Virulence gene profiles and phylogeny of Shiga toxin-positive<i>Escherichia coli</i>strains isolated from FDA regulated foods during 2010-2017

González-Escalona, Narjol; Kase, Julie A.

doi:10.1101/461327

Cited by 4 publications

(8 citation statements)

References 78 publications

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“…Many E. coli strains are not harmful to humans and will pose no risk for public health. Thus, obtaining the completely closed genome of any potential STEC will provide an accurate characterization of all virulence genes it possesses to allow a prediction of potential health risk (25). In our study, the de novo assembly of the complete MAG for EDL933 (either in fragments or completely closed) was achieved in all samples with levels above 10 5 CFU/ml, which was almost equivalent to the limit of detection by qPCR, but with the added benefit of complete genome characterization critically needed during outbreak and traceback investigations.…”

Section: Discussionmentioning

confidence: 99%

“…We again used the spiked E. coli EDL933 genome as reference for in silico analysis (Bioproject number PRJNA639799). We have previously reported a set of 94 virulence genes (25) and the EDL933 genome showed the presence of 23 of those genes (Table 4). Among those genes were esp, tccP, nle genes, tir, and 20 toxB.…”

Section: Virulence Gene Identificationmentioning

confidence: 97%

“…determined by batch screening in Ridom SeqSphere+ software v 7.0.6 (Ridom) using the genes deposited in the NCBI Pathogen Detection Reference Gene Catalog (https://www.ncbi.nlm.nih.gov/pathogens/isolates#/refgene/) and described in Gonzalez-Escalona and Kase (2019) (25).…”

Section: In Silico Identification Of Virulence Genes the Presence Ofmentioning

confidence: 99%

“…WGS is rapidly changing the approach to foodborne illnesses and outbreak investigations (21). WGS is being used to monitor and identify foodborne pathogens (22,23) and the presence of antimicrobial resistance or virulence genes (24,25). Specific information on serotype and pathogenicity as it relates to phylogenic relationships is increasingly important in outbreak scenarios (26,27).…”

Section: Introductionmentioning

confidence: 99%

“…Specific information on serotype and pathogenicity as it relates to phylogenic relationships is increasingly important in outbreak scenarios (26,27). Some of the virulence genes detected by WGS mediate attachment and colonization of STECs and can be found in the locus of enterocyte effacement (LEE), including intimin (eae) and type 3 secretion system (TTSS) effector proteins (esp, esc, tir), non-LEE effectors (nleA, nleB, nleC), and other putative virulence genes (ehxA, etpD, subA, toxB, saa) (4,25,28,29).…”

Section: Introductionmentioning

confidence: 99%

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Precision long-read metagenomics sequencing for food safety by detection and assembly of Shiga toxin-producingEscherichia coliin irrigation water

Maguire

Kase

Roberson

et al. 2020

Preprint

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Shiga toxin-producing Escherichia coli (STEC) contamination of agricultural water might be an important factor to recent foodborne illness and outbreaks involving leafy greens. Whole genome sequencing generation of closed bacterial genomes plays an important role in source tracking. We aimed to determine the limits of detection and classification of STECs by qPCR and nanopore sequencing using enriched irrigation water artificially contaminated with E. coli O157:H7 (EDL933). We determined the limit of STEC detection by qPCR to be 30 CFU/reaction, which is equivalent to 105 CFU/ml in the enrichment. By using Oxford Nanopore's EPI2ME WIMP workflow and de novo assembly with Flye followed by taxon classification with a k-mer analysis software (Kraken), E. coli O157:H7 could be detected at 103 CFU/ml (68 reads) and a complete fragmented E. coli O157:H7 metagenome-assembled genome (MAG) was obtained at 105-108 CFU/ml. Using a custom script to extract the E. coli reads, a completely closed MAG was obtained at 107-108 CFU/ml and a complete, fragmented MAG was obtained at 105-106 CFU/ml. In silico virulence detection for E. coli MAGs for 105-108 CFU/ml showed that the virulotype was indistinguishable from the spiked E. coli O157:H7 strain. We further identified the bacterial species in the un-spiked enrichment, including antimicrobial resistance genes, which could have important implications to food safety. We propose this workflow could be used for detection and complete genomic characterization of STEC from a complex microbial sample and could be applied to determine the limit of detection and assembly of other foodborne bacterial pathogens.

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Section: Discussionmentioning

confidence: 99%

Section: Virulence Gene Identificationmentioning

confidence: 97%

Section: In Silico Identification Of Virulence Genes the Presence Ofmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Precision long-read metagenomics sequencing for food safety by detection and assembly of Shiga toxin-producingEscherichia coliin irrigation water

Maguire

Kase

Roberson

et al. 2020

Preprint

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Impact of DNA extraction on whole genome sequencing analysis for characterization and relatedness of Shiga toxin-producing Escherichia coli isolates

Nouws

Bogaerts

Verhaegen

et al. 2020

Sci Rep

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Whole genome sequencing (WGS) has proven to be the ultimate tool for bacterial isolate characterization and relatedness determination. However, standardized and harmonized workflows, e.g. for DNA extraction, are required to ensure robust and exchangeable WGS data. Data sharing between (inter)national laboratories is essential to support foodborne pathogen control, including outbreak investigation. This study evaluated eight commercial DNA preparation kits for their potential influence on: (i) DNA quality for Nextera XT library preparation; (ii) MiSeq sequencing (data quality, read mapping against plasmid and chromosome references); and (iii) WGS data analysis, i.e. isolate characterization (serotyping, virulence and antimicrobial resistance genotyping) and phylogenetic relatedness (core genome multilocus sequence typing and single nucleotide polymorphism analysis). Shiga toxin-producing Escherichia coli (STEC) was selected as a case study. Overall, data quality and inferred phylogenetic relationships between isolates were not affected by the DNA extraction kit choice, irrespective of the presence of confounding factors such as EDTA in DNA solution buffers. Nevertheless, completeness of STEC characterization was, although not substantially, influenced by the plasmid extraction performance of the kits, especially when using Nextera XT library preparation. This study contributes to addressing the WGS challenges of standardizing protocols to support data portability and to enable full exploitation of its potential. Whole genome sequencing (WGS) is being or has already been implemented internationally in foodborne pathogen surveillance and outbreak investigations 1-4. Thanks to its single nucleotide resolution, WGS provides superior isolate characterization and (sub)typing. This enables increased pathogen insight, and linking of human illness to specific foods or production environments with an unprecedented level of confidence 5. There are however several challenges to be considered to fully exploit its potential, including the standardization and harmonization of laboratory and computational workflows. Several consortia 6-9 are currently addressing

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Whole Genome Sequencing of Extended-Spectrum- and AmpC- β-Lactamase-Positive Enterobacterales Isolated From Spinach Production in Gauteng Province, South Africa

et al. 2021

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The increasing occurrence of multidrug-resistant (MDR) extended-spectrum β-lactamase- (ESBL) and/or AmpC β-lactamase- (AmpC) producing Enterobacterales in irrigation water and associated irrigated fresh produce represents risks related to the environment, food safety, and public health. In South Africa, information about the presence of ESBL/AmpC-producing Enterobacterales from non-clinical sources is limited, particularly in the water–plant-food interface. This study aimed to characterize 19 selected MDR ESBL/AmpC-producing Escherichia coli (n=3), Klebsiella pneumoniae (n=5), Serratia fonticola (n=10), and Salmonella enterica (n=1) isolates from spinach and associated irrigation water samples from two commercial spinach production systems within South Africa, using whole genome sequencing (WGS). Antibiotic resistance genes potentially encoding resistance to eight different classes were present, with blaCTX-M-15 being the dominant ESBL encoding gene and blaACT-types being the dominant AmpC encoding gene detected. A greater number of resistance genes across more antibiotic classes were seen in all the K. pneumoniae strains, compared to the other genera tested. From one farm, blaCTX-M-15-positive K. pneumoniae strains of the same sequence type 985 (ST 985) were present in spinach at harvest and retail samples after processing, suggesting successful persistence of these MDR strains. In addition, ESBL-producing K. pneumoniae ST15, an emerging high-risk clone causing nosocomical outbreaks worldwide, was isolated from irrigation water. Known resistance plasmid replicon types of Enterobacterales including IncFIB, IncFIA, IncFII, IncB/O, and IncHI1B were observed in all strains following analysis with PlasmidFinder. However, blaCTX-M-15 was the only β-lactamase resistance gene associated with plasmids (IncFII and IncFIB) in K. pneumoniae (n=4) strains. In one E. coli and five K. pneumoniae strains, integron In191 was observed. Relevant similarities to human pathogens were predicted with PathogenFinder for all 19 strains, with a confidence of 0.635–0.721 in S. fonticola, 0.852–0.931 in E. coli, 0.796–0.899 in K. pneumoniae, and 0.939 in the S. enterica strain. The presence of MDR ESBL/AmpC-producing E. coli, K. pneumoniae, S. fonticola, and S. enterica with similarities to human pathogens in the agricultural production systems reflects environmental and food contamination mediated by anthropogenic activities, contributing to the spread of antibiotic resistance genes.

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Virulence gene profiles and phylogeny of Shiga toxin-positiveEscherichia colistrains isolated from FDA regulated foods during 2010-2017

Cited by 4 publications

References 78 publications

Precision long-read metagenomics sequencing for food safety by detection and assembly of Shiga toxin-producingEscherichia coliin irrigation water

Precision long-read metagenomics sequencing for food safety by detection and assembly of Shiga toxin-producingEscherichia coliin irrigation water

Impact of DNA extraction on whole genome sequencing analysis for characterization and relatedness of Shiga toxin-producing Escherichia coli isolates

Whole Genome Sequencing of Extended-Spectrum- and AmpC- β-Lactamase-Positive Enterobacterales Isolated From Spinach Production in Gauteng Province, South Africa

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