Molecular Gene Profiling of Clostridium botulinum Group III and Its Detection in Naturally Contaminated Samples Originating from Various European Countries

Woudstra, Cédric; Maréchal, Caroline Le; Souillard, Rozenn; Bayon-Auboyer, Marie-Hélène; Anniballi, Fabrizio; Auricchio, Bruna; Medici, Dario De; Bano, Luca; Koene, Miriam; Sansonetti, Marie-Hélène; Desoutter, Denise; Hansbauer, Eva-Maria; Dorner, Martin B.; Dorner, Brigitte G.; Fach, Patrick

doi:10.1128/aem.03915-14

Cited by 15 publications

(15 citation statements)

References 25 publications

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“…Detection of the encoding genes for botulinum neurotoxin (BoNT) types A, B, E, and F and a group III target was performed using real‐time PCR with a Bio‐Rad CFX96 thermal cycler using published primers and probes (Fach, Micheau, Mazuet, Perelle, & Popoff, ; Woudstra et al, ). Each PCR reaction included a total volume of 25 µl, containing 5 µl of DNA template, 10 µl of Perfecta Tough mix (Quanta; VWR, Fontenay, France) and a final concentration of 600 nmol/L for primers and 400 nmol/L for probes.…”

Section: Methodsmentioning

confidence: 99%

Evaluation of the occurrence of sporulating and nonsporulating pathogenic bacteria in manure and in digestate of five agricultural biogas plants

et al. 2019

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The number of agricultural biogas plants has been increasing in the past decades in some European countries. Digestates obtained after anaerobic digestion (AD) of manure are usually spread on agricultural land; however, their hygiene status regarding pathogens posing public health and/or animal health challenges has been poorly characterized up to now in France. In this study, three replicates of manure and digestate were collected from five farm biogas plants receiving animal manure in order to assess the occurrence and concentrations of sporulating (Clostridium botulinum, Clostridioides difficile, Clostridium perfringens) and nonsporulating (Listeria monocytogenes, thermotolerant Campylobacter spp., Salmonella, Escherichia coli, enterococci) bacteria. Concentrations of E. coli, enterococci, and C. perfringens in digestates ranged from 102 to 104, 104 to 105, and <103 to 7 × 105 CFU/g, respectively. Salmonella and C. difficile were detected in manure and digestate from the five biogas plants at concentrations ranging from <1.3 to >7 × 102 MPN/g and from 1.3 to 3 × 102 MPN/g, respectively. Thermotolerant Campylobacter, detected in all the manures, was only found in two digestates at a concentration of cells ranging from <10 to 2.6 × 102 CFU/g. Listeria monocytogenes and C. botulinum were detected in three manures and four digestates. The bacterial counts of L. monocytogenes and C. botulinum did not exceed 3 × 102 and 14 MPN/g, respectively. C. botulinum type B was detected at very low level in both the manure and digestate of farm biogas plants with no botulism history. The levels of pathogenic bacteria in both manure and digestate suggested that some bacteria can persist throughout AD.

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Section: Methodsmentioning

confidence: 99%

Evaluation of the occurrence of sporulating and nonsporulating pathogenic bacteria in manure and in digestate of five agricultural biogas plants

et al. 2019

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Section: Real-time Pcrmentioning

confidence: 99%

“…The real-time PCR, primers, and probes in this study were used according to Woudstra et al [42]. Real-time PCR with a Bio-Rad CFX96 thermal cycler (Bio-Rad, Marne-La-Coquette, France) was used to detect the C. botulinum BoNT gene and a gene characteristic of C. novyi sensu lato [42]. Each assay was performed in a total volume of 20 µL that contained 5 µL DNA template, 10 µL PerfeCTaqPCR ToughMix (Quantabio, distributed by VWR, Fontenay-sous-Bois, France), and a final concentration of 600 nM for primers and 400 nM for probes.…”

Section: Real-time Pcrmentioning

confidence: 99%

“…The pellet obtained after DNA extraction using InstaGene matrix (Bio-Rad, Marne-la-Coquette, France) was homogenized using a vortex and 100 µL per plate was streaked over a pre-reduced Egg Yolk Agar (EYA) medium comprising 4% Blood Agar Base No 2 (Oxoid, Dardilly, France), 1.5% Bacto Agar (VWR, Fontenay-sous-Bois, France), and extemporaneously-prepared 10% egg yolk emulsion. Five colonies per plate positive for lipase and lecithinase activities [27] were individually collected after 24 h, 48 h, and 72 h of incubation at 37 • C in an anaerobic chamber, and cultured each time in 1 mL of TPGY broth at 37 • C in the anaerobic chamber (A35 Don Whitley distributed by Biomérieux, Bruz, France) for at least 24 h. DNA was extracted from 800 µL of each culture using the InstaGene matrix (Bio-Rad, Marne-la-Coquette, France) and the ntnh gene and a gene characteristic of C. novyi sensu lato [42] was detected using real-time PCR as previously described [24]. The remaining 200 µL was stored in the anaerobic chamber until PCR results were obtained.…”

Section: Isolation On Egg Yolk Agarmentioning

confidence: 99%

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Development of An Innovative and Quick Method for the Isolation of Clostridium botulinum Strains Involved in Avian Botulism Outbreaks

Gratiet

Poëzévara

Rouxel

et al. 2020

Toxins

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Avian botulism is a serious neuroparalytic disease mainly caused by a type C/D botulinum neurotoxin produced by Clostridium botulinum group III, one of the entwined bacterial species from the Clostridium novyi sensu lato genospecies. Its isolation is very challenging due to the absence of selective media and the instability of the phage carrying the gene encoding for the neurotoxin. The present study describes the development of an original method for isolating C. botulinum group III strains. Briefly, this method consists of streaking the InstaGene matrix extraction pellet on Egg Yolk Agar plates and then collecting the colonies with lipase and lecithinase activities. Using this approach, it was possible to isolate 21 C. novyi sensu lato strains from 22 enrichment broths of avian livers, including 14 toxic strains. This method was successfully used to re-isolate type C, D, C/D, and D/C strains from liver samples spiked with five spores per gram. This method is cheap, user-friendly, and reliable. It can be used to quickly isolate toxic strains involved in avian botulism with a 64% success rate and C. novyi sensu lato with a 95% rate. This opens up new perspectives for C. botulinum genomic research, which will shed light on the epidemiology of avian botulism.

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“…Animal botulism is considered an emerging disease in Europe, notably in poultry production ( 2 ), where it could lead to significant economic losses ( 3 ). The actual development of molecular tools allows rapid detection ( 4 ) and characterization ( 5 ) of the strains involved in an outbreak. We previously showed ( 4 ) that animal botulism in Europe is mainly due to mosaic type C/D for avian species, and type D/C for bovine.…”

Section: Genome Announcementmentioning

confidence: 99%

Draft Genome Sequences of 17 French Clostridium botulinum Group III Strains

Woudstra¹,

Maréchal²,

Souillard³

et al. 2015

Genome Announc

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Molecular Gene Profiling of Clostridium botulinum Group III and Its Detection in Naturally Contaminated Samples Originating from Various European Countries

Cited by 15 publications

References 25 publications

Evaluation of the occurrence of sporulating and nonsporulating pathogenic bacteria in manure and in digestate of five agricultural biogas plants

Evaluation of the occurrence of sporulating and nonsporulating pathogenic bacteria in manure and in digestate of five agricultural biogas plants

Development of An Innovative and Quick Method for the Isolation of Clostridium botulinum Strains Involved in Avian Botulism Outbreaks

Draft Genome Sequences of 17 French Clostridium botulinum Group III Strains

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