Nutrition & Food Science

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Author: search.filters.author.Chen, Zhao

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    Benchmarking Long-Read Assemblers for Genomic Analyses of Bacterial Pathogens Using Oxford Nanopore Sequencing
    (MDPI, 2020-12-01) Chen, Zhao; Erickson, David L.; Meng, Jianghong
    Oxford Nanopore sequencing can be used to achieve complete bacterial genomes. However, the error rates of Oxford Nanopore long reads are greater compared to Illumina short reads. Long-read assemblers using a variety of assembly algorithms have been developed to overcome this deficiency, which have not been benchmarked for genomic analyses of bacterial pathogens using Oxford Nanopore long reads. In this study, long-read assemblers, namely Canu, Flye, Miniasm/Racon, Raven, Redbean, and Shasta, were thus benchmarked using Oxford Nanopore long reads of bacterial pathogens. Ten species were tested for mediocre- and low-quality simulated reads, and 10 species were tested for real reads. Raven was the most robust assembler, obtaining complete and accurate genomes. All Miniasm/Racon and Raven assemblies of mediocre-quality reads provided accurate antimicrobial resistance (AMR) profiles, while the Raven assembly of Klebsiella variicola with low-quality reads was the only assembly with an accurate AMR profile among all assemblers and species. All assemblers functioned well for predicting virulence genes using mediocre-quality and real reads, whereas only the Raven assemblies of low-quality reads had accurate numbers of virulence genes. Regarding multilocus sequence typing (MLST), Miniasm/Racon was the most effective assembler for mediocre-quality reads, while only the Raven assemblies of Escherichia coli O157:H7 and K. variicola with low-quality reads showed positive MLST results. Miniasm/Racon and Raven were the best performers for MLST using real reads. The Miniasm/Racon and Raven assemblies showed accurate phylogenetic inference. For the pan-genome analyses, Raven was the strongest assembler for simulated reads, whereas Miniasm/Racon and Raven performed the best for real reads. Overall, the most robust and accurate assembler was Raven, closely followed by Miniasm/Racon.
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    Persistence of Salmonella enterica and Enterococcus faecium NRRL B-2354 on Baby Spinach Subjected to Temperature Abuse after Exposure to Sub-Lethal Stresses
    (MDPI, 2021-09-10) Chen, Zhao; Meng, Jianghong
    The exposure of foodborne pathogens such as Salmonella enterica to a sub-lethal stress may protect bacterial cells against distinct stresses during the production of leafy greens, which can constitute potential health hazards to consumers. In this study, we evaluated how the prior exposure of S. enterica to sub-lethal food processing-related stresses influenced its subsequent persistence on baby spinach under cold (4 °C for 7 days) and temperature abuse (37 °C for 2 h + 4 °C for 7 days) conditions. We also compared the survival characteristics of pre-stressed S. enterica and Enterococcus faecium NRRL B-2354 as its surrogate on baby spinach. A cocktail of three S. enterica serovars, as well as S. Typhimurium ATCC 14028 wild type and its ΔrpoS mutant, and E. faecium NRRL B-2354, was first exposed to sub-lethal desiccation, oxidation, heat shock, and acid stresses. Afterward, baby spinach was inoculated with unstressed or pre-stressed cells at 7.0 log CFU/sample unit, followed by 7-day storage under cold and temperature abuse conditions. The unstressed S. enterica (fresh cells in sterile 0.85% saline) decreased rapidly within the first day and thereafter persisted around 5.5 log CFU/sample unit under both conditions. The desiccation-stressed S. enterica showed the highest bacterial counts (p < 0.05) compared to other conditions. The unstressed S. enterica survived better (p < 0.05) than the oxidation- and acid-stressed S. enterica, while there were no significant differences (p > 0.05) between the unstressed and heat-shocked S. enterica. Unlike the wild type, temperature abuse did not lead to the enhanced survival of the ΔrpoS mutant after exposure to desiccation stress, indicating that the rpoS gene could play a critical role in the persistence of desiccation-stressed S. enterica subjected to temperature abuse. E. faecium NRRL B-2354 was more persistent (p < 0.05) than the pre-stressed S. enterica under both conditions, suggesting its use as a suitable surrogate for pre-stressed S. enterica by providing a sufficient safety margin. Our results demonstrate the merit of considering the prior exposure of foodborne pathogens to sub-lethal stresses when validating the storage conditions for leafy greens.
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    Critical Assessment of Short-Read Assemblers for the Metagenomic Identification of Foodborne and Waterborne Pathogens Using Simulated Bacterial Communities
    (MDPI, 2022-12-06) Chen, Zhao; Meng, Jianghong
    Metagenomics offers the highest level of strain discrimination of bacterial pathogens from complex food and water microbiota. With the rapid evolvement of assembly algorithms, defining an optimal assembler based on the performance in the metagenomic identification of foodborne and waterborne pathogens is warranted. We aimed to benchmark short-read assemblers for the metagenomic identification of foodborne and waterborne pathogens using simulated bacterial communities. Bacterial communities on fresh spinach and in surface water were simulated by generating paired-end short reads of Illumina HiSeq, MiSeq, and NovaSeq at different sequencing depths. Multidrug-resistant Salmonella Indiana SI43 and Pseudomonas aeruginosa PAO1 were included in the simulated communities on fresh spinach and in surface water, respectively. ABySS, IDBA-UD, MaSuRCA, MEGAHIT, metaSPAdes, and Ray Meta were benchmarked in terms of assembly quality, identifications of plasmids, virulence genes, Salmonella pathogenicity island, antimicrobial resistance genes, chromosomal point mutations, serotyping, multilocus sequence typing, and whole-genome phylogeny. Overall, MEGHIT, metaSPAdes, and Ray Meta were more effective for metagenomic identification. We did not obtain an optimal assembler when using the extracted reads classified as Salmonella or P. aeruginosa for downstream genomic analyses, but the extracted reads showed consistent phylogenetic topology with the reference genome when they were aligned with Salmonella or P. aeruginosa strains. In most cases, HiSeq, MiSeq, and NovaSeq were comparable at the same sequencing depth, while higher sequencing depths generally led to more accurate results. As assembly algorithms advance and mature, the evaluation of assemblers should be a continuous process.
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    Benchmarking hybrid assembly approaches for genomic analyses of bacterial pathogens using Illumina and Oxford Nanopore sequencing
    (Springer Nature, 2020-09-14) Chen, Zhao; Erickson, David L.; Meng, Jianghong
    We benchmarked the hybrid assembly approaches of MaSuRCA, SPAdes, and Unicycler for bacterial pathogens using Illumina and Oxford Nanopore sequencing by determining genome completeness and accuracy, antimicrobial resistance (AMR), virulence potential, multilocus sequence typing (MLST), phylogeny, and pan genome. Ten bacterial species (10 strains) were tested for simulated reads of both mediocre- and low-quality, whereas 11 bacterial species (12 strains) were tested for real reads. Unicycler performed the best for achieving contiguous genomes, closely followed by MaSuRCA, while all SPAdes assemblies were incomplete. MaSuRCA was less tolerant of low-quality long reads than SPAdes and Unicycler. The hybrid assemblies of five antimicrobial-resistant strains with simulated reads provided consistent AMR genotypes with the reference genomes. The MaSuRCA assembly of Staphylococcus aureus with real reads contained msr(A) and tet(K), while the reference genome and SPAdes and Unicycler assemblies harbored blaZ. The AMR genotypes of the reference genomes and hybrid assemblies were consistent for the other five antimicrobial-resistant strains with real reads. The numbers of virulence genes in all hybrid assemblies were similar to those of the reference genomes, irrespective of simulated or real reads. Only one exception existed that the reference genome and hybrid assemblies of Pseudomonas aeruginosa with mediocre-quality long reads carried 241 virulence genes, whereas 184 virulence genes were identified in the hybrid assemblies of low-quality long reads. The MaSuRCA assemblies of Escherichia coli O157:H7 and Salmonella Typhimurium with mediocre-quality long reads contained 126 and 118 virulence genes, respectively, while 110 and 107 virulence genes were detected in their MaSuRCA assemblies of low-quality long reads, respectively. All approaches performed well in our MLST and phylogenetic analyses. The pan genomes of the hybrid assemblies of S. Typhimurium with mediocre-quality long reads were similar to that of the reference genome, while SPAdes and Unicycler were more tolerant of low-quality long reads than MaSuRCA for the pan-genome analysis. All approaches functioned well in the pan-genome analysis of Campylobacter jejuni with real reads. Our research demonstrates the hybrid assembly pipeline of Unicycler as a superior approach for genomic analyses of bacterial pathogens using Illumina and Oxford Nanopore sequencing.