Theses and Dissertations from UMD
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Item WHOLE GENOME SEQUENCING ANALYSIS ON SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157:H7 FROM CATTLE FED WITH DIFFERENT DIETARY PROTEIN CONCENTRATIONS(2017) YANG, XUN; MENG, JIANGHONG; Food Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Escherichia coli serotype O157:H7 was first recognized in 1982 as a human pathogen associated with outbreaks of bloody diarrhea in the United States and is now considered a major cause of foodborne infections because of its high hospitalization rate. Cattle is the major reservoir of E. coli O157:H7. Cattle harbor E. coli O157:H7 in the hindgut and shed the organisms in the feces, which serves as a source of contamination of food and water. It is hypothesized that dietary ingredients that reach the hindgut are likely to affect colonization and fecal shedding of STEC. Increased flow of dietary ingredients (starch, fiber, protein, and lipid) are likely to alter ecology of the hindgut, resulting in altered pH and fermentation products, which could have a positive or negative impact on E. coli O157:H7. The objectives of this study are to investigate E. coli O157:H7 populations in fecal shedding of cattle. The cattle in this study were fed with diets with different levels of ruminally-degradable and –undegradable protein. A total of 286 E. coli O157:H7 isolates were recovered from feces of 576 crossbred calves at the Clayton Livestock Research Center in Clayton, New Mexico. The organisms were sequenced using Illumina Miseq system. De novo assembly of raw reads was performed using SPAdes and SNPs analysis of the isolates was conducted using kSNP3. Virulence factor Database (VFDB), created by the MOH key laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, was used as a reference for BLAST. The results indicated that increased flow of undegradable protein may increase the shedding of E. coli O157. However, the effect of ractopamine was still unknown. Three clades were identified among the E. coli O157:H7 isolates, including clades 6, 7, and 8, most of which belong to clade 8 (205 of 286). 49588 SNPs were found according to kSNP3. 19043 SNPs were identified as core SNPs. The phylogenetic analysis showed that the E. coli O157 isolates which collected from neighboring Pens were more closely to each other.Item Genomic analysis of bacteriophages from non-O157 shiga toxin-producing Escherichia coli(2015) Tang, Shuai; Meng, Jianghong; Food Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Shiga toxin-producing Escherichia coli (STEC) is the fiercest pathotype among all diarrheic E. coli. STEC O157:H7 has been a predominant serotype for STEC in the United States. However, an increasing number of cases of infections by STEC other than O157 have been reported in recent years. Shiga toxin (Stx) is the most important virulence factor of STEC and is encoded by stx, which is introduced into STEC genome by bacteriophages through gene transduction. A detailed understanding about Stx bacteriophages is necessary to reveal the emergence and pathogenicity of STEC. The very unstable genomes of Stx bacteriophages result in a dynamic phenotypic versatility including virulence, host cell repertoire and tolerance to adversities. Sequencing technology enables us to generate genomic sequence data of bacteriophages at an affordable cost. The project aimed at obtaining genomic DNA sequences of Stx bacteriophages of non-O157 STEC isolates of diverse serotypes. Thirteen bacteriophages were successfully induced from 83 STEC isolates of serotypes O74, O111, O121, O130, O163, O179 and O183. The bacteriophage DNA samples were collected and sequenced using MiSeq Desktop Sequencer (Illumina®, Inc). Automatically assembled sequences were manually compared to E. coli genome sequence available from NCBI (NC_000913.3) to verify the reliability of the sequencing results. Nine verified bacteriophage sequences were aligned to two Stx bacteriophage genomes of NCBI (NC_000924.1 and NC_018846.1) and visible alignment results were obtained. A phylogenetic relationship of the nine phages and the two reference sequences was constructed and gene profiles of each sample sequences were identified. The comparative analysis indicated that recombination events occurred in probacteriophages showed traces. Similarity of bacteriophage genomes correlated to the serotypes of host bacteria based on the comparison of phylogenetic tree and STEC serotypes. Gene identification results showed that nucleotide variance does not show region specificity, silent mutations are frequent in housekeeping genes and virulence genes are conservative among phage samples.Item ALTERNATIVE APPROACHES IN MOLECULAR CHARACTERIZATION OF FOODBORNE PATHOGENS: SHIGA TOXIN-PRODUCING Escherichia coli AND Salmonella SEROTYPES(2014) Toro Ibaceta, Magaly Alejandra; Meng, Jianghong; Food Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Shiga toxin-producing E. coli (STEC) and Salmonella enterica subspecies enterica (S. enterica) are two major foodborne pathogens. They cause almost 1.5 million of cases of disease each year in the US. Due to their public health impact, development of new methods for their detection and identification are top priority. This research focused on identifying alternative molecular methods and markers for the identification of STEC and Salmonella. First, a suspension array was developed to simultaneously identify the seven most prevalent STEC (O26, O45, O103, O111, O121, O145, and O157) in the US. The panel targeted genes wzx or wzy and Shigatoxin genes. Testing and optimization employed four to eleven isolates of each serotype in the panel. STEC fluorescence values were 30 to >270 times greater than those of negative controls, demonstrating the method's effectiveness for the molecular serotyping of STEC. STEC strains (n=194) of 43 serotypes were examined for clustered regularly interspaced short palindromic repeats (CRISPR) arrays to study relatedness among serotypes. A subset of strains (n=81) was analyzed for cas and virulence genes to determine a possible relationship. CRISPR spacer content correlated well with serotypes, although some strains with different serogroup but the same H type shared identical arrays (O26:H11, O103:H11, and O111:H11). cas and virulence genes were not associated, but strains with greater probability of causing outbreaks and disease showed fewer spacers than those less likely to cause them (p<0.05). Therefore, CRISPR array content correlated well with STEC serotype, and CRISPR-cas systems were inversely related to strain virulence potential. Finally, the CRISPR arrays of 221 S. enterica of 53 serotypes were analyzed to define their relationship. CRISPR-cas systems of 50 S. enterica serotype Bareilly (S. Bareilly) were analyzed to resolve intra-serotype variations. CRISPR arrays correlated well with serotypes, although some serotypes displayed more than one type of array (e.g. S. Bareilly). Additionally, CRISPR-cas system elements reflected S. Bareilly phylogeny, but the array content was not linked to food vehicle or isolate's geographical origin. In conclusion, CRISPR array are useful for designing molecular serotyping assays, but a range of strains should be included to account for variation in S. enterica.