Journal articles: 'Pathogen enrichment'

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MURAKAMI, TAKU. "Filter-Based Pathogen Enrichment Technology for Detection of Multiple Viable Foodborne Pathogens in 1 Day." Journal of Food Protection 75, no. 9 (September 1, 2012): 1603–10. http://dx.doi.org/10.4315/0362-028x.jfp-12-039.

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Conventional foodborne pathogen assays currently used in the food industry often require long culture enrichments to increase pathogen levels so they can be detected. Even using sensitive real-time PCR assays, culture enrichment at least overnight is necessary especially for detection of pathogens with slow growth rates such as Listeria monocytogenes. To eliminate this cumbersome enrichment step and detect minute amounts of pathogens within 1 day, filter-based pathogen enrichment technology was developed utilizing a unique combination of glass fiber depth filter and porous filter aid materials to efficiently separate pathogens from food homogenates and avoid filter clogging by food particles. After pathogen immobilization in depth filters, only viable pathogens were selectively collected in a small volume of growth medium via microbial multiplication and migration; nonviable pathogens remained inside the filters. By assaying viable pathogens using real-time PCRs, multiple species of foodborne pathogens were detected, including L. monocytogenes, Salmonella enterica, and Escherichia coli O157:H7, at around 1 CFU/ml or 1 CFU/g in various food samples. This filter-based pathogen enrichment technology is a unique bacterial enrichment alternative to the conventional culture enrichment step and can significantly shorten the time necessary to obtain assay results.

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Lee, Justin S., Ryan S. Mackie, Thomas Harrison, Basir Shariat, Trey Kind, Timo Kehl, Martin Löchelt, Christina Boucher, and Sue VandeWoude. "Targeted Enrichment for Pathogen Detection and Characterization in Three Felid Species." Journal of Clinical Microbiology 55, no. 6 (March 22, 2017): 1658–70. http://dx.doi.org/10.1128/jcm.01463-16.

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ABSTRACT Traditional diagnostic assays often lack sensitivity and can be difficult to multiplex across many pathogens. Next-generation sequencing (NGS) can overcome some of these problems but has limited application in the detection of low-copy-number pathogens in complex samples. Targeted genome capture (TGC) utilizes oligonucleotide probes to enrich specific nucleic acids in heterogeneous extracts and can therefore increase the proportion of NGS reads for low-abundance targets. While earlier studies have demonstrated the utility of this technology for detection of novel pathogens in human clinical samples, the capacity and practicality of TGC-NGS in a veterinary diagnostic setting have not yet been evaluated. Here we report the use of TGC-NGS assays for the detection and characterization of diverse feline pathogen taxa. We detected 31 pathogens comprising nine pathogen taxa in 28 felid samples analyzed. This included 20 pathogens detected via traditional PCR and 11 additional pathogens that had not been previously detected in the same samples. Most of the pathogens detected were sequenced at sufficient breadth and depth to confidently classify them at the species or subspecies level. Target nucleic acids were enriched from a low of 58-fold to 56 million-fold relative to host nucleic acids. Despite the promising performance of these assays, a number of pathogens detected by conventional PCR or serology were not isolated by TGC-NGS, suggesting that further validation is required before this technology can be used in lieu of quality-controlled standard assays. We conclude that TGC-NGS offers great potential as a broad multiplex pathogen characterization assay in veterinary diagnostic and research settings.

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HUANG, SHISHI, TAY BOON HUI, HYUN-GYUN YUK, and QIANWANG ZHENG. "Development of an Effective Two-Step Enrichment Process to Enhance Bax System Detection of Healthy and Injured Salmonella Enteritidis in Liquid Whole Egg and Egg Yolk." Journal of Food Protection 83, no. 3 (February 14, 2020): 397–404. http://dx.doi.org/10.4315/0362-028x.jfp-19-280.

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ABSTRACT The BAX system for pathogen detection has been highly accurate in a variety of food products. However, false-negative results have been reported for the detection of pathogens in liquid egg products because of failed pathogen resuscitation and the existence of inhibitory components. In this study, a short-time enrichment step was used to simultaneously resuscitate the target cells to the detection level and to dilute the inhibitory components to reduce detection interference. The MP medium (BAX system) enabled faster multiplication of healthy Salmonella cells than did buffered peptone water (BPW) in tested liquid whole egg and egg yolk. However, MP failed to resuscitate heat-injured cells even after 24 h of incubation. Therefore, MP was replaced with BPW as the enrichment broth for the BAX system. However, the use of BPW for a one-step enrichment was not effective for removal of PCR inhibitors in egg yolk, and unstable detection results were obtained. To improve detection accuracy, a second step of enrichment with brain heart infusion was added. This two-step enrichment process shortened the enrichment time to 14 h and greatly increased the number of samples in which the pathogen was detected during the same enrichment time, especially in the liquid egg yolk samples. The validation study revealed 100% diagnostic accuracy of the two-step enrichment process plus the BAX system. These results indicate that a two-step enrichment process added to the BAX system can improve the detection of pathogenic Salmonella in liquid egg products. HIGHLIGHTS

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Kim, Hyochin, and Arun K. Bhunia. "SEL, a Selective Enrichment Broth for Simultaneous Growth of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes." Applied and Environmental Microbiology 74, no. 15 (June 6, 2008): 4853–66. http://dx.doi.org/10.1128/aem.02756-07.

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ABSTRACT Multipathogen detection on a single-assay platform not only reduces the cost for testing but also provides data on the presence of pathogens in a single experiment. To achieve this detection, a multipathogen selective enrichment medium is essential to allow the concurrent growth of pathogens. SEL broth was formulated to allow the simultaneous growth of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes. The results were compared to those obtained with the respective individual selective enrichment broths, Rappaport-Vassiliadis (RV) for S. enterica, modified E. coli broth with 20 mg of novobiocin/liter for E. coli O157:H7, and Fraser broth for L. monocytogenes, and a currently used universal preenrichment broth (UPB). The growth of each pathogen in SEL inoculated at 101 or 103 CFU/ml was superior to that in the respective individual enrichment broth, except in the case of RV, in which Salmonella cells inoculated at both concentrations grew equally well. In mixed-culture experiments with cells of the three species present in equal concentrations or at a 1:10:1,000 ratio, the overall growth was proportional to the initial inoculation levels; however, the growth of L. monocytogenes was markedly suppressed when cells of this species were present at lower concentrations than those of the other two species. Further, SEL was able to resuscitate acid- and cold-stressed cells, and recovery was comparable to that in nonselective tryptic soy broth containing 6% yeast extract but superior to that in the respective individual selective broths. SEL promoted the growth of all three pathogens in a mixture in ready-to-eat salami and in turkey meat samples. Moreover, each pathogen was readily detected by a pathogen-specific immunochromatographic lateral-flow or multiplex PCR assay. Even though the growth of each pathogen in SEL was comparable to that in UPB, SEL inhibited greater numbers of nontarget organisms than did UPB. In summary, SEL was demonstrated to be a promising new multiplex selective enrichment broth for the detection of the three most prominent food-borne pathogens by antibody- or nucleic acid-based methods.

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Furtwängler, Anja, Judith Neukamm, Lisa Böhme, Ella Reiter, Melanie Vollstedt, Natasha Arora, Pushpendra Singh, et al. "Comparison of target enrichment strategies for ancient pathogen DNA." BioTechniques 69, no. 6 (December 2020): 455–59. http://dx.doi.org/10.2144/btn-2020-0100.

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In ancient DNA research, the degraded nature of the samples generally results in poor yields of highly fragmented DNA; targeted DNA enrichment is thus required to maximize research outcomes. The three commonly used methods – array-based hybridization capture and in-solution capture using either RNA or DNA baits – have different characteristics that may influence the capture efficiency, specificity and reproducibility. Here we compare their performance in enriching pathogen DNA of Mycobacterium leprae and Treponema pallidum from 11 ancient and 19 modern samples. We find that in-solution approaches are the most effective method in ancient and modern samples of both pathogens and that RNA baits usually perform better than DNA baits.

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ZHAO, TONG, and MICHAEL P. DOYLE. "Evaluation of Universal Preenrichment Broth for Growth of Heat-Injured Pathogens." Journal of Food Protection 64, no. 11 (November 1, 2001): 1751–55. http://dx.doi.org/10.4315/0362-028x-64.11.1751.

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Universal preenrichment broth (UPB) was developed to enable enrichment of injured foodborne pathogens of different genera simultaneously in lieu of having to undergo separate simultaneous enrichment cultures for subsequent detection or isolation of each pathogen. Enrichment conditions in UPB for growth of injured pathogens to populations that will enable pathogen detection by rapid immuno-based or polymerase chain reaction (PCR)-based assays have not been defined. Hence, studies were done to determine recovery and growth rates of heat-injured Escherichia coli O157:H7, Salmonella enterica ser. Typhimurium, Salmonella enterica ser. Enteritidis, and Listeria monocytogenes in UPB. Bacterial cells were heat injured in tryptic phosphate broth at 57.2°C and inoculated at populations of ca. 0.17 to 63 injured cells per ml with raw ground beef, fresh chicken, lettuce, and environmental sponge samples. Enrichment cultures were sampled at 1, 2, 3, 4, 5, 6, and 24 h at 37°C postinoculation, and pathogens were enumerated on appropriate selective media. Results revealed that recovery and growth of pathogens during the first 6 h of enrichment were not sufficient to ensure adequate numbers of bacteria (>103 CFU/ml) for detection by most immunoassays or PCR assays. Cells often required 3 to 4 h for recovery before growth was initiated. Salmonella Typhimurium, Salmonella Enteritidis, E. coli O157:H7, or L. monocytogenes cell populations in enrichment cultures with ground beef or lettuce at 6 h were 0.5 to 2.9 log10 CFU/ml. At 24 h of incubation, cell counts of enrichment samples for the three pathogens from all food and environmental sponge samples ranged from 4.0 to 8.3 log10 CFU/ml. Enrichment in UPB at 37°C of foods or environmental sponge samples containing heat-injured cells of Salmonella Typhimurium, Salmonella Enteritidis, E. coli O157:H7, or L. monocytogenes reliably provides at 24 h of incubation—but not at 6 h—sufficient cell populations for detection by rapid immunoassay or PCR assay procedures that can detect at least 4 log10 CFU/ml. These results raise questions regarding the sensitivity of rapid detection methods that employ an abbreviated enrichment protocol of 6 h or less.

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Parichehr, Moezi, Kargar Mohammad, Doosti Abbas, and Khoshneviszadeh Mehdi. "Developing a multiplex real-time PCR with a new pre-enrichment to simultaneously detect four foodborne bacteria in milk." Future Microbiology 14, no. 10 (July 2019): 885–98. http://dx.doi.org/10.2217/fmb-2019-0044.

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Aim: The aim of this study is to formulate a new single nonselective pre-enrichment medium (ELSS) that can support the concurrent growth of four major foodborne pathogens containing E. coli O157: H7, L. monocytogenes, S. aureus and S. enterica serovar Entertidis to develop a multiplex TaqMan Real-time PCR (mRT-PCR). Methods: The mRT-PCR with a new pre-enrichment was carried out for simultaneous detection and quantification of these foodborne bacteria. Results: By using mRT-PCR after 16 h pre-enrichment in ELSS, the detection limit of each pathogen was 1 CFU/25 ml contaminated milk, as well as inclusivity and exclusivity reached 100%. Conclusion: The mRT-PCR assay with pre-enrichment step is a fast and reliable technique for detecting single or multiple pathogens in food products.

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Hayashi, Masahiro, Tatsuya Natori, Sayoko Kubota-Hayashi, Machiko Miyata, Kiyofumi Ohkusu, Keiko Kawamoto, Hisao Kurazono, Souichi Makino, and Takayuki Ezaki. "A New Protocol to Detect Multiple Foodborne Pathogens with PCR Dipstick DNA Chromatography after a Six-Hour Enrichment Culture in a Broad-Range Food Pathogen Enrichment Broth." BioMed Research International 2013 (2013): 1–10. http://dx.doi.org/10.1155/2013/295050.

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A quick foodborne pathogen screening method after six-hour enrichment culture with a broad-range food pathogen enrichment broth is described. Pathogenic factors ofSalmonella enterica,Shigellaspp., enteroinvasiveEscherichia coli, and enterohemorrhagicE. coliare amplified with a cocktail primer and rapid polymerase chain reaction (PCR), which finishes amplification in 30 min. The PCR amplicon was differentiated with a dipstick DNA chromatography assay in 5–10 min. Starting from a four- to six-hour enrichment culture, this assay was finished within 45 min. Detection sensitivity of this protocol was less than 2.5 CFU/25 g forS. entericaand 3.3 CFU/25 g for enterohemorrhagicE. coliin spiked ground meat experiments.

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TORTORELLO, M. L., K. F. REINEKE, D. S. STEWART, and R. B. RAYBOURNE. "Comparison of Methods for Determining the Presence of Escherichia coli O157:H7 in Apple Juice." Journal of Food Protection 61, no. 11 (November 1, 1998): 1425–30. http://dx.doi.org/10.4315/0362-028x-61.11.1425.

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Six methods were compared for detection of three strains of Escherichia coli O157:H7 in enrichments of inoculated apple juice. Juice was inoculated at levels varying from 0.1 to 100 CFU/ml and centrifuged after ovemight storage at 4°C, and pellets were incubated at 37°C in nonselective enrichment broth. At hourly intervals between 5 and 10 h and at 24 h, the enrichments were tested for E. coli O157:H7 by direct fluorescent antibody (DFA), antibody-direct epifluorescent filter technique (Ab-DEFT), direct selective plating on sorbitol MacConkey agar (SMA), immunomagnetic separation coupled to either selective plating (IMS-SMA) or the polymerase chain reaction (IMS-PCR), and flow cytometry (FC). The most consistent detection of 0.1 CFU/ml of the slowest growing strain of the pathogen was provided by the IMS-SMA and IMS-PCR after 8 h of enrichment. The time required for detection at the level of 0.1 CFU/ml for each assay was Ab-DEFT, 11 h; IMS-PCR, 16 h; FC, 24 h; IMS-SMA, 32 h; and SMA, 48 h. Absolute detection limits (without enrichment) were: IMS-PCR, 103 CFU/ml; Ab-DEFT and IMS-SMA, 104 CFU/ml; SMA, 105 CFU/ml; and DFA, 106 CFU/ml. Recovery of the pathogen (10 CFU/ml) in apple juice after 28 days of 4°C storage was possible by means of an 8-h enrichment and Ab-DEFT, IMS-PCR, or IMS-SMA.

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Hahm, Byoung-Kwon, Hyochin Kim, Atul K. Singh, and Arun K. Bhunia. "Pathogen enrichment device (PED) enables one-step growth, enrichment and separation of pathogen from food matrices for detection using bioanalytical platforms." Journal of Microbiological Methods 117 (October 2015): 64–73. http://dx.doi.org/10.1016/j.mimet.2015.07.016.

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Mester, Patrick, Martin Wagner, and Peter Rossmanith. "Molecular Enrichment for Qualitative Molecular Pathogen Detection in Food." Food Analytical Methods 11, no. 5 (December 4, 2017): 1251–56. http://dx.doi.org/10.1007/s12161-017-1103-z.

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Chen, Juan, Junni Tang, Arun K. Bhunia, Cheng Tang, Changting Wang, and Hui Shi. "Development of a multi-pathogen enrichment broth for simultaneous growth of five common foodborne pathogens." Journal of General and Applied Microbiology 61, no. 6 (2015): 224–31. http://dx.doi.org/10.2323/jgam.61.224.

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Masud, Numair, Amy Ellison, Edward C. Pope, and Jo Cable. "Cost of a deprived environment – increased intraspecific aggression and susceptibility to pathogen infections." Journal of Experimental Biology 223, no. 20 (September 17, 2020): jeb229450. http://dx.doi.org/10.1242/jeb.229450.

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ABSTRACTA lack of environmental enrichment can be severely detrimental to animal welfare. For terrestrial species, including humans, barren environments are associated with reduced cognitive function and increased stress responses and pathology. Despite a clear link between increased stress and reduced immune function, uncertainty remains on how enrichment might influence susceptibility to disease. For aquatic vertebrates, we are only now beginning to assess enrichment needs. Enrichment deprivation in fish has been linked to increased stress responses, agonistic behaviour, physiological changes and reduced survival. Limited data exist, however, on the impact of enrichment on disease resistance in fish, despite infectious diseases being a major challenge for global aquaculture. Here, using a model vertebrate host–parasite system, we investigated the impact of enrichment deprivation on susceptibility to disease, behaviour and physiology. Fish in barren tanks showed significantly higher infection burdens compared with those in enriched enclosures and they also displayed increased intraspecific aggression behaviour. Infections caused hosts to have significantly increased standard metabolic rates compared with uninfected conspecifics, but this did not differ between enriched and barren tanks. This study highlights the universal physiological cost of parasite infection and the biological cost (increased susceptibility to infection and increased aggression) of depriving captive animals of environmental enrichment.

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Ebeling, Anne, Alex T. Strauss, Peter B. Adler, Carlos A. Arnillas, Isabel C. Barrio, Lori A. Biederman, Elizabeth T. Borer, et al. "Nutrient enrichment increases invertebrate herbivory and pathogen damage in grasslands." Journal of Ecology 110, no. 2 (October 31, 2021): 327–39. http://dx.doi.org/10.1111/1365-2745.13801.

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Thilliez, Gaetan J. A., Miles R. Armstrong, Tze‐Yin Lim, Katie Baker, Agathe Jouet, Ben Ward, Cock Oosterhout, et al. "Pathogen enrichment sequencing (PenSeq) enables population genomic studies in oomycetes." New Phytologist 221, no. 3 (October 5, 2018): 1634–48. http://dx.doi.org/10.1111/nph.15441.

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Israeli, Ofir, Efi Makdasi, Inbar Cohen-Gihon, Anat Zvi, Shirley Lazar, Ohad Shifman, Haim Levy, David Gur, Orly Laskar, and Adi Beth-Din. "A rapid high-throughput sequencing-based approach for the identification of unknown bacterial pathogens in whole blood." Future Science OA 6, no. 6 (July 1, 2020): FSO476. http://dx.doi.org/10.2144/fsoa-2020-0013.

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High-throughput DNA sequencing (HTS) of pathogens in whole blood samples is hampered by the high host/pathogen nucleic acids ratio. We describe a novel and rapid bacterial enrichment procedure whose implementation is exemplified in simulated bacteremic human blood samples. The procedure involves depletion of the host DNA, rapid HTS and bioinformatic analyses. Following this procedure, Y. pestis, F. tularensis and B. anthracis spiked-in samples displayed an improved host/pathogen DNA ratio of 2.5–5.9 orders of magnitude, in samples with bacteria spiked-in at 103–105 CFU/ml. The procedure described in this study enables rapid and detailed metagenomic profiling of pathogens within 8–9 h, circumventing the challenges imposed by the high background present in the bacteremic blood and by the unknown nature of the sample.

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Biniaz, Yaser, Ahmad Tahmasebi, Aminallah Tahmasebi, Benedicte Albrectsen, Péter Poczai, and Alireza Afsharifar. "Transcriptome Meta-Analysis Identifies Candidate Hub Genes and Pathways of Pathogen Stress Responses in Arabidopsis thaliana." Biology 11, no. 8 (August 1, 2022): 1155. http://dx.doi.org/10.3390/biology11081155.

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Following a pathogen attack, plants defend themselves using multiple defense mechanisms to prevent infections. We used a meta-analysis and systems-biology analysis to search for general molecular plant defense responses from transcriptomic data reported from different pathogen attacks in Arabidopsis thaliana. Data from seven studies were subjected to meta-analysis, which revealed a total of 3694 differentially expressed genes (DEGs), where both healthy and infected plants were considered. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis further suggested that the DEGs were involved in several biosynthetic metabolic pathways, including those responsible for the biosynthesis of secondary metabolites and pathways central to photosynthesis and plant–pathogen interactions. Using network analysis, we highlight the importance of WRKY40, WRKY46 and STZ, and suggest that they serve as major points in protein–protein interactions. This is especially true regarding networks of composite-metabolic responses by pathogens. In summary, this research provides a new approach that illuminates how different mechanisms of transcriptome responses can be activated in plants under pathogen infection and indicates that common genes vary in their ability to regulate plant responses to the pathogens studied herein.

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Conrad, Cheyenne C., Kim Stanford, Tim A. McAllister, James Thomas, and Tim Reuter. "Competition during enrichment of pathogenicEscherichia colimay result in culture bias." FACETS 1, no. 1 (March 1, 2017): 114–26. http://dx.doi.org/10.1139/facets-2016-0007.

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Deadly outbreaks and illnesses due to Shiga toxin-producing Escherichia coli (STEC) occur worldwide; however, the cultivation methods required for adequate monitoring and traceback investigations are inefficient at best. Detection of STEC relies heavily on enrichment; yet no standard media or protocols exist. Furthermore, whether enrichment may bias detection of multiple STEC serogroups from complex samples is unknown. Thus, 14 STEC strains of serogroups O157 and the top six non-O157s (O26, O45, O103, O111, O121, and O145) were enriched in pairs for 6–78 h in broth and evaluated by quantitative polymerase chain reaction (qPCR). Here we show that a conventional 6-h enrichment protocol did not result in intra-species culture bias for the isolates tested. However, subsequent enrichments often produced biased cultures, with differences in the qPCR gene copy number ≥2 log10apparent in 12%, 38%, and 52% of competitions after 30, 54, and 78 h of consecutive enrichments, respectively. Some strains were able to prevail and (or) out-compete the opponent strain in 100% of competitions. Our results suggest that culture bias should be considered and (or) evaluated further due to the potential implications during routine pathogen screening and outbreak investigations.

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Sommermann, Loreen, Doreen Babin, Jan Helge Behr, Soumitra Paul Chowdhury, Martin Sandmann, Saskia Windisch, Günter Neumann, et al. "Long-Term Fertilization Strategy Impacts Rhizoctonia solani–Microbe Interactions in Soil and Rhizosphere and Defense Responses in Lettuce." Microorganisms 10, no. 9 (August 26, 2022): 1717. http://dx.doi.org/10.3390/microorganisms10091717.

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The long-term effects of agricultural management such as different fertilization strategies on soil microbiota and soil suppressiveness against plant pathogens are crucial. Therefore, the suppressiveness of soils differing in fertilization history was assessed using two Rhizoctonia solani isolates and their respective host plants (lettuce, sugar beet) in pot experiments. Further, the effects of fertilization history and the pathogen R. solani AG1-IB on the bulk soil, root-associated soil and rhizosphere microbiota of lettuce were analyzed based on amplicon sequencing of the 16S rRNA gene and ITS2 region. Organic fertilization history supported the spread of the soil-borne pathogens compared to long-term mineral fertilization. The fertilization strategy affected bacterial and fungal community composition in the root-associated soil and rhizosphere, respectively, but only the fungal community shifted in response to the inoculated pathogen. The potential plant-beneficial genus Talaromyces was enriched in the rhizosphere by organic fertilization and presence of the pathogen. Moreover, increased expression levels of defense-related genes in shoots of lettuce were observed in the soil with organic fertilization history, both in the absence and presence of the pathogen. This may reflect the enrichment of potential plant-beneficial microorganisms in the rhizosphere, but also pathogen infestation. However, enhanced defense responses resulted in retarded plant growth in the presence of R. solani (plant growth/defense tradeoff).

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OLIVEIRA, TEREZA C. R. M., SHAI BARBUT, and MANSEL W. GRIFFITHS. "A Robotic DNA Purification Protocol and Real-Time PCR for the Detection of Campylobacter jejuni in Foods." Journal of Food Protection 68, no. 10 (October 1, 2005): 2131–35. http://dx.doi.org/10.4315/0362-028x-68.10.2131.

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Primers designed to amplify a Campylobacter jejuni cadF gene sequence were used in an SYBR Green I real-time PCR assay as an alternative to conventional bacteriological methods for the rapid detection of C. jejuni in foods. Twenty-five grams of chicken skin (breast and thigh) was contaminated by adding approximately 1, 10, or 50 CFU of C. jejuni ATCC 35560. Twenty-five grams of pork and 25-ml aliquots of milk were also inoculated with 1 and 10 CFU of the pathogen. The samples were incubated in Bolton broth for different periods at 37 and 42°C under microaerophilic conditions. Using a commercial robotic DNA purification system, DNA was extracted and purified from 1-ml aliquots of the enrichment cultures before and after centrifugation of the 250-ml enrichment broth at 15,900 × g for 10 min at 4°C. The DNA was used as the template in a real-time PCR assay. C. jejuni was detected after 12 h of enrichment from samples inoculated with about 50 CFU/25-g sample. After centrifugation, an enrichment step of 8 h was sufficient to allow detection of pathogen in samples inoculated with 10 CFU/25 g. However, 24 h of enrichment was necessary to detect pathogen in samples inoculated with approximately 1 CFU/25 g. The real-time PCR protocol developed in this study significantly reduced the detection time of C. jejuni in foods.

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Liu, Huifang, Geun Su Noh, Yange Luan, Zhen Qiao, Bonhan Koo, Yoon Ok Jang, and Yong Shin. "A Sample Preparation Technique Using Biocompatible Composites for Biomedical Applications." Molecules 24, no. 7 (April 3, 2019): 1321. http://dx.doi.org/10.3390/molecules24071321.

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Infectious diseases, especially pathogenic infections, are a growing threat to public health worldwide. Since pathogenic bacteria usually exist in complex matrices at very low concentrations, the development of technology for rapid, convenient, and biocompatible sample enrichment is essential for sensitive diagnostics. In this study, a cucurbit[6]uril (CB) supermolecular decorated amine-functionalized diatom (DA) composite was fabricated to support efficient sample enrichment and in situ nucleic acid preparation from enriched pathogens and cells. CB was introduced to enhance the rate and effectiveness of pathogen absorption using the CB–DA composite. This novel CB–DA composite achieved a capture efficiency of approximately 90% at an Escherichia coli concentration of 106 CFU/mL within 3 min. Real-time PCR analyses of DNA samples recovered using the CB–DA enrichment system showed a four-fold increase in the early amplification signal strength, and this effective method for capturing nucleic acid might be useful for preparing samples for diagnostic systems.

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JARVIS, KAREN G., CHIUN-KANG HSU, JAMES B. PETTENGILL, JOHN IHRIE, HIREN KARATHIA, NUR A. HASAN, and CHRISTOPHER J. GRIM. "Microbiome Population Dynamics of Cold-Smoked Sockeye Salmon during Refrigerated Storage and after Culture Enrichment." Journal of Food Protection 85, no. 2 (February 1, 2021): 238–53. http://dx.doi.org/10.4315/jfp-21-228.

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ABSTRACT Cold-smoked salmon is a ready-to-eat seafood product of high commercial importance. The processing and storage steps facilitate the introduction, growth, and persistence of foodborne pathogens and spoilage bacteria. The growth of commensal bacteria during storage and once the product is opened also influence the quality and safety of cold-smoked salmon. Here we investigated the microbial community through targeted 16S rRNA gene and shotgun metagenomic sequencing as means to better understand the interactions among bacteria in cold-smoked salmon. Cold-smoked salmon samples were tested over 30 days of aerobic storage at 4°C and cultured at each time point in a buffered Listeria enrichment broth (BLEB) commonly used to detect Listeria in foods. The microbiomes were composed of Firmicutes and Proteobacteria, namely, Carnobacterium, Brochothrix, Pseudomonas, Serratia, and Psychrobacter. Pseudomonas species were the most diverse species, with 181 taxa identified. In addition, we identified potential homologs to 10 classes of bacteriocins in microbiomes of cold-smoked salmon stored at 4°C and corresponding BLEB culture enrichments. The findings presented here contribute to our understanding of microbiome population dynamics in cold-smoked salmon, including changes in bacterial taxa during aerobic cold storage and after culture enrichment. This may facilitate improvements to pathogen detection and quality preservation of this food. HIGHLIGHTS

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Berber, Engin, Nurettin Çanakoğlu, İbrahim Sözdutmaz, Emrah Simsek, Neslihan Sursal, Gencay Ekinci, Serkan Kökkaya, et al. "Seasonal and Age-Associated Pathogen Distribution in Newborn Calves with Diarrhea Admitted to ICU." Veterinary Sciences 8, no. 7 (July 9, 2021): 128. http://dx.doi.org/10.3390/vetsci8070128.

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Calf mortality constitutes a substantial loss for agriculture economy-based countries and is also a significant herd problem in developed countries. However, the occurrence and frequency of responsible gastro-intestinal (GI) pathogens in severe newborn diarrhea is still not well known. We aimed to determine the seasonal and age-associated pathogen distribution of severe diarrhea in newborn calves admitted to the intensive care unit (ICU) of Erciyes University animal hospital over a year. Fecal samples were collected during the ICU admissions, and specimens were subjected to a diarrheal pathogen screening panel that included bovine coronavirus (BCoV), Cryptosporidium spp., ETEC K99+, and bovine rotavirus, using RT-PCR and conventional PCR methods. Further isolation experiments were performed with permissive cell cultures and bacterial enrichment methods to identify the clinical importance of infectious pathogen shedding in the ICU. Among the hospitalized calves aged less than 45 days old, the majority of calves originated from small farms (85.9%). The pathogen that most frequently occurred was Cryptosporidium spp. (61.5%) followed by rotavirus (56.4%). The frequency of animal admission to ICU and GI pathogen identification was higher during the winter season (44.9%) when compared to other seasons. Most calves included in the study were 1–6 days old (44.9%). Lastly, co-infection with rotavirus and Cryptosporidium spp. occurred more frequently than other dual or multi-infection events. This study was the first to define severe diarrhea—causing GI pathogens from ICU admitted newborn calves in Turkey.

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van Belkum, Alex, Carina Almeida, Benjamin Bardiaux, Sarah V. Barrass, Sarah J. Butcher, Tuğçe Çaykara, Sounak Chowdhury, et al. "Host-Pathogen Adhesion as the Basis of Innovative Diagnostics for Emerging Pathogens." Diagnostics 11, no. 7 (July 14, 2021): 1259. http://dx.doi.org/10.3390/diagnostics11071259.

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Infectious diseases are an existential health threat, potentiated by emerging and re-emerging viruses and increasing bacterial antibiotic resistance. Targeted treatment of infectious diseases requires precision diagnostics, especially in cases where broad-range therapeutics such as antibiotics fail. There is thus an increasing need for new approaches to develop sensitive and specific in vitro diagnostic (IVD) tests. Basic science and translational research are needed to identify key microbial molecules as diagnostic targets, to identify relevant host counterparts, and to use this knowledge in developing or improving IVD. In this regard, an overlooked feature is the capacity of pathogens to adhere specifically to host cells and tissues. The molecular entities relevant for pathogen–surface interaction are the so-called adhesins. Adhesins vary from protein compounds to (poly-)saccharides or lipid structures that interact with eukaryotic host cell matrix molecules and receptors. Such interactions co-define the specificity and sensitivity of a diagnostic test. Currently, adhesin-receptor binding is typically used in the pre-analytical phase of IVD tests, focusing on pathogen enrichment. Further exploration of adhesin–ligand interaction, supported by present high-throughput “omics” technologies, might stimulate a new generation of broadly applicable pathogen detection and characterization tools. This review describes recent results of novel structure-defining technologies allowing for detailed molecular analysis of adhesins, their receptors and complexes. Since the host ligands evolve slowly, the corresponding adhesin interaction is under selective pressure to maintain a constant receptor binding domain. IVD should exploit such conserved binding sites and, in particular, use the human ligand to enrich the pathogen. We provide an inventory of methods based on adhesion factors and pathogen attachment mechanisms, which can also be of relevance to currently emerging pathogens, including SARS-CoV-2, the causative agent of COVID-19.

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Su, Lv, Lifan Zhang, Duoqian Nie, Eiko E. Kuramae, Biao Shen, and Qirong Shen. "Bacterial Tomato Pathogen Ralstonia solanacearum Invasion Modulates Rhizosphere Compounds and Facilitates the Cascade Effect of Fungal Pathogen Fusarium solani." Microorganisms 8, no. 6 (May 27, 2020): 806. http://dx.doi.org/10.3390/microorganisms8060806.

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Soil-borne pathogen invasions can significantly change the microbial communities of the host rhizosphere. However, whether bacterial Ralstonia solanacearum pathogen invasion influences the abundance of fungal pathogens remains unclear. In this study, we combined high-throughput sequencing, qPCR, liquid chromatography and soil culture experiments to analyze the rhizosphere fungal composition, co-occurrence of fungal communities, copy numbers of functional genes, contents of phenolic acids and their associations in healthy and bacterial wilt-diseased tomato plants. We found that R. solanacearum invasion increased the abundance of the soil-borne pathogen Fusarium solani. The concentrations of three phenolic acids in the rhizosphere soil of bacterial wilt-diseased tomato plants were significantly higher than those in the rhizosphere soil of healthy tomato plants. In addition, the increased concentrations of phenolic acids significantly stimulated F. solani growth in the soil. Furthermore, a simple fungal network with fewer links, nodes and hubs (highly connected nodes) was found in the diseased tomato plant rhizosphere. These results indicate that once the symptom of bacterial wilt disease is observed in tomato, the roots of the wilt-diseased tomato plants need to be removed in a timely manner to prevent the enrichment of other fungal soil-borne pathogens. These findings provide some ecological clues for the mixed co-occurrence of bacterial wilt disease and other fungal soil-borne diseases.

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Kadimisetty, Karteek, Kun Yin, Aoife M. Roche, Yanjie Yi, Frederic D. Bushman, Ronald G. Collman, Robert Gross, Liang Feng, and Changchun Liu. "An integrated self-powered 3D printed sample concentrator for highly sensitive molecular detection of HIV in whole blood at the point of care." Analyst 146, no. 10 (2021): 3234–41. http://dx.doi.org/10.1039/d0an02482a.

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Zhao, Peng, Jiajin Zhang, Wei Zhang, Dong Zhao, Yi Ma, Changjun Hou, Laichun Lu, and Danqun Huo. "Fabrication of a novel hydrogel-based microfluidic chip and its application in pathogen analysis." Analytical Methods 13, no. 43 (2021): 5240–46. http://dx.doi.org/10.1039/d1ay01522b.

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Korsnes, Kjetil, Ove Nicolaisen, Cecilie K. Skår, Audun H. Nerland, and Øivind Bergh. "Bacteria in the gut of juvenile cod Gadus morhua fed live feed enriched with four different commercial diets." ICES Journal of Marine Science 63, no. 2 (January 1, 2006): 296–301. http://dx.doi.org/10.1016/j.icesjms.2005.10.012.

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AbstractAtlantic cod, Gadus morhua L., larvae were fed rotifers, Brachionus plicatilis and Artemia franciscana enriched on four different commercial media, using the manufacturers' protocols. Pooled samples of 20 cod larvae were homogenized, diluted, and plated out on Petri dishes. The number of colony-forming units per larva was estimated, and the dominant strains subsequently sampled for sequencing of 16S rDNA. Bacteria showing high sequence similarity to a pathogen characteristic of cod and other fish species, Listonella anguillarum, were present in all four groups. Other taxa present among the dominating bacterial colonies were Pseudoalteromonas sp., and Vibrio sp. However, these bacteria could be assigned to genera only. The different enrichments probably affected the number of colony-forming bacteria per millilitre in the enrichment cultures as well as in the larval gastrointestinal (GI) tract. Also, the composition of the microbiota associated with the larval GI tract was probably affected by the enrichment media.

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Dudenhöffer, Jan‐Hendrik, Stefan Scheu, and Alexandre Jousset. "Systemic enrichment of antifungal traits in the rhizosphere microbiome after pathogen attack." Journal of Ecology 104, no. 6 (July 26, 2016): 1566–75. http://dx.doi.org/10.1111/1365-2745.12626.

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Lee, Sei Won, Young Ae Kang, Choong Eun Jin, Ho Cheol Kim, Geun Su Noh, Hyo Joo Lee, Joung Ha Park, Yong Seo Koo, Yong Shin, and Sung-Han Kim. "Gene-based diagnosis of tuberculosis with a new-generation pathogen enrichment technique." European Respiratory Journal 55, no. 3 (December 5, 2019): 1901885. http://dx.doi.org/10.1183/13993003.01885-2019.

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KOSTIĆ, TANJA, BEATRIX STESSL, MARTIN WAGNER, and ANGELA SESSITSCH. "Microarray Analysis Reveals the Actual Specificity of Enrichment Media Used for Food Safety Assessment." Journal of Food Protection 74, no. 6 (June 1, 2011): 1030–34. http://dx.doi.org/10.4315/0362-028x.jfp-10-388.

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Microbial diagnostic microarrays are tools for simultaneous detection and identification of microorganisms in food, clinical, and environmental samples. In comparison to classic methods, microarray-based systems have the potential for high throughput, parallelism, and miniaturization. High specificity and high sensitivity of detection have been demonstrated. A microbial diagnostic microarray for the detection of the most relevant bacterial food- and waterborne pathogens and indicator organisms was developed and thoroughly validated. The microarray platform based on sequence-specific end labeling of oligonucleotides and the phylogenetically robust gyrB marker gene allowed a highly specific (resolution on genus and/or species level) and sensitive (0.1% relative and 104 CFU absolute sensitivity) detection of the target pathogens. In initial challenge studies of the applicability of microarray-based food analysis, we obtained results demonstrating the questionable specificity of standardized culture-dependent microbiological detection methods. Taking into consideration the importance of reliable food safety assessment methods, comprehensive performance assessment is essential. Results demonstrate the potential of this new pathogen diagnostic microarray to evaluate culture-based standard methods in microbiological food analysis.

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Caruso, Paola, Jose Luis Palomo, Edson Bertolini, BelÃ¯Â¿Â½n Ã¯Â¿Â½lvarez, MarÃ¯Â¿Â½a M. LÃ¯Â¿Â½pez, and Elena G. Biosca. "Seasonal Variation of Ralstonia solanacearum Biovar 2 Populations in a Spanish River: Recovery of Stressed Cells at Low Temperatures." Applied and Environmental Microbiology 71, no. 1 (January 2005): 140–48. http://dx.doi.org/10.1128/aem.71.1.140-148.2005.

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ABSTRACT The presence of Ralstonia solanacearum biovar 2 in the watercourses of European countries is increasing, but little is known about its ecology in aquatic habitats. The detection of this pathogen in 2000 in one Spanish river led us to study its population density at different locations on the river over a period of 3 years. During 2000 and 2001, the pathogen was recovered at low densities (10 to 80 CFU/ml) by direct plating on modified SMSA agar from water samples at 14Ã¯Â¿Â½C or higher, but its isolation was usually unsuccessful at temperatures below 9Ã¯Â¿Â½C. To monitor the pathogen's abundance in winter, we used two liquid selective media for enrichment (at 29 and 35Ã¯Â¿Â½C) and compared them by using spiked river water samples: modified Wilbrink broth (MWB) was more efficient than modified SMSA broth for double-antibody-sandwich indirect enzyme-linked immunosorbent assay (DASI-ELISA) detection of R. solanacearum. Enrichment in MWB at both temperatures allowed us to recover R. solanacearum cells that were nonculturable on solid media up to 25 days after their entry into the viable but nonculturable state. When we applied this technique to water samples during the cold months of 2001 and 2002, we obtained the best detection results by the most-probable-number method after enrichment at 35Ã¯Â¿Â½C with MWB. The enrichment protocol was combined with DASI-ELISA and validated by Co-PCR to detect both naturally and artificially starved and cold-stressed cells in water, which were still infective. Overall, the data from this study demonstrate the effects of temperature variation on the population and culturability of R. solanacearum cells on solid media and their survival at low temperatures.

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Arenas, Ailan F., Gladys E. Salcedo, and Jorge E. Gomez-Marin. "R Script Approach to Infer Toxoplasma Infection Mechanisms From Microarrays and Domain-Domain Protein Interactions." Bioinformatics and Biology Insights 11 (January 1, 2017): 117793221774725. http://dx.doi.org/10.1177/1177932217747256.

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Pathogen-host protein-protein interaction systems examine the interactions between the protein repertoires of 2 distinct organisms. Some of these pathogen proteins interact with the host protein system and may manipulate it for their own advantages. In this work, we designed an R script by concatenating 2 functions called rowDM and rowCVmed to infer pathogen-host interaction using previously reported microarray data, including host gene enrichment analysis and the crossing of interspecific domain-domain interactions. We applied this script to the Toxoplasma-host system to describe pathogen survival mechanisms from human, mouse, and Toxoplasma Gene Expression Omnibus series. Our outcomes exhibited similar results with previously reported microarray analyses, but we found other important proteins that could contribute to toxoplasma pathogenesis. We observed that Toxoplasma ROP38 is the most differentially expressed protein among toxoplasma strains. Enrichment analysis and KEGG mapping indicated that the human retinal genes most affected by Toxoplasma infections are those related to antiapoptotic mechanisms. We suggest that proteins PIK3R1, PRKCA, PRKCG, PRKCB, HRAS, and c-JUN could be the possible substrates for differentially expressed Toxoplasma kinase ROP38. Likewise, we propose that Toxoplasma causes overexpression of apoptotic suppression human genes.

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LUCHANSKY, JOHN B., ANNA C. S. PORTO, F. MORGAN WALLACE, and JEFFREY E. CALL. "Recovery of Listeria monocytogenes from Vacuum-Sealed Packages of Frankfurters: Comparison of the U.S. Department of Agriculture (USDA) Food Safety and Inspection Service Product Composite Enrichment Method, the USDA Agricultural Research Service (ARS) Product Composite Rinse Method, and the USDA-ARS Package Rinse Method†‡." Journal of Food Protection 65, no. 3 (March 1, 2002): 567–70. http://dx.doi.org/10.4315/0362-028x-65.3.567.

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This study compared three methods for the recovery of Listeria monocytogenes from commercially prepared and vacuum-packaged frankfurters that were inoculated with a five-strain mixture of this pathogen at averages of 22 and 20,133 CFU per package over three trials. The presence and levels of the pathogen were determined by (i) the U.S. Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS) product composite enrichment method, involving the selective enrichment of a 25-g composite of product and the subsequent plating of this product onto selective agar plates; (ii) the USDA Agricultural Research Service (ARS) product composite rinse method, involving the rinsing of a 25-g composite of product with 0.1% peptone water and the subsequent plating of a portion of the rinse fluid directly onto selective agar plates; and (iii) the USDA-ARS package rinse method, involving the use of 25 ml of 0.1% peptone water to rinse the entire contents of a package and the subsequent plating of a portion of the rinse fluid directly onto selective agar plates. For packages inoculated with 20,133 CFU, L. monocytogenes was recovered at a frequency (percentage of packages positive) of 100% by each of the three methods. The pathogen was recovered at efficiencies (percentages of recovery of L. monocytogenes) of 43 and 94% with the USDA-ARS product rinse method and the USDA-ARS package rinse method, respectively. For packages inoculated with 22 CFU, L. monocytogenes was recovered at frequencies of 17, 10, and 100% by the USDA-FSIS product composite enrichment method, the USDA-ARS product composite rinse method, and the USDA-ARS package rinse method, respectively. The pathogen was recovered at efficiencies of 20 and 95% with the USDA-ARS product composite rinse method and the USDA-ARS package rinse method, respectively. In a related study, the USDA-ARS package rinse method was the only method that detected the pathogen in 60 packages from each of five brands of frankfurters purchased from local grocery stores. These data establish that the USDA-ARS package rinse method is markedly more sensitive, as well as demonstrably more rapid and facile, than either the approved USDA-FSIS product composite enrichment method or the USDA-ARS product composite rinse method in determining the presence or absence of L. monocytogenes and establishing the levels of the pathogen that may be on the surface of ready-to-eat foods such as frankfurters.

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Lee, Seon-Yeong, Feixiong Chen, and Tae Yoon Lee. "Tryptamine-functionalized magnetic nanoparticles for highly sensitive detection of Salmonella typhimurium." Analyst 146, no. 8 (2021): 2559–66. http://dx.doi.org/10.1039/d0an02458a.

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Folgueiras-González, Alba, Robin van den Braak, Martin Deijs, Lia van der Hoek, and Ad de Groof. "A Versatile Processing Workflow to Enable Pathogen Detection in Clinical Samples from Organs Using VIDISCA." Diagnostics 11, no. 5 (April 27, 2021): 791. http://dx.doi.org/10.3390/diagnostics11050791.

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In recent years, refined molecular methods coupled with powerful high throughput sequencing technologies have increased the potential of virus discovery in clinical samples. However, host genetic material remains a complicating factor that interferes with discovery of novel viruses in solid tissue samples as the relative abundance of the virus material is low. Physical enrichment processing methods, although usually complicated, labor-intensive, and costly, have proven to be successful for improving sensitivity of virus detection in complex samples. In order to further increase detectability, we studied the application of fast and simple high-throughput virus enrichment methods on tissue homogenates. Probe sonication in high EDTA concentrations, organic extraction with Vertrel™ XF, or a combination of both, were applied prior to chromatography-like enrichment using Capto™ Core 700 resin, after which effects on virus detection sensitivity by the VIDISCA method were determined. Sonication in the presence of high concentrations of EDTA showed the best performance with an increased proportion of viral reads, up to 9.4 times, yet minimal effect on the host background signal. When this sonication procedure in high EDTA concentrations was followed by organic extraction with Vertrel™ XF and two rounds of core bead chromatography enrichment, an increase up to 10.5 times in the proportion of viral reads in the processed samples was achieved, with reduction of host background sequencing. We present a simple and semi-high-throughput method that can be used to enrich homogenized tissue samples for viral reads.

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Parsons, Cameron, Midya Jahanafroozi, and Sophia Kathariou. "Requirement of lmo1930, a Gene in the Menaquinone Biosynthesis Operon, for Esculin Hydrolysis and Lithium Chloride Tolerance in Listeria monocytogenes." Microorganisms 7, no. 11 (November 8, 2019): 539. http://dx.doi.org/10.3390/microorganisms7110539.

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Listeria monocytogenes is a foodborne pathogen that is widely distributed in nature, having been isolated from a variety of sources such as soil, water, plant matter, and animals. In addition, L. monocytogenes is often detected in the regular sampling of food and food processing environments. The most common method for detecting L. monocytogenes is the use of selective enrichments. Both lithium chloride and esculin, in combination with ferric ammonium citrate, are utilized in several of the most commonly-employed selective enrichment schemes for L. monocytogenes. Here we report that transposon-based inactivation of lmo1930, one of the genes in the menaquinone biosynthesis operon, via transposon mutagenesis severely impaired the ability of L. monocytogenes to grow in the presence of lithium chloride or hydrolyze esculin, and conferred reduced growth and colony size. All phenotypes were restored upon genetic complementation. Thus, strains of L. monocytogenes with mutations leading to inactivation of lmo1930 may evade many commonly-used selective enrichment protocols employed in the detection of L. monocytogenes.

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Milton, A. A. P., G. Bhuvana Priya, K. M. Momin, M. Angappan, Samir Das, and S. Ghatak. "An appraisal of various pathogen detection methods in eggs and poultry." Issue 2 (November - December) 1, no. 2 (December 1, 2020): 93–97. http://dx.doi.org/10.51128/jfas.2020.a017.

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Abstract: To ensure safety in egg and poultry products, timely detection of pathogenic microbes is of paramount importance. This review offers an appraisal of different routinely used and novel emerging pathogen detection methods in egg, poultry and their products. Timely detection of pathogens is decisive to curtail outbreak risks, reduce hospitalisation, and provide product assurance. It will also reduce the cost of holding food products in cold storage and reduces product recalls. Some crucial issues need to be taken care of in choosing or developing a foodborne pathogen detection method. They are requirement of costly or sophisticated equipment, portability, trained personnel, viable but non-culturable bacteria (may give false-negative results), dead microbes (may give false-positive results), stressed or sub-lethally damaged bacteria and slow-growing microbes (require enrichment). In this review, the focus has been given on culture-based methods, nucleic acid-based methods, immunological methods and biosensor based methods. Keywords: Egg; poultry; detection methods; product assurance; safety.

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Vong, Linda, Lee J. Pinnell, Pekka Määttänen, C. William Yeung, Eberhard Lurz, and Philip M. Sherman. "Selective enrichment of commensal gut bacteria protects againstCitrobacter rodentium-induced colitis." American Journal of Physiology-Gastrointestinal and Liver Physiology 309, no. 3 (August 1, 2015): G181—G192. http://dx.doi.org/10.1152/ajpgi.00053.2015.

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The intestinal microbiota plays a key role in shaping the host immune system. Perturbation of gut microbial composition, termed dysbiosis, is associated with an increased susceptibility to intestinal pathogens and is a hallmark of a number of inflammatory, metabolic, and infectious diseases. The prospect of mining the commensal gut microbiota for bacterial strains that can impact immune function represents an attractive strategy to counteract dysbiosis and resulting disease. In this study, we show that selective enrichment of commensal gut lactobacilli protects against the murine pathogen Citrobacter rodentium, a well-characterized model of enteropathogenic and enterohemorrhagic Escherichia coli infection. The lactobacilli-enriched bacterial culture prevented the expansion of Gammaproteobacteria and Actinobacteria and was associated with improved indexes of epithelial barrier function (dextran flux), transmissible crypt hyperplasia, and tissue inflammatory cytokine levels. Moreover, cultivation of gut bacteria from Citrobacter rodentium-infected mice reveals the differential capacity of bacterial subsets to mobilize neutrophil oxidative burst and initiate the formation of weblike neutrophil extracellular traps. Our findings highlight the beneficial effects of a lactobacilli -enriched commensal gut microenvironment and, in the context of an intestinal barrier breach, the ability of neutrophils to immobilize both commensal and pathogenic bacteria.

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Waller, David F., and Steven A. Ogata. "Quantitative Immunocapture PCR Assay for Detection of Campylobacter jejuni in Foods." Applied and Environmental Microbiology 66, no. 9 (September 1, 2000): 4115–18. http://dx.doi.org/10.1128/aem.66.9.4115-4118.2000.

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ABSTRACT The rapid detection of food-borne bacterial pathogens as part of a quality control program is necessary for the maintenance of a safe food supply. In this report, we present our findings for an immunocapture PCR method for the detection of Campylobacter jejuni in foods. The method permits direct detection of the pathogen without an enrichment step and can be performed in approximately 8 h. Assay results are quantitative, and one cell in a milliliter sample can be detected. Application of the method to spiked milk samples and chicken skin washes did not affect the sensitivity of the assay.

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LETELLIER, ANN, S. MESSIER, and S. QUESSY. "Prevalence of Salmonella spp. and Yersinia enterocolitica in Finishing Swine at Canadian Abattoirs." Journal of Food Protection 62, no. 1 (January 1, 1999): 22–25. http://dx.doi.org/10.4315/0362-028x-62.1.22.

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The prevalence of Salmonella spp. and Yersinia enterocolitica in finishing swine was evaluated using samples of cecal material. Samples were taken at six different slaughterhouses from 1420 healthy, 5-month-old pigs, raised by 223 producers in Québec (1009 samples), Ontario (283), and Manitoba, Canada (128). Two different broth media (Rappaport-Vassiliadis and Tetrathionate brilliant green) were used for the selective enrichment of Salmonella spp. The recovery of Y. enterocolitica was done by a cold enrichment technique, followed by plating on a selective media (cefsulodin-irgasan-novobiocin agar). Prevalence (with a 95% confidence interval) of Salmonella spp. and Y. enterocolitica were, respectively, 5.2% (4.0 to 6.4%) and 20.9% (18.8 to 23.0%). Overall, 24.6% of the animals tested were positive for one or both of these pathogens. Since only a few herds (2.8%) appeared to be highly contaminated by Salmonella spp., efforts should be undertaken in priority to control this pathogen in those herds.

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Khanna, Nina, Claudia Stuehler, Barbara Conrad, Carsten Berges, Sarah Lurati, Eike Kallmeyer, Sven Krappmann, Hermann Einsele, and Max S. Topp. "Generation of a Multiple Pathogen-Specific T Cell Product for Adoptive Immunotherapy Based on Activation-Dependent Expression of CD154." Blood 116, no. 21 (November 19, 2010): 2326. http://dx.doi.org/10.1182/blood.v116.21.2326.2326.

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Abstract Abstract 2326 In patients undergoing hematopoietic stem cell transplantation (HSCT) infectious complications are frequent causing substantial morbidity and mortality. Adoptive T cell therapy specific for single pathogens has previously shown to efficiently control viral and fungal infections but approaches targeting multiple pathogens are limited to T cells generated with EBV transformed B cells that are genetically modified expressing multiple viral antigens. Infections are often experienced by different viral and fungal pathogens such as cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus (AdV), Aspergillus fumigatus (AF) and Candida albicans (CA) that show a wide spectrum of memory T cell frequencies. Those with a low precursor frequency are not suitable for selection methods based on the secretion of cytokines such as IFN-g. As CMV seropositivity among HSCT donors may range only between 30–40% and immunity to the other pathogens can be detected simultaneously in more than 85% of HSCT donors we focused on the generation of a multi-specific T cell product for EBV, AdV, AF and CA for easy transfer under current regulatory requirements. We aimed to develop a simple protocol which (i) is able to enrich T cells specific for pathogens with low precursor frequency and (ii) allows simultaneous expansion of multiple pathogen-specific T cells in a single culture. We determined if CD154, which is transiently expressed on antigen stimulated CD4+ but also to a lesser extend on CD8+ T cells, would be a potential candidate for selection of pathogen-specific T cells. For stimulation we used peptide pools for AdV hexon protein, EBV latent membrane protein 2 (LMP2) and CA mannose protein 65 (MP65) as well as one AF immune dominant epitope derived from the Crf1 protein. To select and expand antigen-specific T cells, we stimulated PBMC for 16 hours, separated them by CD154+ MicroBead Kit (Miltenyi) and co-cultured them with irradiated autologous PBMC with IL-2, IL-7 and IL-15 for 14 days. The isolated cells were on average 0.62% of the starting fraction and could be expanded 20- to 145-fold. The median frequency of AdV-specific T cells increased from day 1 to day 14 87-fold from 30 to 2620 spot forming counts (SFC)/2×105 cells, for EBV 229-fold from 15 to 3430 SFC/2×105 cells and for CA 960-fold from 3 to 2400 SFC/2×105 cells assessed by IFN-γ ELISPOT. AF-specific T cells that were undetectable in PBMC increased to a median of 2260 SFC/2×105 cells. Although isolation of CD154+ cells favors enrichment of CD4+ T cells, a low fraction of virus-specific CD8+ T cells were simultaneously expanded. Next, we tested the efficacy of the CD154-based enrichment for the generation of multi pathogen-specific T cell lines reactive to all 4 pathogens. Selection and expansion was comparable, there was however a notable shift in the frequencies of T cells specific for different antigens in multi pathogen-specific cultures compared to single lines. The median increase of AdV-and CA- specific T cell lines was comparable (2345 SFC/2×105 and 3205 SFC/2×105 cells) but the frequencies for EBV (575 SFC/2×105 cells) as well as for AF (465 SFC/2×105 cells) were diminished in multi-specific lines. Nevertheless, lysis of LCL pulsed with LMP2 or AdV peptide pools was efficient with 72% and 36% by single and 30% and 45% by multi-specific T cell lines (at an E:T ratio of 20:1) as assessed by 51Cr-release assay. The single and multi pathogen-specific T cell lines generated by peptides responded to endogenously processed antigens and were able to specifically proliferate upon antigen stimulation. In contrast, T cell-mediated allo-reactivity was almost abrogated when compared to the starting population. In conclusion, we established a simple expansion protocol for selection, expansion and enrichment of allo-depleted single and multiple pathogen-specific CD4+ and CD8+ T cells specific for AdV, EBV, AF and CA that may further expand if the T cells are stimulated by their native antigen in vivo. This expansion protocol may form the basis for adoptive immunotherapy trials in HSCT recipients at risk for multiple infectious complications. This study has been supported by a grant of BayImmunet. Disclosures: No relevant conflicts of interest to declare.

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Leach, Kelly M., Joyce M. Stroot, and Daniel V. Lim. "Same-Day Detection of Escherichia coli O157:H7 from Spinach by Using Electrochemiluminescent and Cytometric Bead Array Biosensors." Applied and Environmental Microbiology 76, no. 24 (October 29, 2010): 8044–52. http://dx.doi.org/10.1128/aem.01990-10.

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ABSTRACT Contamination of fresh produce with Escherichia coli O157:H7 and other pathogens commonly causes food-borne illness and disease outbreaks. Thus, screening for pathogens is warranted, but improved testing procedures are needed to allow reproducible same-day detection of low initial contamination levels on perishable foods, and methods for detecting numerous pathogens in a single test are desired. Experimental procedures were developed to enable rapid screening of spinach for E. coli O157:H7 by using multiplex-capable immunological assays that are analyzed using biosensors. Detection was achieved using an automated electrochemiluminescent (ECL) assay system and a fluorescence-based cytometric bead array. Using the ECL system, less than 0.1 CFU of E. coli O157:H7 per gram of spinach was detected after 5 h of enrichment, corresponding to 6.5 h of total assay time. Using the cytometric bead array, less than 0.1 CFU/g was detected after 7 h of enrichment, with a total time to detection of less than 10 h. These results illustrate that both biosensor assays are useful for rapid detection of E. coli O157:H7 on produce in time frames that are comparable to or better than those of other testing formats. Both methods may be useful for multiplexed pathogen detection in the food industry and other testing situations.

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GOVARIS (Α. ΓΚΟΒΑΡΗΣ), A., D. K. PAPAGEORGIOU (Δ.Κ. ΠΑΠΑΓΕΩΡΓΙΟΥ), and K. PAPATHEODOROU (Κ. ΠΑΠΑΘΕΟΔΩΡΟΥ). "Survival of Escherichia coli 0157:H7 in cultured butter during storage." Journal of the Hellenic Veterinary Medical Society 53, no. 2 (January 31, 2018): 147. http://dx.doi.org/10.12681/jhvms.15371.

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The survival of E. coli 0157:H7 was examined in 3 types of butter: unsalted, salted with 0.46% and 0.93% salt. The butter samples were inoculated with a low (c.a. 3.14 log CFU/g) or high concentration (c.a. 4.80 log CFU/g) inoculum of the pathogen and were stored at 4°C and 12°C. The contaminated butter samples were stored for 2 months at 4°C and up to visible spoilage (20-26 days) at 12° C. By the end of the storage at 4°C, the tests with the high inoculum of the pathogen revealed that populations of E. coli 0157:H7 were decreased by 2.26 log CFU/g in the unsalted and in 0.46% salted types of butter, and by 2.74 log CFU/g in the 0.93% salted type of butter, while the tests with the low inoculum of the pathogen revealed that populations of E. coli Ol57:H7 decreased by 1.81 log CFU/g in the unsalted type of butter and were detectable in the other types of butter only after enrichment. By the end of the storage at 12°C, the tests with the high inoculum of the pathogen revealed that populations of E coli Ol57:H7 decreased by 2.71 and 3.17 log CFU/g in the unsalted and 0.46% salted types of butter, after 20 and 22 days, respectively and were detectable, in the 0.93% salted type of butter after 26 days, only after enrichment, while the tests with the low inoculum of the pathogen revealed that populations of E. coli O l 57:H7 were decreased by 1.88 log CFU/g in the unsalted type of butter after 20 days, and were detectable, in the 0.46% and 0.93% salted types of butter after 22 and 24 days, respectively, only after enrichment. The initial pH of all butter samples (5.18±0.01) decreased slightly (0.06-0.10) during storage at 4°C, and more (0.48 - 0.54) during storage at 12°C.

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Pettengill, James B., Eugene McAvoy, James R. White, Marc Allard, Eric Brown, and Andrea Ottesen. "Using metagenomic analyses to estimate the consequences of enrichment bias for pathogen detection." BMC Research Notes 5, no. 1 (2012): 378. http://dx.doi.org/10.1186/1756-0500-5-378.

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Dao, Thuy Nguyen Thi, Eun Yeong Lee, Bonhan Koo, Choong Eun Jin, Tae Yoon Lee, and Yong Shin. "A microfluidic enrichment platform with a recombinase polymerase amplification sensor for pathogen diagnosis." Analytical Biochemistry 544 (March 2018): 87–92. http://dx.doi.org/10.1016/j.ab.2017.12.030.

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ERICKSON, MARILYN C., JYE-YIN LIAO, ALISON S. PAYTON, PETER W. COOK, HENK C. DEN BAKKER, JESUS BAUTISTA, and JUAN CARLOS DÍAZ-PÉREZ. "Survival of Salmonella enterica and Escherichia coli O157:H7 Sprayed onto the Foliage of Field-Grown Cabbage Plants." Journal of Food Protection 82, no. 3 (February 26, 2019): 479–85. http://dx.doi.org/10.4315/0362-028x.jfp-18-326.

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ABSTRACT To reduce the number of cabbage pathogen outbreaks, it is essential to understand the fate of enteric pathogens that contaminate plants in the field. To assist in that effort, two independent trials were conducted with a red cultivar (cv. Red Dynasty) and a green cultivar (cv. Bravo F1) of field-grown cabbage (Brassica oleracea var. capitata). In the first trial, plants with small heads were sprayed with an inoculum containing both attenuated Salmonella enterica Typhimurium and Escherichia coli O157:H7 (5.0 log CFU/mL). Initial pathogen levels (ca. 3.9 log CFU per head), determined through plate count enumeration (limit of detection was 1.3 log CFU/g), dropped precipitously such that 2 days later, they could not be detected by enrichment culture in 22 to 35% of the heads. However, subsequent declines were at a slower rate; no differences were observed between red and green cabbage heads (P > 0.05), and heads were still positive for the pathogens 22 days after being sprayed with the inoculum. As a result, the logistic model revealed that for every 2 days contaminated cabbage heads remained in the field, the probability of finding a positive sample decreased by a factor of 1.1 (95% confidence interval from 1.0 to 1.2, P = 0.0022) and 1.2 (95% confidence interval from 1.0 to 1.4, P ≤ 0.0001) for Salmonella and E. coli O157:H7, respectively. In the second trial occurring 2 weeks later, plants with medium red or green cabbage heads were sprayed with an inoculum at a dose of 3.5 log CFU/mL. A similar decay in prevalence over time occurred for green cabbage as in trial 1; however, pathogen decline in red cabbage was less in trial 2 than in trial 1. The extended persistence of pathogens in cabbage heads exhibited in both trials infers that harvest of contaminated cabbage destined for raw consumption is risky. Additional field studies are necessary to determine whether similar pathogen fates occur in other regions or climates and to clarify the effect of the maturity of red cabbage on pathogen inactivation.

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GUERINI, MICHAEL N., JOSEPH M. BOSILEVAC, and MOHAMMAD KOOHMARAIE. "Rapid Enrichment Strategy for Isolation of Listeria from Bovine Hide, Carcass, and Meat Samples†." Journal of Food Protection 70, no. 1 (January 1, 2007): 53–57. http://dx.doi.org/10.4315/0362-028x-70.1.53.

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Since the outbreak of foodborne illness linked to Escherichia coli O157:H7 bacteria in ground beef in the early 1980s, the beef processing industry has focused on increasing the safety of beef products by implementing procedures for surveying live cattle, carcasses, and beef products for bacterial pathogens. Effective methods are in place for screening cattle and beef products for the presence of E. coli O157:H7 contamination, and recent work has established the acceptability of these methods for surveillance of Salmonella. In keeping with the need to continually improve the food safety of beef products, new work investigating pathogen prevalence now includes surveillance for Listeria monocytogenes. Tryptic soy broth (TSB) has been documented as a robust nonselective medium for the enrichment of both E. coli and Salmonella from bovine hide, carcass, and meat samples. The University of Vermont modification medium is most often used as the primary enrichment medium for surveillance of Listeria spp. In this study, samples from bovine hides (n = 50), preevisceration carcasses (n = 50), and beef trim (n = 193) were used to evaluate TSB as a primary enrichment medium for the isolation of Listeria spp., including L. monocytogenes. No significant difference (P > 0.05) between TSB and the University of Vermont modification medium was observed when all three sample types underwent primary enrichment for the isolation of Listeria spp. Furthermore, the standard secondary enrichment ratio for Fraser broth used for Listeria recovery can be modified to accommodate a high-throughput method for processing multiple samples.

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CARLIN, CATHARINE R., SAMANTHA S. LAU, RACHEL A. CHENG, ARIEL J. BUEHLER, ZEINA KASSAIFY, and MARTIN WIEDMANN. "Validation Using Diverse, Difficult-to-Detect Salmonella Strains and a Dark Chocolate Matrix Highlights the Critical Role of Strain Selection for Evaluation of Simplified, Rapid PCR-Based Methods Offering Next-Day Time to Results." Journal of Food Protection 83, no. 8 (April 2, 2020): 1374–86. http://dx.doi.org/10.4315/jfp-20-066.

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ABSTRACT Modifications to pathogen detection kits to accomplish simplified protocols with reduced time to results may impact method performance, particularly when combining shortened enrichment times and simplified enrichment procedures. We used Salmonella detection in dark chocolate as a model to test the impact of different enrichment times (minimum and maximum validated times) and procedures on detection of low levels of difficult-to-detect Salmonella strains, for three PCR kits that were AOAC International Performance Tested Method certified for detection of Salmonella spp. in dark chocolate. Initial inclusivity studies with pure cultures showed that all three kits detected 70 of 70 Salmonella spp. strains at 1 log above the theoretical limit of detection, with some strains yielding later cycle threshold values or having variable detection among technical replicates, indicating reduced assay performance for these strains. Based on these data, we selected a S. enterica subsp. enterica serovar Poona strain as well as three non-subsp. enterica strains to test the ability of the three kits to detect Salmonella in dark chocolate inoculated at low levels (0.06 to 1.18 most probable number per 25 g). With primary enrichment in skim milk at 35°C, detection frequency for all assays did not significantly differ from the reference method for both the minimum and maximum validated enrichment times. However, a pilot study that used primary enrichment in buffered peptone water at 42°C yielded significantly fewer positive samples (13 of 80) than were obtained with the U.S. Food and Drug Administration Bacteriological Analytical Manual method using enrichment in skim milk at 35°C (40 of 80 positive samples); strains representing subsp. houtenae and salamae were detected in significantly fewer chocolate samples than enrichment with skim milk. Our data indicate that continued efforts to simplify rapid pathogen detection kits may reduce kit performance in a way that can only be detected with stringent evaluation protocols that are designed to identify kit failure modes. HIGHLIGHTS

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FREUND, SUSAN M., JOHN A. KOBURGER, and CHENG-I. WEI. "Enhanced Recovery of Plesiomonas shigelloides following an Enrichment Technique1." Journal of Food Protection 51, no. 2 (February 1, 1988): 110–12. http://dx.doi.org/10.4315/0362-028x-51.2.110.

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Enrichment techniques using five broths (gram-negative broth, alkaline peptone water, tetrathionate broth without iodine and two Plesiomonas broths) were compared to direct plating methods using freshwater samples to determine their ability to increase the isolation rate of Plesiomonas shigelloides, a suspected food and waterbome pathogen. Tetrathionate broth consistently gave significantly (p<0.05) greater recovery of P. shigelloides than the other four broths tested as well as by direct plating. Incubation of the enrichment broths at 40°C also resulted in significantly higher recovery of Plesiomonas than at 35°C. It is therefore suggested that for routine monitoring of P. shigelloides, tetrathionate broth incubated at 40°C be used for enrichment before plating.

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Journal articles on the topic 'Pathogen enrichment'

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