Differentiating Botulinum Neurotoxin-Producing Clostridia with a Simple, Multiplex PCR Assay

Williamson, Charles H.  D.; Vazquez, Adam J.; Hill, Karen; Smith, Theresa J.; Nottingham, Roxanne; Stone, Nathan E.; Sobek, Colin J.; Cocking, Jill H.; Fernández, Rafael A.; Caballero, Patricia A.; Leiser, Owen P.; Keim, Paul; Sahl, Jason W.; Björkroth, Johanna

doi:10.1128/aem.00806-17

Title: Differentiating Botulinum Neurotoxin-Producing Clostridia with a Simple, Multiplex PCR Assay

Journal Article · Fri Jul 21 00:00:00 EDT 2017 · Applied and Environmental Microbiology

DOI:https://doi.org/10.1128/aem.00806-17· OSTI ID:1626064

Williamson, Charles H. D. ^[1]; Vazquez, Adam J. ^[1]; Hill, Karen ^[2]; Smith, Theresa J. ^[3]; Nottingham, Roxanne ^[1]; Stone, Nathan E. ^[1]; Sobek, Colin J. ^[1]; Cocking, Jill H. ^[4]; Fernández, Rafael A. ^[5]; Caballero, Patricia A. ^[5]; Leiser, Owen P. ^[1];

^[1]; Sahl, Jason W. ^[1]; Björkroth, Johanna ^[6]

Northern Arizona University, Flagstaff, Arizona, (United States). Pathogen and Microbiome Institute
Los Alamos National Lab. (LANL), Los Alamos, NM (United States). Bioscience Division
United States Army Medical Research Institute of Infectious Diseases, Frederick, Maryland (United States). Molecular and Translational Sciences Division
Northern Arizona University, Flagstaff, Arizona (United States). Informatics, Computing and Cyber Systems
Área Microbiología, Departamento de Patología, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Centro Universitario, Mendoza (Argentina)
Univ. of Helsinki (Finland)

Diverse members of the genus Clostridium produce botulinum neurotoxins (BoNTs), which cause a flaccid paralysis known as botulism. While multiple species of clostridia produce BoNTs, the majority of human botulism cases have been attributed to Clostridium botulinum groups I and II. Recent comparative genomic studies have demonstrated the genomic diversity within these BoNT-producing species. This report introduces a multiplex PCR assay for differentiating members of C. botulinum group I, C. sporogenes, and two major subgroups within C. botulinum group II. Coding region sequences unique to each of the four species/subgroups were identified by in silico analyses of thousands of genome assemblies, and PCR primers were designed to amplify each marker. The resulting multiplex PCR assay correctly assigned 41 tested isolates to the appropriate species or subgroup. A separate PCR assay to determine the presence of the ntnh gene (a gene associated with the botulinum neurotoxin gene cluster) was developed and validated. The ntnh gene PCR assay provides information about the presence or absence of the botulinum neurotoxin gene cluster and the type of gene cluster present (hapositive [ha⁺] or orfX⁺). The increased availability of whole-genome sequence data and comparative genomic tools enabled the design of these assays, which provide valuable information for characterizing BoNT-producing clostridia. The PCR assays are rapid, inexpensive tests that can be applied to a variety of sample types to assign isolates to species/subgroups and to detect clostridia with botulinum neurotoxin gene (bont) clusters. IMPORTANCE: Diverse clostridia produce the botulinum neurotoxin, one of the most potent known neurotoxins. In this study, a multiplex PCR assay was developed to differentiate clostridia that are most commonly isolated in connection with human botulism cases: C. botulinum group I, C. sporogenes, and two major subgroups within C. botulinum group II. Since BoNT-producing and nontoxigenic isolates can be found in each species, a PCR assay to determine the presence of the ntnh gene, which is a universally present component of bont gene clusters, and to provide information about the type (ha⁺ or orfX⁺) of bont gene cluster present in a sample was also developed. The PCR assays provide simple, rapid, and inexpensive tools for screening uncharacterized isolates from clinical or environmental samples. The information provided by these assays can inform epidemiological studies, aid with identifying mixtures of isolates and unknown isolates in culture collections, and confirm the presence of bacteria of interest.

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Research Organization:: Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)

Sponsoring Organization:: USDOE; U.S. Department of Homeland Security

Grant/Contract Number:: AC52-06NA25396; HSHQDC-16-C-B0013

OSTI ID:: 1626064

Journal Information:: Applied and Environmental Microbiology, Vol. 83, Issue 18; ISSN 0099-2240

Publisher:: American Society for MicrobiologyCopyright Statement

Country of Publication:: United States

Language:: English

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Genetic Characterization and Comparison of Clostridium botulinum Isolates from Botulism Cases in Japan between 2006 and 2011 Kenri, Tsuyoshi; Sekizuka, Tsuyoshi; Yamamoto, Akihiko Applied and Environmental Microbiology, Vol. 80, Issue 22 https://doi.org/10.1128/aem.02134-14	journal	September 2014
Genetic Diversity of Clostridium sporogenes PA 3679 Isolates Obtained from Different Sources as Resolved by Pulsed-Field Gel Electrophoresis and High-Throughput Sequencing Schill, Kristin M.; Wang, Yun; Butler, Robert R. Applied and Environmental Microbiology, Vol. 82, Issue 1 https://doi.org/10.1128/aem.02616-15	journal	October 2015
Flagellin Diversity in Clostridium botulinum Groups I and II: a New Strategy for Strain Identification Paul, Catherine J.; Twine, Susan M.; Tam, Kevin J. Applied and Environmental Microbiology, Vol. 73, Issue 9 https://doi.org/10.1128/aem.02623-06	journal	March 2007
Neurotoxin Gene Profiling of Clostridium botulinum Types C and D Native to Different Countries within Europe Woudstra, Cedric; Skarin, Hanna; Anniballi, Fabrizio Applied and Environmental Microbiology, Vol. 78, Issue 9 https://doi.org/10.1128/aem.07568-11	journal	February 2012
Multiplex PCR Assay for Detection and Identification of Clostridium botulinum Types A, B, E, and F in Food and Fecal Material Lindström, Miia; Keto, Riikka; Markkula, Annukka Applied and Environmental Microbiology, Vol. 67, Issue 12 https://doi.org/10.1128/aem.67.12.5694-5699.2001	journal	December 2001
Distribution of Botulinum Toxin-Producing Clostridia in Soils of Argentina Lúquez, Carolina; Bianco, María I.; de Jong, Laura I. T. Applied and Environmental Microbiology, Vol. 71, Issue 7 https://doi.org/10.1128/aem.71.7.4137-4139.2005	journal	July 2005
Laboratory Diagnostics of Botulism Lindström, Miia; Korkeala, Hannu Clinical Microbiology Reviews, Vol. 19, Issue 2 https://doi.org/10.1128/cmr.19.2.298-314.2006	journal	April 2006

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The orphan germinant receptor protein GerXAO (but not GerX3b) is essential for L-alanine induced germination in Clostridium botulinum Group II Brunt, Jason; Carter, Andrew T.; Pye, Hannah V. Scientific Reports, Vol. 8, Issue 1 https://doi.org/10.1038/s41598-018-25411-x	journal	May 2018
Functional detection of botulinum neurotoxin serotypes A to F by monoclonal neoepitope-specific antibodies and suspension array technology Von Berg, Laura; Stern, Daniel; Pauly, Diana Robert Koch-Institut https://doi.org/10.25646/6162	text	January 2019
Proteomic analysis of four Clostridium botulinum strains identifies proteins that link biological responses to proteomic signatures Deatherage Kaiser, Brooke L.; Hill, Karen K.; Smith, Theresa J. PLOS ONE, Vol. 13, Issue 10 https://doi.org/10.1371/journal.pone.0205586	journal	October 2018