反向间接胶乳凝集试验法检测粪便A型产气荚膜梭菌肠毒素的结果及评价

本文以１０种５２株供试菌分别与７个不同年龄组的健康人粪便混合,共配成３６４份模拟标本,采用反向间接胶乳凝集（ＲＰＬＡ）试验法与生物学试验法（小鼠致死试验、豚鼠皮肤血管透性因子试验,Ｖｅｒｏ细胞毒性试验）检测各标本中的Ａ型产气荚膜梭菌肠毒素（简称Ｃｐ－Ｅｎｔ）。除产气荚膜梭菌之外的其他菌种培养液２３８份标本（３４株）,ＲＰＬＡ与生物学试验结果完全一致,均为阴性。产气荚膜梭菌１２６份标本（１８株）中有７０份标本的ＲＰＬＡ同生物学诸法完全一致地检出了Ｃｐ－Ｅｎｔ．有１株７份标本（ＣｐＮＣＴＣ８７９７）的ＲＰＬＡ为阳性,而各生物学试验却均为阴性,该菌株经ＰＣＲ检查证明确为肠毒素原性产气荚膜梭菌,表明ＲＰＬＡ比生物学试验法更灵敏。有５株（ＣｐＮＣＴｃ８２３８,ＣｐＮＣＴＣ１０６１１,ＣｐＮＣＴＣ１０６１４,ＣｐＮＣＴＣ１０６１２,ＣｐＬ－５２）３５份标本ＲＰＬＡ与各生物学试验结果均为阴性,但经ＰＣＲ检吉证明该５株菌均为肠毒素原性产气荚膜梭菌,后经超声波破碎菌体提取物对其中部分菌株进行试验的结果仍然显示了ＲＰＬＡ与生物学法的一致性。有２株（ＣｐＮＣＴＣ８６８６,ＣｐＮＣＴＣ８４４９）１４份标本的所有结果均为阴性,ＰＣＲ     

Typing of sheep clinical isolates and identification of enterotoxigenic Clostridium perfringens strains by classical methods and by polymerase chain reaction (PCR).

A simple procedure was developed to identify toxitypes of Clostridium perfringens of different origins. Ninety strains of C. perfringens were identified by classical bacteriological methods, typing of the strains was done by a seroneutralisation test on mice. Production of enterotoxin was tested and all strains were analysed by PCR using gene of toxin alpha, gene of toxin E, gene of toxin beta and gene of enterotoxin. Simple amplification (amplifying one gene), and duplex and triplex amplification (amplifying two and three genes simultaneously) were performed. In the conditions of the experiment, the PCR method has proved efficacious. The specificity and sensitivity are excellent and superior to those of the classical methods. The prophylaxis of enterotoxaemia in animals is achieved by vaccination, the PCR technique can thus become a first-choice tool for the identification and typing of the C. perfringens strains which initiate these diseases. In turn, this would simplify the development of vaccines adapted to the epidemiological situation.     

Intestinal flora of patients with suspected antibiotic associated diarrhea (AAD). I. Clostridium perfringens

Stool samples of 158 patients suspected of antibiotic-associated diarrhoea (AAD) were studied. Toxin A of C. difficile and enterotoxin of C. perfringens were detected in stool samples by immunoenzymatic assays and PCR. In 35 stool samples toxin A of C. difficile was detected and in 48 cases (30%) C. difficile strains were cultured from 21 stool samples (13%). The presence of the cpe gene of C. perfringens, enabling the production of enterotoxin, could not be detected by PCR, both in stool samples and in isolated strains, using ent 1 and ent 2 primer pairs. C. difficile and C. perfringens were isolated from the same stool samples in 4 cases. From stool samples of two patients with AAD C. perfringens strains, thermoresistant spores were cultured.     

Enterotoxin Production by Lecithinase-positive and Lecithinase-negative Clostridium perfringens Isolated from Food Poisoning Outbreaks and other sources

Strains of Clostridium perfringens from a variety of sources were examined for their ability to produce enterotoxin in vitro. Fifty-six of 65 (86%) strains isolated from separate outbreaks of food poisoning were found to be enterotoxigenic, only two of 174 strains from other sources produced enterotoxin. The ability to produce this toxin was not confined to particular serotypes: types frequently encountered as the cause of outbreaks were also isolated as enterotoxin-negative strains from faeces, minced beef and meat carcasses. Loss of toxigenicity was also observed in different serotypes. Five strains of lecithinase-negative Cl. perfringens produced high levels of enterotoxin. Four strains of Clostridium plagarum failed to produce enterotoxin although they were serologically typable with the Cl. perfringens antisera.     

Incidence of Enterotoxigenic Clostridium perfringens in Healthy Humans in relation to the Enhancement of Enterotoxin Production by Heat Treatment

Enterotoxin production was greatly enhanced in two of five food poisoning strains of Clostridium perfringens subjected to heat treatment prior to incubation in Duncan and Strong sporulation medium. Heating was carried out on three successive cultures of each strain, the optimum temperature for treatment being 85 °C for one strain and 95 °C for another: on each occasion cultures were heated for 20 min. The triple heat treatment procedure was used in testing strains of Cl. perfringens isolated from faeces of healthy human subjects for production of enterotoxin. Eleven of 35 (31%) individuals were found to be carriers of enterotoxigenic strains, the isolates producing more than 0·1 μ/ml of enterotoxin. Six of the 11 enterotoxigenic strains were killed by heating at 95 °C but one isolate produced more enterotoxin following treatment at this temperature than after heating at 75 °C.     

Prevalence and characterization of Clostridium perfringens from spices in Argentina

Spices can present high microbial counts and Clostridium perfringens, Bacillus cereus, Salmonella and Shigella, among others have been isolated from spices. C. perfringens is an important pathogen agent causing, among other diseases, enteritis in humans caused by C. perfringens enterotoxin (CPE) which causes human food poisoning and enterotoxemia in domestic animals. The aims of the present work were (i) to establish the hygienic sanitary quality of some spices in San Luis, Argentina; (ii) to determine the presence of C. perfringens in these spices by means of the most probable number (MPN) and count on plate methods; (iii) to characterize the enterotoxigenic strains of C. perfringens by PCR and immunological methods such as reverse passive latex agglutination (RPLA) and (iv) to type by PCR C. perfringens strains isolated. A total of 115 samples of spices, 67 of which were purchased in local retail stores and 48 domestically collected were analysed. Total aerobe counts on tryptone glucose yeast extract agar medium of the 115 samples were between <10 and 10(6) CFU/g. The colifecal counts using Mac Conkey broth of the 115 samples were <4-10(3)CFU/g, with 28 samples (24.34%) exceeding the limit established by the Spanish Alimentary Code (10 CFU/g) while 2 samples (1.73%) had a sulfite reducing anaerobe load above standard limits. A total of 14 C. perfringens strains (12.17%) were isolated and characterized from 115 samples by the standard biochemical tests. Four of which (28.60%) turned out to be enterotoxigenic by PCR and RPLA. In order to type C. perfringens strains based on their main toxins, the 14 strains were analysed by PCR. All strains belonged to type A. All RPLA positive strains were cpe(+) by PCR. The percentage of enterotoxigenic strains was more elevated that those reported in other studies for this type of sample. These results indicate that sanitary conditions in different production stages of species must be improved to reduce health hazards. The high percentage (24.34%) of samples with colifecal values above standard limits is an indication of deficient sanitary conditions. These results suggest the need to provide legislation on the sanitary and hygienic quality of spices in our country.     

The suitability of Tórtora's medium for the production of enterotoxin in Clostridium perfringens strains

Examination of 200 samples from soil and the same number of samples from healthy human feces yielded 49 (24.5%) and 105 (52.5%) strains of heat-resistant Clostridium perfringens respectively. Fourteen (7.0%) strains isolated from soil and 37 (18.5%) from feces synthesized enterotoxin, as demonstrated by Tórtora's method, at sufficient levels to permit its detection by mouse lethality, microslide double gel diffusion or counterimmunoelectrophoresis tests. By using the Duncan-Strong (DS) method, only four (2%) enterotoxigenic strains from soil and 14 (7.0%) from feces were obtained. The supernatant fluid from two enterotoxigenic-negative strains grown in DS medium gave a false-positive reaction when they were injected intravenously into mice. Tórtora's medium was preferable because a larger number of isolated strains produced spores and enterotoxin to permit their recognition as enterotoxigenic strains.     

Direct detection of Clostridium perfringens enterotoxin in patients' stools during an outbreak of food poisoning

An outbreak of diarrhoea in a hotel affected 25 time keepers attending the 1997 Mediterranean Games. Epidemiological investigation implicated a 'pasta al ragù' consumed at the hotel's restaurant and Clostridium perfringens food poisoning was identified by direct detection of C. perfringens enterotoxin in patients' stools. This report confirms that a careful evaluation of epidemiological features, together with the availability of direct and rapid laboratory methods, may lead to a prompt identification of C. perfringens food poisoning.     

Enterotoxigenicity and genetic relatedness of Clostridium perfringens isolates from retail foods in the United States

Clostridium perfringens is a leading cause of bacterial food-borne illness in countries where consumption of meat and poultry is high. For example, each year in the United States, this organism is the second or third most common cause of confirmed cases of food-borne illness. Surveys of the incidence of this organism in retail foods were done in the 1960s without regard to whether isolates were enterotoxigenic. It is now known that not all strains of this organism possess the enterotoxin gene responsible for illness. We examined the incidence of this organism in 131 food samples from retail food stores in an area of the northeastern United States. Forty isolates were obtained by using the iron milk method at 45 degrees C, with confirmation by use of motility nitrate and lactose gelatin media. The presence of the C. perfringens enterotoxin (cpe) and alpha toxin (cpa) genes was determined by PCR using previously published primer sequences. All isolates possessed cpa. None of the isolates were identified as carrying the cpe gene by this method or by another method using a digoxigenin-labeled gene probe. Consistent with these results, none of the sporulating-cell extracts contained enterotoxin as determined by reverse passive latex hemagglutination. Pulsed-field gel electrophoresis was used to determine the genetic relatedness of the isolates. About 5% of the isolates were considered to be closely related (2- to 3-band difference). The others were considered to be unrelated to one another. The results demonstrate the rarity of cpe(+) strains in retail foods and the genetic diversity among nonoutbreak strains.     

Investigation of animal botulism outbreaks by PCR and standard methods

Abstract A double PCR procedure is proposed for identification of Clostridium botulinum C and D. This method consists of a first PCR amplification with a degenerate primer pair able to amplify a 340 bp common DNA fragment from botulinum neurotoxin (BoNT) C1 and D genes, followed by two subsequent PCR amplifications with two primer pairs specific for BoNT/C1 and D respectively (198 bp DNA fragment). This method was found to be specific for C. botulinum C and D, amongst 81 strains of C. botulinum and 21 different species of other Clostridium and bacteria tested. The detection limit ranged from 10 to 10³ bacteria in the reaction volume according to the C. botulinum C and D strains. In 160 naturally contaminated animal and food samples submitted to a 48 h enrichment culture, the double PCR showed an 89.4% correlation rate with the standard mouse bioassay. A clear distinction between botulism type C and D was obtained. The double PCR provides a reliable alternative for detection and identification of C. botulinum C and D in clinical and food samples.     

Multiple typing techniques applied to a Clostridium perfringens food poisoning outbreak.

Twenty-one stool specimens obtained from persons implicated in two food poisoning outbreaks at the same institution in Smith Falls, Ontario, were examined for Clostridium perfringens. Ninety-two colonies of Cl. perfringens (3-5 per stool specimen) were typed with antisera, bacteriocins and by plasmid analysis. They were also tested for the in vitro production of bacteriocin and enterotoxin. Sixteen of the 21 stool specimens were tested directly for enterotoxin. This was detected in 13, five of which were from individuals listed as 'asymptomatic' food handlers. The predominant strain isolated from 15 of the 21 stool samples produced bacteriocin and enterotoxin in vitro, contained no plasmids, and was of a common bacteriocin type and serotype.     

Detection and quantification of four species of the genus Clostridium in infant feces

To determine the composition of Clostridium in the feces of infants approximately 30 days old, we have developed a detection and quantification method of Clostridium paraputrificum, Clostridium perfringens, Clostridium tertium, and Clostridium difficile by species-specific primers. C. perfringens and C. difficile were detected in four fecal samples from 22 infants (18.2%), whereas C. paraputrificum was detected in three samples (16.7%). C. tertium was detected in two samples (9.1%). Moreover, the occurrences of the four species in bottle-and mix-fed infants were relatively higher than in breast-fed infants (P< 0.05). Subsequently, positive samples detected by nested PCR (polymerase chain reaction) were subjected to realtime PCR. The results showed that the numbers of C. paraputrificum, C. perfringens, C. tertium, and C. difficile ranged from about 1x10(5) to 3x10(7) cells/g wet feces.     

Multiple typing techniques applied to a Clostridium perfringens food poisoning outbreak

D.E. MAHONY, R. AHMED AND S.G. JACKSON, 1992. Twenty-one stool specimens obtained from persons implicated in two food poisoning outbreaks at the same institution in Smith Falls, Ontario, were examined for Clostridium perfringens. Ninety-two colonies of Cl. perfringens (3–5 per stool specimen) were typed with antisera, bacteriocins and by plasmid analysis. They were also tested for the in vitro production of bacteriocin and enterotoxin. Sixteen of the 21 stool specimens were tested directly for enterotoxin. This was detected in 13, five of which were from individuals listed as 'asymptomatic' food handlers. The predominant strain isolated from 15 of the 21 stool samples produced bacteriocin and enterotoxin in vitro , contained no plasmids, and was of a common bacteriocin type and serotype.     

Duplex shuttle PCR for differential diagnosis of budgerigar fledgling disease and psittacine beak and feather disease

Two common viral diseases in psittacine birds including budgerigar fledgling disease (BFD), generally called avian polyomavirus (APV) infection, and psittacine beak and feather disease (PBFD) have similar clinical manifestations characterized by feather disorders. A duplex shuttle PCR was developed for detection of APV and PBFD virus (PBFDV). Two pairs of oligonucleotide primers were designed to amplify a 298-bp fragment of the t/T antigen region of APV genome and a 495-bp fragment of the capsid protein region encoded by open reading frame (ORF) C1 of PBFDV genome, respectively. In the present study, APV and PBFDV were detected simultaneously in one tube by duplex shuttle PCR using these two pairs of primers. The detection limits were 2 viral copies of APV and 3 viral copies of PBFDV. In the clinical application, we detected 16 APV-positive, 15 PBFDV-positive, and 3 mixed infected samples in 39 samples examined. Sequences of the amplified products were read. The t/T antigen region was conserved in the APV-positive samples as expected. ORF C1 of PBFDV genome showed diversity. Phylogenic analysis indicated that PBFDV ORF C1 consisted of 6 clusters which were related to subfamilies of psittacine birds. Our duplex shuttle PCR could be a useful method for differential diagnosis and molecular epidemiology of BFD and PBFD.     

Use of inosine-containing oligonucleotide primers for enzymatic amplification of different alleles of the gene coding for heat-stable toxin type I of enterotoxigenic Escherichia coli.

A method which employs the polymerase chain reaction (PCR) to identify Escherichia coli strains containing the estA gene was developed. This gene codes for heat-stable enterotoxin type I. The use of an inosine-containing pair of amplification primers allowed the amplification of a specific 175-bp DNA fragment from several different estA alleles. The amplified fragments were identified and distinguished by allele-specific oligonucleotide hybridization and characterized by restriction endonuclease analysis. An extension of the classical two-primer PCR proved to be a very simple and rapid method to identify and characterize the estA alleles. Besides the inosine-containing pair of primers, which recognized all described alleles, additional oligonucleotides were used as primers. The sequence of each of these primers was allele specific, and each was amplification compatible with one of the inosine-containing primers. Thus, in one PCR the 175-bp fragment typical for all estA alleles and an allele-specific fragment of different size were produced. These fragments could be separated by agarose gel electrophoresis and were recognized by ethidium bromide staining. Twenty-seven E. coli strains were tested with this amplification system. The presence or lack of the genetic information for production of heat-stable enterotoxin type I was perfectly consistent with the ability of these strains to produce this enterotoxin, as determined by enzyme-linked immunosorbent assay.     

PCR and gene probe identification of botulinum neurotoxin A-, B-, E-, F-, and G-producing Clostridium spp. and evaluation in food samples.

A degenerate primer pair was selected to amplify specifically a 260-bp DNA fragment from Clostridium botulinum types A, B, E, F, and G, and five individual probes allowed identification of each toxinotype by hybridization of the PCR products. The 72 strains of different Clostridium species tested and 11 other bacterial species commonly found in food samples gave an amplification product. This assay was able to detect 1 C. botulinum type A or B and 10 C. botulinum type E strains per reaction. With 184 artificially contaminated food samples, after an 18-h enrichment step, the sensitivity was 10 bacteria per g of sample and the correlation with the mouse bioassay reached 95.6%.     

A survey of Clostridium perfringens enterotoxin antibody in human and animal sera in western Canada

Sera from human, cattle, sheep, swine, and horse populations in western Canada were tested for the presence of Clostridium perfringens enterotoxin antibody by the passive hemagglutination (PHA) test, supplemented by an immunodiffusion test and by counterimmunoelectrophoresis. A total of 224 human, 345 cattle, 165 sheep, 620 swine, and 768 horse serum samples were examined. Low-titer reactions in the PHA test were detected in human, cattle, horse, and swine sera, in that order, with no titers demonstrated in sheep. The titers in human sera ranged up to 1:128 and three of these samples were also positive in the other two serological tests. The significance of this antibody is not clear, but it is suggested that the low prevalence of the antibody may reflect a low prevalence of enterotoxigenic C. perfringens strains in western Canada. Such serological surveys may be applicable to epidemiological studies involving enterotoxigenic C perfringens.     

Enterotoxin-producing Bacteroides fragilis (ETBF) Strains in Stool Samples Submitted for Testing ofClostridium difficile and its Toxins

Fifty faecal samples of patients suspected of having diarrhoea associated with Clostridium difficile were studied. Toxins of C. difficile were tested in vivo directly from the faecal sample using Toxin Detection Kits (Oxoid) to detect toxin A and primers for detection genes of Toxin A and B in a PCR test. The same samples were tested for B. fragilis enterotoxin gene directly from the faecal sample using special primers and a PCR test. Samples were inoculated onto selective media for C. difficile (CCCA) and B. fragilis (BBE) for isolation of bacteria.In vitro Toxin A of C. difficile in culture was tested using a C. difficile toxin A immunoassay (Oxoid, U.K. test and Toxin B of C. difficile was tested by using the McCoy cell line. C. difficile toxin A and B genes were determined in DNA of isolated strains using special primers and a PCR reaction. The enterotoxin production in B. fragilis strains was tested on the human carcinoma cell line HT29/C1. The presence of fragilysin gene was detected using a special pair of primers and a PCR reaction. Toxinogenic strains of C. difficile and enterotoxigenic Bacteroides fragilis (ETBF) strains were isolated from the same samples.     

Enterotoxigenic profiles and polymerase chain reaction detection of Bacillus cereus group cells and B. cereus strains from foods and food-borne outbreaks

Bacillus cereus is one of the important food pathogens. Since B. cereus group cells, such as B. cereus, B. thuringiensis, B. anthracis and B. mycoides, share many phenotypical properties and a high level of chromosomal sequence similarity, it is interesting to investigate the virulence profiles for B. cereus group cells, including B. cereus strains isolated from foods and samples associated with food-poisoning outbreaks. For this investigation, the presence of enterotoxin genes, such as those of haemolysin BL, B. cereus enterotoxin T and enterotoxin FM, were assayed by polymerase chain reaction (PCR) methods. Meanwhile, their enterotoxin activities were assayed using the BCET-RPLA kit, haemolytic patterns on sheep blood agar and their cytotoxicity to Chinese hamster ovary (CHO) cells. Results showed that there were 12 enterotoxigenic profiles for the 98 B. cereus group strains collected. In addition, if any of the three types of enterotoxins was present in the B. cereus group cells, these cells were shown to be cytotoxic to the CHO cells. Similar enterotoxigenic profiles could be found among strains of B. cereus, B. mycoides and B. thuringiensis. Thus, all B. cereus group strains may be potentially toxigenic and the detection of these cells in foods is important. We thus designed PCR primers, termed Ph1/Ph2, from the sphingomyelinase gene of B. cereus cells. These primers were specific for all B. cereus group strains and could be used for the detection of B. cereus cells contaminated in food samples.     

Inhibitory effects of collagen on the PCR for detection of Clostridium perfringens

It is essential to identify specific food components that inhibit PCR in order to increase the sensitivity of the PCR method for rapid detection of pathogens contaminating a food. We found that collagen, a major component of several foods, inhibited PCR. The inhibitory action of collagen on PCR could be partially reversed by adjusting the concentration of magnesium ion in the reaction mixture and by the use of various DNA extraction methods to remove the collagen from the DNA. Also, the source of thermostable DNA polymerase was affected by the presence of collagen. These results suggest the need to optimize the extraction and assay conditions for rapid detection of enterotoxigenic Clostridium perfringens by PCR with respect to the kind of food being analyzed.