Isolation and enumeration of Clostridium botulinum by direct inoculation of infant fecal specimens on egg yolk agar and Clostridium botulinum isolation media

Glasby, Constance; Hatheway, Charles L.

doi:10.1128/jcm.21.2.264-266.1985

Cited by 13 publications

(6 citation statements)

References 5 publications

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“…Examination of multiple stool specimens and enumeration of lipase-positive organisms on selective egg yolk agar (8) may improve the reliability of the investigations. These methods would show that the infant is actually colonized by the organism and would lessen the probability of encountering an intermittent specimen in which low numbers of the organism might result in negative tests.…”

Section: Discussionmentioning

confidence: 99%

“…Confirmation of the diagnosis of infant botulism relies on the detection of botulinal toxin or Clostridium botulinum in the stool (10); serum from these patients is rarely reported to have detectable levels of toxin. Stools from affected infants usually contain moderate to high toxin levels (101 to 105 mouse lethal doses per g; Centers for Disease Control [CDC], unpublished observations), and they readily yield C. botulinum by direct streaking of specimens on agar medium (8) or from broth enrichment cultures (10). Stool specimens obtained in some cases weeks and even months after the onset of illness continue to be positive for the organism and its toxin (2).…”

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Examination of feces and serum for diagnosis of infant botulism in 336 patients

Hatheway

McCroskey

1987

J Clin Microbiol

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In the 12-year period 1975 to 1987, feces from 336 infants were examined for botulinal neurotoxin and Clostridium botulinum. All the infants had illnesses which prompted their physicians to consider infant botulism in the diagnosis. Stool specimens from 113 of the infants yielded organisms that produced botulinal neurotoxins assumed to be responsible for the illness. The types of botulinal toxin in the confirmed cases were distributed as follows: 38 A, 69 B, 2 atypical B, 1 E, 1 F, 1 A + B, and 1 B + F. The type A and B toxins in a single infant were produced by two different strains of organism, and the type B and F toxins in another infant were produced by a single strain. The physiological characteristics of all the isolated toxigenic organisms except two were consistent with those of group I (proteolytic) C. botulinum. The toxigenic isolate from the infant with type E botulism was identified as C. butyricum, and that from the infant with type F botulism was identified as C. barati. Toxin of the same type as produced by the isolated organisms was identified in feces of 98 of 111 culture-positive infants. Botulinal toxin was identified in the serum of 9 of 67 culture-positive infants (8 of 22 infants with type A organisms; 1 of 43 infants with type B organisms; neither of 2 infants with A + B or atypical type B organisms). Botulinal toxin was not detected in feces (206 infants) or in serum (114 infants) of the culture-negative infants. The culture-positive infants had clinical features and a course of illness consistent with those of infant botulism. Most of the culture-negative infants probably had illnesses other than botulism, but specimens might have been obtained late in some infants' illnesses, when the organism had disappeared.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Examination of feces and serum for diagnosis of infant botulism in 336 patients

Hatheway

McCroskey

1987

J Clin Microbiol

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“…Otkrivanje gena koji kodira proizvodnju toksina u bakterijskim sporama je od velikog značaja u slučaju da uzorci potiču iz životne sredine odnosno u uzorcima gde se primenjuje obogaćenje koje može dovesti do rasta konkurentnih mikroorganizama koji mogu kočiti rast ciljnog mikroorganizma (156). PCR metodologijom se mogu obrađivati uzorci koji potiču iz životne sredine, hrane, biološkog materijala, crevnog sadržaja i uzorci fekalnog porekla (80,106,131,135 metode gde se pored izolacije radi i brojanje kolonija na pločama ili u epruvetama i izražava kao broj formiranih bakterija po gramu/mililitru -CFU/g(ml) (68,124). Jedna od korišćenih metoda je i metoda utvrđivanja najverovatnijeg mogućeg broja (most probable number), MPN u uzorcima koji su reagovali pozitivno.…”

Section: Clostridium Botulinum I Bont-aunclassified

The investigation of the presence of Clostridium botulinum in honey and honey bee samples

Matović¹

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47%) meda i jednom (1,64%) uzorku medonosnih pčela. Broj spora u pozitivnim uzorcima meda bio je od 20/kg do 204/kg, a u uzorku medonosnih pčela 110/kg.U jednom uzorku meda detektovane su spore B tipa C. botulinum, u jednom uzorku je detektovan E tip, u dva uzorka su detektovane spore istovremeno dva tipa C.botulinum (A, E) i u jednom uzorku istovremeno tri tipa C. botulinum (A, B, E). Spore Clostridium botulinum tipa E dokazane su u jednom uzorku medonosnih pčela. U istim uzorcima meda, odnosno medonosnih pčela, klasičnim kvalitativno-kvantitativnim metodama nije dokazano prisustvo spora Clostridium botulinum.Detekcija spora Clostridium botulinum direktno iz neobrađenih uzoraka meda i medonosnih pčela, bez predobogaćenja, nije moguća primenom metoda PCR i klasičnih metoda mikrobiologije. Klasične metode mikrobiologije, uključujući i metodu SRPS ISO 15213:2011, nisu podesne za detekciju spora Clostridium botulinum u uzorcima meda. Zbog niske osetljivosti navedene metode daju lažno negativne rezultate. U uzorku meda koji je služio kao pozitivna kontrola i koji je prethodno namerno kontaminiran referentnim sojem Clostridium botulinum NCTC 7272, PCR metodom može da se dokaže jedna spora/g meda.U cilju detekcije spora Clostridium botulinum u uzorcima meda i medonosnih pčela primenom metode PCR, za svaki ispitujući uzorak moraju se izvoditi višestruka ponovljanja, ispitivanja jednog istog uzorka, zbog malog broja i/ili neravnomerne raspoređenosti spora u uzorcima. Kjučne reči: Med; Medonosna pčela; Clostridium botulinum; PCR Naučna oblast: Veterinarska medicina Uža naučna oblast: Mikrobiologija namirnica UDK broj: 638:579The investigation of the presence of Clostridium botulinum in honey and honey bee samples

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“…Enumeration of C. botulinum was conventionally performed by using a pourplate procedure (Hauschild and Hilsheimer, 1977;Glasby and Hatheway, 1985) or the most probable number method (MPN) combined with MBA or PCR (Hielm et al, 1996). Recently, real-time PCR, which is based on the quantification of specific amplified DNA, has been applied for the quantitative detection of C. botulinum type A (Yoon et al, 2005) and type E (Kimura et al, 2001) in food and of Clostridium sp.…”

Section: Quantification Of C Botulinummentioning

confidence: 99%

Incidence of Clostridium botulinum Spores in Honey and Infant Food Samples Collected from Vietnam and Germany

An¹

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2.3.2.6 Link to sudden infant death syndrome (SIDS) 2.3.2.7 Risk factors for infant botulism 2.3.3 Wound botulism 2.3.4 Hidden botulism 2.3.5 Inadvertent botulism 3 OBJECTIVES 4 MATERIALS AND METHODS 4.1 Bacterial strains and culture conditions 4.2 Media 4.3 Honey and infant food samples 4.4 Spore production 4.5 Enumeration procedure 4.6 Procedures for detection of C. botulinum spores in honey and infant foods 4.7 Preparation of honey and infant food samples 4.8 Preparation of spiked honey samples for enumeration of C. botulinum spores 4.9 Preparation of spiked infant food samples for detection of C. botulinum spores 4.10 DNA isolation 4.11 PCR procedure 4.12 Mouse bioassay v 4.13 Restriction enzyme analysis of PCR products 4.14 Purification of PCR fragments for sequencing 4.15 Sequencing analysis 4.16 Isolation of C. botulinum from PCR-positive samples 4.17 Polyacrylamide gel electrophoresis (PAGE) 4.18 Statistical analysis 5 RESULTS 5.1 Spore production 5.2 Enumeration of C. botulinum spores in spiked honey samples 5.3 Incidence of C. botulinum spores in honey and infant food samples 5.3.1 Incidence of C. botulinum spores in honey and infant foods purchased in Vietnam 5.3.2 Incidence of C. botulinum spores in honey and infant foods purchased in Germany 5.4 Mouse bioassays 5.5 Isolation of C. botulinum from PCR-positive samples 5.6 Incidence of C. botulinum spores in artificially inoculated honey and infant foods 5.7 Restriction enzyme analysis 5.8 Sequence analysis 6 DISCUSSION 6.1 Spore production 6.2 Enumeration of C. botulinum spores in spiked honey samples 6.3 Incidence of C. botulinum spores in honey and infant food samples collected from Vietnam and Germany 6.4 PCR analyses 6.5 Mouse bioassays vi 6.6 Isolation of C. botulinum from PCR-positive samples 6.7 Incidence of C. botulinum spores in artificially inoculated honey and infant foods 6.8 Restriction enzyme analysis 6.9 DNA sequencing analysis

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Isolation and enumeration of Clostridium botulinum by direct inoculation of infant fecal specimens on egg yolk agar and Clostridium botulinum isolation media

Cited by 13 publications

References 5 publications

Examination of feces and serum for diagnosis of infant botulism in 336 patients

Examination of feces and serum for diagnosis of infant botulism in 336 patients

The investigation of the presence of Clostridium botulinum in honey and honey bee samples

Incidence of Clostridium botulinum Spores in Honey and Infant Food Samples Collected from Vietnam and Germany

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