Inoculum Size of Clostridium botulinum 56A Spores Influences Time-to-Detection and Percent Growth-Positive Samples

The influence of inoculum size on the growth kinetics of Clostridium botulinum 56A and percentage of growth‐positive samples was studied in a complete factorial design with factors of inoculum size (1, 100, or 10,000 spores), pH, and sodium‐chloride concentration. Growth was followed hourly as change in A620. Polynomial regression was used to analyze the data. The time‐to‐detection and percent growth‐positive samples were significantly affected by inoculum size and its quadratic term. When inoculum size increased from 1 to 100 spores/sample, the percent growth‐positive samples increased, and the time‐to‐detection decreased. When the inoculum was 1000 spores/sample or higher, there was little additional effect. Inoculum size might influence results through simple probability or quorum sensing. The maximum growth rate was independent of inoculum levels.     

Shelf Life and Clostridium botulinum Toxin Development during Storage of Modified Atmosphere-packaged Fresh Catfish Fillets

Shelf life (onset of sensory spoilage) and potential for toxin production by Clostridium botulinum type E in retail type packages of fresh catfish fillets in high barrier film were investigated under selected atmospheres when stored under refrigeration and temperature-abuse conditions. Shelf life of fillets in all atmospheres decreased with increase of storage temperature from 4°C to 16°C. Trimethylamine content associated with onset of spoilage was different for each storage temperature and atmosphere. Surface pH and K-values were not good indicators of onset of sensory spoilage. Toxin development coincided with sensory spoilage at 16°C storage for fillets packaged in either atmosphere. At 4°C, none of the MA-packaged fillets became toxic, even after 37 days of sensory spoilage.     

新疆豆制品生产环境中肉毒梭菌的分离鉴定

从新疆伊犁察布查尔县的土壤中分离到1株优势菌,该分离株生长特性与A型肉毒梭菌(Clostridiumbotulinum type A)一致,为革兰氏阳性粗大杆菌,在EYA培养基上菌落边缘呈锯齿状,表面粗糙不规则,有较大的乳浊环。根据分离株的形态、生理生化特性,结合琼脂糖凝胶凝胶电泳图像、16SrDNA V6-V8序列(GenBank登录号:JN248616)与系统发育树分析,将其鉴定为A型肉毒梭菌。     

Isolation of obligate anaerobic rumen bacteria capable of degrading the neurotoxin β‐ODAP (β‐N‐oxalyl‐L‐α,β‐diaminopropionic acid) as evaluated by a liquid chromatography/biosensor analysis system

Six pure strains of obligate anaerobes capable of degrading the toxin β‐N‐oxalyl‐L ‐α, β‐diaminopropionic acid (β‐ODAP) contained in grass pea (Lathyrus sativus) have been isolated from cow rumen. The new isolates were identified as Megasphaera elsdenii (five different genotypes) and Clostridium bifermentans using 16S rDNA analysis. The β‐ODAP degrading efficiency of the isolates was evaluated by measuring the amount of β‐ODAP in the growth medium, which contained β‐ODAP as the only carbon source, before and after incubation with the microbes. The method of analysis was liquid chromatography employing bioelectrochemical detection. The biosensor is based on co‐immobilising two enzymes, glutamate oxidase (GlOx) and horseradish peroxidase (HRP), on the end of a spectrographic graphite electrode. β‐ODAP is oxidised by GlOx to form H₂O₂, which in turn is bioelectrocatalytically reduced by HRP through a mediated reaction using a polymeric mediator incorporating Os^{2 + /3+} functionalities rapidly shuttling electrons with the electrode_giving rise to the analytical signal. On the basis of this analysis system, the new isolates are capable of utilising β‐ODAP as sole carbon source to a maximum of 90–95% within 5 days with concomitant increase in cell protein. Copyright © 2005 Society of Chemical Industry     

Microbial risks in mild hot smoking of fish

Hot smoked fish should fulfill the requirements set up for ready-to-eat foods. The total bacterial count on the raw material is 10(3) to 10(5) CFU per cm2 of skin and 10(2) to 10(9) CFU per gram of intestines. Contamination with pathogenic bacteria is very low, mainly with Clostridium botulinum, Listeria monocytogenes, Staphylococcus aureus, and Vibrio parahaemolyticus. In the premises of low hygiene, the contamination may increase due to unsanitary procedures, rotation of assigned duties of workers, and airborne microorganisms during packing of the product. Hot smoking in mild conditions at temperature in the fish not exceeding 65 degrees C and low concentration of salt does not inactivate all pathogens or inhibit bacteria during storage. Thus the required safety can be obtained only by using very fresh fish handled in hygienic conditions, controlling the processing and the plant hygiene in critical control points, and chilling of the product to about 2 degrees C. Most critical are the hygienic conditions in handling of the product after smoking. The use of preservatives for extending the shelf life of smoked fish is being investigated. High-quality shelf life of mild hot smoked mackerel at about 2 degrees C is at least 3 weeks.     

Detection of VAMP Proteolysis by Tetanus and Botulinum Neurotoxin Type B In Vivo with a Cleavage-Specific Antibody

Tetanus and Botulinum type B neurotoxins are bacterial metalloproteases that specifically cleave the vesicle-associated membrane protein VAMP at an identical peptide bond, resulting in inhibition of neuroexocytosis. The minute amounts of these neurotoxins commonly used in experimental animals are not detectable, nor is detection of their VAMP substrate sensitive enough. The immune detection of the cleaved substrate is much more sensitive, as we have previously shown for botulinum neurotoxin type A. Here, we describe the production in rabbit of a polyclonal antibody raised versus a peptide encompassing the 13 residues C-terminal with respect to the neurotoxin cleavage site. The antibody was affinity purified and found to recognize, with high specificity and selectivity, the novel N-terminus of VAMP that becomes exposed after cleavage by tetanus toxin and botulinum toxin type B. This antibody recognizes the neoepitope not only in native and denatured VAMP but also in cultured neurons and in neurons in vivo in neurotoxin-treated mice or rats, suggesting the great potential of this novel tool to elucidate tetanus and botulinum B toxin activity in vivo.     

Detection of C. Botulinum Types in Honey by mPCR

The aim of this study was to determine the prevalence of Clostridium botulinum in honey samples using conventional methods and multiplex PCR (mPCR). A total number of 150 honey samples were randomly collected from apiaries, retail shops, weekly open bazaars, and supermarkets in Samsun, Turkey. Of 150 honey samples, 4 (2.6%) were positive for the botulinum neurotoxin gene by mPCR analysis. A total of 4 C. botulinum isolates were obtained from the mPCR positive samples, of which 3 were type A and 1 was type B. No samples were positive regarding the type E and type F neurotoxin genes. This is the first report of type A and type B spores of C. botulinum being detected and isolated in Turkey. This study revealed that some honey samples may present a potential hazard for food borne and infant botulism.     

Sporulation Strategies and Potential Role of the Exosporium in Survival and Persistence of Clostridium botulinum

Clostridium botulinum produces the botulinum neurotoxin that causes botulism, a rare but potentially lethal paralysis. Endospores play an important role in the survival, transmission, and pathogenesis of C. botulinum. C. botulinum strains are very diverse, both genetically and ecologically. Group I strains are terrestrial, mesophilic, and produce highly heat-resistant spores, while Group II strains can be terrestrial (type B) or aquatic (type E) and are generally psychrotrophic and produce spores of moderate heat resistance. Group III strains are either terrestrial or aquatic, mesophilic or slightly thermophilic, and the heat resistance properties of their spores are poorly characterized. Here, we analyzed the sporulation dynamics in population, spore morphology, and other spore properties of 10 C. botulinum strains belonging to Groups I–III. We propose two distinct sporulation strategies used by C. botulinum Groups I–III strains, report their spore properties, and suggest a putative role for the exosporium in conferring high heat resistance. Strains within each physiological group produced spores with similar characteristics, likely reflecting adaptation to respective environmental habitats. Our work provides new information on the spores and on the population and single-cell level strategies in the sporulation of C. botulinum.     

生孢梭菌芽孢萌发条件的优化

本文研究了不同温度、时间和pH值条件下,生孢梭菌芽孢萌发所需的最佳条件.本研究将活化传代好的生孢梭菌菌液培养7 d,多次离心后,加入无菌水制成生孢梭菌芽孢悬浮液,处理温度为70℃～90℃,处理时间为5～25 min,处理pH值为4～8,用平板计数法观察计算不同条件处理后的芽孢萌发率,用响应面优化法对影响生孢梭菌芽孢萌发...