DOT-enzyme linked immunosorbent assay for detection of Clostridium perfringens type A enterotoxin

A procedure, which we have termed DOT-ELISA, to detect Clostridium perfringens type A enterotoxin on nitrocellulose paper is described. Seventy eight preparations from 39 cultures of C. perfringens type A were tested simultaneously by this and by Plate-ELISA methods. The results were comparable. DOT-ELISA detected as little as 0.02 micrograms of purified enterotoxin and 0.13 micrograms of enterotoxin in cell-free culture supernatant. As little as 0.02 micrograms purified enterotoxin mixed with human faeces could be detected specifically. The method is simple and does not require an ELISA reader.     

Detection of staphylococcal enterotoxin B in spiked food samples

Contamination of food with infectious agents, intentional or not, is a global concern with far-reaching economic and social impact. Staphylococcal enterotoxins are a major cause of food poisoning, but most methods for the identification of these agents in food require extensive pretreatment or concentration of the sample prior to analysis. The array biosensor was developed as a portable device for the simultaneous analysis of multiple complex samples for multiple targets with minimal sample preparation. In this study, we use an array biosensor to expand and improve on a staphylococcal enterotoxin B (SEB) assay with the ultimate intent of incorporating testing for SEB into a battery of sensitive and convenient assays for food safety validation. In addition to buffer studies, six different types of food samples, including beverages, homogenates of fruit and meat, and carcass washings, were spiked with SEB, incubated for at least 2 h to permit antigen sequestration, and assayed. For all samples, there were differences in fluorescence intensity, but 0.5 ng of SEB per ml could be detected in <20 min with little if any pretreatment and no sample preconcentration.     

Novel insights into the epidemiology of Clostridium perfringens type A food poisoning

Clostridium perfringens food poisoning ranks among the most common gastrointestinal diseases in developed countries. The disease is caused by C. perfringens enterotoxin (CPE) encoded by cpe and produced by less than 5% of C. perfringens type A strains. Molecular epidemiological research in the past 15 years has focused on the reservoirs and routes of cpe-positive C. perfringens aiming to clarify the role and epidemiology of chromosomal and plasmid-borne cpe-carrying strains. This literature review highlights novel aspects in the epidemiology of CPE-mediated diseases. We suggest that (1) chromosomal and plasmid-borne cpe-carrying C. perfringens strains are genetically and epidemiologically distinct and have adapted to different environments; (2) not only chromosomal but also plasmid-borne cpe-carrying C. perfringens strains cause food poisonings; (3) other CPE-mediated diseases, such as antibiotic-associated and sporadic diarrhea, associated with plasmid-borne cpe-positive strains, may be food-related; (4) the role of animals as the main reservoir of cpe-positive C. perfringens needs to be reconsidered; (5) humans serve as an important reservoir of cpe-positive C. perfringens, introducing a contamination risk into foods through handling; and (6) the current standard procedures to diagnose C. perfringens food poisoning fail to detect and isolate many C. perfringens strains, distorting the epidemiological understanding of C. perfringens food poisoning.     

Detection of Clostridium perfringens enterotoxin gene by the polymerase chain reaction amplification procedure.

The polymerase chain reaction (PCR) procedure was evaluated to see if it is a simple and rapid method to detect Clostridium perfringens enterotoxin gene. The method, involving the use of two synthesised primers and gene amplification by the PCR procedure, detects a DNA fragment of 364 base pairs of C. perfringens enterotoxin gene by gel electrophoresis. The enterotoxin gene of strains was detected by use of purified chromosomal DNA. The supernatant of sporulating cultures in a sporulating medium was able to be used as template DNA. Template DNA can be obtained by merely culturing the strain in DS medium, a sporulating medium, for 48 h at 37 degrees C. All C. perfringens strains showing positive results in the PCR procedure were demonstrated to produce enterotoxin by a conventional method and all strains showing negative results were enterotoxin negative. To detect the enterotoxin gene in stool specimens by the PCR procedure, the specimen was heat-treated for 10 min at 90 degrees C and cultured in a sporulating medium, the supernatant of which was used as template DNA. From the stool specimens showing positive results in the PCR procedure by this method, enterotoxigenic C. perfringens was isolated from the heat-treated specimens. Thus, it is possible to detect enterotoxigenic C. perfringens in stools without isolation of the organism.     

A probabilistic analysis of Clostridium perfringens growth during food service operations

The purpose of this study was threefold: first, the study was designed to illustrate the use of data and information collected in food safety surveys in a quantitative risk assessment. In this case, the focus was on the food service industry; however, similar data from other parts of the food chain could be similarly incorporated. The second objective was to quantitatively describe and better understand the role that the food service industry plays in the safety of food. The third objective was to illustrate the additional decision-making information that is available when uncertainty and variability are incorporated into the modelling of systems.     

外送餐引起产气荚膜梭菌食物中毒流行病学调查分析

目的查明发生在某工厂的食源性疾病暴发的可疑食品、致病因子及危险因素等,并对事件调查过程中暴露出的问题进行探讨,为今后类似事件的防控和调查提供参考依据。方法病例定义为于2019年3月3日～3月4日期间在M工厂加班职工中发生腹痛、腹泻(≥3次/24 h)或呕吐症状之一者,运用描述性和分析性流行病学方法开展病例访谈和回顾性研究。收集病例的粪便标本、剩余食品和相关环境样品进行病原分离和采用聚合酶链式反应(PCR)检测阳性菌株的毒素基因。结果检索到病例106名,罹患率为73.6%(106/144),临床症状以腹泻(78.3%,83/106)、腹痛(78.3%,83/106)为主,部分半腹部胀气、胀痛(9.4%,10/106),无发热;流行曲线为点源暴露模式,潜伏期为2～22 h,可疑餐次为2019年3月3日的午餐;单因素分析和Logistic回归分析结果显示,发病与红烧鱼块[相对危险度(RR)=1.55, 95%置信区间(95%CI):1.29～1.85]、蒜苗肉丝(RR=1.26, 95%CI:1.01～1.57)和雪菜烧鸭血(RR=1.47, 95%CI:1.16～1.87)有关;在3份肛拭子、3份环境样品中检出产气荚膜梭菌,菌株和2份剩余食品均检测出α毒素和产气荚膜梭菌肠毒素(CPE)基因。送餐的D企业在加工经营中存在着被细菌污染并繁殖的条件。结论本次事件是因食用某供餐企业提供的配送餐引起的产气荚膜梭菌食物中毒,外送餐做好后应迅速冷却、低温储存,不能立即进食的,在食前要充分加热。     

Influence of peptone source on sporulation of Clostridium perfringens type A

Clostridium perfringens is a foodborne disease agent that produces a sporulation-specific enterotoxin. To produce enterotoxin for experimental purposes or spores for challenge or physiological studies, the use of a convenient sporulation medium is required. The most commonly used is Duncan-Strong medium. Few isolates sporulate at high levels in this medium. We investigated the effectiveness of peptones from a variety of sources on the sporulation of this organism compared with the peptone in the original formulation, proteose peptone (control). Seven strains were used to screen 32 peptones, with starch or raffinose as the carbohydrate source. In most cases, raffinose was more effective than starch in stimulating sporulation, confirming our previous study. Two promising peptones, potato peptone, and Proteose Peptone no. 3, were selected and tested against 49 additional enterotoxin-positive and -negative strains, with raffinose as the carbohydrate. For 49 strains, 5 sporulated best (>10%) in the control peptone, 6 sporulated best in Peptone no. 3, and 23 sporulated best in the potato peptone. Of the 23 strains, 16 sporulated at levels 25% more than the control peptone. The increase in sporulation rates was reflected in the enterotoxin and heat-resistant spore levels. The methylxanthines caffeine and theobromine were effective in increasing the sporulation of less than half of 19 enterotoxin-positive strains. Our results suggest that the replacement of proteose peptone with potato peptone be considered if difficulty in obtaining spores of specific strains of C. perfringens is encountered.     

Inhibitory effects of nisin against Clostridium perfringens food poisoning and nonfood-borne isolates

The enterotoxigenic Clostridium perfringens type A is the causative agent of C. perfringens type A food poisoning (FP) and nonfood-borne (NFB) human gastrointestinal diseases. Due to its ability to form highly resistant endospores, it has become a great concern to the meat industry to produce meat free of C. perfringens. In this study, we evaluated the antimicrobial effect of nisin against C. perfringens FP and NFB isolates. No inhibitory effect of nisin was observed against germination of spores of both FP and NFB isolates in laboratory medium. However, nisin effectively arrested outgrowth of germinated spores of C. perfringens in rich medium. Interestingly, germinated spores of NFB isolates possessed higher resistance to nisin than that of FP isolates. Furthermore, nisin exhibited inhibitory effect against vegetative growth of both FP and NFB isolates in laboratory medium, with vegetative cells of NFB isolates showing higher resistance than that of FP isolates. However, the antimicrobial activity of nisin against C. perfringens was significantly decreased in a meat model system. In conclusion, although nisin showed inhibitory effect against spore outgrowth and vegetative cells of C. perfringens FP and NFB isolates in laboratory conditions, no such effect was observed against C. perfringens spores inoculated into a meat model system.     

Combination of immunomagnetic separation and polymerase chain reaction for the simultaneous detection of Listeria monocytogenes and Salmonella spp. in food samples.

A method that combined the immunomagnetic separation (IMS) technique and the multiplex polymerase chain reaction (PCR) method (i.e., the IMS-mPCR method) was developed for simultaneous detection of Listreria monocytogenes and Salmonella spp. in food samples. When only the multiplex PCR method was used, it was found that if cell numbers of each of the two target organisms (L. monocytogenes and Salmonella spp.) were above the detection limit, but differed by more than 2 logs-e.g., n x 10(7) to n x 10(4) or n x 10(6) to n x 10(3)--the organism presenting the lower numbers might go undetected. Following the enrichment step with universal preenrichment (UP) broth, if an IMS method using equal quantities of anti-Listeria and anti-Salmonella immunomagnetic beads was performed prior to PCR, both pathogens could be detected unambiguously. Such results could be obtained for target organisms in food samples, such as milk, dairy, and meat products, if similar enrichment and IMS steps were performed prior to PCR.     

饮用天然矿泉水中产气荚膜梭菌检测方法的改良

将饮用天然矿泉水中产气荚膜梭菌检测国标方法GB 8538-2016《食品安全国家标准饮用天然矿泉水检验方法》作一些改良,以使检测结果更加准确可靠。用产气荚膜梭菌CICC22949人工污染的水样通过孔径为0.22μm的无菌滤膜过滤,分别将胰胨-亚硫酸盐-环丝氨酸琼脂(tryptose-sulfite-cycloserine agar,TSC)培养基和亚硫酸盐-多黏菌素-磺胺嘧啶琼脂(sulfite-polymyxin-sulphadiazxine agarr,SPS)培养基采用双层培养基覆盖法进行试验。改良后的方法在滤膜上有黑色可疑菌落,而国标方法滤膜上的菌落无黑色。TSC培养基上目标菌计数值显著高于SPS培养基计数值(P<0.05)。用新鲜配制的TSC培养基经双层培养基覆盖改良后的方法检测产气荚膜梭菌,现象更明显,结果更可靠,更适合饮用天然矿泉水中产气荚膜梭菌的检验。     

Variations in Recovery of Clostridium perfringens on Commercial Sulfite-Polymyxin-Sulfadiazine (SPS) Agar

SUMMARY— Liquid cultures of several strains of Clostridium perfringens type A were diluted with phosphate buffer and enumerated on laboratory-prepared sulfite-polymyxin-sulfa-diazine (SPS) agar, four lots of commercial SPS agar, and on non-selective media. Recoveries on laboratory-prepared SPS agar and on one lot of commercial SPS agar were the same as on non-selective media. Recoveries on three commercial lots of SPS agar were significantly lower.
Replacement of phosphate buffer with 0.1% peptone allowed quantitative recoveries of C. perfringens on three out of four lots of commercial SPS agar, irrespective of whether the cells were grown in liquid cultures or on meat and meat products.     

Inhibition of growth,enterotoxin production,and spore formation of Clostridium perfringens by extracts of medicinal plants

The extracts of 14 plants used in the traditional medicine of Mexico were evaluated for their effects on the growth, spore formation, and enterotoxin production of Clostridium perfringens type A. The extracts of Psidium guajava L., Haemotoxylon brasiletto, and Euphobia prostata were the most effective inhibitors of growth, spore formation, and enterotoxin production. No enterotoxins were detected when extracts were added to the media at less than the MIC for growth.     

Screening bovine carcass sponge samples for Escherichia coli O157 using a short enrichment coupled with immunomagnetic separation and a polymerase chain reaction-based (BAX) detection stept

A bovine carcass sponge sample screening protocol for detecting Escherichia coli O157:H7 was composed of a short selective enrichment followed by an immunomagnetic separation (IMS) and target detection using the BAX E. coli O157 polymerase chain reaction assay. This screening protocol was compared to a culture-based method for detection of the organism in carcass sponge samples. Enriched samples were subjected to IMS; the bead suspension was divided and plated on selected media or stored at -20 degrees C, then subjected to BAX analysis. The results showed a high degree of agreement between the plating method and the BAX system. Fifty-two of the 59 culture-positive samples were also positive using the BAX system (88.1% sensitivity). Of the 76 samples that appeared negative for the presence of E. coli O157:H7 by the culture method, 66 were determined as negative using the BAX system (86.8% specificity). Four of the 10 samples found negative by the initial culture method and positive by the BAX method were subsequently found to be culture positive upon reanalysis. Based on these data, the BAX system combined with a short, selective enrichment and IMS may be a rapid, reliable, and simple method to screen for E. coli O157:H7 in carcass sponge samples. Our data indicate that optimization and subsequent testing of this protocol for use as a carcass screening tool are warranted.     

Prevalence of Clostridium botulinum types B,E and F in faecal samples from Swedish cattle

Faeces were collected from 60 cows at three slaughterhouses situated in southern and central Sweden. The faecal samples were collected during two sampling periods over the year, summer and winter. All samples were analysed for the presence of Clostridium botulinum spores, according to a combined selection and enrichment PCR procedure. One PCR assay was specific for part of the type B neurotoxin gene, while the other assay was specific for both type E and F neurotoxin genes. The prevalence of C. botulinum in Swedish cattle was established to be 73% for non-proteolytic type B and less than 5% for types E and F. Twenty-eight (64%) of the positive faecal samples had a spore load of less than 4 spores/g. Statistical analysis (ANOVA) showed that seasonal variation (summer and winter) had a significant effect on the prevalence of C. botulinum type B in cattle, whereas the effect of geographical location of rearing of the cattle (southern and central Sweden) was less significant.     

聚缬氨酸修饰电极测定食品中柠檬黄

在pH=8.0的磷酸盐缓冲溶液中,利用电化学方法制备了聚缬氨酸修饰电极。研究了柠檬黄在此电极上的电化学行为,建立了测定柠檬黄的新方法。在pH 6.0的磷酸盐缓冲溶液中,柠檬黄在聚缬氨酸修饰电极上的还原峰电流与其浓度在6.0×10-8².9×10-5mol/L内呈良好线性关系,检出限为2.0×10-8mol/L。     

A simple and highly sensitive spectrophotometric determination of Cr(VI) in food samples by using 3,4-dihydroxybenzaldehydeisonicotinoylhydrazone (3,4-DHBINH)

A simple, rapid, sensitive and inexpensive method has been developed for the determination of trace amounts of chromium(VI) using 3,4-dihydroxybenzaldehydeisonicotinoylhydrazone (3,4-DHBINH). The metal ion gives a yellow coloured complex with 3,4-DHBINH in acetate buffer of pH 5.5 with 1:1 (metal:ligand) composition. The complex shows a maximum absorption at 400 nm. Beer’s law is obeyed in the range 0.5–7.7 ppm of Cr(VI). The molar absorptivity, Sandell’s sensitivity and detection limit were found to be 1.35 × 10⁴ L mol⁻¹ cm⁻¹, 0.0075 μg cm⁻² and 0.0045 μg mL⁻¹, respectively. The correlation co-efficient and regression co-efficient of the Cr(VI)–3,4-DHBINH complex were 0.99 and 0.12, respectively. Major cations and anions did not show any interference. The developed method has been successfully applied for the analysis of Cr(VI) in food samples (leafy vegetables), comparing the results simultaneously with those obtained using an Atomic Absorption Spectrophotometer, whereby the validity of the method has been tested.     

Stimulation of Gas Production and Growth of Clostridium perfringens Type A (No. 3624) by Legumes

SUMMARY: Dry beans and other legumes contain an unidentified factor which stimulates rapid growth and gas production by Clostridium perfringens, Type A. This factor may be related to the flatus-inducing properties of dry beans. it is suggested that flatus gases are the product of accelerated gas production by the intestinal anaerobe. Gas production and growth of C. perfringens were inhibited by some of the same antibiotics that are known to block flatulence in higher animals. Hydrogen and carbon dioxide, the major constituents in flatus gases, were also found to be the primary gases collected over cultures of the anaerobe grown in a synthetic medium. Bland foods, such as rice and barley, evoked minimal responses. Pure carbohydrates including lactose, raffinose, stachyose and starch had no effect on gas production when the organism was grown in a complete basal medium containing glucose. An assay procedure has been developed for measuring the response of the microorganism to various substrates. This procedure should facilitate isolation, purification and characterization of the unknown factor. If a direct relationship can be established between this factor and the flatulence factor in dry beans, the assay procedure should find applications in establishing a flatus index for foods and aid in the development of nonflatulent food products.     

Identification and characterization of Clostridium perfringens using single target DNA microarray chip

A DNA microarray method was developed to identify the presence of toxin genes: encoding beta toxin (cpb), epsilon toxin (etx), enterotoxin (cpe), alpha toxin (cpa), and iota toxin (iA) in Clostridium perfringens. To build the DNA chip, each gene sequence was represented by one approximately 22-bp amino-modified oligonucleotide printed twice on aldehyde-coated slides. Multiplex PCR with Cy3 and Cy5-dCTP derivatized fluorescent nucleotides was used to label five genes and fluorescent probes were prepared. The PCR probes were denatured and single-strand-labeled DNAs were separated and purified using magnetic beads. The presence of toxin genes in C. perfringens was detected by hybridization of amplified ssDNA probes to oligonucleotides on the chip representing one target sequence of each toxin gene. The DNA chip was able to identify eight strains of C. perfringens.     

Amplified fragment length polymorphism (AFLP) analysis of Clostridium perfringens for epidemiological typing

Thirty-five Clostridium perfringens isolates from patients and foods implicated in seven outbreaks of suspected Cl. perfringens food poisoning together with five unrelated incidents were analysed by serotyping and amplified fragment length polymorphism (AFLP). Despite minor band differences, AFLP was found to be highly reproducible and 16 different profiles (each unique to the 12 incidents) were recognised. The results from both serotyping and AFLP analysis identified exactly the same groups of related cultures. It is concluded that AFLP can provide a rapid, sensitive and reproducible method for the typing of Cl. perfringens for outbreak investigation.