Three Dimensional Vero Cell-Platform for Rapid and Sensitive Screening of Shiga-Toxin Producing Escherichia coli

Shiga-toxin producing Escherichia coli (STEC) is a serious public health concern. Current Vero cell assay, although sensitive, is lengthy and requires 48–72 h to assess STEC presence in a sample. In this study, we investigated if Vero cells in a three-dimensional (3D) platform would provide improved sensitivity for rapid screening of STEC. Vero cells (epithelial kidney cell line) were grown as a monolayer (2D) or in a collagen-matrix (3D) and exposed to Shiga-toxin (Stx) preparation or STEC cells that were pre-exposed to antibiotics (mitomycin C, ciprofloxacin, or polymyxin B) for toxin induction. Lactate dehydrogenase (LDH) release from Vero cells was used as a biomarker for cytotoxicity. Modified tryptic soy broth (mTSB) as enrichment broth containing mitomycin C (2 μg/ml) or ciprofloxacin (100 ng/ml) significantly induced Stx production, which was further confirmed by the dot-immunoblot assay. The 3D Vero platform detected STEC after 6 h post-infection with cytotoxicity values ranging from 33 to 79%, which is considerably faster than the traditional 2D platform, when tested with STEC. The cytotoxicity for non-Stx producing bacteria, Salmonella, Listeria, Citrobacter, Serratia, and Hafnia was found to be below the cytotoxicity cutoff value of 15%. The detection limit for the 3D Vero cell assay was estimated to be 107 CFU/ml for bacteria and about 32 ng/ml for Stx in 6 h. STEC-inoculated ground beef samples (n = 27) resulted in 38–46% cytotoxicity, and the bacterial isolates (n = 42) from ground beef samples were further confirmed to be stx1 and stx2 positive in a multiplex PCR yielding a very low false-positive result. This 3D cell-based screening assay relies on mammalian cell pathogen interaction that can complement other molecular techniques for the detection of cell-free Stx or STEC cells from food samples for early detection and prevention

Food-Associated Stress Primes Foodborne Pathogens for the Gastrointestinal Phase of Infection

The incidence of foodborne outbreaks and product recalls is on the rise. The ability of the pathogen to adapt and survive under stressful environments of food processing and the host gastrointestinal tract may contribute to increasing foodborne illnesses. In the host, multiple factors such as bacteriolytic enzymes, acidic pH, bile, resident microflora, antimicrobial peptides, and innate and adaptive immune responses are essential in eliminating pathogens. Likewise, food processing and preservation techniques are employed to eliminate or reduce human pathogens load in food. However, sub-lethal processing or preservation treatments may evoke bacterial coping mechanisms that alter gene expression, specifically and broadly, resulting in resistance to the bactericidal insults. Furthermore, environmentally cued changes in gene expression can lead to changes in bacterial adhesion, colonization, invasion, and toxin production that contribute to pathogen virulence. The shared microenvironment between the food preservation techniques and the host gastrointestinal tract drives microbes to adapt to the stressful environment, resulting in enhanced virulence and infectivity during a foodborne illness episode

Secreted Listeria Adhesion Protein (Lap) Influences Lap-Mediated Listeria monocytogenes Paracellular Translocation Through Epithelial Barrier

Background Listeria adhesion protein (Lap), an alcohol acetaldehyde dehydrogenase (lmo1634) promotes bacterial paracellular translocation through epithelial cell junctions during gastrointestinal phase of infection. Secreted Lap is critical for pathogenesis and is mediated by SecA2 system; however, if strain dependent variation in Lap secretion would affect L. monocytogenes paracellular translocation through epithelial barrier is unknown. Methods Amounts of Lap secretion were examined in clinical isolates of L. monocytogenes by cell fractionation analysis using Western blot. Quantitative reverse transcriptase PCR (qRT-PCR) was used to verify protein expression profiles. Adhesion and invasion of isolates were analyzed by in vitro Caco-2 cell culture model and paracellular translocation was determined using a trans-well model pre-seeded with Caco-2 cells. Results Western blot revealed that expression of Lap in whole cell preparation of isolates was very similar; however, cell fractionation analysis indicated variable Lap secretion among isolates. The strains showing high Lap secretion in supernatant exhibited significantly higher adhesion (3.4 - 4.8% vs 1.5 - 2.3%, P \u3c 0.05), invasion and paracellular translocation in Caco-2 cells than the low secreting isolates. In cell wall fraction, Lap level was mostly uniform for both groups, while Lap accumulated in cytosol in low secreting strains indicating that Lap distribution in cellular compartments is a strain-dependent phenomenon, which may be controlled by the protein transport system, SecA2. ΔsecA2 mutants showed significantly reduced paracellular translocation through epithelial barrier (0.48 ± 0.01 vs 0.24 ± 0.02, P \u3c 0.05). qRT-PCR did not show any discernible variation in laptranscript levels in either high or low secreting isolates. Conclusion This study revealed that secreted Lap is an important determinant in Lap-mediated L. monocytogenes translocation through paracellular route and may serve as an indicator for pathogenic potential of an isolate

Salmonella enterica serovar Typhimurium adhesion and cytotoxicity during epithelial cell stress is reduced by Lactobacillus rhamnosus GG

<p>Abstract</p> <p>Background</p> <p>Physiological stressors may alter susceptibility of the host intestinal epithelium to infection by enteric pathogens. In the current study, cytotoxic effect, adhesion and invasion of <it>Salmonella enterica </it>serovar Typhimurium (<it>S</it>. Typhimurium) to Caco-2 cells exposed to thermal stress (41°C, 1 h) was investigated. Probiotic bacteria have been shown to reduce interaction of pathogens with the epithelium under non-stress conditions and may have a significant effect on epithelial viability during infection; however, probiotic effect on pathogen interaction with epithelial cells under physiological stress is not known. Therefore, we investigated the influence of <it>Lactobacillus rhamnosus </it>GG and <it>Lactobacillus gasseri </it>on <it>Salmonella </it>adhesion and <it>Salmonella</it>-induced cytotoxicity of Caco-2 cells subjected to thermal stress.</p> <p>Results</p> <p>Thermal stress increased the cytotoxic effect of both <it>S</it>. Typhimurium (P = 0.0001) and nonpathogenic <it>E. coli </it>K12 (P = 0.004) to Caco-2 cells, and resulted in greater susceptibility of cell monolayers to <it>S</it>. Typhimurium adhesion (P = 0.001). Thermal stress had no significant impact on inflammatory cytokines released by Caco-2 cells, although exposure to <it>S</it>. Typhimurium resulted in greater than 80% increase in production of IL-6 and IL-8. Blocking <it>S</it>. Typhimurium with anti-ShdA antibody prior to exposure of <it>Salmonella </it>decreased adhesion (P = 0.01) to non-stressed and thermal-stressed Caco-2 cells. Pre-exposure of Caco-2 cells to <it>L. rhamnosus </it>GG significantly reduced <it>Salmonella</it>-induced cytotoxicity (P = 0.001) and <it>Salmonella </it>adhesion (P = 0.001) to Caco-2 cells during thermal stress, while <it>L. gasseri </it>had no effect.</p> <p>Conclusion</p> <p>Results suggest that thermal stress increases susceptibility of intestinal epithelial Caco-2 cells to <it>Salmonella </it>adhesion, and increases the cytotoxic effect of <it>Salmonella </it>during infection. Use of <it>L. rhamnosus </it>GG as a probiotic may reduce the severity of infection during epithelial cell stress. Mechanisms by which thermal stress increases susceptibility to <it>S</it>. Typhimurium colonization and by which <it>L. rhamnosus </it>GG limits the severity of infection remain to be elucidated.</p

Lactobacillus delbrueckii ssp. bulgaricus B-30892 can inhibit cytotoxic effects and adhesion of pathogenic Clostridium difficile to Caco-2 cells

<p>Abstract</p> <p>Background</p> <p>Probiotic microorganisms are receiving increasing interest for use in the prevention, treatment, or dietary management of certain diseases, including antibiotic-associated diarrhea (AAD). <it>Clostridium difficile </it>is the most common cause of AAD and the resulting <it>C. difficile </it>– mediated infection (CDI), is potentially deadly. <it>C. difficile </it>associated diarrhea (CDAD) is manifested by severe inflammation and colitis, mostly due to the release of two exotoxins by <it>C. difficile </it>causing destruction of epithelial cells in the intestine. The aim of this study was to determine the effect of probiotic bacteria <it>Lactobacillus delbrueckii </it>ssp. <it>bulgaricus </it>B-30892 (LDB B-30892) on <it>C. difficile</it>-mediated cytotoxicity using Caco-2 cells as a model.</p> <p>Methods</p> <p>Experiments were carried out to test if the cytotoxicity induced by <it>C. difficile-</it>conditioned-medium on Caco-2 cells can be altered by cell-free supernatant (CFS) from LDB B-30892 in different dilutions (1:2 to 1:2048). In a similar experimental setup, comparative evaluations of other probiotic strains were made by contrasting the results from these strains with the results from LDB B-30892, specifically the ability to affect <it>C. difficile </it>induced cytotoxicity on Caco-2 monolayers. Adhesion assays followed by quantitative analysis by Giemsa staining were conducted to test if the CFSs from LDB B-30892 and other probiotic test strains have the capability to alter the adhesion of <it>C. difficile </it>to the Caco-2 monolayer. Experiments were also performed to evaluate if LDB B-30892 or its released components have any bactericidal effect on <it>C. difficile</it>.</p> <p>Results and discussion</p> <p>Co-culturing of LDB B-30892 with <it>C. difficile </it>inhibited the <it>C. difficile-</it>mediated cytotoxicity on Caco-2 cells. When CFS from LDB B-30892-<it>C. difficile </it>co-culture was administered (up to a dilution of 1:16) on Caco-2 monolayer, there were no signs of cytotoxicity. When CFS from separately grown LDB B-30892 was mixed with the cell-free toxin preparation (CFT) of separately cultured <it>C. difficile</it>, the LDB B-30892 CFS was inhibitory to <it>C. difficile </it>CFT-mediated cytotoxicity at a ratio of 1:8 (LDB B-30892 CFS:<it>C. difficile </it>CFT). We failed to find any similar inhibition of <it>C. difficile-</it>mediated cytotoxicity when other probiotic organisms were tested in parallel to LDB B-30892. Our data of cytotoxicity experiments suggest that LDB B-30892 releases one or more bioactive component(s) into the CFS, which neutralizes the cytotoxicity induced by <it>C. difficile</it>, probably by inactivating its toxin(s). Our data also indicate that CFS from LDB B-30892 reduced the adhesion of <it>C. difficile </it>by 81%, which is significantly (<it>P </it><0.01) higher than all other probiotic organisms tested in this study.</p> <p>Conclusion</p> <p>This study reveals the very first findings that <it>Lactobacillus delbrueckii </it>ssp. <it>bulgaricus </it>B-30892 (LDB B-30892) can eliminate <it>C. difficile</it>-mediated cytotoxicity, using Caco-2 cells as a model. The study also demonstrates that LDB B-30892 can reduce the colonization of <it>C. difficile </it>cells in colorectal cells. More study is warranted to elucidate the specific mechanism of action of such reduction of cytotoxicity and colonization.</p

Bacterial Biofilms and Their Implications in Pathogenesis and Food Safety

Biofilm formation is an integral part of the microbial life cycle in nature. In food processing environments, bacterial transmissions occur primarily through raw or undercooked foods and by cross-contamination during unsanitary food preparation practices. Foodborne pathogens form biofilms as a survival strategy in various unfavorable environments, which also become a frequent source of recurrent contamination and outbreaks of foodborne illness. Instead of focusing on bacterial biofilm formation and their pathogenicity individually, this review discusses on a molecular level how these two physiological processes are connected in several common foodborne pathogens such as Listeria monocytogenes, Staphylococcus aureus, Salmonella enterica and Escherichia coli. In addition, biofilm formation by Pseudomonas aeruginosa is discussed because it aids the persistence of many foodborne pathogens forming polymicrobial biofilms on food contact surfaces, thus significantly elevating food safety and public health concerns. Furthermore, in-depth analyses of several bacterial molecules with dual functions in biofilm formation and pathogenicity are highlighted

Antilisterial and Antibiofilm Activities of Pediocin and LAP Functionalized Gold Nanoparticles

In this study, we synthesized and assessed the antilisterial and antibiofilm properties of a novel gold nanocomposite, functionalized with antimicrobial peptide, Pediocin AcH and Listeria adhesion protein (LAP) for targeted inactivation of L. monocytogenes. The gold nanoparticle (GNP) and the gold nanocomposites (GNP-Pediocin-LAP) were characterized using spectroscopic and transmission electron microscopy (TEM) and their effect on human enterocyte-like Caco-2 cells were assessed by lactate dehydrogenase (LDH)-based cytotoxicity and inhibition of Listeria adhesion assay. The antilisterial and antibiofilm activities of nanocomposites on L. monocytogenes were determined by a plating method. TEM image analysis indicated that the size of GNP and the gold nanoconjugates to be about 20 and 40 nm, respectively; and spectroscopy indicated the successful loading of proteins onto citrate-stabilized GNPs. Gold nanocomposites were non-toxic and significantly reduced L. monocytogenes adhesion to Caco-2 cells. Relative to the GNP-Pediocin conjugate, the GNP-Pediocin-LAP conjugate showed 11.1% higher zone of inhibition in agar diffusion assay, and higher reduction (1.5 log10 CFU/mL) in L. monocytogenes counts. Same preparation also showed 24 and 31% more reduction in Listeria counts in biofilms after 24 and 48 h of incubation, respectively. Nanocomposites were also highly effective in decontamination of L. monocytogenes on a miniature industrial conveyor system. Altogether, co-action of Pediocin and the LAP functionalized on GNP (GNP-Pediocin-LAP), demonstrated higher antilisterial and antibiofilm activities compared to the Pediocin functionalized GNP or Pediocin alone suggesting GNP can provide a platform to load multiple proteins for surface decontamination of L. monocytogenes in industrial settings

Effects of Dielectrophoresis on Growth, Viability and Immuno-reactivity of Listeria monocytogenes

Dielectrophoresis (DEP) has been regarded as a useful tool for manipulating biological cells prior to the detection of cells. Since DEP uses high AC electrical fields, it is important to examine whether these electrical fields in any way damage cells or affect their characteristics in subsequent analytical procedures. In this study, we investigated the effects of DEP manipulation on the characteristics of Listeria monocytogenes cells, including the immuno-reactivity to several Listeria-specific antibodies, the cell growth profile in liquid medium, and the cell viability on selective agar plates. It was found that a 1-h DEP treatment increased the cell immuno-reactivity to the commercial Listeria species-specific polyclonal antibodies (from KPL) by ~31.8% and to the C11E9 monoclonal antibodies by ~82.9%, whereas no significant changes were observed with either anti-InlB or anti-ActA antibodies. A 1-h DEP treatment did not cause any change in the growth profile of Listeria in the low conductive growth medium (LCGM); however, prolonged treatments (4 h or greater) caused significant delays in cell growth. The results of plating methods showed that a 4-h DEP treatment (5 MHz, 20 Vpp) reduced the viable cell numbers by 56.8–89.7 %. These results indicated that DEP manipulation may or may not affect the final detection signal in immuno-based detection depending on the type of antigen-antibody reaction involved. However, prolonged DEP treatment for manipulating bacterial cells could produce negative effects on the cell detection by growth-based methods. Careful selection of DEP operation conditions could avoid or minimize negative effects on subsequent cell detection performance

Novel PCR Assays Complement Laser Biosensor-Based Method and Facilitate Listeria Species Detection from Food

The goal of this study was to develop the Listeria species-specific PCR assays based on a house-keeping gene (lmo1634) encoding alcohol acetaldehyde dehydrogenase (Aad), previously designated as Listeriaadhesion protein (LAP), and compare results with a label-free light scattering sensor, BARDOT (bacterial rapid detection using optical scattering technology). PCR primer sets targeting the lap genes from the species ofListeria sensu stricto were designed and tested with 47 Listeria and 8 non-Listeria strains. The resulting PCR primer sets detected either all species of Listeria sensu stricto or individual L. innocua, L. ivanovii and L. seeligeri, L. welshimeri, and L. marthii without producing any amplified products from other bacteria tested. The PCR assays with Listeria sensu stricto-specific primers also successfully detected all species of Listeria sensu stricto and/or Listeria innocua from mixed culture-inoculated food samples, and each bacterium in food was verified by using the light scattering sensor that generated unique scatter signature for each species of Listeria tested. The PCR assays based on the house-keeping gene aad (lap) can be used for detection of either all species of Listeria sensu stricto or certain individual Listeria species in a mixture from food with a detection limit of about 104 CFU/mL