotaxis of E. coli in targeting lung cancer.

Use of gamma radiation for inactivating Salmonella spp., Escherichia coli O157:H7 and Listeria monocytogenes in tahini halva.

Tahini halva is a traditional sweet product that is consumed with bread in different countries. It is a low water activity (aw) product basically made by mixing and cooking tahini, sugar, citric acid and Saponaria officinalis root extract together. Tahini halva maybe contaminated with foodborne pathogens during any stage of production from tahini and other raw ingredients, workers, environment or contact surfaces. The objectives of the study were to i) investigate the efficacy of gamma radiation to inactivate Salmonella spp., Escherichia coli O157:H7 and Listeria monocytogenes in tahini halva, ii) evaluate the effect of pre-irradiation storage (0, 7 and 30 days at 21 °C) of tahini halva on the sensitivity of these microorganisms toward gamma radiation, and iii) evaluate the effect of post-irradiation storage of tahini halva for up to 6 months on the their survival characteristics. Tahini halva samples were inoculated with Salmonella spp., E. coli O157:H7 and L. monocytogenes separately then stored at 21 °C for 0, 7 and 30 days prior to irradiation at 0-4 KGy and for up to 6 months after irradiation at 4 KGy. Salmonella spp. were the most irradiation resistance among the tested microorganisms. Irradiation (0.8-4.0 KGy) reduced the bacteria in samples stored for 0, 7 and 30 days pre-irradiation in the range of 0.43-2.11, 0.45-2.68 and 0.52-2.7 log10 CFU/g for Salmonella spp., 0.55-3.08, 0.66-3.00 and 0.60-2.80 log10 CFU/g for E. coli O157:H7, and 0.69-2.96, 0.86-4.30, 0.62-3.29 log10 CFU/g for L. monocytogenes, respectively. The D10-value, the irradiation dose needed to inactivate 1 log10 of pathogen, was 1.83, 1.47 and 1.50 KGy for Salmonella spp., 1.28, 1.32 and 1.48 KGy for E. coli O157:H7, and 1.33, 0.94 and 1.27 KGy for L. monocytogenes in pre-irradiation stored samples for 0, 7 and 30 days, respectively. Post-irradiation storage was efficient in decreasing the levels of the microorganisms ca. ≥2 log10 CFU/g in the first month and to undetected level after the second month of storage but enrichment results showed that Salmonella spp. and L. monocytogenes were detected in the samples until of the end of storage period. The study demonstrates that gamma radiation can be applied to inactivate of foodborne pathogens in tahini halva. Irradiation dose at 4 KGy can reduce Salmonella spp., E. coli O157:H7 and L. monocytogenes in tahini halva by 2-3 log10 CFU/g. Storage of tahini halva before or after irradiation may reduce the risk of foodborne pathogens in the product.

Individual and combined efficacies of mild heat and ultraviolet-c radiation against Escherichia coli O157:H7, Salmonella enterica, and Listeria monocytogenes in coconut liquid endosperm.

This study determined the inactivation kinetic parameters of selected pathogens in heat, ultraviolet-C and combined heat-UV-C treated coconut liquid endosperm. Separate cocktails of Escherichia coli O157:H7, Salmonella enterica serovars, and Listeria monocytogenes strains were inoculated into coconut liquid endosperm (pH 5.15, TSS 4.4oBx, TA 0.062% malic acid, extinction coefficient (ε) at 254 nm of 0.0154 cm-1) for inactivation studies. Result showed that all organisms generally exhibited a log-linear heat inactivation behavior (R2 0.81-0.99). The E. coli O157:H7 cocktail (D55 = 19.75 min, D57 = 10.79 min, D60 = 3.38 min, and D63 = 0.46 min) was found to be significantly more resistant (P > 0.05) than the tested cocktail of L. monocytogenes (D55 = 11.68 min, D57 = 4.53 min, D60 = 1.82 min and D63 = 0.26 min) and S. enterica cocktail (D55 = 3.08 min, D57 = 2.60 min, D60 = 0.89 min and D63 = 0.25 min). Despite the differences in DT values, computed z values for L. monocytogenes cocktail (5.12 ±â€¯0.43 °C) and E. coli O157:H7 cocktail (4.95 ±â€¯0.12 °C) were not significantly different (P > 0.05), but were both significantly (P < 0.05) lower than that of S. enterica cocktail (7.10 ±â€¯0.15 °C). All test organisms also exhibited a generally log-linear UV-C inactivation behavior (R2 0.90-0.99) with E. coli O157:H7 cocktail (DUV-C = 25.26 mJ/cm2) demonstrating greatest resistance to UV-C than S. enterica (DUV-C = 24.65 mJ/cm2) and L. monocytogenes (DUV-C = 17.30 mJ/cm2) cocktails. The D55 values of each organism cocktail were used to calculate for the 3-log reduction heating process schedules, during which UV-C treatments were simultaneously applied. Lethal rates (F values) calculations in the combined processes revealed that within the 3-log reduction heating processes, co-exposure of UV-C resulted in 5.62 to 6.20 log reductions in the test organism populations. Heating caused 69.3, 97.2, and 67.4% of the reduction in E. coli O157:H7, S. enterica and L. monocytogenes cocktails, respectively. These results can be used as baseline data in the establishment of mild heat treatment in combination with UV-C process schedules for coconut liquid endosperm and other similar products.

Blend of organic acids and medium chain fatty acids prevents the inflammatory response and intestinal barrier dysfunction in mice challenged with enterohemorrhagic Escherichia coli O157:H7.

Impaired epithelial barrier function disrupts immune homeostasis and increases inflammation in intestines, leading to many intestinal diseases. The blend of organic acids (OAs) and medium chain fatty acids (MCFAs) has been shown to have synergistic bactericidal effect. In this study, we demonstrated that two blends of OAs and MCFAs (OM1 and OM2) could prevent the inflammatory response and intestinal barrier dysfunction in enterohemorrhagic Escherichia coli (EHEC)-infected mice. Treatments of OM1 and OM2 significantly reduced the body weight loss and production of IL-6 and TNF-α induced by EHEC. Mice treated with OM1 and OM2 showed decrease in serum D-lactic concentration, DAO activity and bacterial transfer to liver and spleen. Furthermore, OM1 and OM2 increased the expression of tight junction proteins occludin and ZO-1, mucus protein MUC-2, and host defense peptides mBD1, mBD2 and mBD3. Finally, OM1 and OM2 increased the population of Lactobacillus spp. and Bifidobacterium spp., but decreased that of E. coli in the cecum. These findings indicate that OM1 and OM2 may be used to develop a prophylactic agent for intestinal inflammation and injury in enteric pathogen infection.

Inactivation of Escherichia coli and Staphylococcus aureus on contaminated perilla leaves by Dielectric Barrier Discharge (DBD) plasma treatment.

This study focused on sterilization methods for the reduction of microorganisms on perilla leaves by cylinder type Dielectric Barrier Discharge (DBD) plasma with underwater bubbler treatment. S. aureus and E. coli in a suspension were reduced to less than 3.4 and 0.5 log CFU/ml after the plasma treatment for 3 min, respectively. On the perilla leaves, they were also reduced to 4.8 and 1.6 log CFU/ml after the plasma treatment, respectively. The S. aureus and E. coli bacterial cell wall was damaged by the plasma treatment evident by scanning electron microscopic analysis. The observed infrared bands of the FTIR spectra demonstrated changes in protein, lipid, polysaccharide, polyphosphate group and other carbohydrate functionalities of plasma treated bacteria and untreated bacterial cell membranes. The degradation of the constituent bonds of the bacterial cell membrane by RONS generated from plasma destroys the DNA, RNA, and proteins within the cell, and may eventually cause cell death. In this study, H2O2 (13.68 µM) and NO3 (138 µM), which are the main factors generated by plasma, proved to have a bactericidal effect by inducing lipid peroxidation of bacterial cell membranes. In conclusion, cylinder type DBD plasma with underwater bubbler can be used as an environmentally friendly food disinfection device in cleaning processes of the food industry.