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

Abstract

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 valuablemore » 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.« less

Authors:
 [1];  [1];  [2];  [3];  [1];  [1];  [1];  [4];  [5];  [5];  [1]; ORCiD logo [1];  [1];  [6]
  1. Northern Arizona University, Flagstaff, Arizona, (United States). Pathogen and Microbiome Institute
  2. Los Alamos National Lab. (LANL), Los Alamos, NM (United States). Bioscience Division
  3. United States Army Medical Research Institute of Infectious Diseases, Frederick, Maryland (United States). Molecular and Translational Sciences Division
  4. Northern Arizona University, Flagstaff, Arizona (United States). Informatics, Computing and Cyber Systems
  5. Área Microbiología, Departamento de Patología, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Centro Universitario, Mendoza (Argentina)
  6. Univ. of Helsinki (Finland)
Publication Date:
Research Org.:
Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)
Sponsoring Org.:
USDOE; U.S. Department of Homeland Security
OSTI Identifier:
1626064
Grant/Contract Number:  
AC52-06NA25396; HSHQDC-16-C-B0013
Resource Type:
Accepted Manuscript
Journal Name:
Applied and Environmental Microbiology
Additional Journal Information:
Journal Volume: 83; Journal Issue: 18; Journal ID: ISSN 0099-2240
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Biotechnology & Applied Microbiology; Microbiology

Citation Formats

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., and Björkroth, Johanna. Differentiating Botulinum Neurotoxin-Producing Clostridia with a Simple, Multiplex PCR Assay. United States: N. p., 2017. Web. doi:10.1128/aem.00806-17.
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. Differentiating Botulinum Neurotoxin-Producing Clostridia with a Simple, Multiplex PCR Assay. United States. https://doi.org/10.1128/aem.00806-17
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., and Björkroth, Johanna. Fri . "Differentiating Botulinum Neurotoxin-Producing Clostridia with a Simple, Multiplex PCR Assay". United States. https://doi.org/10.1128/aem.00806-17. https://www.osti.gov/servlets/purl/1626064.
@article{osti_1626064,
title = {Differentiating Botulinum Neurotoxin-Producing Clostridia with a Simple, Multiplex PCR Assay},
author = {Williamson, Charles H. D. and Vazquez, Adam J. and Hill, Karen and Smith, Theresa J. and Nottingham, Roxanne and Stone, Nathan E. and Sobek, Colin J. and Cocking, Jill H. and Fernández, Rafael A. and Caballero, Patricia A. and Leiser, Owen P. and Keim, Paul and Sahl, Jason W. and Björkroth, Johanna},
abstractNote = {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.},
doi = {10.1128/aem.00806-17},
journal = {Applied and Environmental Microbiology},
number = 18,
volume = 83,
place = {United States},
year = {Fri Jul 21 00:00:00 EDT 2017},
month = {Fri Jul 21 00:00:00 EDT 2017}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Figures / Tables:

FIG 1 FIG 1: Phylogenies differentiating BoNT-producing bacteria and heat maps of BSR values. (A) BoNT-producing clostridia were differentiated with a phylogeny inferred using an alignment of rpoB genes. (B) Members of C. botulinum group I, C. sporogenes, and members of C. botulinum group II were further delineated with core genome SNPmore » phylogenies. The SNP phylogenies differentiate C. botulinum group I from C. sporogenes and separate C. botulinum group II into two major subgroups. The heat maps alongside the core genome SNP phylogenetic trees indicate the BSR values of the markers identified with LS-BSR. BSR values are used as a measure of the relatedness of coding region sequences between genomes. Blue boxes indicate the presence of the coding region sequence in a genome assembly, while gray boxes indicate that the coding region sequence is absent.« less

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Works referenced in this record:

Botulinum Neurotoxin Is Shielded by NTNHA in an Interlocked Complex
journal, February 2012


BayesHammer: Bayesian clustering for error correction in single-cell sequencing
journal, January 2013

  • Nikolenko, Sergey I.; Korobeynikov, Anton I.; Alekseyev, Max A.
  • BMC Genomics, Vol. 14, Issue Suppl 1
  • DOI: 10.1186/1471-2164-14-S1-S7

rrnDB: improved tools for interpreting rRNA gene abundance in bacteria and archaea and a new foundation for future development
journal, November 2014

  • Stoddard, Steven F.; Smith, Byron J.; Hein, Robert
  • Nucleic Acids Research, Vol. 43, Issue D1
  • DOI: 10.1093/nar/gku1201

Comparative genomic analyses reveal broad diversity in botulinum-toxin-producing Clostridia
journal, March 2016


Versatile and open software for comparing large genomes
journal, January 2004

  • Kurtz, Stefan; Phillippy, Adam; Delcher, Arthur L.
  • Genome Biology, Vol. 5, Issue 2, R12
  • DOI: 10.1186/gb-2004-5-2-r12

BLAT---The BLAST-Like Alignment Tool
journal, March 2002


Phylogeny and taxonomy of the food-borne pathogen Clostridium botulinum and its neurotoxins
journal, January 1998


Phylogenetic analysis of Clostridium botulinum type A by multi-locus sequence typing
journal, August 2008


Global Occurrence of Infant Botulism, 1976-2006
journal, July 2008


Mechanisms of food processing and storage-related stress tolerance in Clostridium botulinum
journal, May 2015


High prevalence of Clostridium botulinum types A and B in honey samples detected by polymerase chain reaction
journal, January 2002

  • Nevas, Mari; Hielm, Sebastian; Lindström, Miia
  • International Journal of Food Microbiology, Vol. 72, Issue 1-2
  • DOI: 10.1016/S0168-1605(01)00615-8

More than 50% of Clostridium difficile Isolates from Pet Dogs in Flagstaff, USA, Carry Toxigenic Genotypes
journal, October 2016


NASP: an accurate, rapid method for the identification of SNPs in WGS datasets that supports flexible input and output formats
journal, August 2016

  • Sahl, Jason W.; Lemmer, Darrin; Travis, Jason
  • Microbial Genomics, Vol. 2, Issue 8
  • DOI: 10.1099/mgen.0.000074

Base-Calling of Automated Sequencer Traces Using Phred.  I. Accuracy Assessment
journal, March 1998

  • Ewing, Brent; Hillier, LaDeana; Wendl, Michael C.
  • Genome Research, Vol. 8, Issue 3
  • DOI: 10.1101/gr.8.3.175

Pentaplexed Quantitative Real-Time PCR Assay for the Simultaneous Detection and Quantification of Botulinum Neurotoxin-Producing Clostridia in Food and Clinical Samples
journal, April 2010

  • Kirchner, Sebastian; Krämer, K. Melanie; Schulze, Martin
  • Applied and Environmental Microbiology, Vol. 76, Issue 13
  • DOI: 10.1128/AEM.02490-09

Trimmomatic: a flexible trimmer for Illumina sequence data
journal, April 2014


Characteristics of Clostridium botulinum Type F Isolated from the Pacific Coast of the United States
journal, January 1967


Clostridium botulinum Type F: Isolation from Venison Jerky
journal, January 1972


FastTree 2 – Approximately Maximum-Likelihood Trees for Large Alignments
journal, March 2010


Genetic Characterization of the Exceptionally High Heat Resistance of the Non-toxic Surrogate Clostridium sporogenes PA 3679
journal, April 2017


Prodigal: prokaryotic gene recognition and translation initiation site identification
journal, March 2010


Botulinum neurotoxins: genetic, structural and mechanistic insights
journal, June 2014

  • Rossetto, Ornella; Pirazzini, Marco; Montecucco, Cesare
  • Nature Reviews Microbiology, Vol. 12, Issue 8
  • DOI: 10.1038/nrmicro3295

ART: a next-generation sequencing read simulator
journal, December 2011


Simple Method for Detection of Clostridium botulinum Type A to F Neurotoxin Genes by Ploymerase Chain Reaction
journal, January 1996


BactQuant: An enhanced broad-coverage bacterial quantitative real-time PCR assay
journal, January 2012


Infant Botulism
journal, October 1976


IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies
journal, November 2014

  • Nguyen, Lam-Tung; Schmidt, Heiko A.; von Haeseler, Arndt
  • Molecular Biology and Evolution, Vol. 32, Issue 1
  • DOI: 10.1093/molbev/msu300

Base-Calling of Automated Sequencer Traces Using Phred. II. Error Probabilities
journal, March 1998


Recommended Mass Spectrometry-Based Strategies to Identify Botulinum Neurotoxin-Containing Samples
journal, May 2015


A framework for variation discovery and genotyping using next-generation DNA sequencing data
journal, April 2011

  • DePristo, Mark A.; Banks, Eric; Poplin, Ryan
  • Nature Genetics, Vol. 43, Issue 5
  • DOI: 10.1038/ng.806

Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees
journal, April 2016

  • Letunic, Ivica; Bork, Peer
  • Nucleic Acids Research, Vol. 44, Issue W1
  • DOI: 10.1093/nar/gkw290

Impact of Clostridium botulinum genomic diversity on food safety
journal, August 2016


Prokka: rapid prokaryotic genome annotation
journal, March 2014


Universal and specific quantitative detection of botulinum neurotoxin genes
journal, January 2010


PrimerProspector: de novo design and taxonomic analysis of barcoded polymerase chain reaction primers
journal, February 2011


The large-scale blast score ratio (LS-BSR) pipeline: a method to rapidly compare genetic content between bacterial genomes
journal, January 2014

  • Sahl, Jason W.; Caporaso, J. Gregory; Rasko, David A.
  • PeerJ, Vol. 2
  • DOI: 10.7717/peerj.332

SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing
journal, May 2012

  • Bankevich, Anton; Nurk, Sergey; Antipov, Dmitry
  • Journal of Computational Biology, Vol. 19, Issue 5
  • DOI: 10.1089/cmb.2012.0021

PCR assay for differentiating between Group I (proteolytic) and Group II (nonproteolytic) strains of Clostridium botulinum
journal, May 2008


phangorn: phylogenetic analysis in R
journal, December 2010


Genomes, neurotoxins and biology of Clostridium botulinum Group I and Group II
journal, May 2015


Basic local alignment search tool
journal, October 1990

  • Altschul, Stephen F.; Gish, Warren; Miller, Webb
  • Journal of Molecular Biology, Vol. 215, Issue 3, p. 403-410
  • DOI: 10.1016/S0022-2836(05)80360-2

Ultrafast Approximation for Phylogenetic Bootstrap
journal, February 2013

  • Minh, B. Q.; Nguyen, M. A. T.; von Haeseler, A.
  • Molecular Biology and Evolution, Vol. 30, Issue 5
  • DOI: 10.1093/molbev/mst024

The Genome Analysis Toolkit: A MapReduce framework for analyzing next-generation DNA sequencing data
journal, July 2010


Analysis of a unique Clostridium botulinum strain from the Southern hemisphere producing a novel type E botulinum neurotoxin subtype
journal, January 2012

  • Raphael, Brian H.; Lautenschlager, Matthew; Kalb, Suzanne R.
  • BMC Microbiology, Vol. 12, Issue 1
  • DOI: 10.1186/1471-2180-12-245

Implications of Genome-Based Discrimination between Clostridium botulinum Group I and Clostridium sporogenes Strains for Bacterial Taxonomy
journal, June 2015

  • Weigand, Michael R.; Pena-Gonzalez, Angela; Shirey, Timothy B.
  • Applied and Environmental Microbiology, Vol. 81, Issue 16
  • DOI: 10.1128/AEM.01159-15

Genomic and physiological variability within Group II (non-proteolytic) Clostridium botulinum
journal, January 2013


Distribution of Clostridium botulinum Type E Strains in Nunavik, Northern Quebec, Canada
journal, November 2012

  • Leclair, Daniel; Farber, Jeffrey M.; Doidge, Bill
  • Applied and Environmental Microbiology, Vol. 79, Issue 2
  • DOI: 10.1128/AEM.05999-11

Presence of Clostridium botulinum spores in Matricaria chamomilla (chamomile) and its relationship with infant botulism
journal, February 2008


Hazard and control of group II (non-proteolytic) Clostridium botulinum in modern food processing
journal, April 2006


MUSCLE: multiple sequence alignment with high accuracy and high throughput
journal, March 2004

  • Edgar, R. C.
  • Nucleic Acids Research, Vol. 32, Issue 5, p. 1792-1797
  • DOI: 10.1093/nar/gkh340

Biodiversity of Clostridium botulinum Type E Associated with a Large Outbreak of Botulism in Wildlife from Lake Erie and Lake Ontario
journal, November 2010

  • Hannett, George E.; Stone, Ward B.; Davis, Stephen W.
  • Applied and Environmental Microbiology, Vol. 77, Issue 3
  • DOI: 10.1128/AEM.01578-10

Pilon: An Integrated Tool for Comprehensive Microbial Variant Detection and Genome Assembly Improvement
journal, November 2014


Fast algorithms for large-scale genome alignment and comparison
journal, June 2002


A detailed analysis of 16S ribosomal RNA gene segments for the diagnosis of pathogenic bacteria
journal, May 2007

  • Chakravorty, Soumitesh; Helb, Danica; Burday, Michele
  • Journal of Microbiological Methods, Vol. 69, Issue 2
  • DOI: 10.1016/j.mimet.2007.02.005

Jalview Version 2--a multiple sequence alignment editor and analysis workbench
journal, January 2009


Improving draft assemblies by iterative mapping and assembly of short reads to eliminate gaps
journal, January 2010


Historical and current perspectives on Clostridium botulinum diversity
journal, May 2015


Genetic Diversity among Botulinum Neurotoxin-Producing Clostridial Strains
journal, November 2006

  • Hill, K. K.; Smith, T. J.; Helma, C. H.
  • Journal of Bacteriology, Vol. 189, Issue 3
  • DOI: 10.1128/JB.01180-06

Characteristics of Clostridium botulinum Type F Isolated from the Pacific Coast of the United States
journal, November 1967


Outgrowth and toxin production of nonproteolytic type B Clostridium botulinum at 3.3 to 5.6 C.
journal, January 1967


PCR and gene probe identification of botulinum neurotoxin A-, B-, E-, F-, and G-producing Clostridium spp. and evaluation in food samples.
journal, January 1995


PCR for detection of Clostridium botulinum type C in avian and environmental samples.
journal, January 1996


Clostridium botulinum Type F: Isolation from Venison Jerky
journal, August 1972


Isolation of Clostridium botulinum from Honey
journal, February 1979


Ancient genomes reveal social and genetic structure of Late Neolithic Switzerland
journal, April 2020

  • Furtwängler, Anja; Rohrlach, A. B.; Lamnidis, Thiseas C.
  • Nature Communications, Vol. 11, Issue 1
  • DOI: 10.1038/s41467-020-15560-x

Why does the Aβ peptide of Alzheimer share structural similarity with antimicrobial peptides?
journal, March 2020

  • Pastore, Annalisa; Raimondi, Francesco; Rajendran, Lawrence
  • Communications Biology, Vol. 3, Issue 1
  • DOI: 10.1038/s42003-020-0865-9

Draft Genome Sequence of Comamonas aquatilis Strain LK (= CSUR P6418 = CECT 9772), Isolated from the Planarian Schmidtea mediterranea
journal, February 2021

  • Kangale, Luis Johnson; Levasseur, Anthony; Raoult, Didier
  • Microbiology Resource Announcements, Vol. 10, Issue 5
  • DOI: 10.1128/mra.00297-20

Draft Genome Sequence of Medusavirus Stheno, Isolated from the Tatakai River of Uji, Japan
journal, January 2021

  • Yoshida, Koki; Zhang, Ruixuan; Garcia, Kimberly G.
  • Microbiology Resource Announcements, Vol. 10, Issue 1
  • DOI: 10.1128/mra.01323-20

Characterization and functional analysis of phytoene synthase gene family in tobacco
journal, January 2021


Comparison of 15 dinoflagellate genomes reveals extensive sequence and structural divergence in family Symbiodiniaceae and genus Symbiodinium
journal, April 2021


Infant Botulism: Three Cases in a Small Town
journal, October 1986


BayesHammer: Bayesian clustering for error correction in single-cell sequencing
text, January 2012


Complete genome analysis of a novel iflavirus from a leaf beetle, Aulacophora lewisii
journal, October 2020


Impact of Clostridium botulinum genomic diversity on food safety
journal, August 2016


Hazard and control of group II (non-proteolytic) Clostridium botulinum in modern food processing
journal, April 2006


PCR assay for differentiating between Group I (proteolytic) and Group II (nonproteolytic) strains of Clostridium botulinum
journal, May 2008


A detailed analysis of 16S ribosomal RNA gene segments for the diagnosis of pathogenic bacteria
journal, May 2007

  • Chakravorty, Soumitesh; Helb, Danica; Burday, Michele
  • Journal of Microbiological Methods, Vol. 69, Issue 2
  • DOI: 10.1016/j.mimet.2007.02.005

Historical and current perspectives on Clostridium botulinum diversity
journal, May 2015


Mechanisms of food processing and storage-related stress tolerance in Clostridium botulinum
journal, May 2015


Genomes, neurotoxins and biology of Clostridium botulinum Group I and Group II
journal, May 2015


A framework for variation discovery and genotyping using next-generation DNA sequencing data
journal, April 2011

  • DePristo, Mark A.; Banks, Eric; Poplin, Ryan
  • Nature Genetics, Vol. 43, Issue 5
  • DOI: 10.1038/ng.806

Botulinum neurotoxins: genetic, structural and mechanistic insights
journal, June 2014

  • Rossetto, Ornella; Pirazzini, Marco; Montecucco, Cesare
  • Nature Reviews Microbiology, Vol. 12, Issue 8
  • DOI: 10.1038/nrmicro3295

Commensal Pseudomonas protect Arabidopsis thaliana from a coexisting pathogen via multiple lineage-dependent mechanisms
journal, December 2021


Rhizosphere bacteriome structure and functions
journal, February 2022


CXCR4 signaling directs Igk recombination and the molecular mechanisms of late B lymphopoiesis
journal, September 2019

  • Mandal, Malay; Okoreeh, Michael K.; Kennedy, Domenick E.
  • Nature Immunology, Vol. 20, Issue 10
  • DOI: 10.1038/s41590-019-0468-0

Acapsular Staphylococcus aureus with a non-functional agr regains capsule expression after passage through the bloodstream in a bacteremia mouse model
journal, August 2020

  • Suligoy, Carlos M.; Díaz, Rocío E.; Gehrke, Ana-Katharina
  • Scientific Reports, Vol. 10, Issue 1
  • DOI: 10.1038/s41598-020-70671-1

Climatic oscillations in Quaternary have shaped the co-evolutionary patterns between the Norway spruce and its host-associated herbivore
journal, October 2020


Multiplex profiling of serum proteins in solution using barcoded antibody fragments and next generation sequencing
journal, July 2020

  • Brofelth, Mattias; Ekstrand, Anna Isinger; Gour, Shashank
  • Communications Biology, Vol. 3, Issue 1
  • DOI: 10.1038/s42003-020-1068-0

Phylogeny and taxonomy of the food-borne pathogen Clostridium botulinum and its neurotoxins
journal, January 1998


Effect of pH and NaCl on growth from spores of non‐proteolytic Clostridium botulinum at chill temperature
journal, February 1997


PrimerProspector: de novo design and taxonomic analysis of barcoded polymerase chain reaction primers
journal, February 2011


Fast algorithms for large-scale genome alignment and comparison
journal, June 2002


MUSCLE: multiple sequence alignment with high accuracy and high throughput
journal, March 2004

  • Edgar, R. C.
  • Nucleic Acids Research, Vol. 32, Issue 5, p. 1792-1797
  • DOI: 10.1093/nar/gkh340

Phylogenetic analysis of Clostridium botulinum type A by multi-locus sequence typing
journal, August 2008


Base-Calling of Automated Sequencer Traces Using Phred. II. Error Probabilities
journal, March 1998


Simple Method for Detection of Clostridium botulinum Type A to F Neurotoxin Genes by Ploymerase Chain Reaction
journal, January 1996


Genetic Diversity of the Flagellin Genes of Clostridium botulinum Groups I and II
journal, April 2013

  • Woudstra, Cedric; Lambert, Dominic; Anniballi, Fabrizio
  • Applied and Environmental Microbiology, Vol. 79, Issue 13
  • DOI: 10.1128/aem.00686-13

Clostridium botulinum Group II Isolate Phylogenomic Profiling Using Whole-Genome Sequence Data
journal, June 2015

  • Weedmark, K. A.; Mabon, P.; Hayden, K. L.
  • Applied and Environmental Microbiology, Vol. 81, Issue 17
  • DOI: 10.1128/aem.01155-15

Genetic Characterization and Comparison of Clostridium botulinum Isolates from Botulism Cases in Japan between 2006 and 2011
journal, September 2014

  • Kenri, Tsuyoshi; Sekizuka, Tsuyoshi; Yamamoto, Akihiko
  • Applied and Environmental Microbiology, Vol. 80, Issue 22
  • DOI: 10.1128/aem.02134-14

Genetic Diversity of Clostridium sporogenes PA 3679 Isolates Obtained from Different Sources as Resolved by Pulsed-Field Gel Electrophoresis and High-Throughput Sequencing
journal, October 2015

  • Schill, Kristin M.; Wang, Yun; Butler, Robert R.
  • Applied and Environmental Microbiology, Vol. 82, Issue 1
  • DOI: 10.1128/aem.02616-15

Flagellin Diversity in Clostridium botulinum Groups I and II: a New Strategy for Strain Identification
journal, March 2007

  • Paul, Catherine J.; Twine, Susan M.; Tam, Kevin J.
  • Applied and Environmental Microbiology, Vol. 73, Issue 9
  • DOI: 10.1128/aem.02623-06

Neurotoxin Gene Profiling of Clostridium botulinum Types C and D Native to Different Countries within Europe
journal, February 2012

  • Woudstra, Cedric; Skarin, Hanna; Anniballi, Fabrizio
  • Applied and Environmental Microbiology, Vol. 78, Issue 9
  • DOI: 10.1128/aem.07568-11

Multiplex PCR Assay for Detection and Identification of Clostridium botulinum Types A, B, E, and F in Food and Fecal Material
journal, December 2001


Distribution of Botulinum Toxin-Producing Clostridia in Soils of Argentina
journal, July 2005

  • Lúquez, Carolina; Bianco, María I.; de Jong, Laura I. T.
  • Applied and Environmental Microbiology, Vol. 71, Issue 7
  • DOI: 10.1128/aem.71.7.4137-4139.2005

Characteristics of Clostridium botulinum Type F Isolated from the Pacific Coast of the United States
journal, November 1967


Clostridium botulinum Type F: Isolation from Venison Jerky
journal, August 1972


Laboratory Diagnostics of Botulism
journal, April 2006


Outgrowth and toxin production of nonproteolytic type B Clostridium botulinum at 3.3 to 5.6 C.
journal, January 1967


PCR for detection of Clostridium botulinum type C in avian and environmental samples.
journal, January 1996


Isolation of Clostridium botulinum from Honey
journal, February 1979


Universal and specific quantitative detection of botulinum neurotoxin genes
journal, January 2010


BactQuant: An enhanced broad-coverage bacterial quantitative real-time PCR assay
journal, January 2012


Improving draft assemblies by iterative mapping and assembly of short reads to eliminate gaps
journal, January 2010


Comparative genomic analyses reveal broad diversity in botulinum-toxin-producing Clostridia
journal, March 2016


FastTree 2 – Approximately Maximum-Likelihood Trees for Large Alignments
journal, March 2010


More than 50% of Clostridium difficile Isolates from Pet Dogs in Flagstaff, USA, Carry Toxigenic Genotypes
journal, October 2016


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  • Sahl, Jason W.; Caporaso, J. Gregory; Rasko, David A.
  • PeerJ, Vol. 2
  • DOI: 10.7717/peerj.332

Works referencing / citing this record:

Functional detection of botulinum neurotoxin serotypes A to F by monoclonal neoepitope-specific antibodies and suspension array technology
journal, April 2019


Proteomic analysis of four Clostridium botulinum strains identifies proteins that link biological responses to proteomic signatures
journal, October 2018


Figures/Tables have been extracted from DOE-funded journal article accepted manuscripts.