Precision long-read metagenomics sequencing for food safety by detection and assembly of Shiga toxin-producing<i>Escherichia coli</i>in irrigation water

Maguire, Meghan; Kase, Julie A.; Roberson, Dwayne; Muruvanda, Tim; Brown, Eric W.; Allard, Marc W.; Musser, Steven M.; González-Escalona, Narjol

doi:10.1101/2020.07.17.209718

Cited by 4 publications

(8 citation statements)

References 56 publications

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“…Several E. coli O serotypes are closely associated with human diseases with high morbidity and mortality rates. For example, the Shiga toxin-producing E. coli (STEC) O157:H7 is a well-known pathogenic clone that causes hemolytic-uremic syndrome and foodborne illnesses [29]. The "big six" non-O157 STEC strains (O26, O45, O103, O111, O121, and O145) cause less severe infections than O157.…”

Section: Discussionmentioning

confidence: 99%

Two Enteropathogenic Escherichia coli Strains Representing Novel Serotypes and Investigation of Their Roles in Adhesion

Wang

Jiao²,

Zhang

et al. 2021

J. Microbiol. Biotechnol.

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Enteropathogenic Escherichia coli (EPEC), which belongs to the attaching and effacing diarrheagenic E. coli strains, is a major causative agent of life-threatening diarrhea in infants in developing countries. Most EPEC isolates correspond to certain O serotypes; however, many strains are nontypeable. Two EPEC strains, EPEC001 and EPEC080, which could not be serotyped during routine detection, were isolated. In this study, we conducted an in-depth characterization of their putative O-antigen gene clusters (O-AGCs) and also performed constructed mutagenesis of the O-AGCs for functional analysis of O-antigen (OAg) synthesis. Sequence analysis revealed that the occurrence of O-AGCs in EPEC001 and E. coli O132 may be mediated by recombination between them, and EPEC080 and E. coli O2/O50 might acquire each O-AGC from uncommon ancestors. We also indicated that OAgknockout bacteria were highly adhesive in vitro, except for the EPEC001 wzy derivative, whose adherent capability was less than that of its wild-type strain, providing direct evidence that OAg plays a key role in EPEC pathogenesis. Together, we identified two EPEC O serotypes in silico and experimentally, and we also studied the adherent capabilities of their OAgs, which highlighted the fundamental and pathogenic role of OAg in EPEC.

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Section: Discussionmentioning

confidence: 99%

Two Enteropathogenic Escherichia coli Strains Representing Novel Serotypes and Investigation of Their Roles in Adhesion

Wang

Jiao²,

Zhang

et al. 2021

J. Microbiol. Biotechnol.

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“…This is achievable by sample enrichment where low levels of target are ampli ed many fold, but herewith we aimed to establish the extent to which virulotyping is possible in agricultural water. We obtained species-level detection of E. coli with less than 150 reads, but we were unable to make accurate strain-level and virulotype identi cation, which requires a complete genome (9,32,49).…”

Section: Discussionmentioning

confidence: 99%

“…Nanopore sequencing is, however, not typically used as a screening tool due to its high cost compared to other means of detection like qPCR. We have previously established that the limit of complete, fragmented assembly for STECs by nanopore sequencing is 10 5 CFU/ml (49). This is achievable by sample enrichment where low levels of target are ampli ed many fold, but herewith we aimed to establish the extent to which virulotyping is possible in agricultural water.…”

Section: Discussionmentioning

confidence: 99%

Metagenomic survey of agricultural water using long read sequencing: considerations for a successful analysis

Maguire

Kase

Brown

et al. 2021

Preprint

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Background Leafy greens are responsible for nearly half of the produce-related Shiga toxin-producing Escherichia coli (STEC) outbreaks in the United States and recent investigations have implicated agricultural water as a potential source. Current FDA detection protocols require extensive analysis time. Development of methods for rapid detection of STECs in the field are imperative to maintain food safety. We aimed to use Oxford Nanopore rapid sequencing kits for an in-field determination of agricultural water microbiome and possible detection and characterization of STECs strain(s) in these samples. Results We tested the performance of the nanopore rapid sequencing kit (RAD004) for fast microbiome determination using the well characterized ZymoBIOMICS mock microbial community and the number of reads for each identified species was present in the expected proportion. Rapid sequencing kit (LRK001 and RAD004) library preparation of DNA extracted from agricultural water resulted in poor nanopore sequencing reactions, with low output (0.3–1.7 M reads), a high proportion of failed reads (50–60%), and highly sheared DNA before and after a magnetic bead clean up. To improve performance, we prepared a DNA library with the ligation kit (LSK109), which includes multiple cleaning steps, reducing inherent inhibitors and producing a better outcome (2.2 M reads, 15% failed reads). Metagenomic composition of four sample sites determined using the ligation kit, showed a highly diverse bacterial community for each site, with 11 total genera identified, including Synechococcus and Cyanobium. No definitive presence of STEC could be confirmed in any of the sites. Approximately 100 reads from each site (0.02% of total reads) were identified as Escherichia coli, but the specific strain or their virulence genes could not be detected. Sites 9, 10, and 12 were found to be positive for STEC presence by microbiological techniques after enrichment. Conclusions The rapid sequencing kits can be appropriate for genus or species level microbial identification, but we recommend the use of the ligation kit for increased sequencing depth and removal of contaminants in agricultural water. However, we were not able to identify any STEC strains in these nanopore microbiome samples, due to low initial concentrations. The results from this pilot study provide preliminary evidence that MinION sequencing of agricultural water using the ligation kit has the potential to be used for rapid microbiome determination in the field with optimal results for water quality surveillance.

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“…Different long-read sequencing technologies exist, among which the most commonly used were commercialized by Pacific Biosciences (PacBio) and Oxford Nanopore Technology (ONT) [13,14]. The MinION platform, released in 2014 by Oxford Nanopore Technology, known for its portability and its ability to generate very long reads, has been increasingly tested for food-borne pathogen detection [15][16][17][18][19][20].…”

Section: Introductionmentioning

confidence: 99%

A step forward for Shiga toxin-producing Escherichia coli identification and characterization in raw milk using long-read metagenomics

et al. 2022

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Shiga toxin-producing Escherichia coli (STEC) are a cause of severe human illness and are frequently associated with haemolytic uraemic syndrome (HUS) in children. It remains difficult to identify virulence factors for STEC that absolutely predict the potential to cause human disease. In addition to the Shiga-toxin (stx genes), many additional factors have been reported, such as intimin (eae gene), which is clearly an aggravating factor for developing HUS. Current STEC detection methods classically rely on real-time PCR (qPCR) to detect the presence of the key virulence markers (stx and eae). Although qPCR gives an insight into the presence of these virulence markers, it is not appropriate for confirming their presence in the same strain. Therefore, isolation steps are necessary to confirm STEC viability and characterize STEC genomes. While STEC isolation is laborious and time-consuming, metagenomics has the potential to accelerate the STEC characterization process in an isolation-free manner. Recently, short-read sequencing metagenomics have been applied for this purpose, but assembly quality and contiguity suffer from the high proportion of mobile genetic elements occurring in STEC strains. To circumvent this problem, we used long-read sequencing metagenomics for identifying eae-positive STEC strains using raw cow's milk as a causative matrix for STEC food-borne outbreaks. By comparing enrichment conditions, optimizing library preparation for MinION sequencing and generating an easy-to-use STEC characterization pipeline, the direct identification of an eae-positive STEC strain was successful after enrichment of artificially contaminated raw cow's milk samples at a contamination level as low as 5 c.f.u. ml−1. Our newly developed method combines optimized enrichment conditions of STEC in raw milk in combination with a complete STEC analysis pipeline from long-read sequencing metagenomics data. This study shows the potential of the innovative methodology for characterizing STEC strains from complex matrices. Further developments will nonetheless be necessary for this method to be applied in STEC surveillance.

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

Cited by 4 publications

References 56 publications

Two Enteropathogenic Escherichia coli Strains Representing Novel Serotypes and Investigation of Their Roles in Adhesion

Two Enteropathogenic Escherichia coli Strains Representing Novel Serotypes and Investigation of Their Roles in Adhesion

Metagenomic survey of agricultural water using long read sequencing: considerations for a successful analysis

A step forward for Shiga toxin-producing Escherichia coli identification and characterization in raw milk using long-read metagenomics

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