Using hydrochloric acid and bile resistance for optimized detection and isolation of Shiga toxin-producing Escherichia coli (STEC) from sprouts

Lamparter, Marina C.; Seemann, A; Hobe, Carolin; Schuh, Elisabeth

doi:10.1016/j.ijfoodmicro.2020.108562

Cited by 2 publications

(1 citation statement)

References 48 publications

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“…Strain EDL933 (stx1a, stx2a, eae) was used as positive control. Dsb or ntb2 plasmid [33][34][35][36] was used as an inhibition control. The prepared samples were amplified using the CFX96 real-time detection system (BioRad) with the following program: 10 min at 95 °C (5 °C s −1 ); followed by 39 cycles of 15 s at 95 °C (2 °C s −1 ) and 60 s at 60 °C (2 °C s −1 ); and a final step of 30 s at 40 °C (5 °C s −1 ).…”

Section: Real-time Pcr Analysis Of Enriched Raw Milkmentioning

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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Section: Real-time Pcr Analysis Of Enriched Raw Milkmentioning

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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Clonal spread of non-O157 Shiga toxigenic Escherichia coli O21:H25 in raw water buffalo milks

Uyanik,

Gücükoğlu,

Gürler

et al. 2023

Journal of Applied Microbiology

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Aims This study was conducted to investigate the presence of Shiga toxin-producing O157 and non-O157 E. coli in raw water buffalo milk, as well as to determine the virulence gene profiles, phylogroups, sequence types, and serotypes of the isolated strains. Methods and results A total of 200 hand-milked raw water buffalo milk samples were collected from 200 different water buffaloes over a period of three months from 20 different farms. Isolation of STEC was performed using CHROMagar STEC. Presence of stx1, stx2, and eaeA genes were investigated by mPCR. Phylogroups and sequence types of E. coli strains were determined by Clermont phylotyping and MLST. Serotyping was performed using PCR or WGS. According to the results, two milk samples obtained from two different farms were found as STEC-positive. All Stx-positive E. coli isolates belonged to phylogenetic group A and were assigned to ST10. WGS results indicated that serotype of two isolates was O21:H25 and average nucleotide identity was detected at 99.99%. Thirteen additional registered E. coli O21:H25 assembled WGS data were obtained from EnteroBase and a phylogenetic tree was constructed. Conclusions With this study, the presence of stx2 harboring E. coli O21:H25 in milk was identified for the first time. Although the identified serotype is considered a non-pathogen seropathotype, we conclude it could play an important role in the environmental circulation of Stx-phages and consequently contribute to the emergence of new STEC-related outbreaks.

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Using hydrochloric acid and bile resistance for optimized detection and isolation of Shiga toxin-producing Escherichia coli (STEC) from sprouts

Cited by 2 publications

References 48 publications

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

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

Clonal spread of non-O157 Shiga toxigenic Escherichia coli O21:H25 in raw water buffalo milks

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