Print to detect: a rapid and ultrasensitive phage-based dipstick assay for foodborne pathogens.

Foodborne pathogens are a burden to the economy and a constant threat to public health. The ability to rapidly detect the presence of foodborne pathogens is a vital component of any strategy towards establishing a safe and secure food supply chain. Bacteriophages (phages) are viruses capable of infecting and replicating within bacteria in a strain-specific manner. The ubiquitous and selective nature of phages makes them ideal for the detection and biocontrol of bacteria. Therefore, the objective of this research was to develop and test a phage-based paper dipstick biosensor for the detection of various foodborne pathogens in food matrices. The first step was to identify the best method for immobilizing phages on paper such that their biological activity (infectivity) was preserved. It was found that piezoelectric inkjet printing resulted in lower loss of phage infectivity when compared with other printing methods (namely gravure and blade coating) and that ColorLok paper was ideally suited to create functional sensors. The phage-based bioactive papers developed with use of piezoelectric inkjet printing actively lysed their target bacteria and retained this antibacterial activity for up to 1 week when stored at room temperature and 80% relative humidity. These bioactive paper strips in combination with quantitative real-time PCR were used for quantitative determination of target bacteria in broth and food matrices. A phage dipstick was used to capture and infect Escherichia coli O157:H7, E. coli O45:H2, and Salmonella Newport in spinach, ground beef and chicken homogenates, respectively, and quantitative real-time PCR was used to detect the progeny phages. A detection limit of 10-50 colony-forming units per millilitre was demonstrated with a total assay time of 8 h, which was the duration of a typical work shift in an industrial setting. This detection method is rapid and cost-effective, and may potentially be applied to a broad range of bacterial foodborne pathogens. Graphical abstract ᅟ.

Towards rapid on-site phage-mediated detection of generic Escherichia coli in water using luminescent and visual readout.

Wild-type T4 bacteriophage and recombinant reporter lac Z T4 bacteriophage carrying the ß-galactosidase gene were used for detection of generic Escherichia coli by monitoring the release of ß-galactosidase upon phage-mediated cell lysis. The reaction was performed on a paper-based portable culture device to limit the diffusion of reagents and, hence, increase the sensitivity of the assay, and to avoid handling large sample volumes, making the assay suitable for on-site analysis. Chromogenic (chlorophenol red-ß-D-galactopyranoside, CPRG) and bioluminescent (6-O-ß-galactopyranosyl-luciferin, Beta-Glo(®)) ß-galactosidase substrates were tested in the assay. Water samples were first filtered through 0.45-µm pore size filters to concentrate bacteria. The filters were then placed into the paper-based device containing nutrient medium and incubated at 37 °C for 4 h. Bacteriophage with the respective indicator substrate was added to the device, and signal (color, luminescence) development was recorded with a digital camera, luminometer, or luminescence imaging device. It was demonstrated that as low as 40 or <10 colony-forming units (cfu) ml(-1) of E. coli can be detected visually within 8 h when wild-type T4 bacteriophage or recombinant lacZ T4 bacteriophage were used in the assay, respectively. Application of the bioluminescent ß-galactosidase substrate allowed reliable detection of <10 cfu ml(-1) within 5.5 h. The specificity of the assay was demonstrated using a panel of microorganisms including Aeromonas hydrophila, Enterobacter cloacae, E. coli, and Salmonella Typhimurium.

Bacteriophages for detection and control of bacterial pathogens in food and food-processing environment.

This chapter presents recent advances in bacteriophage research and their application in the area of food safety. Section 1 describes general facts on phage biology that are relevant to their application for control and detection of bacterial pathogens in food and environmental samples. Section 2 summarizes the recently acquired data on application of bacteriophages to control growth of bacterial pathogens and spoilage organisms in food and food-processing environment. Section 3 deals with application of bacteriophages for detection and identification of bacterial pathogens. Advantages of bacteriophage-based methods are presented and their shortcomings are discussed. The chapter is intended for food scientist and food product developers, and people in food inspection and health agencies with the ultimate goal to attract their attention to the new developing technology that has a tremendous potential in providing means for producing wholesome and safe food.

Photodynamic treatment: a new efficient alternative for surface sanitation.

A novel and promising technology-photodynamic treatment (PDT), aimed for surface cleaning and sanitation in food industry-is presented. It is based on the treatment of surfaces with nontoxic dyes (photosensitizers), followed by illumination of the surface with regular white light. The method is currently used in the medical field and was proved to have wide specificity against a variety of bacterial and viral pathogens as well as against yeasts and protozoa. An additional advantage of this approach is that development of resistance of microorganisms to PDT was shown to be unlikely. The theoretical basis of light-induced antimicrobial treatment is described, followed by examples of its application for the cleaning and disinfection of surfaces. All available information supports the idea that PDT could offer a very efficient and cost-effective way to combat microbial contamination of foods. The advantages and pitfalls of the technique are discussed. Directions of future research needed for bringing the technology to commercial reality are identified.

Characterization of immune-active peptides obtained from milk fermented by Lactobacillus helveticus.

The objectives of this research were to confirm the effect of compounds derived from milk fermented by Lactobacillus helveticus (LH-2) on the nonspecific host defence system, and isolate and characterize the active peptides that mediate the immune response. The cell-free supernatant obtained from the fermented milk and its fractions were tested in vitro for immuno-modulating activity using murine macrophages (RAW 264.7 cell line). Cytokine production (Interleukin-6 (IL-6), Tumor Necrosis Factor-alpha (TNF-alpha), and Interleukin-1beta (IL1-beta)), nitric oxide (NO) production and phagocytosis were used as biomarkers. Macrophages stimulated with cell-free supernatant of fermented milk showed higher production of cytokines and NO compared with macrophages stimulated with LPS (Lipopolysaccharide) and a commercial immunomodulator derived from beta-casein (f54-59). Phagocytosis was observed by macrophages stimulated with the supernatant. Two of nine fractions collected from the supernatant using size exclusion chromatography produced the highest response when used to stimulate macrophages. The results of the dose-response study of the effect of the fraction with the highest stimulation effect on the production of TNF-alpha showed a direct correlation between protein concentration and TNF-alpha release. The fraction contained four novel peptides, three derived from the hydrolysis of beta-casein and one from the hydrolysis of alpha-lactalbumin. These results confirm that fermentation of milk by Lactobacillus helveticus (LH-2) results in the production of specific peptides capable of modulating macrophage activity.

Photodynamic treatment: a novel method for sanitation of food handling and food processing surfaces.

The photodynamic bactericidal effect of the photoactive dyes acriflavine neutral, rose bengal, phloxine B, and malachite green (oxalate salt) at concentrations of 5 to 5,000 microg/ml against two gram-negative strains (Escherichia coli LJH 128 and Salmonella Typhimurium C1058), two gram-positive strains (Bacillus sp. C578 and Listeria monocytogenes LJH 375), and yeast (Saccharomyces cerevisiae C1172) was investigated. Incubation of the investigated bacteria with acriflavine neutral under illumination resulted in a significant reduction in cell numbers compared with dark incubation. Rose bengal caused a significant killing effect for bacteria incubated both in the dark and under illumination. Malachite green was active against gram-positive bacteria under illumination and did not affect gram-negative bacteria or yeasts. Incubation with phloxine B resulted in a significant decline in cell numbers for gram-positive bacteria, both in the dark and under illumination; gram-negative bacteria and yeasts were unaffected. Conjugation of rose bengal and phloxine B with poly(vinyl amine) resulted in an enhanced bactericidal effect during both dark and light incubation. This was explained by electrostatic interaction of the polymer with the cell surface, which resulted in closer contact of the photoactive dye and cell. No killing effect was observed for yeasts incubated with dye conjugates. Filter paper treated with dye-poly(vinyl amine) conjugates showed high photodynamic bactericidal activity against the bacterial strains, but not against the yeasts. The extent of bacterial killing depended on the nature and concentration of the dye conjugate and the type of microorganism. The presented data suggest that a photodynamic approach for constructing "self-decontaminating" materials has potential.

Improved bioluminescent assay of somatic cell counts in raw milk.

Somatic cell count (SCC) in milk is considered to be a valuable indicator of cow mastitis. For assessment of SCC in milk, the bioluminescent assay based on determination of ATP from somatic cells ([ATPsom]) in milk was proposed earlier. However, this assay is still not widely used in practice owing to lower reliability compared with conventional methods such as direct microscopy and flow cytometry. We revised the bioluminescent SCC assay and developed a simple protocol based on determination of the total non-bacterial ATP concentration in milk. It was shown that the novel ATP-releasing agent Neonol-10 (oxy-ethylated iso-nonyl phenol) has superior performance providing 100% lysis of somatic cells while not disrupting bacterial cells of milk at a concentration of 1.5% w/w. There was high correlation (R2=0.99) between measured bioluminescence and SCC as measured by direct microscopy. The observed detection limit of the bioluminescent milk SCC assay was as low as 900 cell/ml, time of analysis was 2-3 min per sample. The proposed method has high potential for on-site mastitis diagnostics.

Rapid methods to assess sanitizing efficacy of benzalkonium chloride to Listeria monocytogenes biofilms.

Different methods were used to investigate biofilm growth including crystal violet staining, ATP bioluminescence and total viable count. Seven strains of Listeria monocytogenes and 8 of their derivative strains were screened for their capacity to form biofilms. Both adaptation to benzalkonium chloride (BC) and curing of plasmids did not significantly affect biofilm-forming ability. The strains of L. monocytogenes belonging to serotype 1 formed biofilms significantly better as compared to serotype 4 (P=0.0003). To estimate the efficacy of BC for biofilm elimination the best and the poorest biofilm-formers were used (C719 and LJH 381). It was observed that, L. monocytogenes strain C719 in biofilms is at least 1000 times more resistant to BC than in planktonic form. Cells present in biofilms were shown to recover and grow after BC treatment thus providing a source of recontamination. It was shown that ATP bioluminescence provides good correlation with bacterial counts of L. monocytogenes in biofilms. Staining with crystal violet, on the contrary, did not correlate with bacterial growth in biofilms in the presence of high concentrations of BC but provided information on the concentration of bacterial cells, both live and dead, attached to the surface. ATP bioluminescence was found to be a reliable method for rapid estimation of the efficacy of sanitizers for biofilm disinfection. Crystal violet staining, on the other hand, was shown to be a suitable method to monitor removal of biofilms. Our investigation showed that for Listeria biofilms concentrations of BC higher then 10 mg/ml should be applied for at least 30 min to kill almost all the live cells in biofilms. However, this concentration was still not enough to remove biofilms from the surface of plastic.

Illuminating cellular physiology: recent developments.

Bioluminescent methods are gaining more and more attention among scientists due to their sensitivity, selectivity and simplicity; coupled with the fact that the bioluminescence can be monitored both in vitro and in vivo. Since the discovery of bioluminescence in the 19th century, enzymes involved in the bioluminescent process have been isolated and cloned. The bioluminescent reactions in several different organisms have also been fully characterized and used as reporters in a wide variety of biochemical assays. From the 1990s it became clear that bioluminescence can be detected and quantified directly from inside a living cell. This gave rise to numerous possibilities for the in vivo monitoring of intracellular processes non-invasively using bioluminescent molecules as reporters. This review describes recent developments in the area of bioluminescent imaging for cell biology. Newly developed imaging methods allow transcriptional/translational regulation, signal transduction, protein-protein interaction, oncogenic transformation, cell and protein trafficking, and target drug action to be monitored in vivo in real-time with high temporal and spatial resolution; thus providing researchers with priceless information on cellular functions. Advantages and limitations of these novel bioluminescent methods are discussed and possible future developments identified.

Method for assessment of functional affinity of antibodies for live bacteria.

A rapid and convenient method for assessment of functional affinity of antibodies against live bacteria is described. When a combination of immunomagnetic separation (IMS) with bioluminescent or fluorescent genetic labelling of the cells was employed, the method showed good correlation with plate count. However, the use of reporter bacteria allowed results to be obtained within 1 h compared with days using conventional methods. Due to its lower detection limit, the bioluminescent assay performed better than the fluorescent assay. Antibody affinities for Escherichia coli O157:H7 and Salmonella enteritidis were examined at different environmental conditions such as pH 3-7, temperature 4-25 degrees C, and sodium chloride concentrations 0-5% and compared with sensitivities of ELISA.

In vivo assessment of effect of fermented milk diet on course of infection in mice with bioluminescent Salmonella.

A novel method for assessing the effect of fermented milk and its components on the course of Salmonella infection in live mice is described. Following a period of feeding with whole fermented milk (group W), a cell-free fraction of fermented milk (group S), or saline (group C, control), mice were challenged by oral gavage with a bioluminescent strain of Salmonella Enteritidis. Colonization of the gastrointestinal tract and subsequent infection could be followed by bioluminescent imaging of live mice with a cooled slow-scan CCD camera. Each group of mice was fed for 7 days with the appropriate product. On the eighth day, all mice were orally infected with 10(6) Salmonella cells. On the sixth day after infection, mice in groups W and C showed evidence of disease. No bioluminescent signal was observed for any of the mice in group S. The physical condition of the mice in groups W and S was normal, but some deterioration in the health of the mice in the control group (group C) occurred. On the eighth day after infection, a weak bioluminescence signal was observed for the mice in group W, but still no signal was observed for any of the mice in group S. Mice in both of these groups appeared normal, but the mice in group C showed strong evidence of infection and marked deterioration in their physical condition accompanied by a bioluminescence signal. This method allows the assessment of the effects of potential nutraceutical agents on the course of infection by foodborne pathogens in live animals in real time.