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  • Azinheiro, Sarah; Kant, Krishna; Shahbazi, Mohammad-Ali; Garrido-Maestu, Alejandro; Prado, Marta; Dieguez, Lorena (2020)
    Rapid and sensitive detection of foodborne pathogens in food industry is of high importance in day-to-day practice to ensure safe food. To address this issue, multiple foodborne pathogens are targeted for rapid identification based in DNA amplification. A 3D PDMS sponge was fabricated using salt crystals as scarifying mold and functionalized with a ligand, apolipoprotein-H (ApoH), to test bacterial capturing for both Gram positive (L. monocytogenes) and negative bacteria (Salmonella spp.), in a microfluidic device. Pure culture of both pathogens in a range of ∼10–105 CFU/mL were tested and the application of the developed automated pre-concentration protocol in real samples was verified using spiked surface samples after swab sampling. Bacterial DNA was extracted directly from the sponge and used for Real Time quantitative Polymerase Chain Reaction (qPCR) detection. The sponges did not show any significant resistance to sample flow and could easily be incorporated in a microfluidic device. A capture efficiency above 70% was observed for both targeted (Gram positive and Gram negative) pathogens and a Limit of Detection (LoD) in the range of 103 and 104 CFU/mL was obtained for Salmonella spp. and L. monocytogenes, respectively. Using this approached, we are able to perform multiplexed (Gram positive and Gram negative) capturing and reduce the enrichment time compared to the gold standard plate culture (over 1-day) method. The use of a 3D sponge for direct capturing of multiplexed pathogen on microfluidic device, followed by qPCR detection is an efficient and versatile method to stratify the presence of bacteria. This approach and methodology has potential to be integrated in full automatized device and used as point of need (PoN) system for foodborne pathogen stratification in food packaging/production industries.
  • Zhang, Ji; Ketola, Tarmo; Örmälä, Anni-Maria; Mappes, Johanna; Laakso, Jouni (2014)
  • Kapp, Karmen; Orav, Anne; Roasto, Mati; Raal, Ain; Püssa, Tõnu; Vuorela, Heikki; Tammela, Päivi; Vuorela, Pia (2020)
    Mint flavorings are widely used in confections, beverages, and dairy products. For the first time, mint flavoring composition of mint candies and food supplements (n=45), originating from 16 countries, as well as their antibacterial properties, was analyzed. The flavorings were isolated by Marcussons type micro-apparatus and analyzed by GC-MS. The total content of the mint flavoring hydrodistilled extracts was in the range of 0.01-0.9%. The most abundant compounds identified in the extracts were limonene, 1,8-cineole, menthone, menthofuran, isomenthone, menthol and its isomers, menthyl acetate. The antimicrobial activity of 13 reference substances and 10 selected mint flavoring hydrodistilled extracts was tested on Escherichia coli and Staphylococcus aureus by broth dilution method. Linalool acetate and (-)-carvone, as most active against both bacteria, had the lowest MIC (90) values. (+)-Menthyl acetate, (-)-menthyl acetate, and limonene showed no antimicrobial activity. Three of the tested extracts had antimicrobial activity against E. coli and 8 extracts against S. aureus . Their summary antimicrobial activity was not always in concordance with the activities of respective reference substances.
  • Ran, Li; Wan, Xing; Takala, Timo; Saris, Per (2021)
    The yeastSaccharomyces boulardiiis well known for its probiotic effects such as treating or preventing gastrointestinal diseases. Due to its ability to survive in stomach and intestine,S. boulardiicould be applied as a vehicle for producing and delivering bioactive substances of interest to human gut. In this study, we cloned the genelecCencoding the antilisterial peptide leucocin C from lactic acid bacteriumLeuconostoc carnosuminS. boulardii. The constructedS. boulardiistrain secreted a peptide, which had molecular weight corresponding to leucocin C in SDS-PAGE. The peptide band inhibitedListeria monocytogenesin gel overlay assay. Likewise, concentratedS. boulardiiculture supernatant inhibited the growth ofL. monocytogenes. The growth profile and acid tolerance of the leucocin C secretingS. boulardiiwere similar as those of the strain carrying the empty vector. We further demonstrated that the cells of the leucocin C producingS. boulardiiefficiently killedL. monocytogenes, also without antibiotic selection pressure. These results showed that antilisterial activity could be added to the arsenal of probiotic activities ofS. boulardii, demonstrating its potential as a carrier for therapeutics delivery.
  • Fernandez, Maria; Hudson, John Andrew; Korpela, Riitta; de los Reyes-Gavilsn, Clara G. (2015)
  • Nowak, J.; Visnovsky, S. B.; Cruz, C. D.; Fletcher, G. C.; van Vliet, A. H. M.; Hedderley, D.; Butler, R.; Flint, S.; Palmer, J.; Pitman, A. R. (2021)
    Aims To understand the genetics involved in surface attachment and biofilm formation ofListeria monocytogenes. Methods and Results Anin vitroscreen of a Himar1 transposon library ofL. monocytogenesstrain 15G01 identified three transposants that produced significantly different biofilm levels when compared to the wild-type strain; two mutants exhibited enhanced biofilm formation and one produced less biofilm biomass than the wild-type. The mutant 15G01mprF::Himar1, which had a transposon insertion in themprFgene, was selected for further analysis. The mutant produced a more densely populated biofilm on solid surfaces such as stainless steel and polystyrene, as determined using scanning electron and light microscopy. The 15G01mprF::Himar1 mutant remained viable in biofilms, but showed an increase in sensitivity to the cationic antimicrobial gallidermin. The mutant also displayed reduced invasiveness in CaCo-2 intestinal cells, suggesting virulence properties are compromised by the inactivation ofmprF. Conclusions Biofilm formation and gallidermin resistance ofL. monocytogenesis influenced bymprF, but this trait is associated with a compromise in invasiveness. Significance and Impact of the Study The presence of pathogenic microorganisms in the food processing environment can cause a significant problem, especially when these microorganisms are established as biofilms. This study shows that the inactivation of themprFgene results in enhanced biofilm formation and abiotic surface attachment ofL. monocytogenes.
  • Lehto, Marja; Kuisma, Risto Martti Johannes; Kymäläinen, Hanna-Riitta; Mäki, Maarit (2018)
    The effect of decontamination methods on fresh-cut vegetable washing waters was evaluated. NEW, ClO2, organic acid-based product FPW, and UV-C were tested with and without an interfering carrot juice of 1% (IS), on Yersinia enterocolitica and Yersinia pseudotuberculosis, Escherichia coli, and yeast Candida lambica. The use of ClO2 (50 ppm active chlorine) resulted in >4 log reduction of Y. enterocolitica, Y. pseudotuberculosis, E. coli and >3 log reduction of C. lambica. The antibacterial effect of NEW was less effective in the presence of IS when compared with ClO2. The inactivation of C. lambica by FPW reached a maximum of 2.8 log cfu/mL (concentration 0.125%), but the antimicrobial effect was delayed by the IS. The effect of FPW on E. coli was significantly reduced by 1% IS. The inactivation of E. coli and C. lambica with UV-C IS decreased the inactivation and lengthened its time. Filtration improved the effect of UV-C inactivation. Practical applicationsWhen chemical decontamination methods were used in fresh-cut vegetable processing, the presence of organic matter in process water increased the reaction times and the need for higher concentrations of the chemical decontamination and the time of physical decontamination. Yersinia required longer inactivation times than E. coli. When UV-C is used for decontamination of process waters, waters should be filtered to enhance the disinfection efficacy.
  • Laaksonen, Sauli; Oksanen, Antti; Julmi, Jerome; Zweifel, Claudio; Fredriksson-Ahomaa, Maria; Stephan, Roger (2017)
    Background: Various food-producing animals were recognized in recent years as healthy carriers of bacterial pathogens causing human illness. In northern Fennoscandia, the husbandry of semi-domesticated reindeer (Rangifer tarandus tarandus) is a traditional livelihood and meat is the main product. This study determined the presence of selected foodborne pathogens, methicillin-resistant Staphylococcus aureus (MRSA), and extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in healthy semi-domesticated reindeer at slaughter in northern Finland and Norway. Results: All 470 reindeer fecal samples tested negative for Salmonella spp., whereas L. monocytogenes was detected in 3%, Yersinia spp. in 10%, and Shiga toxins genes (stx1 and/or stx2) in 33% of the samples. Listeria monocytogenes isolates belonged to the serotype 1/2a (14/15) and 4b, Yersinia spp. were identified mainly as Y. kristensenii (30/46) and Y. enterocolitica (8/46), and stx2 predominated among the Shiga toxin genes (stx2 alone or in combination with stx1 was found in 25% of the samples). With regard to the frequency and distribution of stx1/stx2, striking differences were evident among the 10 different areas of origin. Hence, reindeer could constitute a reservoir for Shiga toxin-producing E. coli (STEC), but strain isolation and characterization is required for verification purposes and to assess the potential human pathogenicity of strains. On the other hand, the favorable antibiotic resistance profiles (only 5% of 95 E. coli isolates were resistant to one or more of the tested antibiotics) and the absence of MRSA and ESBL-producing Enterobacteriaceae (when applying selective methods) suggest only a limited risk of transmission to humans. Conclusions: Healthy semi-domesticated reindeer in northern Finland and Norway can be carriers of certain bacterial foodborne pathogens. Strict compliance with good hygiene practices during any step of slaughter (in particular during dehiding and evisceration) is therefore of central importance to avoid carcass contamination and to prevent foodborne pathogens from entering the food chain.
  • Zarei, Mehdi; Rahimi, Saeid; Saris, Per Erik Joakim; Yousefvand, Amin (2022)
    In order to develop strategies for preventing biofilm formation in the dairy industry, a deeper understanding of the interaction between different species during biofilm formation is necessary. Bacterial strains of the P. fluorescens group are known as the most important biofilm-formers on the surface of dairy processing equipment that may attract and/or shelter other spoilage or pathogenic bacteria. The present study used different strains of the P. fluorescens group as background microbiota of milk, and evaluated their interaction with Staphylococcus aureus, Bacillus cereus, Escherichia coli O157:H7, and Salmonella Typhimurium during dual-species biofilm formation on stainless steel surfaces. Two separate scenarios for dual-species biofilms were considered: concurrent inoculation of Pseudomonas and pathogen (CI), and delayed inoculation of pathogen to the pre-formed Pseudomonas biofilm (DI). The gram-positive pathogens used in this study did not form dual-species biofilms with P. fluorescens strains unless they were simultaneously inoculated with Pseudomonas strains. E. coli O157:H7 was able to form dual-species biofilms with all seven P. fluorescens group strains, both in concurrent (CI) and delayed (DI) inoculation. However, the percentage of contribution varied depending on the P. fluorescens strains and the inoculation scenario. S. Typhimurium contributed to biofilm formation with all seven P. fluorescens group strains under the CI scenario, with varying degrees of contribution. However, under the DI scenario, S. Typhimurium did not contribute to the biofilm formed by three of the seven P. fluorescens group strains. Overall, these are the first results to illustrate that the strains within the P. fluorescens group have significant differences in the formation of mono-or dual-species biofilms with pathogenic bacteria. Furthermore, the possibility of forming dual-species biofilms with pathogens depends on whether the pathogens form the biofilm simultaneously with the P. fluorescens group strains or whether these strains have already formed a biofilm.
  • Dahlsten, Elias; Isokallio, Marita; Somervuo, Panu; Lindström, Miia; Korkeala, Hannu (2014)
  • Moreno-Cinos, Carlos; Sassetti, Elisa; Salado, Irene G.; Witt, Gesa; Benramdane, Siham; Reinhardt, Laura; Cruz, Cristina D.; Joossens, Jurgen; Van der Veken, Pieter; Brötz-Oesterhelt, Heike; Tammela, Päivi Sirpa Marjaana; Winterhalter, Mathias; Gribbon, Philip; Windshügel, Björn; Augustyns, Koen (2019)
    Increased Gram-negative bacteria resistance to antibiotics is becoming a global problem, and new classes of antibiotics with novel mechanisms of action are required. The caseinolytic protease subunit P (ClpP) is a serine protease conserved among bacteria that is considered as an interesting drug target. ClpP function is involved in protein turnover and homeostasis, stress response, and virulence among other processes. The focus of this study was to identify new inhibitors of Escherichia coli ClpP and to understand their mode of action. A focused library of serine protease inhibitors based on diaryl phosphonate warheads was tested for ClpP inhibition, and a chemical exploration around the hit compounds was conducted. Altogether, 14 new potent inhibitors of E. coli ClpP were identified. Compounds 85 and 92 emerged as most interesting compounds from this study due to their potency and, respectively, to its moderate but consistent antibacterial properties as well as the favorable cytotoxicity profile.