Quantitative Interaction Effects of Carbon Dioxide, Sodium Chloride, and Sodium Nitrite on Neurotoxin Gene Expression in Nonproteolytic
            <i>Clostridium botulinum</i>
            Type B

Lövenklev, Maria; Artin, Ingrid; Hagberg, Oskar; Borch, Elisabeth; Holst, Elisabet; Rådström, Peter

doi:10.1128/aem.70.5.2928-2934.2004

Cited by 50 publications

(30 citation statements)

References 28 publications

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“…Another approach to ensure that only live cells are detected is reverse-transcription PCR (RT-PCR), where gene expression rather than the gene itself is detected. Reverse transcription-PCR protocols for the botB and botE genes have been described (136,137,146). In a case of a human botulism outbreak, however, laborious procedures to obtain high-quality RNA may prove to be too time-consuming.…”

Section: Molecular Detection Of Clostridium Botulinummentioning

confidence: 99%

“…Furthermore, the requirements for selection of reagents, reaction standardization, and sample pretreatment are higher than those for conventional PCR. Quantitative detection of the botB gene has been employed for research purposes (136,137), whereas a recent application allowed the quantitative detection of C. botulinum type A spores spiked in canned corn and sausage (223) ( Table 3). In this method, DNA is extracted directly from spores and no enrichment steps are employed.…”

Section: Molecular Detection Of Clostridium Botulinummentioning

confidence: 99%

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Laboratory Diagnostics of Botulism

2006

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SUMMARY Botulism is a potentially lethal paralytic disease caused by botulinum neurotoxin. Human pathogenic neurotoxins of types A, B, E, and F are produced by a diverse group of anaerobic spore-forming bacteria, including Clostridium botulinum groups I and II, Clostridium butyricum, and Clostridium baratii. The routine laboratory diagnostics of botulism is based on the detection of botulinum neurotoxin in the patient. Detection of toxin-producing clostridia in the patient and/or the vehicle confirms the diagnosis. The neurotoxin detection is based on the mouse lethality assay. Sensitive and rapid in vitro assays have been developed, but they have not yet been appropriately validated on clinical and food matrices. Culture methods for C. botulinum are poorly developed, and efficient isolation and identification tools are lacking. Molecular techniques targeted to the neurotoxin genes are ideal for the detection and identification of C. botulinum, but they do not detect biologically active neurotoxin and should not be used alone. Apart from rapid diagnosis, the laboratory diagnostics of botulism should aim at increasing our understanding of the epidemiology and prevention of the disease. Therefore, the toxin-producing organisms should be routinely isolated from the patient and the vehicle. The physiological group and genetic traits of the isolates should be determined.

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Section: Molecular Detection Of Clostridium Botulinummentioning

confidence: 99%

Section: Molecular Detection Of Clostridium Botulinummentioning

confidence: 99%

Laboratory Diagnostics of Botulism

2006

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“…Interestingly, there was a hierarchy of expression signal; at all CO 2 concentrations CBO1974-1978 Ͼ CBO0123-0125 Ͼ CBO2797-2795. (5,13,15,20). Previous studies using proteolytic C. botulinum reported a delay in neurotoxin formation with headspace con- centrations in excess of 45% CO 2 (11,12,17,18), although a further report indicated that neurotoxin formation was unaffected by sparging with 100% CO 2 (30).…”

Section: Resultsmentioning

confidence: 96%

“…However, strains of nonproteolytic C. botulinum types B and E, despite having increased lag time and reduced growth rate (13,15), display an increase in both neurotoxin gene expression and neurotoxin formation with higher CO 2 concentrations (5,20). The purpose of the present study was to quantify the effect of CO 2 concentration on the growth, relative gene expression, and neurotoxin production by proteolytic C. botulinum type A1 strain ATCC 3502.…”

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confidence: 97%

Effects of Carbon Dioxide on Growth of ProteolyticClostridium botulinum, Its Ability To Produce Neurotoxin, and Its Transcriptome

Artin

Mason

et al. 2010

Appl Environ Microbiol

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The antimicrobial gas carbon dioxide is frequently used in modified atmosphere packaging. In the present study, the effects of CO 2 (10 to 70%, vol/vol) on gene expression (measured using quantitative reverse transcription-PCR and a whole-genome DNA microarray) and neurotoxin formation (measured using an enzyme-linked immunosorbent assay [ELISA]) by proteolytic Clostridium botulinum type A1 strain ATCC 3502 were studied during the growth cycle. Interestingly, in marked contrast to the situation with nonproteolytic C. botulinum types B and E, CO 2 had little effect on any of these parameters. At all CO 2 concentrations, relative expression of neurotoxin cluster genes peaked in the transition between exponential and stationary phases, with evidence of a second rise in expression in late stationary phase. Microarray analysis enabled identification of coding sequences whose expression profiles matched those of the neurotoxin cluster. Further research is needed to determine whether these are connected to neurotoxin formation or are merely growth phase associated.Proteolytic Clostridium botulinum and nonproteolytic C. botulinum are the two neurotoxigenic clostridia most frequently associated with food-borne botulism (22,23,24), an intoxication caused by consumption of contaminated food containing preformed botulinum neurotoxin. The botulinum neurotoxins are the most potent toxins known, with as little as 30 ng being potentially fatal (24, 25). There are seven botulinum neurotoxin subtypes (A to G) and also a number of subtypes, of which subtype A1 has received the most attention to date (25 Carbon dioxide is frequently used as an antimicrobial gas in modified atmosphere packaging (MAP) of food (14,22,23). However, strains of nonproteolytic C. botulinum types B and E, despite having increased lag time and reduced growth rate (13, 15), display an increase in both neurotoxin gene expression and neurotoxin formation with higher CO 2 concentrations (5, 20). The purpose of the present study was to quantify the effect of CO 2 concentration on the growth, relative gene expression, and neurotoxin production by proteolytic C. botulinum type A1 strain ATCC 3502. Relative expression of cntA/A1 and cntR was measured using a new quantitative reverse transcription-PCR (qRT-PCR) method, and a DNA microarray was used to study the transcriptome. MATERIALS AND METHODSCulture conditions and growth parameters. Overnight cultures of proteolytic C. botulinum type A1 strain ATCC 3502 were grown in TPY medium flushed with the appropriate gas mixture (10%, 35%, or 70% CO 2 ; 10% H 2 ; and the balance N 2 [by vol]) and used to inoculate flasks of similarly treated TPY-C broth (5). The initial pH values of the media were 6.8 to 6.4, reflecting the increasing CO 2 concentration. Cultures were grown at 37°C in an anaerobic workstation with the same atmosphere, each in triplicate. Samples for RNA and neurotoxin measurement were withdrawn at four to nine time points during the experiment. Maximum specific growth rates [(OD)] of the change in optical...

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“…Some researchers have reported the detection and/or quantification of C. botulinum neurotoxin genes for type A 23) , for types A, B, and E 24), 25) , for types A and B 26) , and for type E 27) . Other real-time PCR approaches, which involve quantification of RNA expression, were reported for types A and E 28) , for type B 29), 30) , and for type D 31) . These representative studies did not use duplex PCR quantification methods.…”

Section: Introductionmentioning

confidence: 99%

Quantitative Duplex PCR of Clostridium botulinum Types A and B Neurotoxin Genes

Kasai

Kimura²,

Tajima³

et al. 2007

J. Food Hyg. Soc. Jpn

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A duplex quantitative polymerase chain reaction (PCR) assay for Clostridium botulinum types A and B was developed. The sensitivity and specificity of the assay were verified by using 6 strains of type A, 7 strains of type B, and 14 genera of 42 non-C. botulinum types A and B strains, including C. botulinum types C, D, E, F, and G. In pure culture, the detection limit was 10 2 CFU/ mL for type A and 10 3 CFU/mL for type B. In mushroom broth, increases in the amounts of C. botulinum types A and B could be monitored separately (the quantifiable range was 10 2 to 10 6 for type A and 10 2 to 10 7 for type B) from each sample that contained a large number of background bacteria, and toxin could be detected much earlier than with mouse assay. These results suggest that duplex quantitative PCR methods are useful to detect and quantify C. botulinum types A and/ or B toxin genes.

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Quantitative Interaction Effects of Carbon Dioxide, Sodium Chloride, and Sodium Nitrite on Neurotoxin Gene Expression in Nonproteolytic Clostridium botulinum Type B

Cited by 50 publications

References 28 publications

Laboratory Diagnostics of Botulism

Laboratory Diagnostics of Botulism

Effects of Carbon Dioxide on Growth of ProteolyticClostridium botulinum, Its Ability To Produce Neurotoxin, and Its Transcriptome

Quantitative Duplex PCR of Clostridium botulinum Types A and B Neurotoxin Genes

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