Academic literature on the topic 'Pathogen enrichment'

Horizontal gene transfer enables acquisition and dissemination of novel traits including antibiotic resistance and virulence among bacteria. Frequently such traits are gained through the acquisition of clusters of functionally related genes, often referred to as genomic islands (GIs). Quantifying horizontal flow of GIs and assessing their contributions to the emergence and evolution of novel metabolic traits in bacterial organisms are central to understanding the evolution of bacteria in general and the evolution of pathogenicity and antibiotic resistance in particular, a focus of this dissertation study. Methods for GI detection have also evolved with advances in sequencing and bioinformatics, however, comprehensive assessment of these methods has been lacking. This motivated us to assess the performance of current methods for identifying islands on broad datasets of well-characterized bacterial genomes and synthetic genomes, and leverage this information to develop a novel approach that circumvents the limitations of the current state-of-the-art in GI detection. The main findings from our assessment studies were 1) the methods have complementary strengths, 2) a gene-clustering method utilizing codon usage bias as the discriminant criterion, namely, JS-CB, is most efficient in localizing genomic islands, specifically the well-studied SCCmec resistance island in methicillin resistant Staphylococcus aureus (MRSA) genomes, and 3) in general, the bottom up, gene by gene analysis methods, are inherently limited in their ability to decipher large structures such as GIs as single entities within bacterial genomes. We adapted a top-down approach based on recursive segmentation and agglomerative clustering and developed a GI prediction tool, GEMINI, which combined compositional features with segment context information to localize GIs in the Liverpool epidemic strain of Pseudomonas aeruginosa. Application of GEMINI to the genome of P. aeruginosa LESB58 demonstrated its ability to delineate experimentally verified GIs in the LESB58 genome. GEMINI identified several novel islands including pathogenicity islands and revealed the mosaic structure of several LESB58 harbored GIs. A new GI identification approach, CAFE, with broad applicability was developed. CAFE incorporates biological information encoded in a genome within the statistical framework of segmentation and clustering to more robustly localize GIs in the genome. CAFE identifies genomic islands lacking markers by virtue of their association with genomic islands with markers originating from the same source. This is made possible by performing marker enrichment and phyletic pattern analyses within the integrated framework of recursive segmentation and clustering. CAFE compared favorably with frequently used methods for genomic island detection on synthetic test datasets and on a test-set of known islands from 15 well-characterized bacterial species. These tools can be readily adapted for cataloging GIs in just sequenced, yet uncharacterized genomes.

I investigated effects of environmental change on disease, and effects of disease on ecosystems, using a freshwater zooplankton host and its fungal parasite. This research involved lake surveys, manipulative experiments, and mathematical models. My results indicate that ecosystem characteristics such as habitat structure, nutrient availability, and quality of a host’s resources (here, phytoplankton) can affect the spread of disease. For example, a survey of epidemics in lakes revealed direct and indirect links between habitat structure and epidemic size, where indirect connections were mediated by non-host species. Then, in a mesocosm experiment in a lake, manipulations of habitat structure and nutrient availability interactively affected the spread of disease, and nutrient enrichment increased densities of infected hosts. In a separate laboratory experiment, poor quality resources were shown to decrease parasite transmission rate by altering host foraging behavior. My experimental results also suggest that disease can affect ecosystems through effects on host densities and host traits. In the mesocosm experiment, the parasite indirectly increased abundance of algal resources by decreasing densities of the zooplankton host. Disease in the experimental zooplankton populations also impacted nutrient stoichiometry of algae, which could entail a parasite-mediated shift in food quality for grazers such as the host. Additionally, I showed that infection dramatically reduces host feeding rate, and used a dynamic epidemiological model to illustrate how this parasite-mediated trait change could affect densities of resources and hosts, as well as the spread of disease. I discuss the implications of these ecosystem–disease interactions in light of ongoing changes to habitat and nutrient regimes in freshwater ecosystems.

<p>Availability of genome sequences for numerous bacterial species comprising of different bacterial strains allows elucidation of species and strain specific adaptations that facilitate their survival in widely fluctuating micro-environments and enhance their pathogenic potential. Different bacterial species use different strategies in their pathogenesis and the pathogenic potential of a bacterial species is dependent on its genomic complement of virulence factors. A bacterial virulence factor, within the context of this study, is defined as any endogenous protein product encoded by a gene that aids in the adhesion, invasion, colonization, persistence and pathogenesis of a bacterium within a host. Anecdotal evidence suggests that bacterial virulence genes are undergoing diversifying evolution to counteract the rapid adaptability of its host&rsquo<br>s immune defences. Genome sequences of pathogenic bacterial species and strains provide unique opportunities to study the action of diversifying selection operating on different classes of bacterial genes.</p>

Challenges encountered in pathogen identification and detection include the genetic heterogeneity of strains within species of some foodborne pathogens, isolation of injured cells, mixed strains or mixed species contamination of foods, and differentiation between viable and dead cells. The first objective of this research was to evaluate an isolation medium that was based on time-delayed release (5 to 6 h) of selective agents in tablet format to a modified Listeria recovery enrichment broth (mLRB) medium for enhanced and rapid recovery of injured Listeria. The second objective involved the use of Fourier transform infrared (FT-IR) spectroscopy and chemometric analysis for the differentiation of: Listeria monocytogenes epidemic clones (ECs); viable versus heat-killed populations; different mixed strains and mixed species of Listeria; and different injury treatments and repair in Listeria populations. Nitrite- or acid-injured Listeria at approximately 10 CFU/ml were recovered in mLRB medium, and cell populations enumerated at various times (12 to 48 h) of incubation at 37oC. Analysis of variance revealed that acid-injured Listeria populations in mLRBS6 (mLRB plus the selective agents at 6 h) were significantly higher (P < 0.05) than those in mLRBS0 (mLRB plus the selective agents at 0 h) at 24 h; however, the differences in populations on these two media were not significant for nitrite-injured Listeria. Cell populations of four strains of Listeria recovered in mLRBTD (mLRB plus the time-delayed release tablets of the selective agents) were significantly higher than when those strains were enriched in the U.S. Food and Drug Administration (FDA), International Organization for Standardization (ISO), and U.S. Department of Agriculture (USDA) broths at 24 h. Comparison between artificially contaminated milk and meat samples with a four-strain cocktail of Listeria resulted in cell populations that were significantly higher (P < 0.05) on mLRBTD for contaminated meat than on mLRBTD for contaminated milk at 24 h. FT-IR spectroscopy in the mid-infrared region (4000 to 600 cm-1) and chemometrics was successfully applied to discriminate L. monocytogenes strains belonging to the same EC (ECII or ECIV) (100% accurate spectral classification), intact and heat-killed populations of each EC strain (100% accurate spectral classification), and spectral wavenumbers 1650 to 1390 cm-1 were used to differentiate heat-killed from intact populations. FT-IR spectroscopy and chemometrics in the wavelength region 1800 to 900 cm-1 could successfully discriminate different mixed strains of L. monocytogenes (98.15% accurate spectral classification) and different mixed species of L. monocytogenes and L. innocua (92.06% accurate spectral classification) from individual strains; Wavelength range 1800 to 900 cm-1 was successfully used to discriminate between intact, acid-injured, and heat-injured Listeria, with repaired cells from acid and heat treatments clustering closer to intact cells (93.33% of spectra accurately classified). Delayed-addition of selective agents to broth medium improves recovery of injured Listeria by allowing repair time, could minimize contamination through manual addition of selective agents, and saves analyst time; FT-IR spectroscopy is a highly discriminatory and reproducible technique that can be used for the differentiation of strains and various physiological states of Listeria.

The advent of next-generation sequencing (NGS) has accelerated biological research by providing the opportunity to connect genome level sequence information to function. One key application of NGS has been analysis of gene expression and variation by implementing ways to recapitulate RNA level differences accurately. However, for studies that require accurate estimates of closely related variants or isoforms, as in case of evolutionary dynamics of viral quasispecies, each of the available sequencing platforms have critical limitations. In this thesis work, we have developed methods to eliminate three of the significant limitations of current NGS platforms. These new methods tackle (i) Inability to reconstruct individual whole viral genomes due to genome fragmentation during sequence library preparations in short-read platforms (ii) High sequencing errors associated with 1D Oxford Nanopore sequencing platform and (iii) High background levels of host RNA hampering the detection of pathogen or novel RNA species in metagenomics studies. Our three novel workflows, namely, Single RNA sequencing (sRNA-Seq), TelN proteolemerase based nanopore 2D sequencing (T2D-NanoSeq), and Direct RNA amplification (D-RAMP), are designed to resolve each of these three limitations respectively. We have demonstrated each of the workflows, benchmarked them using synthetic samples and measured their efficiency. Our first method, sRNA-Seq, can report sequences distantly separated on a single RNA molecule with > 10% recovery and 87% specificity. On the other hand, T2D-NanoSeq, has 40% efficiency in covalently linking both forward and reverse sequences of a duplex DNA that enables tandem reads of both strands which can allow us to reduce sequencing errors. Finally, our third method can generate short DNA probes against target (host) RNA molecules from a physical sample of the RNA and has demonstrated a ~5 fold decrease in the target RNA compared to non-targeted RNA. Overall, these approaches provide improved sequencing alternatives for quantitative and accurate RNA sequencing.

碩士<br>國立臺灣海洋大學<br>食品科學系<br>95<br>First part of this study is to develop a combination of multiple primers for the major food poisoning pathogens: Bacillus cereus、Campylobacter jejuni, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella spp., Shigella spp., Staphylococcus aureus and Vibrio parahaemolyticus, and further to investigate the feasibility of applying those primer combination for rapid detection the presence of those pathogens by PCR. The second part of this study is to develop a PCR enrichment technique that can rapidly identify the presence of target pathogen at low number of cells and at a shorter period of incubation time as compared to traditional enrichment procedures. Escherichia coli O157:H7, and Vibrio parahaemolyticus were selected for this part of study. Results indicated that the production of non-specific PCR products will obstruct the interpretation of DNA electrophoretograms with multiple PCR primer combination, and thus make individual identification of those pathogens impossible. Possible approaches to resolve these inferences including categorized primer pair and combination design, Ta (annealing temperature) selection, and target pathogen grouping were suggested for further studies. In PCR enrichment experiment, results suggested that the detection limit can be reduced from 102~103 CFU/ml for traditional selective enrichment procedures to as low as a single cell for both Escherichia coli O157:H7 and Vibrio parahaemolyticus by PCR enrichment technique. Since PCR enrichment technique tends to cause non-specific signal and different primers need respective Ta, it is not suitable for applying on various pathogenic microbes. However, PCR enrichment technique will still be a potential, low costing, time saving, and sensitive methology while the problems of annealing conditions and non-specific PCR reactions are solved.

The access of pathogenic microorganisms to the human body can compromise the health of the individual, causing several clinical manifestations. Helicobacter pylori and Campylobacter are two important gastrointestinal pathogens. H. pylori infection is one of the most common human infections, whose treatment includes the administration of antibiotics, namely fluoroquinolones (FQ). However, there has been an increasing resistance of H. pylori to FQ, which can lead to treatment failures, making it important not only to detect the bacterium but also to define its resistance profile. Campylobacter is currently considered the leading cause of bacterial foodborne illnesses, usually associated with the consumption of raw meat. The detection of pathogenic microorganisms can be achieved by conventional culture techniques or by molecular methods, namely immunological tests or nucleic acid detection. Biomode is an innovative company that develops and commercializes rapid diagnostic methods based on Nucleic Acid Mimic (NAM) – fluorescence in situ hybridization (FISH) technology, which enables the rapid detection of microorganisms, through the hybridization of complementary fluorescent probes with specific sequences present in the target microorganism. In this context, the present work focused on two applications of NAM-FISH. In the clinical area, the main objective was the development of a method for the detection of H. pylori and its resistance to FQ. For this purpose, Peptide Nucleic Acid (PNA) and Locked Nucleic Acid (LNA)/2'OMe probes were designed for the detection of mutations that cause resistance. In order to cover the most prevalent mutations, as well as the wildtype phenotype, 5 LNA/2'OMe probes were selected. In the area of food safety, the objective was the optimization of a PNA-FISH method for Campylobacter detection in food samples. A preliminary testing of the inclusivity/exclusivity of the Campylobacter probe resulted in the detection of two non-target microorganisms, H. cinaedi and H. pamatensis. To optimize the procedure, samples of raw broiler meat inoculated artificially with C. jejuni were used. Prior to PNA -FISH, a new step was introduced in which the enriched samples are subjected to a centrifugation (10 000 g), followed by resuspension in 0.1% Triton X-100, in order to reduce the strong autofluorescence shown in samples without any treatment. The possibility of a two-step sample enrichment was also tested, however, this approach did not show advantages compared to the one-step procedure. Additionally, a robustness test, required by the AOAC International to obtain product certification, was performed, which showed that the variation of the parameters of the time and temperature of hybridization influence the performance of the method, showing that the PNA-FISH conditions must be strictly controlled. The results obtained in this study showed that the PNA-FISH method is suitable for the rapid detection of Campylobacter in food samples. With this work, it is concluded that although the two NAM-FISH based methods are a promising alternative for the detection of H. pylori and Campylobacter, they both require optimization.<br>O acesso de microrganismos patogénicos ao corpo humano pode comprometer a saúde do indivíduo, provocando variadas manifestações clínicas. Helicobacter pylori e Campylobacter são dois importantes patógenos gastrointestinais. A infeção por H. pylori é uma das infeções humanas mais comuns, cujo tratamento inclui a administração de antibióticos, nomeadamente fluoroquinolonas (FQ). Contudo, tem-se verificado um aumento da resistência de H. pylori a FQ, o que pode resultar em falhas no tratamento, tornando-se importante não só a deteção da bactéria, mas também a definição do seu perfil de resistência. Campylobacter é atualmente considerada a principal causa de doenças transmitidas por alimentos, encontrando-se normalmente associada ao consumo de carne crua. A deteção de microrganismos patogénicos pode ser alcançada por técnicas de cultura convencionais ou por métodos moleculares, nomeadamente testes imunológicos ou de deteção de ácidos nucleicos. A Biomode é uma empresa inovadora que desenvolve e comercializa métodos de diagnóstico rápidos baseados na tecnologia de hibridação fluorescente in situ (FISH) de ácidos nucleicos mímicos (NAM), NAM-FISH, que possibilita a deteção rápida de microrganismos, através da hibridação de sondas fluorescentes complementares com sequências específicas presentes no microrganismo alvo. Neste contexto, o presente trabalho teve como foco duas aplicações do NAM-FISH. Na área clínica, o objetivo principal foi o desenvolvimento de um método para a deteção de H. pylori e da resistência a FQ. Para tal, procedeu-se ao desenho de sondas de Peptide Nucleic Acid (PNA) e Locked Nucleic Acid (LNA)/2’OMe para a deteção de mutações causadoras da resistência. De forma a cobrir as mutações mais prevalentes, bem como o fenótipo wild-type, foram selecionadas 5 sondas de LNA/2’OMe. Na área da segurança alimentar, foi objetivo a otimização de um método de PNA-FISH para a deteção de Campylobacter em amostras alimentares. Um ensaio preliminar da inclusividade/exclusividade da sonda Campylobacter resultou na deteção de dois microrganismos não-alvo, H. cinaedi e H. pamatensis. Para a otimização do procedimento, foram utilizadas amostras de carne de frango crua, inoculadas artificialmente com C. jejuni. Antes do PNAFISH, foi introduzido um novo passo, no qual as amostras enriquecidas são sujeitas a uma centrifugação (10 000 g), seguida de ressuspensão em 0.1% Triton X-100, com o objetivo de reduzir a autofluorescência forte visualizada em amostras sem qualquer tratamento. Testou-se também a possibilidade de um enriquecimento das amostras em dois passos, no entanto, esta abordagem não demonstrou vantagens comparativamente ao procedimento em um passo. Efetuou-se ainda um teste de robustez, requerido pela AOAC International para a obtenção de certificação de produto, que revelou que a variação dos parâmetros do tempo e temperatura de hibridação influenciam a performance do método, pelo que as condições do PNA-FISH devem ser rigorosamente controladas. Os resultados obtidos neste estudo mostraram que o método PNAFISH é adequado para a rápida deteção de Campylobacter em amostras alimentares. Com este trabalho conclui-se que, embora os dois métodos baseados em NAM-FISH sejam uma opção promissora para a deteção de H. pylori e Campylobacter, ambos necessitam de otimização futura.<br>Mestrado em Biotecnologia

A new system which offers a low-cost and high performance means for detecting four bacterial pathogens Aeromonas salmonicida, Tenacibaculum maritimum, Lactococcus garvieae and Yersinia ruckeri in covertly infected farmed salmonid fish has been developed. The system couples Selective Enrichment Culture (SEC), Polymerase Chain Reaction (PCR) and Enzyme Hybridization Assay (EHA) technologies to provide streamlined high-throughput sample processing suitable for large scale surveillance and monitoring programs. The SEC media used for the technology were those developed by T.Wilson and J.Carson for the Cooperative Research Centre (CRC) for Aquaculture Ltd, except for the A. salmonicida medium which was developed using information obtained during the CRC project and by performing Minimum Inhibitory Concentration (MIG) assays with 32 antimicrobial agents that were not previously tested. Laboratory sensitivity of the A. salmonicida medium was 100% as determined by Most Probable Number (MPN) analysis and specificity of the medium was 97%. Bacterial DNA and RNA were extracted from the SEC media using guanidinium isothiocyanate (GuSCN) and Whatman Polyfiltronics GF/B 96-well Uni-filter plates. Sensitivity of the system as determined by PCR was between 1 and 16 CFU per 200 μI of selective-enrichment medium for DNA and between 1 and 9 CFU per 200 μI of selective-enrichment medium for RNA. The use of vacuum filtration and the 96-well glass microfibre filter plate allowed for rapid high-throughput and inexpensive extraction. Due to the high sensitivity of PCR, the technology is prone to false-positive results due to amplicon carry-over. To help prevent the occurrence of these erroneous results the photochemical IP-10 was added to the procedure. IP-10 inactivates PCR amplicons rendering them incapable of re-amplification in subsequent PCR reactions. Nucleolink ™ PCR-enzyme hybridization strips were used to perform sensitive streamlined (RT)PCR-EHA. For each bacterium 4 fg of pure target DNA or RNA was detected. With this system only one tube per sample from cDNA to EHA was required, decreasing the cost and time involved in sample transfer and decreasing the risk of cross-contamination between samples. In laboratory trials the final SEC-(RT-)PCR-EHA system proved to be very sensitive with 1-16 CFU from selective-enrichment culture detected. Specificity of the system was >99%. The system was also rapid with the 96-well nucleic acid extraction to EHA results achieved in as little as 8 hours. Test validation with field samples was achieved by determining test specificity and sensitivity from an epidemiological perspective. During this testing the sensitivity and specificity of the system matched that obtained using purified nucleic acids in the laboratory. Validation was conducted using a total of 10 fish trials using fish from different environments and from different sample sources. The system described here uses two types of technology, PCR and RT-PCR. The SEC-PCR-EHA system detects genomic DNA from the target pathogen, demonstrating evidence of infection, past or present and is ideal for use in surveillance programs and for quarantine. The SEC-RT-PCR-EHA system detects ribosomal RNA from the target pathogens. This system gives a more accurate indication of the presence of live bacteria and therefore of live covert infection and is useful when monitoring changes in the disease status of a population of fish over a short period of time. The system was developed using inexpensive materials instead of proprietary products, and disposable equipment usage was minimised in an effort to keep the system low-cost. The sensitivity of the system was maximised to ensure the detection of covertly infected fish and specificity of the system was as good as the PCR primers would allow. The system utilises 96-well technology to minimise the volume of reagents and to enable streamlined sample processing using a multichannel pipette. In summary, a low-cost, high-performance and streamlined highthroughput sampling system has been developed.