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Foodborne bacteria have persisted as a significant threat to public health and to the food and agriculture industry. Due to the widespread impact of these pathogens, there has been a push for the development of strategies that can rapidly detect foodborne bacteria on-site. Shiga toxin-producing E. coli strains (such as E. coli O157:H7, E. coli O121, and E. coli O26) from contaminated food have been a major concern. They carry genes stx1 and/or stx2 that produce two toxins, Shiga toxin 1 and Shiga toxin 2, which are virulent proteins. In this work, we demonstrate the development of a rapid test based on an isothermal recombinase polymerase amplification reaction for two Shiga toxin genes in a single reaction. Results of the amplification reaction are visualized simultaneously for both Shiga toxins on a single lateral flow paper strip. This strategy targets the DNA encoding Shiga toxin 1 and 2, allowing for broad detection of any Shiga toxin-producing bacterial species. From sample to answer, this method can achieve results in approximately 35 min with a detection limit of 10 CFU/mL. This strategy is sensitive and selective, detecting only Shiga toxin-producing bacteria. There was no interference observed from non-pathogenic or pathogenic non-Shiga toxin-producing bacteria. A detection limit of 10 CFU/mL for Shiga toxin-producing E. coli was also obtained in a food matrix. This strategy is advantageous as it allows for timely identification of Shiga toxin-related contamination for quick initial food contamination assessments.
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Metagenomics as a Public Health Risk Assessment Tool in a Study of Natural Creek Sediments Influenced by Agricultural and Livestock Runoff: Potential and Limitations
ABSTRACT Little is known about the public health risks associated with natural creek sediments that are affected by runoff and fecal pollution from agricultural and livestock practices. For instance, the persistence of foodborne pathogens such as Shiga toxin-producing Escherichia coli (STEC) originating from these practices remains poorly quantified. Towards closing these knowledge gaps, the water-sediment interface of two creeks in the Salinas River Valley of California was sampled over a 9-month period using metagenomics and traditional culture-based tests for STEC. Our results revealed that these sediment communities are extremely diverse and have functional and taxonomic diversity comparable to that observed in soils. With our sequencing effort (∼4 Gbp per library), we were unable to detect any pathogenic E. coli in the metagenomes of 11 samples that had tested positive using culture-based methods, apparently due to relatively low abundance. Furthermore, there were no significant differences in the abundance of human- or cow-specific gut microbiome sequences in the downstream impacted sites compared to that in upstream more pristine (control) sites, indicating natural dilution of anthropogenic inputs. Notably, the high number of metagenomic reads carrying antibiotic resistance genes (ARGs) found in all samples was significantly higher than ARG reads in other available freshwater and soil metagenomes, suggesting that these communities may be natural reservoirs of ARGs. The work presented here should serve as a guide for sampling volumes, amount of sequencing to apply, and what bioinformatics analyses to perform when using metagenomics for public health risk studies of environmental samples such as sediments. IMPORTANCE Current agricultural and livestock practices contribute to fecal contamination in the environment and the spread of food- and waterborne disease and antibiotic resistance genes (ARGs). Traditionally, the level of pollution and risk to public health are assessed by culture-based tests for the intestinal bacterium Escherichia coli . However, the accuracy of these traditional methods (e.g., low accuracy in quantification, and false-positive signal when PCR based) and their suitability for sediments remain unclear. We collected sediments for a time series metagenomics study from one of the most highly productive agricultural regions in the United States in order to assess how agricultural runoff affects the native microbial communities and if the presence of Shiga toxin-producing Escherichia coli (STEC) in sediment samples can be detected directly by sequencing. Our study provided important information on the potential for using metagenomics as a tool for assessment of public health risk in natural environments.
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- Award ID(s):
- 1831582
- PAR ID:
- 10175325
- Date Published:
- Journal Name:
- Applied and Environmental Microbiology
- Volume:
- 86
- Issue:
- 6
- ISSN:
- 0099-2240
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1128/AEM.02525-19
