A Cautionary Report for Pathogen Identification Using Shotgun Metagenomics; A Comparison to Aerobic Culture and Polymerase Chain Reaction for Salmonella enterica Identification

Enrique Doster, Pablo Rovira, Noelle R. Noyes, Brandy A. Burgess, Xiang Yang, Margaret D. Weinroth, Lyndsey Linke, Roberta Magnuson, Christina Boucher, Keith E. Belk, Paul S. Morley

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3 Scopus citations

Abstract

This study was conducted to compare aerobic culture, polymerase chain reaction (PCR), lateral flow immunoassay (LFI), and shotgun metagenomics for identification of Salmonella enterica in feces collected from feedlot cattle. Samples were analyzed in parallel using all four tests. Results from aerobic culture and PCR were 100% concordant and indicated low S. enterica prevalence (3/60 samples positive). Although low S. enterica prevalence restricted formal statistical comparisons, LFI and deep metagenomic sequencing results were discordant with these results. Specifically, metagenomic analysis using k-mer-based classification against the RefSeq database indicated that 11/60 of samples contained sequence reads that matched to the S. enterica genome and uniquely identified this species of bacteria within the sample. However, further examination revealed that plasmid sequences were often included with bacterial genomic sequence data submitted to NCBI, which can lead to incorrect taxonomic classification. To circumvent this classification problem, we separated all plasmid sequences included in bacterial RefSeq genomes and reassigned them to a unique taxon so that they would not be uniquely associated with specific bacterial species such as S. enterica. Using this revised database and taxonomic structure, we found that only 6/60 samples contained sequences specific for S. enterica, suggesting increased relative specificity. Reads identified as S. enterica in these six samples were further evaluated using BLAST and NCBI’s nr/nt database, which identified that only 2/60 samples contained reads exclusive to S. enterica chromosomal genomes. These two samples were culture- and PCR-negative, suggesting that even deep metagenomic sequencing suffers from lower sensitivity and specificity in comparison to more traditional pathogen detection methods. Additionally, no sample reads were taxonomically classified as S. enterica with two other metagenomic tools, Metagenomic Intra-species Diversity Analysis System (MIDAS) and Metagenomic Phylogenetic Analysis 2 (MetaPhlAn2). This study re-affirmed that the traditional techniques of aerobic culture and PCR provide similar results for S. enterica identification in cattle feces. On the other hand, metagenomic results are highly influenced by the classification method and reference database employed. These results highlight the nuances of computational detection of species-level sequences within short-read metagenomic sequence data, and emphasize the need for cautious interpretation of such results.

Original languageEnglish (US)
Article number2499
JournalFrontiers in Microbiology
Volume10
DOIs
StatePublished - Nov 1 2019

Bibliographical note

Funding Information:
The authors thank the participating feedlot and their personnel for their assistance and cooperation in this study. The authors are grateful to Zoetis for contributing the Draxxin antimicrobial used in this study. The authors also thank the University of Colorado Denver Genomics and Microarray Core Facility for their aid in high-throughput sequencing. Funding. This work was supported by the National Cattlemen?s Beef Association through the Beef Checkoff program. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Publisher Copyright:
© Copyright © 2019 Doster, Rovira, Noyes, Burgess, Yang, Weinroth, Linke, Magnuson, Boucher, Belk and Morley.

Keywords

  • PCR
  • Salmonella enterica
  • culture
  • pathogen identification
  • shotgun metagenomics

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