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PCR assay for differentiating between group I (proteolytic) and group II
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(nonproteolytic) strains of Clostridium botulinum
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Running title: PCR for differentiating group I and II C. botulinum
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Short communication
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Elias Dahlsten, Hannu Korkeala, Panu Somervuo, & Miia Lindström*
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Department of Food and Environmental Hygiene, University of Helsinki, Helsinki, Finland
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*
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Miia Lindström
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Department of Food and Environmental Hygiene
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P.O. Box 66
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00014 University of Helsinki
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Finland
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Phone: 358-9-19157107
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Fax: 358-9-19157101
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E-mail: miia.lindstrom@helsinki.fi
Corresponding author:
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ABSTRACT
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Group I (proteolytic) and II (nonproteolytic) C. botulinum are genetically and physiologically distinct
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groups of organisms, with both groups being involved with human botulism. Due to differences in
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spore heat resistance and growth characteristics, the two groups possess different types of human health
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risks through foods, drink, and the environment. The epidemiology of human botulism due to Group I
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and II C. botulinum is poorly understood, largely due to insufficient characterization of disease isolates,
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and warrants thorough outbreak investigation with a particular attention to discrimination between the
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different physiological groups of C. botulinum. In this study, a PCR assay was developed to
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discriminate between Group I and Group II C. botulinum. The assay is based on the fldB associated
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with phenylalanine metabolism in proteolytic clostridia, and employs an internal amplification control
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targeted to conservative regions of 16S rrn in Group I and II C. botulinum. The assay correctly
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identified all 36 Group I and 24 Group II C. botulinum strains, possessing a 100% exclusivity and
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inclusivity. The assay provides a substantial improvement in discriminating between the Group I and II
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C. botulinum, which traditionally is based on a time-consuming and error-prone culture method.
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Differentiation between the physiological groups of C. botulinum is an essential step in investigation of
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human botulism outbreaks, and should be considered as a diagnostic corner-stone in order to improve
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our epidemiological understanding of human botulism.
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Keywords: Clostridium botulinum, Group I, Group II, botulism, epidemiological investigation
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INTRODUCTION
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Clostridium botulinum produces highly potent botulinum neurotoxin, which causes paralytic disease,
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botulism, in humans and animals. The human disease-related C. botulinum strains are divided into four
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toxinotypes (A, B, E, and F) and two metabolic groups (I and II). Group I strains produce one or two of
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toxin types A, B, and F, and Group II strains produce toxin B, E, or F. The two groups differ in their
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genetics, physiology, and epidemiology; thus they cause a variety of forms of botulism in humans.
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The classical foodborne botulism is an intoxication resulting from ingestion of preformed
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neurotoxin with food or drink and involves equally Group I and II C. botulinum. Food botulism
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outbreaks due to the mesophilic Group I are often related to canned foods and vegetables, while the
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psychrotrophic Group II is more frequently associated with packaged and chilled marine foods
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(Lindström, Kiviniemi & Korkeala, 2006). Group I C. botulinum has also been involved in infant,
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intestinal, and wound botulism, which actually are infections as they all employ in vivo outgrowth and
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toxin production from C. botulinum cells or spores (reviewed by Lindström & Korkeala, 2006).
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However, the epidemiological scene is becoming more complex along with observations on Group II
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C. botulinum being involved with infectious forms of botulism, such as wound botulism (Artin,
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Björkman, Cronqvist, Rådström & Holst, 2007). To better understand the obscure epidemiology of the
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different forms of human botulism and their causative agents, increased attention on routine diagnostics
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is warranted.
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Identification of C. botulinum is traditionally based on botulinum neurotoxin production.
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Further characterization of disease isolates has been ignored until these days, which has left a large
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knowledge gap in the epidemiology of botulism. The distinct genetic, phenotypic and metabolic
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characteristics of the different physiological groups (Hutson, Thompson, Lawson, Schocken-Itturino,
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Bottger & Collins, 1993; Hielm, Björkroth, Hyytiä & Korkeala, 1999; Keto-Timonen, Nevas &
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Korkeala, 2005; Hill et al., 2007) suggest the four groups to represent different phylogenetic lineages
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(Collins & East, 1998). Differentiation between Group I and II C. botulinum strains currently relies on
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testing proteolytic activity of Group I but not Group II strains on a casein-based agar. This method
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takes at least 48 h to complete and is prone to error. Amplified fragment length polymorphism analysis
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(Keto-Timonen et al., 2005), ribotyping (Hielm et al. 1999), PCR-restriction fragment length
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polymorphism analysis (Paul et al., 2007) and a method utilizing focal plane array Fourier transform
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infrared (FPA-FTIR) spectroscopy (Kirkwood et al., 2006) have also been demonstrated to distinguish
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between Group I and II strains. Drawbacks to these approaches, however, include the requirement for
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special equipment and being relatively costly and time-consuming for routine clinical use when
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compared to traditional PCR.
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The amino acid phenylalanine (Phe) is utilized by proteolytic clostridia for ATP production
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(Elsden, Hilton & Waller, 1976; Bader, Rauschenbach & Simon, 1982). An enzyme complex
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FldA(I)BC (phenyllactate dehydratase) isolated from C. sporogenes, a close nontoxigenic relative of
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Group I C. botulinum (Sebaihia et al. 2007), has been demonstrated to play a key role in usage of Phe
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for energy production, and the gene cluster fldAIBC encoding the complex has been sequenced
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(Dickert, Pierik & Buckel, 2002). Phe is also required in culture media by Group I C. botulinum in
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amounts exceeding biosynthesis requirements, while nutrient requirements of Group II strains are
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limited to carbohydrates and organic nitrogen compounds (Whitmer & Johnson, 1988). Thus, the
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mechanisms involved in metabolism of Phe can be expected to be present in C. sporogenes and Group I
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C. botulinum, but not in Group II C. botulinum. The presence of the fldAIBC cluster in the recently
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sequenced genome of the proteolytic C. botulinum strain ATCC 3502 has been confirmed (Sebaihia et
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al., 2007). The gene cluster could therefore be used in molecular differentiation between Group I and II
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C. botulinum strains.
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The aim of this study was to establish a PCR assay for differentiation between Group I and II
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C. botulinum isolates. The assay is targeted to the fldAIBC gene cluster and uses an internal
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amplification control targeted to 16S rrn.
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MATERIALS AND METHODS
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Bacterial strains and template preparation. A total of 60 C. botulinum strains from a wide variety of
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sources were included in the analysis (Table 1). The physiological group of the strains was confirmed
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on a casein-based agar. Thirty-six strains represented Group I and 24 strains represented Group II. The
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toxinotype of each strain was confirmed using multiplex PCR (Lindström, Keto, Markkula, Nevas,
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Hielm & Korkeala, 2001).
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Refrigerated spore suspensions were inoculated on egg yolk agar plates and incubated
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anaerobically for 4 d at 30°C (Group II) or 37°C (Group I). Pure colonies were transferred into 10 ml
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of anaerobic tryptone-peptone-glucose-yeast extract broth and incubated anaerobically overnight at
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respective temperatures. One ml of each overnight culture was centrifuged in a 1.5-ml Eppendorf tube
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for 3 min at 13000 G. The cells were resuspended in one milliliter of 10:1 TE (10 mM Tris,
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1 mM EDTA), incubated for 30 min at 37°C and centrifuged for 3 min at 13000 G. The cells were
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resuspended in one ml of sterile water. One µl of heated (95°C, 10 min) cell suspensions were used as
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template in each PCR.
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PCR and agarose gel electrophoresis. Primers (CBEDfldB-f and CBEDfldB-r) targeted to
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gene fldB were designed with Primer3 software (Rozen & Skaletsky, 2000), along with an internal
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amplification control primer pair (CBEDiac-f and CBEDiac-r) targeted to 16S rrn sequences conserved
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in Group I and II C. botulinum (Hutson et al., 1993) (Table 2). The PCR mastermix (reaction volume
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50 µl) contained 10 mM Tris-HCl, 1.5 mM MgCl2, 50 mM KCl, 0.1 % Triton X-100 (DyNAzyme
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Reaction Buffer, Finnzymes, Espoo, Finland), 200 µM each dNTP (dNTP Mix, Finnzymes),
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0.5 µM CBEDfldB primer mix (Oligomer, Helsinki, Finland), 0.1 µM CBEDiac primer mix
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(Oligomer), and two units of DyNAzyme II DNA polymerase (Finnzymes). One microliter of template
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was used per reaction. The PCR cycle contained 28 cycles of denaturation, annealing, and extension at
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95°C for 30 s, 53°C for 30 s, and 72°C for 30 s, respectively, and a final extension step at 72°C for
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5 min. The reactions were performed in a 96-well thermal cycler (DNA Engine; MJ Research,
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Waltham, MA). The PCR products were visualized electrophoretically in 2% agarose gel (I.D.NA
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Agarose, Cambrex Bio Science Rockland, Rockland, ME) stained with ethidium bromide, running at
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90 V for 1 h 45 min. Standard DNA fragments (DNA molecular weight marker VIII; Roche
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Diagnostics, Mannheim, Germany) were used to confirm the sizes of the PCR products.
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RESULTS AND DISCUSSION
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The designed PCR assay gave expected results for all 60 C. botulinum strains: a 552-bp group-specific
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product and a 761-bp internal control product for all 36 Group I strains, and only a 761-bp control
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product for all 24 Group II strains (Fig. 1). Compared with the conventional culture method based on
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casein digestion, this assay is substantially faster, simpler, and more accurate in differentiating between
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C. botulinum Group I and II strains. Traditional PCR is in everyday use in numerous laboratories, thus
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novel PCR assays can be incorporated with little additional effort. It should, however, be noted that the
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designed primers target a gene associated with metabolism of several bacteria; thus the assay cannot be
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utilized in detection or species identification of C. botulinum in clinical, food, or environmental
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samples. Several protocols to accomplish these tasks have been previously described (Lindström &
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Korkeala, 2006). The current assay has been designed to characterize pure cultures, previously
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identified as C. botulinum (type A, B, E or F), into Group I or II. As discussed above, Groups I and II
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C. botulinum strains pose a wide variety of risks to human health. In any case of foodborne, infant,
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intestinal, or wound botulism, an in-depth characterization of the causative organism is of utmost
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importance to increase our understanding of the epidemiology of Group I and II C. botulinum and
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human botulism and ultimately to prevent further outbreaks.
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ACKNOWLEDGEMENTS
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This study was a part of a larger research project financially supported by the Academy of Finland
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(206319), the Finnish Ministry of Agriculture and Forestry (4655/501/2003), the Finnish Funding
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Agency for Technology and Innovation (Tekes) (2431/31/04), and the Walter Ehrström Foundation.
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The authors thank Hanna Korpunen for excellent technical assistance.
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REFERENCES
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Artin, I., Björkman, P., Cronqvist, J., Rådström, P., Holst, E., 2007. First case of type E wound
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botulism diagnosed using real-time PCR. Journal of Clinical Microbiology 45, 3589-3594.
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Bader, J., Rauschenbach, P., Simon, H., 1982. On a hitherto unknown fermentation path of several
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amino acids by proteolytic clostridia. FEBS Letters 140, 67-72.
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Collins, M.D., East, A.K., 1998. Phylogeny and taxonomy of the food-borne pathogen Clostridium
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botulinum and its neurotoxins. Journal of Applied Microbiology 84, 5-17.
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Dickert, S., Pierik, A.J., Buckel, W., 2002. Molecular characterization of phenyllactate dehydratase
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and its initiator from Clostridium sporogenes. Molecular Microbiology 44, 49-60.
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Elsden, S.R., Hilton, M.G., Waller, J.M., 1976. The end products of the metabolism of aromatic amino
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acids by Clostridia. Archives of Microbiology 107, 283-288.
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Hielm, S., Björkroth, J., Hyytiä, E., Korkeala, H., 1999. Ribotyping as an identification tool for
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Clostridium botulinum strains causing human botulism. International Journal of Food Microbiology 47,
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121-131.
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Hill, K.K., Smith, T.J., Helma, C.H., Ticknor, L.O., Foley, B.T., Svensson, R.T., Brown, J.L., Johnson,
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E.A., Smith, L.A., Okinaka, R.T., Jackson, P.J., Marks, J.D., 2007. Genetic diversity among botulinum
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neurotoxin-producing clostridial strains. Journal of Bacteriology 189, 818-832.
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Hutson, R.A., Thompson, D.E., Lawson, P.A., Schocken-Itturino, R.P., Bottger, E.C., Collins, M.D.,
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1993. Genetic interrelationships of proteolytic Clostridium botulinum types A, B, and F and other
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members of the Clostridium botulinum complex as revealed by small-subunit rRNA gene sequences.
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Antonie Van Leeuwenhoek 64, 273-283.
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Keto-Timonen, R., Nevas, M., Korkeala, H., 2005. Efficient DNA Fingerprinting of Clostridium
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botulinum types A, B, E, and F by amplified fragment length polymorphism analysis. Applied and
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Environmental Microbiology 71, 1148-1154.
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Kirkwood, J., Ghetler, A., Sedman, J., Leclair, D., Pagotto, F., Austin, J.W., Ismail, A.A., 2006.
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Differentiation of group I and group II strains of Clostridium botulinum by focal plane array Fourier
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transform infrared spectroscopy. Journal of Food Protection 69, 2377-2383.
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Lindström, M., Keto, R., Markkula, A., Nevas, M., Hielm, S., Korkeala, H., 2001. Multiplex PCR
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assay for detection and identification of Clostridium botulinum types A, B, E, and F in food and fecal
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material. Applied and Environmental Microbiology 67, 5694-5699.
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Lindström, M., Kiviniemi, K., Korkeala, H., 2006. Hazard and control of group II (non-proteolytic)
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Clostridium botulinum in modern food processing. International Journal of Food Microbiology 108, 92-
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Lindström, M., Korkeala, H., 2006. Laboratory diagnostics of botulism. Clinical Microbiology
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Paul, C.J., Twine, S.M., Tam, K.J., Mullen, J.A., Kelly, J.F., Austin, J.W., Logan, S.M., 2007.
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Flagellin diversity in Clostridium botulinum groups I and II: a new strategy for strain identification.
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Applied and Environmental Microbiology 73, 2963-2975.
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programmers. In: Krawetz, S., Misener, S. (Eds.), Bioinformatics Methods and Protocols: Methods in
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FIGURE LEGENDS
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Fig. 1. PCR assay for differentiating between group I (proteolytic) and group II (nonproteolytic) strains
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of Clostridium botulinum. Lanes: 1 and 10, molecular weight marker VIII; 2, C. botulinum group I
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type A; 3, C. botulinum group I type B; 4, C. botulinum group I type F; 5, C. botulinum group I
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type AB; 6, C. botulinum group II type B; 7, C. botulinum group II type E; 8, C. botulinum group II
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type F; and 9, negative control.
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TABLE 1. Clostridium botulinum strains used in this study.
Strain
Origin
Location of isolation
128-16
62A
C5
Hall A (ATCC 3502)
J8
Lindroth RS-3 A
NCTC 7272 A (ATCC 19397)
SL-2A
SL-3A
SL-4A
SL-6A
Smith VPI 1550 A (ATCC 25763)
TA 100/1
Hegarty 213 B (ATCC 7949)
McClung 133-4803 B
SL-1B
SL-5B
126B
M16/18
TA 116/2
NKH 50/1
P 1/3
KV 16/4
P 2/5
M16/5
87/6A
97/10
97/11 (NCFB 3037)
97/12 (NCFB 3036)
97/15 (NCTC 2916)
Langeland F (ATCC 35415)
Swine blood
Cow liver
Vegetable sausage
NDd
Vegetable sausage
Pacific red snapper
ND
ND
ND
ND
ND
Type strain
Honey
Canned shallots
ND
ND
ND
ND
Soil
Honey
Honey
Canned deer meat
Beeswax
Canned deer meat
Soil
Sauteed onion
ND
Infant
Botulism outbreak
Corn
Liver paste
Finland
USA
Finland
USA
Finland
Pacific Coast, USA
ND
ND
ND
ND
ND
ND
Denmark
USA
USA
ND
ND
ND
Finland
Denmark
Norway
Finland
Finland
Finland
Finland
USA
USA
ND
ND
ND
Denmark
PCR result
Proteolytic
for botulinum
activitya
neurotoxinb
+
A
+
A
+
A
+
A
+
A
+
A
+
A
+
A
+
A
+
A
+
A
+
A
+
A
+
B
+
B
+
B
+
B
+
B
+
B
+
B
+
B
+
B
+
B
+
B
+
B
+
AB
+
AB
+
AB
+
AB
+
AB
+
F
PCR result for
metabolic
groupc
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
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TABLE 1 continues.
Strain
Origin
F4VI F
Blue crab viscera
Crab F
Pasteurized crabmeat
Walls FT 42 F (ATCC 25764)
Crab
93/28 (NCTC 10281)
ND
93/31 (FT 14)
ND
Eklund 2B
Marine sediment
Eklund 17B
Marine sediment
Hatheway 706B
Salmon
McClung 2217B (ATCC 17844)
ND
BL 86/17 (Colworth 151)
ND
BL 87/7B (5900 A-T-A-3)
Human
BL 90/4 (Prevot 59)
ND
BL 93/10 (Kapchunka B2)
Fish
Hauschild 31-2570 E
ND
N/341
River lamprey
11/1-1
Whitefish roe
Beluga E
Beluga flipper
Dolman 211 E
Pickled herring
C-51 E
Sealmeat
C-60 E
Dried mutton
CB-S14 E
Fishpond sediment
CB-K35 E
Vendace
CB-K90 E
Chub
Eklund 202F (ATCC 23387)
Marine sediment
Craig 610B8-6F (F56)
Salmon
Hobbs FT 10 F
Marine sediment
86/32 (Colworth 47)
ND
86/33 (Colworth 187)
ND
86/34 (Colworth 195)
ND
a
As observed by the strain’s ability to digest casein.
Location of isolation
York River, UK
USA
UK
ND
ND
Pacific Coast, USA
Pacific Coast, USA
Alaska, USA
ND
ND
ND
ND
ND
USA
Finland
Finland
Alaska, USA
Vancouver, Canada
Greenland, Denmark
Faeroes, Denmark
Finland
Gulf of Bothnia, Finland
Germany
Pacific Coast, USA
Columbia River, USA
UK
ND
ND
ND
PCR result
Proteolytic for botulinum
activitya
neurotoxinb
+
F
+
F
+
F
+
F
+
F
B
B
B
B
B
B
B
B
E
E
E
E
E
E
E
E
E
E
F
F
F
F
F
F
PCR result for
metabolic
groupc
I
I
I
I
I
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
12
218
b
As tested with multiplex PCR for botulinum neurotoxin genes (Lindström et al., 2001).
219
c
As a result of the PCR assay presented here.
220
d
ND, no data available.
221
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222
TABLE 2. Primer pairs designed for differentiating between group I and II Clostridium botulinum strains and for internal amplification
223
control.
Primer
Sequence
(5'-3')
Melting
temperature
(° C)
CBEDfldB-F
ggggagaaaaagttggttgg
57.3
20
50.0
CBEDfldB-R
cgaatccactaaatggtgaagg
58.4
22
45.5
CBEDiac-F
cgacctgagagggtgatcg
61.0
19
63.2
CBEDiac-R
gagctgacgacaaccatgc
58.8
19
57.9
Length
(bases)
GC content Size of product
(%)
(bp)
552
761
224
14