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  • Author or Editor: Á. Belák x
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Fruits and vegetables can be transmission vehicles of human opportunistic and obligate pathogenic bacteria, persisting in inner tissues for shorter or longer periods or colonizing the plants as facultative endophytes. In this study we investigated the ability of commensal E. coli and pathogenic L. monocytogenes strains to internalize sweet pepper seedlings via seed bacterization, as germinating seeds and roots are important infiltration sites for entry of enteric bacteria. By combining cultivation dependent and independent (PCR and FISH-CLSM) techniques we could not detect stably or transiently colonized inoculated bacteria in 6–7 weeks old pepper seedlings, suggesting that there is low risk associated with internalized enteric or human pathogenic bacteria via germinating seeds in sweet pepper.

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Abstract

Previously isolated Pseudomonas lundensis CP-P-5 had antagonistic activity against Salmonella Hartford, Yersinia enterocolitica, and Escherichia coli. In this study, determination of its antagonistic mechanism and potential field of application in food industry was aimed. Using cellophane-test and microcultures of the test strain's cell-free supernatant mixed with the pathogens, our results showed that cells of P. lundensis CP-P-5 and its concentrated cell-free supernatants were effective against the foodborne bacteria, and the supernatants contained more than one compound responsible for inhibitory activity. Searching for the antagonistic compound, NaOH, protease, and heat treatments were done to the supernatants, and proteolytic activity and siderophore production were also tested using the antagonistic strain. Our results support the potential applicability of P. lundensis CP-P-5 as a bioprotective agent against foodborne pathogens in food processing environments.

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Endophytic microorganisms living inside plant tissues might have numerous positive effects on the host plants. Endophytes can promote the growth and yield of the plant, help to remove contaminants from the tissues, and can suppress growth of pathogens; however, some enteric human pathogenic bacteria have also been isolated as endophytes. The aims of our study were the characterisation and identification of endophytic coliform bacteria isolated from different cultivars of sweet pepper (Capsicum annuum var. grossum) using a selective (VRBL) agar medium, and determination of antagonistic interactions between these endophytes and Listeria monocytogenes. The bacterial isolates showed heterogeneity based on their phenotypic and genotypic properties. Results of identification by molecular biological methods also confirmed the presence of different genera/species. When the antagonistic effect of the isolated endophytic bacteria was tested it was found that one isolate — identified as Pseudomonas putida — showed significant inhibition on the growth of Listeria monocytogenes.

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Since genetic recombination is a major factor in the evolution of the cytopathogenic (cp) bovine viral diarrhoea virus (BVDV) biotypes, in this study the cytopathogenicity markers were investigated in the genomes of two cp BVDV strains recently isolated from mucosal disease (MD) cases in Hungary. In the genome of strain H4956, a Jiv-like insertion was found similar to those described in reference strain NADL and in other BVDV 1, BVDV 2 and border disease virus (BDV) strains. The 133 amino acid Jiv-like sequence is inserted at nucleotide position 4984 (amino acid position 1533), 9 nucleotides upstream of that of strain NADL. The insertion showed 96% amino acid sequence identity with the cellular Jiv protein. In the genome of cp BVDV strain H115/PCR, an ubiquitin-containing duplication was found. The duplicated sequence started at nucleotide position 7978 (amino acid 2531) in the NS4B gene. The duplication contained a complete ubiquitin monomer of 76 amino acids and the complete NS3 gene starting at nucleotide position 5153 (amino acid 1589), which corresponds to the first N-terminal amino acid of NS3. The duplication was located further downstream of the known ubiquitin-containing genomic regions of cp BVDV strains, and it consisted of the shortest inserted nucleotide sequence. The insertions and duplication of strains H4956 and H115/PCR further confirmed that recombinations occurring at positions A and B are the most common mechanisms leading to the development of BVDV cytopathogenicity.

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Psychrotrophic Pseudomonas species P. fluorescens, P. fragi and P. lundensis were found as predominant bacteria of chicken meat stored at chill temperature, which showed high level of molecular diversity, while isolates of the psychrotrophic yeasts Candida zeylanoides, Metschnikowia pulcherrima, Rhodotorula glutinis and Rhodotorula mucilaginosa formed clusters of high level similarity within the different species as revealed by RAPD-PCR analysis.Combination of multiplex PCR and sequencing of the rpoB gene resulted correct identification of the Pseudomas isolates, while the routine diagnostic tests led to improper identification in case of half of the isolates, which indicated the extended biochemical and physiological heterogeneity of the food-borne pseudomonads. Majority of P. fluorescens and P. lundensis isolates were strong protease and lipase producers, while P. fragi strains were week or negative from this respect. Proteolytic and lipolytic activities of the isolated yeast strains were species specific and protease production was less frequent than lipolytic activities.

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Bacterial strains with inhibitory effect on Salmonella Hartford, Listeria monocytogenes, Yersinia enterocolitica, and Escherichia coli, respectively, were isolated. Out of the 64 bacteria originated from food processing environments, 20 could inhibit at least one of the tested pathogens, and it was proved that growth decline of the pathogenic bacteria was more remarkable by co-culturing than by using cell-free supernatants of the isolates. Seven different genera (Pseudomonas, Bacillus, Paenibacillus, Macrococcus, Staphylococcus, Serratia, and Rothia) reduced the pathogens’ growth during the time period of analysis, and the strongest inhibitory effect was observed after 24 h between 15 and 30 °C. Sensitivity of the tested human pathogenic bacteria against the inhibitory strains was distinct, as Y. enterocolitica could be inhibited by numerous isolates, while S. Hartford proved to be the most resistant. Our results reveal that the isolated bacteria or their excreted metabolites could hinder pathogen growth when used in sufficient quantities.

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A real-time RT-PCR assay utilising light upon extension fluorogenic primer (LUX RT-PCR) was developed for the rapid and efficient detection of avian influenza viruses (AIV). The assay detected each of the AIV isolates tested (16/16) and gave negative results with heterologous pathogens (17/17). The detection limit of the assay proved to be 10-0.5 EID50/0.2 ml and 101.5 EID50/0.2 ml in allantoic fluid of virus-infected embryonated chicken eggs and in spiked chicken faeces samples, respectively. Based on its specificity, sensitivity and relative simplicity, the LUX RT-PCR assay provides a novel, rapid and cost-effective diagnostic tool for avian influenza surveillance and monitoring programs.

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