Abstract
Bacterial identification and typing are fixtures of microbiology laboratories and are vital aspects of our response mechanisms in the event of foodborne outbreaks and bioterrorist events. Whole genome sequencing (WGS) is leading the way in terms of expanding our ability to identify and characterize bacteria through the identification of subtle differences between genomes (e.g. single nucleotide polymorphisms (SNPs) and insertions/deletions). Modern high-throughput technologies such as pyrosequencing can facilitate the typing of bacteria by generating short-read sequence data of informative regions identified by WGS analyses, at a fraction of the cost of WGS. Thus, pyrosequencing systems remain a valuable asset in the laboratory today. Presented in this chapter are two methods developed in the Amoako laboratory that detail the identification and genotyping of bacterial pathogens. The first targets canonical single nucleotide polymorphisms (canSNPs) of evolutionary importance in Bacillus anthracis, the causative agent of Anthrax. The second assay detects Shiga-toxin (stx) genes, which are associated with virulence in Escherichia coli and Shigella spp., and differentiates the subtypes of stx-1 and stx-2 based on SNP loci. These rapid methods provide end users with important information regarding virulence traits as well as the evolutionary and biogeographic origin of isolates.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Ronaghi M, Uhlén M, Nyrén P (1998) A sequencing method based on real-time pyrophosphate. Science 281:363–365
Amoako KK, Thomas MC, Kong F et al (2012) Rapid detection and antimicrobial resistance gene profiling of Yersinia pestis using pyrosequencing technology. J Microbiol Methods 90:228–234
Janzen TW, Thomas MC, Goji N et al (2015) Rapid detection method for Bacillus anthracis using a combination of multiplexed real-time PCR and pyrosequencing and its application for food biodefense. J Food Prot 78:355–361
Goji N, Mathews A, Huszczynski G et al (2015) A new pyrosequencing assay for rapid detection and genotyping of Shiga toxin, intimin and O157-specific rfbE genes of Escherichia coli. J Microbiol Methods 109:167–179
Van Ert MN, Easterday WR, Simonson TS et al (2007) Strain-specific single-nucleotide polymorphism assays for the Bacillus anthracis Ames strain. J Clin Microbiol 45:47–53
Easterday WR, Van Ert MN, Simonson TS et al (2005) Use of single nucleotide polymorphisms in the plcR gene for specific identification of Bacillus anthracis. J Clin Microbiol 43:1995–1997
Stephens AJ, Huygens F, Inman-Bamber J et al (2006) Methicillin-resistant Staphylococcus aureus genotyping using a small set of polymorphisms. J Med Microbiol 55:43–51
U’Ren JM, Van Ert MN, Schupp JM et al (2005) Use of a real-time PCR TaqMan assay for rapid identification and differentiation of Burkholderia pseudomallei and Burkholderia mallei. J Clin Microbiol 43:5771–5774
Wahab T, Hjalmarsson S, Wollin R et al (2005) Pyrosequencing Bacillus anthracis. Emerg Infect Dis 11:1527–1531
Amoako KK, Janzen TW, Shields MJ et al (2013) Rapid detection and identification of Bacillus anthracis in food using pyrosequencing technology. Int J Food Microbiol 165:319–325
Keim P, Smith KL, Keys C et al (2001) Molecular investigation of the Aum Shinrikyo anthrax release in Kameido, Japan. J Clin Microbiol 39:4566–4567
Vogler AJ, Busch JD, Percy-Fine S et al (2002) Molecular analysis of rifampin resistance in Bacillus anthracis and Bacillus cereus. Antimicrob Agents Chemother 46:511–513
Keim P, Van Ert MN, Pearson T et al (2004) Anthrax molecular epidemiology and forensics: using the appropriate marker for different evolutionary scales. Infect Genet Evol 4:205–213
Pearson T, Busch JD, Ravel J et al (2004) Phylogenetic discovery bias in Bacillus anthracis using single-nucleotide polymorphisms from whole-genome sequencing. Proc Natl Acad Sci U S A 101:13536–13541
Moorhead SM, Dykes GA, Cursons RT (2003) An SNP-based PCR assay to differentiate between Listeria monocytogenes lineages derived from phylogenetic analysis of the sigB gene. J Microbiol Methods 55:425–432
Scheutz F, Teel LD, Beutin L et al (2012) Multicenter evaluation of a sequence-based protocol for subtyping Shiga toxins and standardizing Stx nomenclature. J Clin Microbiol 50:2951–2963
Beutin L, Miko A, Krause G et al (2007) Identification of human-pathogenic strains of Shiga toxin-producing Escherichia coli from food by a combination of serotyping and molecular typing of Shiga toxin genes. Appl Environ Microbiol 73:4769–4775
Persson S, Olsen KE, Ethelberg S et al (2007) Subtyping method for Escherichia coli shiga toxin (verocytotoxin) 2 variants and correlations to clinical manifestations. J Clin Microbiol 45:2020–2024
Beutin L, Kruger U, Krause G et al (2008) Evaluation of major types of Shiga toxin 2E-producing Escherichia coli bacteria present in food, pigs, and the environment as potential pathogens for humans. Appl Environ Microbiol 74:4806–4816
Prager R, Fruth A, Busch U et al (2011) Comparative analysis of virulence genes, genetic diversity, and phylogeny of Shiga toxin 2g and heat-stable enterotoxin STIa encoding Escherichia coli isolates from humans, animals, and environmental sources. Int J Med Microbiol 301(3):181–191
NCBI Resource Coordinators (2014) Database resources of the National Center for Biotechnology Information. Nucleic Acids Res 42:D7–D17
Thomas MC, Shields MJ, Hahn KR et al (2013) Evaluation of DNA extraction methods for Bacillus anthracis spores isolated from spiked food samples. J Appl Microbiol 115(1):156–162
Hahn KR, Janzen TW, Thomas MC et al (2014) Single nucleotide repeat analysis of B. anthracis isolates in Canada through comparison of pyrosequencing and Sanger sequencing. Vet Microbiol 169:228–232
Van Ert MN, Easterday WR, Huynh LY et al (2007) Global genetic population structure of Bacillus anthracis. PLoS One 2:e461
Perna NT, Plunkett G III, Burland V et al (2001) Genome sequence of enterohaemorrhagic Escherichia coli O157:H7. Nature 409:529–533
Acknowledgement
The authors thank Dr. Elizabeth Golsteyn-Thomas and Susan Druhan for providing B. anthracis isolates. We also would like to acknowledge the technical contribution of Kristen Hahn and Zhen Zhong for the B. anthracis pyrosequencing work.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media New York
About this protocol
Cite this protocol
Amoako, K.K., Thomas, M.C., Janzen, T.W., Goji, N. (2017). Rapid SNP Detection and Genotyping of Bacterial Pathogens by Pyrosequencing. In: White, S., Cantsilieris, S. (eds) Genotyping. Methods in Molecular Biology, vol 1492. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6442-0_15
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6442-0_15
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6440-6
Online ISBN: 978-1-4939-6442-0
eBook Packages: Springer Protocols