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Foodborne hepatitis A infections have been considered as a major threat for public health worldwide. Increased incidences of hepatitis A virus (HAV) infection has been associated with growing global trade of food products. Rapid and sensitive detection of HAV in foods is very essential for investigating the outbreaks. Real-time RT-PCR has been most widely used for the detection of HAV by far. However, the technology relies on fluorescence determination of the amplicon and requires sophisticated, high-cost instruments and trained personnel, limiting its use in low resource settings. In this study, a robust, affordable, and simple assay, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay in combination with a bioluminescence-based determination of amplification in real-time (BART), was developed for the detection of HAV in different food matrices, including green onion, strawberry, mussel, and milk. The efficiencies of a one-step RT-LAMP-BART and a two-step RT-LAMP-BART were investigated for the detection of HAV in different food matrices and was compared with that of real-time RT-PCR. The sensitivity of the RT-LAMP-BART assay was significantly affected by Mg 2+ concentration (P < 0.05), in addition to primer quality. The optimal Mg 2+ concentration was 2 mM for one-step RT-LAMP-BART and 4 mM for two-step RT-LAMP-BART. Compared with cartridge-purified primers, HPLC-purified primers could greatly improve the sensitivity of the RT-LAMP-BART assay (P < 0.05). For detecting HAV in different food matrices, the performance of two-step RT-LAMP-BART was comparable with that of real-time RT-PCR and was better than that of one-step RT-LAMP-BART. The detection limit of the two-step RT-LAMP-BART for HAV in green onion, strawberry, mussel, and milk was 8.3 × 10 0 PFU/15 g, 8.3 × 10 1 PFU/50 g, 8.3 × 10 0 PFU/5 g, and 8.3 × 10 0 PFU/40 mL, respectively. The developed RT-LAMP-BART was an effective, simple, sensitive, and robust method for foodborne HAV detection., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Therapies which target quorum sensing (QS) systems that regulate virulence in methicillin-resistant Staphylococcus aureus (MRSA) are a promising alternative to antibiotics. QS systems play a crucial in the regulation of MRSA antibiotic resistance, exotoxin production, antioxidant protection and immune cell evasion, and are therefore attractive therapeutic targets to reduce the virulence of a pathogen. In the present work the the effects of bioactive peptides isolated from two strains of lactic acid bacteria were tested against antibiotic resistance, carotenoid production, resistance to oxidative killing and biofilm structure in two clinical MRSA isolates. The results obtained from fractional-inhibitory concentration assays with bulk and semi-purified bioactive molecules showed a significant synergistic effect increasing cefoxitin mediated killing of MRSA. This was coupled to a six-fold decrease of the major membrane pigment staphyloxanthin, and a 99% increase in susceptibility to oxidative stress mediated killing. Real-time quantitative PCR analysis of the QS-genes agrA and luxS, showed differential expression between MRSA strains, and a significant downregulation of the hemolysin gene hla. Light microscopy and scanning electron microscopy revealed alteration in biofilm formation and clustering behavior. These results demonstrate that bioactive metabolites may be effectively applied in tandem with beta-lactam antibiotics to sensitize MRSA to cefoxitin. Moreover, these results shown that several key QS-controlled virulence mechanisms are diminished by probiotic metabolites., (© 2023. The Author(s).)

Hepatitis A virus (HAV) continues to be a public health concern and has caused large foodborne outbreaks and economic losses worldwide. Rapid detection of HAV in foods can help to confirm the source of outbreaks in a timely manner and prevent more people getting infected. In order to efficiently detect HAV at low levels of contamination in foods, rapid and easy-to-use techniques are required to separate and concentrate viral particles to a small volume. In the current study, HAV particles were eluted from green onion, strawberry, and mussel using glycine buffer (0.05 M glycine, 0.14 M NaCl, 0.2% (v/v) Tween 20, pH 9.0) and suspended viral particles were captured using protamine-coated magnetic nanoparticles (PMNPs). This process caused a selective concentration of the viral particles, which could be followed by quantitative real-time RT-PCR analysis. Results showed that pH, NaCl concentration, and PMNP amount used for the capturing had significant effects on the recovery efficiency of HAV (P < 0.05). The highest recovery rate was obtained at pH 9.0, 0.14 M NaCl, and 50 μL of PMNPs. The optimized PMNP capturing method enabled the rapid capture and concentration of HAV. A sensitive real-time RT-PCR test was developed with detection limits of 8.3 × 10 0  PFU/15 g, 8.3 × 10 1  PFU/50 g, and 8.3 × 10 0  PFU/5 g of HAV in green onion, strawberry, and mussel, respectively. In conclusion, the PMNP method is rapid and convenient in capturing HAV from complex solid food samples and can generate concentrated HAV sample solutions suitable for high-sensitivity real time RT-PCR detection of the virus., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Invasion of Salmonella into host intestinal epithelial cells requires the expression of virulence genes. In this study, cell culture models of human intestinal cells (mucus-producing HT29-MTX cells, absorptive Caco-2 cells, and combined cocultures of the two) were used to determine the effects of Lactococcus lactis subsp. cremoris treatments (exopolysaccharide producing and nonproducing strains) on the virulence gene expression of Salmonella Typhimurium and its mutant lacking the oligopeptide permease subunit A (ΔoppA). During the course of epithelial cell (HT29-MTX, Caco-2, and combined) infection by Salmonella Typhimurium DT104, improved barrier function was reflected by increased transepithelial electrical resistance in cells treated with both strains of L. lactis subsp. cremoris. In addition, virulence gene expression was downregulated, accompanied with lower numbers of invasive bacteria into epithelial cells in the presence of L. lactis subsp. cremoris treatments. Similarly, virulence gene expression of Salmonella was also suppressed when coincubated with overnight cultures of both L. lactis subsp. cremoris strains in the absence of epithelial cells. However, in medium or in the presence of cell cultures, Salmonella lacking the OppA permease function remained virulent. HT29-MTX cells and combined cultures stimulated by Salmonella Typhimurium DT104 showed significantly lower secretion levels of pro-inflammatory cytokine IL-8 after treatment with L. lactis subsp. cremoris cell suspensions. Contrarily, these responses were not observed during infection with S. Typhimurium ΔoppA. Both the exopolysaccharide producing and nonproducing strains of L. lactis subsp. cremoris JFR1 exhibited an antivirulence effect against S. Typhimurium DT104 although no significant difference between the two strains was observed. Our results show that an intact peptide transporter is essential for the suppression of Salmonella virulence genes which leads to the protection of the barrier function in the cell culture models studied.

In this work, the known antiproliferative activity of the untreated milk fat globule membrane (MFGM) against human colon cancer cells was employed to test the hypothesis that the supramolecular structure of the MFGM is of important biological significance. The results indicated that there is a relationship between the extent of thermal denaturation and the loss of antiproliferative capacity. There was also a clear reduction of the biological activity, when the MFGM was treated by hydrolysis using trypsin or phospholipase A 2 , enzymes specific either for the protein or the phospholipids components present in the MFGM. It was concluded that the bioactivity of the MFGM can not be explained only by the presence of bioactive components, but that their structural organization plays a critical role in the antiproliferative activities of the extracts. PRACTICAL APPLICATIONS: The milk fat globule membrane (MFGM) is characterized by a complex composition and structure, with biological significance. It is known that with processing, the composition of the MFGM is modified, due to protein-protein interactions at the interface. In this work, the MFGM was isolated from untreated milk and while maintaining its overall composition, its molecular and supramolecular structures were modified using heating or specific hydrolysis to the protein or phospholipids' components. All targeted modifications affected the bioefficacy of the MFGM against colon cancer cells, thus demonstrating the importance of processing history on the functionality of the MFGM., (© 2019 Wiley Periodicals, Inc.)

Hepatitis A virus (HAV) continues to be the leading cause of viral hepatitis. HAV outbreaks have been linked to the consumption of milk, but methods for HAV detection in milk are very limited. We developed a method to concentrate HAV in milk using protamine-coated iron oxide (Fe 3 O 4 ) magnetic nanoparticles (PMNPs). In this study, protamine was covalently coated on the surface of the MNPs (20-30 nm) by a three-step chemical reaction. The successful linkage of protamine to the MNPs was confirmed by Fourier transform infrared spectroscopy (FTIR), zeta potential, and transmission electron microscopy (TEM). When used for concentrating HAV from 40 mL of milk, 50 μL of PMNPs were added to the sample and mixed for 20 min by gentle rotation, followed by a magnet capture for 30 min. The captured PMNPs were washed with glycine buffer (0.05 M glycine, 0.14 M NaCl, 0.2% (v/v) Tween 20, pH 9.0) and HAV RNA was extracted using the QIAamp MinElute Virus Spin Kit and quantified by real-time RT-PCR. The method showed a detection limit of 8.3 × 10 0  PFU of HAV in milk. The whole concentration procedure could be completed in approximately 50 min. The developed method was simple, inexpensive, and easy-to-perform., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The process of fermentation contributes to the organoleptic properties, preservation, and nutritional benefits of food. Fermented food may interfere with pathogen infections through a variety of mechanisms, including competitive exclusion or improving intestinal barrier integrity. In this study, the effect of milk fermented with Lactococcus lactis ssp. cremoris JFR1 on Salmonella invasion of intestinal epithelial cell cultures was investigated. Epithelial cells (HT29-MTX, Caco-2, and cocultures of the 2) were treated for 1 h with Lactococcus lactis ssp. cremoris JFR1 fermented milk before infection with Salmonella enterica ssp. enterica Typhimurium. Treatment with fermented milk resulted in increased transepithelial electrical resistance, which remained constant for the duration of infection (up to 3 h), illustrating a protective effect. After gentamicin treatment to remove adhered bacterial cells, enumeration revealed a reduction in numbers of intracellular Salmonella. Quantitative reverse-transcription PCR data indicated a downregulation of Salmonella virulence genes hilA, invA, and sopD after treatment with fermented milk. Fermented milk treatment of epithelial cells also exhibited an immunomodulatory effect reducing the production of proinflammatory IL-8. In contrast, chemically acidified milk (glucono delta-lactone) failed to show the same effect on monolayer integrity, Salmonella Typhimurium invasion, and gene expression as well as immune modulation. Furthermore, an oppA knockout mutant of Salmonella Typhimurium infecting treated epithelial cells did not show suppressed virulence gene expression. Collectively, these results suggest that milk fermented with Lactococcus lactis ssp. cremoris JFR1 is effective in vitro in the reduction of Salmonella invasion into intestinal epithelial cells. A functional OppA permease in Salmonella is required to obtain the antivirulence effect of fermented milk., (The Authors. Published by FASS Inc. and Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

Bacteriophages have been envisioned as tools to control a variety of foodborne pathogenic bacteria. Salmonella is a foodborne pathogen that is a threat to public health around the world. Contaminated tomatoes have been associated with several Salmonella outbreaks. Hence, the objective of this work was to identify and characterize different lytic bacteriophages against Salmonella Newport, as one of top ten Salmonella serovars associated with human salmonellosis in North America, and then apply these phages to enhance the safety of cherry tomatoes. Four lytic phages against Salmonella Newport were selected based on their ability to lyse a majority of the 26 screened Salmonella serovars. The selected phages belong to Myoviridae (vB&#95;SnwM&#95;CGG4-1, vB&#95;SnwM&#95;CGG4-2) and Siphoviridae (vB&#95;SnwM&#95;CGG3-1, vB&#95;SnwM&#95;CGG3-2) families. They were found to be stable at different temperatures and pH, have latent periods ranging from 53 to 65 min and burst sizes from 92 to 177. In addition, the two Myoviridae phages have a lower frequency of developing bacteriophage insensitive mutants when compared with the Siphoviridae phages. No significant change in virulence gene expression was observed in the developed bacteriophage insensitive mutants when compared to the parental phage sensitive strain. Furthermore, the vB&#95;SnwM&#95;CGG4-1 genome revealed no homology to virulence or lysogenic genes. A phage cocktail was used to control the growth of S. Newport in broth medium and on contaminated cherry tomato. Complete inhibition of bacterial growth in broth medium was observed at 25 °C for 24 h. In addition, a 4.5 log 10 unit reduction in the bacterial count was observed when applying the phage cocktail onto contaminated tomatoes stored at 22 °C for 3 days. These findings suggest that the isolated phages can be used for biocontrol of S. Newport to improve the safety of ready-to-eat (RTE) produce., (Copyright © 2019. Published by Elsevier B.V.)

Clostridium difficile infection is a range of toxin - mediated intestinal diseases that is often acquired in hospitals and small communities in developed countries. The main virulence factors of C. difficile are two exotoxins, toxin A and toxin B, which damage epithelial cells and manifest as colonic inflammation and mild to severe diarrhea. Inhibiting C. difficile adherence, colonization, and reducing its toxin production could substantially minimize its pathogenicity and lead to faster recovery from the disease. This study investigated the efficacy of probiotic secreted bioactive molecules from Lactobacillus acidophilus La-5, in decreasing C. difficile attachment and cytotoxicity in human epithelial cells in vitro. L. acidophilus La-5 cell-free supernatant (La-5 CFS) was used to treat the hypervirulent C. difficile ribotype 027 culture with subsequent monitoring of cytotoxicity and adhesion. In addition, the effect of pretreating cell lines with La-5 CFS in protecting cells from the cytotoxicity of C. difficile culture filtrate or bacterial cell attachment was examined. La-5 CFS substantially reduced the cytotoxicity and cytopathic effect of C. difficile culture filtrate on HT-29 and Caco-2 cells. Furthermore, La-5 CFS significantly reduced attachment of the C. difficile bacterial cells on both cell lines. It was also found that pretreatment of cell lines with La-5 CFS effectively protected cell lines from cytotoxicity and adherence of C. difficile. Our study suggests that La-5 CFS could potentially be used to prevent and cure C. difficile infection and relapses., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The natural specificity of bacteriophages toward their hosts represents great potential for the development of platforms for the capture and detection of bacterial pathogens. Whole phage can carry reporter genes to alter the phenotype of the target pathogen. Phage can also act as staining agents or the progeny of the infection process can be detected. Alternatively, using phage components as probes offer advantages over whole phage particles, including smaller probe size and resilience to desiccation. Phage structures can be engineered for improved affinity, specificity, and binding properties. However, such concepts require the ability to anchor phage and phage-components onto mechanical supports such as beads or flat surfaces. The ability to orient the anchoring is desired in order to optimize binding efficiency. This chapter presents various methods that have been employed for the attachment of phage and phage components onto support structures such as beads, filters, and sensor surfaces.

The antimicrobial activity of LISTEX P100, Salmonella CG4, and E. coli AG10 bacteriophages were preserved in pullulan-trehalose mixture as dried films and as coatings on food packaging. The phages encapsulated in pullulan-trehalose films were able to retain infectivity for up to 3 months at ambient storage conditions. Various buffers, disaccharides and disaccharide concentrations were investigated to optimize the long-term stability of the phages in the films. It was found that pullulan and trehalose need to be simultaneously present in the film to provide the stabilizing effect and that the presence of buffers that lead to the formation of crystals in the films must be avoided for phage activity to be maintained. Overall, this study describes a method of preserving bacteriophage activity in a dried format that has great potential for use as coatings, which can be used to create antimicrobial surfaces for food preparation and for food preservation.

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.

Background: With the advent of antimicrobial resistance in animal pathogens, novel methods to combat infectious diseases are being sought. Among these, probiotics have been proposed as a means of promoting animal health but problems with their use has been reported. Research has demonstrated that bioactive molecules produced during the growth of certain probiotics interfere with bacterial cell-to-cell communication, which consequently results in an attenuation of virulence in a number of pathogens, including E. coli. The objective of this study was to determine the efficacy of the bioactive molecules, termed proteobiotics, produced by Lactobacillus acidophilus in preventing enterotoxigenic E, coli (ETEC) infection in pigs, which is the etiological agent for enteric colibacillosis, a common disease of nursing and young pigs., Results: To achieve this, piglets were fed a preparation of the bioactive at four levels: 0, 0.5×, 1.0× and 2.0× for 7 days prior to challenge with E. coli K88. There were 36 pigs (18 gilts and 18 barrows) per treatment, resulting in 144 piglets in total for the study. Each pen had 6 piglets (3 gilts and 3 barrows). Only piglets with no physical abnormality or conditions were used in the trial and intact male piglets and ridglings were excluded. The bioactive continued to be fed to the pigs post-challenge. Based of fecal and demeanour scores, pigs fed the low and high dose of the proteobiotic were significanlty less likely to show symptoms of illness than pigs fed no bioactive. While not being significant, the weight gain of pigs given the proteobiotics was improved. At day 4 following challenge, almost 50% of piglets that did not receive the proteobiotic were shedding ETEC in their feces, compared with about 15% of animals receiving the supplement. There was also an indication that the proteobiotics reduced colonization of the ileum by E. coli K88 and improved gut health., Conclusion: This study indicates that the bioactive molecules produced by L. acidophilus reduces incidence of enteric colibacillosis in pigs and their use on farms would help to reduce antibiotic use.

Endophytes are microorganisms that inhabit plant tissues without causing disease. Some endophytes help their hosts to combat pathogens. Here we explored the hypothesis that the plant-derived foods consumed by humans and other animals host endophytes that also antagonize foodborne pathogens or food-rotting agents. Our laboratory previously cultured a library of bacterial endophytes from different members of the maize/corn family (Zea) including wild relatives. Here, 190 of these endophytes were screened for their ability to antagonize four foodborne pathogens (Escherichia coli O157:H7, Listeria monocytogenes, Clostridium perfringens, and Salmonella enterica Newport) and a food spoiling agent (Pseudomonas fluorescens) using dual culture assays. Two Paenibacillus polymyxa endophytes (strains 3C6 and 3G11) were found to inhibit the growth of all five deleterious strains on agar. Using conserved polymerase chain reaction primers and sequencing, both beneficial endophytes were found to encode polymyxin genes, suggesting a potential antibacterial mechanism of action. Polymyxin production by both strains was confirmed using enzyme-linked immunosorbent assay. Strains 3C6 and 3G11 originated, respectively, from the seeds of the wild Central American maize species Zea diploperennis, and the wild ancestor of modern maize, Zea mays ssp parviglumis (Parviglumis). As the latter is the direct ancestor of modern maize, we discuss the role its endophyte(s) may have played in promoting crop domestication by suppressing foodborne pathogens and/or food-spoilage agents.

The innate specificity of bacteriophages toward their hosts makes them excellent candidates for the development of detection assays. They can be used in many ways to detect pathogens, and each has its own advantages and disadvantages. Whole bacteriophages can carry reporter genes to alter the phenotype of the target. Bacteriophages can act as staining agents or the progeny of the infection process can be detected, which further increases the sensitivity of the detection assay. Compared with whole-phage particles, use of phage components as probes offers other advantages: for example, smaller probe size to enhance binding activity, phage structures that can be engineered for better affinity, as well as specificity, binding properties, and robustness. When no natural binding with the target exists, phages can be used as vehicles to identify new protein-ligand interactions necessary for diagnostics. This review comprehensively summarizes many uses of phages as detection tools and points the way toward how phage-based technologies may be improved.

Encapsulation of bacteriophages ("phage") protects phage against environmental deactivation and provides a product that is easy to handle for storage and application with animal feed as an antibiotic alternative. The objective of this study was to evaluate an orally administered, encapsulated phage for efficient phage release in the gastrointestinal tract (GIT) of young chicks receiving feed. An optimized formulation that consisted of 0.8% low molecular weight (MW) alginate, 2% ultra-low molecular weight alginate and 3% whey protein completely released the encapsulated phage within 60 min under simulated intestinal conditions. This product was given to broiler chicks to determine passage time and distribution of the viable phage within the GIT. Following a single oral dose of 10 9 plaque-forming unit (PFU)/chick, the major portion (peak concentration) of the encapsulated phage passed through the chick's GIT and was detected in the feces within 4 h, with low levels being continuously excreted for up to 24 h. In comparison, the passage of free phage through the GIT occurred faster as indicated by a peak concentration in feces after 1.5 h. In assessing the temporal phage distribution, both encapsulated and free phage treatments showed no apparent difference, both having low levels of 10 2 to 10 6 PFU/g of contents along the entire GIT after 1, 2 and 4 h. These low concentrations recovered in vivo led us to examine various exposure conditions (with feed, fecal material, and buffer solutions) that were suspected to have affected phage viability/infectivity during oral delivery, sample recovery, and enumeration by plaque assay. Results showed that the exposure conditions examined did not significantly reduce phage viability and could not account for the observed low phage levels following oral administration in chicks that are on feed. In conclusion, an oral encapsulated phage dose can take more than 4 h to completely move through the GIT of young chicks. Thus, repeated or higher doses may be necessary to attain higher phage concentrations in the GIT., (© Crown copyright 2016.)

Complex oligosaccharides from human milk (HMO) possess an antimicrobial activity and can promote the growth of bifidobacteria such as Bifidobacterium bifidum and Bifidobacterium longum subsp. infantis . In addition, fermentation of carbohydrates by bifidobacteria can result in the production of metabolites presenting an antivirulence effect on several pathogenic bacteria. Whey is rich in complex bovine milk oligosaccharides (BMO) structurally similar to HMO and B. crudilactis , a species of bovine origin, is able to metabolize some of those complex carbohydrates. This study focused on the ability of B. bifidum and B. crudilactis to grow in a culture medium supplemented in 3'-sialyllactose (3'SL) as the main source of carbon, a major BMO encountered in cow milk. Next, the effects of cell-free spent media (CFSM) were tested against virulence expression of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium. Both strains were able to grow in presence of 3'SL, but B. crudilactis showed the best growth (7.92 ± 0.3 log cfu/ml) compared to B. bifidum (6.84 ± 0.9 log cfu/ml). Then, CFSM were tested for their effects on virulence gene expression by ler and hilA promoter activity of luminescent mutants of E. coli and S . Typhimurium, respectively, and on wild type strains of E. coli O157:H7 and S . Typhimurium using RT-qPCR. All CFSM resulted in significant under expression of the ler and hilA genes for the luminescent mutants and ler (ratios of -15.4 and -8.1 respectively) and qseA (ratios of -2.1 and -3.1) for the wild type strain of E. coli O157:H7. The 3'SL, a major BMO, combined with some bifidobacteria strains of bovine or human origin could therefore be an interesting synbiotic to maintain or restore the intestinal health of young children. These effects observed in vitro will be further investigated regarding the overall phenotype of pathogenic agents and the exact nature of the active molecules.

Unlabelled: Bacteriophages present huge potential both as a resource for developing novel tools for bacterial diagnostics and for use in phage therapy. This potential is also valid for bacteriophages specific for Yersinia enterocolitica To increase our knowledge of Y. enterocolitica-specific phages, we characterized two novel yersiniophages. The genomes of the bacteriophages vB&#95;YenM&#95;TG1 (TG1) and vB&#95;YenM&#95;ϕR1-RT (ϕR1-RT), isolated from pig manure in Canada and from sewage in Finland, consist of linear double-stranded DNA of 162,101 and 168,809 bp, respectively. Their genomes comprise 262 putative coding sequences and 4 tRNA genes and share 91% overall nucleotide identity. Based on phylogenetic analyses of their whole-genome sequences and large terminase subunit protein sequences, a genus named Tg1virus within the family Myoviridae is proposed, with TG1 and ϕR1-RT (R1RT in the ICTV database) as member species. These bacteriophages exhibit a host range restricted to Y. enterocolitica and display lytic activity against the epidemiologically significant serotypes O:3, O:5,27, and O:9 at and below 25°C. Adsorption analyses of lipopolysaccharide (LPS) and OmpF mutants demonstrate that these phages use both the LPS inner core heptosyl residues and the outer membrane protein OmpF as phage receptors. Based on RNA sequencing and quantitative proteomics, we also demonstrate that temperature-dependent infection is due to strong repression of OmpF at 37°C. In addition, ϕR1-RT was shown to be able to enter into a pseudolysogenic state. Together, this work provides further insight into phage-host cell interactions by highlighting the importance of understanding underlying factors which may affect the abundance of phage host receptors on the cell surface., Importance: Only a small number of bacteriophages infecting Y. enterocolitica, the predominant causative agent of yersiniosis, have been previously described. Here, two newly isolated Y. enterocolitica phages were studied in detail, with the aim of elucidating the host cell receptors required for infection. Our research further expands the repertoire of phages available for consideration as potential antimicrobial agents or as diagnostic tools for this important bacterial pathogen., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The cerambycid beetles comprise a diverse family that includes many economically important pests of living and dead trees. Pheromone lures have been developed for cerambycids in many parts of the world, but to date, have not been tested in Australia. In this study, we tested the efficacy of several pheromones, identified from North American and European species, as attractants for cerambycids at three sites in southeast Queensland, Australia. Over two field seasons, we trapped 863 individuals from 47 cerambycid species. In the first season, racemic 3-hydroxyhexan-2-one was the most attractive compound among the eight pheromones tested. Subsequently, we aimed to optimize trapping success by combining this compound with other components. However, neither the addition of other pheromone components nor host plant volatiles improved the efficacy of 3-hydroxyhexan-2-one alone. We also tested a generic pheromone blend developed for North American cerambycids, and found that only the combination of this blend with host plant volatiles improved trapping success. The Australian cerambycid fauna is not well known, and effective lures for use in trapping beetles would greatly assist in the study of this important group. Effective semiochemical lures would also have implications for biosecurity through improved monitoring for invasive species., (© The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Due to lack of adequate control methods to prevent contamination in fresh produce and growing consumer demand for natural products, the use of bacteriophages has emerged as a promising approach to enhance safety of these foods. This study sought to control Listeria monocytogenes in cantaloupes and RTE meat and Escherichia coli O104:H4 in alfalfa seeds and sprouts under different storage conditions by using specific lytic bacteriophage cocktails applied either free or immobilized. Bacteriophage cocktails were introduced into prototypes of packaging materials using different techniques: i) immobilizing on positively charged modified cellulose membranes, ii) impregnating paper with bacteriophage suspension, and iii) encapsulating in alginate beads followed by application of beads onto the paper. Phage-treated and non-treated samples were stored for various times and at temperatures of 4°C, 12°C or 25°C. In cantaloupe, when free phage cocktail was added, L. monocytogenes counts dropped below the detection limit of the plating technique (<1 log CFU/g) after 5 days of storage at both 4°C and 12°C. However, at 25°C, counts below the detection limit were observed after 3 and 6h and a 2-log CFU/g reduction in cell numbers was seen after 24h. For the immobilized Listeria phage cocktail, around 1-log CFU/g reduction in the Listeria count was observed by the end of the storage period for all tested storage temperatures. For the alfalfa seeds and sprouts, regardless of the type of phage application technique (spraying of free phage suspension, bringing in contact with bacteriophage-based materials (paper coated with encapsulated bacteriophage or impregnated with bacteriophage suspension)), the count of E. coli O104:H4 was below the detection limit (<1 log CFU/g) after 1h in seeds and about a 1-log cycle reduction in E. coli count was observed on the germinated sprouts by day 5. In ready-to-eat (RTE) meat, LISTEX™ P100, a commercial phage product, was able to significantly reduce the growth of L. monocytogenes at both storage temperatures, 4°C and 10°C, for 25 days regardless of bacteriophage application format (immobilized or non-immobilized (free)). In conclusion, the developed phage-based materials demonstrated significant antimicrobial effect, when applied to the artificially contaminated foods, and can be used as prototypes for developing bioactive antimicrobial packaging materials capable of enhancing the safety of fresh produce and RTE meat., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The aims of this study were to examine the effect of mare's milk on virulence gene expression of Salmonella Typhimurium and observe its potential activity on proliferation of adenocarcinoma Caco-2 cells. Different supernatants of mare's milk, raw or heat-treated at 65°C for 15 s or 30 min, were studied. The changes in hilA gene expression of Salmonella Typhimurium in presence of mare's milk supernatants were assessed using a reporter luminescent strain. A significant decrease in hilA gene expression was observed with all tested supernatants. Virulence gene expression was then assessed using qPCR on a wild-type strain of Salmonella Typhimurium. A significant decrease of hilA and ssrB2 gene expression was observed with raw milk supernatants but not with heat-treated supernatants. The same supernatants were administered to Caco-2 cells to measure their proliferation rate. A significant reduction of proliferative effect was observed only with raw milk supernatants. This study reports that raw mare's milk was able to modulate virulence gene expression of Salmonella Typhimurium and exerts antiproliferative effects on Caco-2 cells. These results may offer new approaches for promoting gastrointestinal health., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Clostridium difficile infection (CDI) is the most prevalent cause of health-care-associated infections. CDI-related health-care costs and deaths are both increasing annually on a global scale. C. difficile have been reported in food products in Canada, Europe, and the United States; however, the systematic transmission of C. difficile between humans and animals is yet to be understood. Because of the limitations of current therapeutic options, there is a need for the development of new patient treatments. Epigallocatechin gallate (EGCG) is a major catechin compound found in green tea extracts and exhibits antioxidant and antimicrobial activities. This study was conducted to investigate the inhibitory effects of EGCG on the expression of virulence genes in C. difficile and in C. difficile-associated diseases by inhibition of quorum sensing. The protein expression of autoinducer-2 (AI-2) was evaluated by AI-2 activity. EGCG at various concentrations had an inhibitory effect on AI-2 production, especially at 10 μg/mL. EGCG also significantly repressed the transcription of virulence genes, including luxS and tcdA, and prolonged the survival of Caenorhabditis elegans infected with C. difficile. Furthermore, treatment with EGCG effectively protected C. difficile-infected mice from C. difficile-induced death. Histological analysis of the colon and cecum of these mice revealed that EGCG protected tissues of the lower intestinal tract from damage. EGCG exerted growth-inhibitory and bactericidal activities on C. difficile in C. difficile-infected mice. Our results suggest that EGCG has significant antipathogenic effects on C. difficile and can be used to prevent or treat C. difficile-associated diseases or C. difficile infections., (© 2015 Institute of Food Technologists®)

The present work evaluated the effect of processing on the antiproliferative activities of milk fat globule membrane (MFGM) extracts. The antiproliferative activity on human adenocarcinoma HT-29 cells of untreated MFGM extracts were compared with those extracted from pasteurized cream, thermally treated cream, or cream subjected to pulsed electrical field (PEF) processing. The PEF with a 37 kV/cm field strength applied for 1,705μs at 50 and 65°C was applied to untreated cream collected from a local dairy. Heating at 50 or 65°C for 3min (the passage time in the PEF chamber) was also tested to evaluate the heating effect during PEF treatments. The MFGM extracted from pasteurized cream did not show an antiproliferative activity. On the other hand, isolates from PEF-treated cream showed activity similar to that of untreated samples. It was also shown that PEF induced interactions between β-lactoglobulin and MFGM proteins at 65°C, whereas the phospholipid composition remained unaltered. This work demonstrates the potential of PEF not only a means to produce a microbiologically safe product, but also as a process preserving the biofunctionality of the MFGM., (Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Based on morphology and comparative nucleotide and protein sequence analysis, a new subfamily of the family Siphoviridae is proposed, named "Jerseyvirinae" and consisting of three genera, "Jerseylikevirus", "Sp3unalikevirus" and "K1glikevirus". To date, this subfamily consists of 18 phages for which the genomes have been sequenced. Salmonella phages Jersey, vB&#95;SenS&#95;AG11, vB&#95;SenS-Ent1, vB&#95;SenS-Ent2, vB&#95;SenS-Ent3, FSL SP-101, SETP3, SETP7, SETP13, SE2, SS3e and wksl3 form the proposed genus "Jerseylikevirus". The proposed genus "K1glikevirus" consists of Escherichia phages K1G, K1H, K1ind1, K1ind2 and K1ind3. The proposed genus "Sp3unalikevirus" contains one member so far. Jersey-like phages appear to be widely distributed, as the above phages were isolated in the UK, Canada, the USA and South Korea between 1970 and the present day. The distinguishing features of this subfamily include a distinct siphovirus morphotype, genomes of 40.7-43.6 kb (49.6-51.4 mol % G+C), a syntenic genome organisation, and a high degree of nucleotide sequence identity and shared proteins. All known members of the proposed subfamily are strictly lytic.

Pulsed electric fields (PEF), heat-assisted PEF (H-PEF), and virulent bacteriophage (VP) are non-thermal techniques for pathogen inactivation in liquids that were investigated individually, and in combination (PEF/VP, H-PEF/VP) to control enterohemorrhagic Escherichia coli (EHEC) O157:H7 in Luria-Bertani broth (LBB) and Ringer's solution (RS). Treated cells were subsequently incubated at refrigeration (4°C) and temperature-abuse conditions (12°C) for 5 days. When EHEC cells grown in LBB were subjected to non-thermal processing and subsequently stored at 12°C for 5 days, reductions in count of between 0.1 and 0.6 log cycles were observed and following storage at 4°C the decrease in counts varied between 0.2 and 1.1 log10 . For bacteria cells suspended in RS values ranged from 0.1 to ≥3.9 log cycles at both storage temperatures. The most effective treatments were H-PEF and H-PEF/VP, both producing a >3.4 log cycle reduction of cells suspended in non-nutrient RS. Analysis of EHEC recovery on selective and non-selective media indicated no occurrence of sub-lethal damage for VP, PEF/VP, and H-PEF/VP-treated cells. The findings indicate that combining PEF and lytic phage may represent a suitable alternative to conventional fluid decontamination following further process optimization., (© 2014 American Institute of Chemical Engineers.)

The stress resistance of bacteria is affected by the physiological status of the bacterial cell and environmental factors such as pH, salts and temperature. In this study, we report on the stress response of Lactobacillus plantarum L67 after four consecutive freeze-thaw cycles. The cold stress response of the cold-shock protein genes (cspC, cspL and cspP) and ATPase activities were then evaluated. The cold stress was adjusted to 5 °C when the bacteria were growing at the mid-exponential phase. A comparative proteomic analysis was performed with two-dimensional gel electrophoresis (2D SDS-PAGE) and a matrix assisted laser desorption/ionization-mass spectrometer. Only 56% of the L. plantarum L67 cells without prior exposure to cold stress survived after four consecutive freeze-thaw cycles. However, 78% of the L. plantarum L67 cells that were treated with cold stress at 5 °C for 6 h survived after freeze-thaw conditions. After applying cold stress to the culture for 6h, the cells were then stored for 60 days at 5 °C, 25 °C and 35 °C separately. The cold-stressed culture of L. plantarum L67 showed an 8% higher viability than the control culture. After applying cold stress for 6h, the transcript levels of two genes (cspP and cspL) were up-regulated 1.4 (cspP) and 1.2 (cspL) times compared to the control. However, cspC was not up-regulated. A proteomic analysis showed that the proteins increased after a reduction of the incubation temperature to 5 °C. The importance of the expression of 13 other relevant proteins was also determined through the study. The exposure of L. plantarum cells to low temperatures aids their ability to survive through subsequent freeze-thaw processes and lyophilization., (Copyright © 2014. Published by Elsevier B.V.)

Aims: To produce light-activated antimicrobial materials composed of the photodynamic dye phloxine B incorporated into paper or cellulose membranes and to investigate ability of these materials to decrease bacterial loads on their surfaces as well as on food surfaces that were in contact with these materials under illumination with regular white light., Methods and Results: Antimicrobial cellulose-based materials with incorporated phloxine B were produced using a layer-by-layer deposition method. Antimicrobial properties of the materials were tested in model systems as well as for decontamination of food and food contact surfaces. Pseudomonas aeruginosa, Listeria monocytogenes and Bacillus anthracis were efficiently killed by exposure of the bacterial suspension to the dye-containing material under illumination with white light, but Salmonella Typhimurium and Escherichia coli O157:H7 were only partially affected. Application of the materials for decontamination of food surfaces artificially contaminated with L. monocytogenes was shown to be ineffective, while the self-decontamination of the material surface by exposure to white light resulted in eradication of L. monocytogenes cells from the material surface., Conclusions: The developed materials showed significant self-decontaminating ability when under illumination; however, decontamination of food surfaces in contact with the developed materials was not achieved., Significance and Impact of the Study: The study demonstrates the antibacterial activity of materials with incorporated photodynamic dyes when under illumination with regular-intensity white light. Possible uses of the light-activated antimicrobial materials for food processing, as food contact surfaces, and surfaces in public areas to prevent cross-contamination are discussed., (© 2014 The Society for Applied Microbiology.)

Background: Bacteriophage vB&#95;YenP&#95;AP5 is a lytic bacteriophage capable of infecting Yersinia enterocolitica strains of serotype O:3, an epidemiologically significant serotype within this bacterial species that causes yersiniosis in humans. This work describes the complete genome sequence of this phage., Results: The genome consists of linear double-stranded DNA of 38,646 bp, with direct terminal repeats of 235 bp in length, and a GC content of 50.7%. There are 45 open reading frames which occupy 89.9% of the genome. Most of the proteins encoded by this virus exhibit sequence similarity to Yersinia phage φYeO3-12 and Salmonella phage φSG-JL2 proteins., Conclusions: Genomic and morphological analyses place the bacteriophage vB&#95;YenP&#95;AP5 in the T7likevirus genus of the subfamily Autographivirinae within the family Podoviridae.

Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease, has a doubling time of 24 hours, making rapid detection very difficult. Mycobacteriophages can be used in the detection of disease-causing mycobacteria such as MAP. Isolation and sequencing the genomes of lytic MAP bacteriophages are important preliminary steps towards designing phage-based rapid detection assays for this bacterium. A simple optimized protocol was developed to allow reproducible production of confluent growth of MAP on plates within four to six weeks of incubation at 30 °C. This protocol was applied to the screening of environmental and fecal samples for bacteriophages inhibiting the growth of MAP. As a result, a lytic phage, vB&#95;MapS&#95;FF47, was isolated from bovine feces. FF47 contains a double-stranded DNA genome ~48 kb in length with 73 protein coding sequences. It does not carry temperate or known virulence genes. This phage was shown to be most closely related to Mycobacterium phage Muddy, isolated in South Africa, and Gordonia phage GTE2; however, it could not infect any of the tested Gordonia, Rhodococcus, or Nocardia spp. that GTE2 could. The protocols that were developed for growth and phage isolation have potential applications in a high-throughput screening for compounds inhibiting the growth of MAP. This work describes the first time that a phage was isolated against M. paratuberculosis.

Cronobacter sakazakii, an opportunistic pathogen found in milk-based powdered infant formulae, has been linked to meningitis in infants, with high fatality rates. A set of phages from various environments were purified and tested in vitro against strains of C. sakazakii. Based on host range and lytic activity, the T4-like phage vB&#95;CsaM&#95;GAP161, which belongs to the family Myoviridae, was selected for evaluation of its efficacy against C. sakazakii. Galleria mellonella larvae were used as a whole-animal model for pre-clinical testing of phage efficiency. Twenty-one Cronobacter strains were evaluated for lethality in G. mellonella larvae. Different strains of C. sakazakii caused 0 to 98% mortality. C. sakazakii 3253, with an LD50 dose of ~2.0×10(5) CFU/larva (24 h, 37 °C) was selected for this study. Larvae infected with a dose of 5×LD50 were treated with phage GAP161 (MOI=8) at various time intervals. The mortality rates were as high as 100% in the groups injected with bacteria only, compared to 16.6% in the group infected with bacteria and treated with phage. Phage GAP161 showed the best protective activity against C. sakazakii when the larvae were treated prior to or immediately after infection. The results obtained with heat-inactivated phage proved that the survival of the larvae is not due to host immune stimulation. These results suggest that phage GAP161 is potentially a useful control agent against C. sakazakii. In addition, G. mellonella may be a useful whole-animal model for pre-screening phages for efficacy and safety prior to clinical evaluation in mammalian models.

Wild-type T4 bacteriophage and recombinant reporter lac Z T4 bacteriophage carrying the β-galactosidase gene were used for detection of generic Escherichia coli by monitoring the release of β-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-β-D-galactopyranoside, CPRG) and bioluminescent (6-O-β-galactopyranosyl-luciferin, Beta-Glo(®)) β-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 β-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.

Lactic acid bacteria (LAB) are of interest because of their potential to modulate immune responses. The effects of LAB range from regulation to stimulation of the immune system. A series of studies were performed in vitro to study the effects of six lactic acid bacteria (LAB), Lactobacillus helveticus LH-2, Lactobacillus acidophilus La-5, La-115, La-116 and La-14, and Lactobacillus salivarius, on maturation and activation of mouse dendritic cells. Production of tumour necrosis factor (TNF)-?, interleukin (IL)-6 and IL-10 by dendritic cells (DCs) was determined after treating cells with live LAB. The expression of DC maturation markers, CD80 and CD40, was also measured using flow cytometry after stimulation with LAB. In addition, the expression of Toll-like receptors (TLRs) 2, 4 and 9 by DCs stimulated with LAB was measured. Our results revealed that LAB act differentially on pro-inflammatory and anti-inflammatory cytokine production and induction of co-stimulatory molecules by DCs. Specifically, L. salivarius was found to be the most effective LAB to induce pro-inflammatory cytokine production and expression of co-stimulatory molecules. Moreover, La-14, La-116 and La-5 induced moderate maturation and activation of DCs. On the other hand, LH-2 and La-115 were the least effective lactobacilli to induce DC responses. The present study also revealed that L. salivarius was able to induce the expression of TLR2, 4 and 9 by DCs. In conclusion, various strains and species of LAB can differentially regulate DC activation and maturation, providing further evidence that these bacteria may have the ability to influence and steer immune responses in vivo.

Cronobacter sakazakii is a Gram-negative pathogen found in milk-based formulae that causes infant meningitis. Bacteriophages have been proposed to control bacterial pathogens; however, comprehensive knowledge about a phage is required to ensure its safety before clinical application. We have characterized C. sakazakii phage vB&#95;CsaM&#95;GAP32 (GAP32), which possesses the second largest sequenced phage genome (358,663bp). A total of 571 genes including 545 protein coding sequences and 26 tRNAs were identified, thus more genes than in the smallest bacterium, Mycoplasma genitalium G37. BLASTP and HHpred searches, together with proteomic analyses reveal that only 23.9% of the putative proteins have defined functions. Some of the unique features of this phage include: a chromosome condensation protein, two copies of the large subunit terminase, a predicted signal-arrest-release lysin; and an RpoD-like protein, which is possibly involved in the switch from immediate early to delayed early transcription. Its closest relatives are all extremely large myoviruses, namely coliphage PBECO4 and Klebsiella phage vB&#95;KleM-RaK2, with whom it shares approximately 44% homologous proteins. Since the homologs are not evenly distributed, we propose that these three phages belong to a new subfamily., (Copyright © 2014. Published by Elsevier Inc.)

Phage-based detection assays have been developed for the detection of viable bacteria for applications in clinical diagnosis, monitoring of water quality, and food safety. The majority of these assays deliver a positive readout in the form of newly generated progeny phages by the bacterial host of interest. Progeny phages are often visualized as plaques, or holes, in a lawn of bacteria on an agar-filled Petri dish; however, this rate-limiting step requires up to 12 h of incubation time. We have previously described an amplification of bacteriophages M13 inside droplets of media suspended in perfluorinated oil; a single phage M13 in a droplet yields 10(7) copies in 3-4 h. Here, we describe that encapsulation of reporter phages, both lytic T4-LacZ and nonlytic M13, in monodisperse droplets can also be used for rapid enumeration of phage. Compartmentalization in droplets accelerated the development of the signal from the reporter enzyme; counting of "positive" droplets yields accurate enumeration of phage particles ranging from 10(2) to 10(6) pfu/mL. For enumeration of T4-LacZ phage, the fluorescent signal appeared in as little as 90 min. Unlike bulk assays, quantification in emulsion is robust and insensitive to fluctuations in environmental conditions (e.g., temperature). Power-free emulsification using gravity-driven flow in the absence of syringe pumps and portable fluorescence imaging solutions makes this technology promising for use at the point of care in low-resource environments. This droplet-based phage enumeration method could accelerate and simplify point-of-care detection of the pathogens for which reporter bacteriophages have been developed.

F-specific (F+) RNA phages are recommended as indicators of fecal contamination and the presence of enteric viruses and as viral surrogates to elucidate the resistance of viruses to adverse conditions or to assess the effectiveness of inactivating processes. Reverse transcription (RT)-PCR methods have been used to detect, quantify, or identify subgroups of F+ RNA phages. However, these methods may overestimate the infectivity of F+ RNA phages in test samples, since the presence of both infectious and inactivated phages (or naked RNA) can lead to positive RT-PCR signals. In this study, we evaluated the ability of an enzyme treatment (ET) with proteinase K and RNase A prior to RNA extraction, followed by RT-PCR, to differentiate infectious and inactivated F+ RNA phages. The results indicated that ET RT-PCR reduced, but did not completely eliminate, false-positive signals encountered with RT-PCR alone. The two-step ET RT-PCR, in which the enzymes were added sequentially, was more effective at reducing false-positive signals than the one-step ET RT-PCR, which involved addition of both enzymes together. Despite its inability to completely eliminate false-positive signals, ET RT-PCR gave more reliable information on the infectivity of F+ RNA phages. Thus, the method is better than RT-PCR alone for detecting F+ RNA phages as indicators to assess the risk of fecal contamination by enteric pathogens or to evaluate the effectiveness of virus-inactivating processes.

Non-thermal processing methods, such as pulsed electric field (PEF) and tangential-flow microfiltration (TFMF), are emerging processing technologies that can minimize the deleterious effects of high temperature short time (HTST) pasteurization on quality attributes of skim milk. The present study investigates the impact of PEF and TFMF, alone or in combination, on color and volatile compounds in skim milk. PEF was applied at 28 or 40 kV/cm for 1122 to 2805 µs, while microfiltration (MF) was conducted using membranes with three pore sizes (lab-scale 0.65 and 1.2 µm TFMF, and pilot-scale 1.4 µm MF). HTST control treatments were applied at 75 or 95 °C for 20 and 45 s, respectively. Noticeable color changes were observed with the 0.65 µm TFMF treatment. No significant color changes were observed in PEF-treated, 1.2 µm TFMF-treated, HTST-treated, and 1.4 µm MF-treated skim milk ( p ≥ 0.05) but the total color difference indicated better color retention with non-thermal preservation. The latter did not affect raw skim milk volatiles significantly after single or combined processing ( p ≥ 0.05), but HTST caused considerable changes in their composition, including ketones, free fatty acids, hydrocarbons, and sulfur compounds ( p < 0.05). The findings indicate that for the particular thermal and non-thermal treatments selected for this study, better retention of skim milk color and flavor components were obtained for the non-thermal treatments.

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to be of major importance in human health. Therefore, these essential polyunsaturated fatty acids have received considerable attention in both human and farm animal nutrition. Currently, fish and fish oils are the main dietary sources of EPA/DHA. To generate sustainable novel sources for EPA and DHA, the 35-kb EPA/DHA synthesis gene cluster was isolated from a marine bacterium, Shewanella baltica MAC1. To streamline the introduction of the genes into food-grade microorganisms such as lactic acid bacteria, unnecessary genes located upstream and downstream of the EPA/DHA gene cluster were deleted. Recombinant Escherichia coli harboring the 20-kb gene cluster produced 3.5- to 6.1-fold more EPA than those carrying the 35-kb DNA fragment coding for EPA/DHA synthesis. The 20-kb EPA/DHA gene cluster was cloned into a modified broad-host-range low copy number vector, pIL252m (4.7 kb, Ery) and expressed in Lactococcus lactis subsp. cremoris MG1363. Recombinant L. lactis produced DHA (1.35 ± 0.5 mg g(-1) cell dry weight) and EPA (0.12 ± 0.04 mg g(-1) cell dry weight). This is believed to be the first successful cloning and expression of EPA/DHA synthesis gene cluster in lactic acid bacteria. Our findings advance the future use of EPA/DHA-producing lactic acid bacteria in such applications as dairy starters, silage adjuncts, and animal feed supplements.

Unlabelled: F + RNA phages are commonly used as indicators of faecal contamination. This study evaluated a fluorescent method for the detection of F + RNA phages based on testing the phage-mediated release of β-galactosidase. Factors that may potentially interfere with phage detection were investigated, and the assay was optimized. Low numbers of F + RNA phages were detected by the fluorescent method coupled with a concentration step using a Disruptor filter. The fluorescent method, when used alone, detected 1 log PFU ml(-1) of F+RNA phages within 3 h, while 0.01 PFU ml(-1) was detected within 5 h when the method was combined with the concentration step. This is the first time to combine a fluorescent method with a filtration step by the use of Disruptor filter for rapid detection of low numbers of F + RNA phages, and this method can be adapted to detect other lytic phages infecting host cells that produce measurable enzyme activity., Significance and Impact of the Study: A fluorescent method coupled with Disruptor filtration was evaluated for the first time to rapidly detect low numbers of F + RNA phages. Rapid detection of F + RNA phages provides an effective way to monitor faecal contamination of environmental water and thus helps prevent contamination of fresh produce via irrigation., (© 2013 The Society for Applied Microbiology.)

Clostridium difficile is a spore-forming, toxin-producing, anaerobic bacterium that colonizes the human gastrointestinal tract. This pathogen causes antibiotic-associated diarrhea and colitis in animals and humans. Antibiotic-associated diseases may be treated with probiotics, and interest is increasing in such uses of probiotics. This study investigated the effect of Lactobacillus strains on the quorum-sensing signals and toxin production of C. difficile. In addition, an in vivo experiment was designed to assess whether Lactobacillus acidophilus GP1B is able to control C. difficile-associated disease. Autoinducer-2 activity was measured for C. difficile using the Vibrio harveyi coupled bioluminescent assay. Cell extract (10μg/mL) of L. acidophilus GP1B exhibited the highest inhibitory activity among 5 to 40μg/mL cell-extract concentrations. Real-time PCR data indicated decreased transcriptional levels in luxS, tcdA, tcdB, and txeR genes in the presence of 10μg/mL of cell extract of L. acidophilus GP1B. Survival rates at 5d for mice given the pathogen alone with L. acidophilus GP1B cell extract or L. acidophilus GP1B were 10, 70, and 80%, respectively. In addition, the lactic acid-produced L. acidophilus GP1B exhibits an inhibitory effect against the growth of C. difficile. Both the L. acidophilus GP1B and GP1B cell extract have significant antipathogenic effects on C. difficile., (Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Campylobacter spp. are among the most commonly reported bacterial causes of acute diarrheal disease in humans worldwide. Potential virulence factors include motility, chemotaxis, colonization ability, adhesion to intestinal cells, invasion and epithelial translocation, intracellular survival, and formation of toxins. Probiotic Lactobacillus and Bifidobacterium strains are known to have an inhibitory effect against the growth of various foodborne pathogens. The objective of this study was to investigate the effect of Lactobacillus acidophilus strain La-5 and Bifidobacterium longum strain NCC2705 cell-free spent media (CFSM) on the expression of virulence genes (cadF, cdtB, flaA, and ciaB) of Campylobacter jejuni strain 81-176 and a luxS mutant, using real-time PCR. Our results demonstrated that the CFSM of both probiotic strains were able to down-regulate the expression of ciaB (ratio of -2.80 and -5.51, respectively) and flaA (ratio of -7.00 and -5.13, respectively) in the wild-type Campylobacter strain. In the luxS mutant, where the activated methyl cycle is disrupted, only the ciaB gene (ratio -7.21) was repressed in the presence of La-5 CFSM. A supplementation of homocysteine to restore the disrupted cycle was able to partially reestablish the probiotic effect of both strains. luxS and the activated methyl cycle might play an active role in the modulation of virulence of C. jejuni by probiotic extracts.

F-specific (F+) RNA phages are widely used as indicators for the presence of fecal contamination and/or enteric viruses in water, and identifying subgroups of F+ RNA phages provides an approach for microbial source tracking. Different survival characteristics of the F+ RNA phage subgroups result in a misinterpretation of their original proportion in water, thus giving misleading information when they are used for microbial source tracking. This study investigated the comparative persistence of subgroups of F+ RNA phages in river water under different conditions. Results suggested that temperature and pH are the major factors affecting the persistence of F+ RNA phages in river water, and organic substances promote phage survival. The comparative persistence patterns of subgroups of F+ RNA phages varied and may bias extrapolation of their initial proportions in surface water. Thus, the characteristics of water should be taken into consideration and the results should be carefully interpreted when F+ RNA phages are used for microbial source tracking.

Lactobacillus helveticus is one of the species of lactic acid bacteria (LAB) most commonly used in the production of fermented milk beverages and some types of hard cheese. The versatile nature of this bacterium is based on its highly efficient proteolytic system consisting of cell-envelope proteinases (CEPs), transport system and intracellular peptidases. Besides use of L. helveticus in cheese processing, the production of fermented milk preparations with health promoting properties has become an important industrial application. Studies have shown that fermented dairy products are able to decrease blood pressure, stimulate the immune system, promote calcium absorption, and exert an anti-virulent effect against pathogens. These beneficial effects are produced by a variety of peptides released during the hydrolysis of milk proteins by the proteolytic system of L. helveticus, which provides the bacterium with its nutritional requirements for growth. In recent years, studies have focused on understanding the factors that affect the kinetics of milk protein hydrolysis by specific strains and have concentrated on the effect of pH, temperature, growth phase, and matrix composition on the bacterial enzymatic system. This review focuses on the role of the proteolytic system of L. helveticus in the production of bioactive compounds formed during fermentation of dairy products. Taking advantage of the powerful proteolytic system of this bacterium opens up future opportunities to search for novel food-derived compounds with potential health promoting properties.

Background: Comparatively little information is available on members of the Myoviridae infecting low G+C content, Gram-positive host bacteria of the family Firmicutes. While numerous Bacillus phages have been isolated up till now only very few Bacillus cereus phages have been characterized in detail., Results: Here we present data on the large, virulent, broad-host-range B. cereus phage vB&#95;BceM&#95;Bc431v3 (Bc431v3). Bc431v3 features a 158,618 bp dsDNA genome, encompassing 239 putative open reading frames (ORFs) and, 20 tRNA genes encoding 17 different amino acids. Since pulsed-field gel electrophoresis indicated that the genome of this phage has a mass of 155-158 kb Bc431v3 DNA appears not to contain long terminal repeats that are found in the genome of Bacillus phage SPO1., Conclusions: Bc431v3 displays significant sequence similarity, at the protein level, to B. cereus phage BCP78, Listeria phage A511 and Enterococcus phage ØEF24C and other morphologically related phages infecting Firmicutes such as Staphylococcus phage K and Lactobacillus phage LP65. Based on these data we suggest that Bc431v3 should be included as a member of the Spounavirinae; however, because of all the diverse taxonomical information has been addressed recently, it is difficult to determine the genus. The Bc431v3 phage contains some highly unusual genes such as gp143 encoding putative tRNAHis guanylyltransferase. In addition, it carries some genes that appear to be related to the host sporulation regulators. These are: gp098, which encodes a putative segregation protein related to FstK/SpoIIIE DNA transporters; gp105, a putative segregation protein; gp108, RNA polymerase sigma factor F/B; and, gp109 encoding RNA polymerase sigma factor G.

The genome of Cronobacter sakazakii podovirus vB&#95;CsaP&#95;GAP227 was fully sequenced. The DNA of this lytic phage consists of 41,796 bp and has a G+C content of 55.7%. Forty-nine open reading frames and no tRNAs were identified. This phage is related to Yersinia phages ϕR8-01 and ϕ80-18 and Aeromonas phage phiAS7.