Enumeration and Characterization of Clostridium perfringens Spores in the Feces of Food Poisoning Patients and Normal Controls

1986 ◽  
Vol 49 (1) ◽  
pp. 23-28 ◽  
Author(s):  
STANLEY M. HARMON ◽  
DONALD A. KAUTTER ◽  
CHARLES L. HATHEWAY
Keyword(s):  
Clostridium Perfringens ◽  
Food Poisoning ◽  
Egg Yolk ◽  
The United States ◽  
Enzyme Linked Immunosorbent Assay ◽  
Minimal Reduction ◽  
Cooked Meat ◽  
Predominant Strain ◽  
Normal Controls

The fecal spore enumeration method for confirming Clostridium perfringens as the cause of food poisoning was evaluated using strains implicated in nine outbreaks in the United States. Confirmed spore counts from 66 stool specimens were made on tryptose-sulfite-cycloserine (TSC) without egg yolk and trypticase soy-sheep blood (TSB) agars. Counts from outbreak stools on TSC agar ranged from 2.0 × 104 to 3.5 × 108 (mean ≥ 1.4 × 106/g) as compared with <103 to 5.0 × 105/g (overall mean 9.5 × 103/g) from normal stools. Similar results were obtained with TSB agar. Isolates from seven of the nine outbreaks were nonhemolytic and produced ≥100 ng enterotoxin/ml in spore broth, as measured by an enzyme-linked immunosorbent assay. Spores in stools from six of the outbreaks were heat-resistant and survived heating for 30 to 60 min at 100°C in cooked meat medium. Strains from the three remaining outbreaks were heat-sensitive and survived heating for only 15 min at 100°C. Enterotoxigenic isolates from all but one of the outbreaks were serotyped. In all instances, the predominant strain in specimens from an outbreak was of the same serotype, indicating that it was the causative strain. Reexamination of five specimens from each of three outbreaks after storage at −20°C for 6 months showed only a minimal reduction in the spore counts.

Expression and characterization of single-chain variable fragment antibody against staphylococcal enterotoxin A in Escherichia coli

2014 ◽  
Vol 60 (11) ◽  
pp. 737-743 ◽  
Author(s):  
Weifeng Chen ◽  
Li Hu ◽  
Aiping Liu ◽  
Jinquan Li ◽  
Fusheng Chen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Food Poisoning ◽  
Staphylococcal Enterotoxin ◽  
Enzyme Linked Immunosorbent Assay ◽  
Staphylococcal Enterotoxin A ◽  
Single Chain Variable Fragment ◽  
Single Chain

The staphylococcal enterotoxins (SEs) are potent gastrointestinal exotoxins synthesized by Staphylococcus aureus, which is responsible for various diseases including septicemia, food poisoning, and toxic shock syndrome, as well as bovine mastitis. Among them, staphylococcal enterotoxin A (SEA) is one of the most commonly present serotypes in staphylococcal food poisoning cases. In this study, the stable hybridoma 3C12 producing anti-SEA monoclonal antibody was established with an equilibrium dissociation constant (KD) of 1.48 × 10−8 mol·L−1, its ScFv-coding genes were obtained and then the anti-SEA single chain variable fragment (ScFv) protein was expressed in Escherichia coli. Characterization of the expressed target ScFv protein was analyzed by sodium dodecyl sulfate – polyacrylamide gel electrophoresis, Western blot, and enzyme-linked immunosorbent assay. The results demonstrated that the recombinant anti-SEA ScFv protein retained a specific binding activity for SEA, and the KD value of the soluble ScFv was about 3.75 × 10−7 mol·L−1. The overall yield of bioactive anti-SEA ScFv in E. coli flask culture was more than 10 mg·L−1.

Food Poisoning Due to Clostridium perfringens in the United States

1983 ◽  
Vol 147 (1) ◽  
pp. 167-170 ◽  
Author(s):  
W. X. Shandera ◽  
C. O. Tacket ◽  
P. A. Blake
Keyword(s):  
United States ◽  
Clostridium Perfringens ◽  
Food Poisoning ◽  
The United States

scholarly journals NanH Is Produced by Sporulating Cultures of Clostridium perfringens Type F Food Poisoning Strains and Enhances the Cytotoxicity of C. perfringens Enterotoxin

mSphere ◽  
2021 ◽  
Vol 6 (2) ◽  
Author(s):  
Jihong Li ◽  
Bruce A. McClane
Keyword(s):  
Clostridium Perfringens ◽  
Virulence Factor ◽  
Mother Cell ◽  
Food Poisoning ◽  
Mature Spore ◽  
The United States ◽  
Content Type ◽  
Intestinal Pathology ◽  
Mother Cells

ABSTRACT Clostridium perfringens type F food poisoning (FP) strains cause one of the most common foodborne illnesses. This FP develops when type F FP strains sporulate in the intestines and produce C. perfringens enterotoxin (CPE), which is responsible for the diarrhea and abdominal cramps of this disease. While C. perfringens can produce up to three different sialidases, the current study surveyed FP strains, which confirmed the results of a previous study that they consistently carry the nanH sialidase gene, often as their only sialidase gene. NanH production was found to be associated with sporulating cultures of the surveyed type F FP strains, including SM101 (a transformable derivative of a FP strain). The sporulation-associated regulation of NanH production by strain SM101 growing in modified Duncan-Strong medium (MDS) was shown to involve Spo0A, but it did not require the completion of sporulation. NanH production was not necessary for either the growth or sporulation of SM101 when cultured in MDS. In those MDS cultures, NanH accumulated in the sporulating mother cell until it was released coincidently with CPE. Since CPE becomes extracellular when mother cells lyse to release their mature spores, this indicates that mother cell lysis is also important for NanH release. The copresence of NanH and CPE in supernatants from lysed sporulating cultures was shown to enhance CPE cytotoxicity for Caco-2 cells. This enhancement was attributable to NanH increasing CPE binding and could be replicated with purified recombinant NanH. These in vitro findings suggest that NanH may be an accessory virulence factor during type F FP. IMPORTANCE Clostridium perfringens type F strains cause the second most common bacterial foodborne illness in the United States. C. perfringens enterotoxin (CPE) is responsible for the diarrhea and cramping symptoms of this food poisoning (FP). Previous studies showed that NanI sialidase can enhance CPE activity in vitro. While many type F FP strains do not produce NanI, they do consistently make NanH sialidase. This study shows that, like CPE, NanH is produced by sporulating type F FP strains and then released extracellularly when their sporulating cells lyse to release their mature spore. NanH was shown to enhance CPE cytotoxicity in vitro by increasing CPE binding to cultured Caco-2 cells. This enhancement could be important because many type F FP strains produce less CPE than necessary (in a purified form) to cause intestinal pathology in animal models. Therefore, NanH represents a potential accessory virulence factor for type F FP.

scholarly journals A rapid qualitative assay for detection of Clostridium perfringens in canned food products

2017 ◽  
Vol 64 (2) ◽  
Author(s):  
Gayatri Ashwinkumar Dave
Keyword(s):  
Phospholipase C ◽  
Clostridium Perfringens ◽  
Colorimetric Assay ◽  
Food Poisoning ◽  
The United States ◽  
Necrotic Enteritis ◽  
Yellow Colour ◽  
Negative Results ◽  
Reporter Assays ◽  
Food Borne Infections

Clostridium perfringens (MTCC 1349) is a Gram-positive, anaerobic, endospore forming, and rod-shaped bacterium. This bacterium produces a variety of toxins under strict anaerobic environment. C. perfringens can grow at temperatures ranging between 20°C and 50°C. It is the major causetive agent for gas gangrene, cellulitis, septicemia, necrotic enteritis and food poisoning, which are common toxin induced conditions noted in human and animals. C. perfringens can produce produce four major types of toxins that are used for the classification of strains, classified under type A–E. Across the globe many countries, including the United States, are affected by C. perfringens food poisonings where it is ranked as one of the most common causes of food borne infections. To date, no direct one step assay for the detection of C. perfringens has been developed and only few methods are known for accurate detection of C. perfringens. Long detection and incubation time is the major consideration of these reporter assays. The prensent study proposes a rapid and reliable colorimetric assay for the detection of C. perfringens. In principale, this assay detects the para nitrophenyl (yellow colour end product) liberated due to the hydrolysis of paranitrophenyl phosphetidyl choline (PNPC) through phospholipase C (lecithinase). Constitutive secretion of phospholipase C is a charactristic feature of C. perfringens. This assay detects the presence of the extracellular lecithinse through the PNPC impragnated impregnated probe. The probe is impregnated with peranitrophenyl phosphotidyl choline ester, which is colourless substrate used by lecithinase. The designed assay is specific towards PNPC and detectes very small quantites of lecithinase under conditions used. The reaction is substrate specific, no cross reaction was observed upon incubation with other substrates. In addition, this assay gave negative results with other clostridium strains, no cross reactions were observed with other experimental strains like C. tetani, C. botulinum, C. acetobutyricum, Bacillus subtilis, and Escherichia coli. This assay is extramly rapid and provides reliable and reproducible results within one hour of incubation at 37°C.

scholarly journals Development and Application of a Mouse Intestinal Loop Model To Study theIn VivoAction of Clostridium perfringens Enterotoxin

2011 ◽  
Vol 79 (8) ◽  
pp. 3020-3027 ◽  
Author(s):  
Justin A. Caserta ◽  
Susan L. Robertson ◽  
Juliann Saputo ◽  
Archana Shrestha ◽  
Bruce A. McClane ◽  
...  
Keyword(s):  
Clostridium Perfringens ◽  
Cell Damage ◽  
Gastrointestinal Symptoms ◽  
Food Poisoning ◽  
The United States ◽  
Intestinal Lumen ◽  
Intestinal Loop ◽  
Loop Model ◽  
Internal Organs ◽  
Content Type

ABSTRACTClostridium perfringensenterotoxin (CPE) is responsible for causing the gastrointestinal symptoms ofC. perfringenstype A food poisoning, the second most commonly identified bacterial food-borne illness in the United States. CPE is produced by sporulatingC. perfringenscells in the small intestinal lumen, where it then causes epithelial cell damage and villous blunting that leads to diarrhea and cramping. Those effects are typically self-limiting; however, severe outbreaks of this food poisoning, particularly two occurring in psychiatric institutions, have involved deaths. Since animal models are currently limited for the study of the CPE action, a mouse ligated intestinal loop model was developed. With this model, significant lethality was observed after 2 h in loops receiving an inoculum of 100 or 200 μg of CPE but not using a 50-μg toxin inoculum. A correlation was noted between the overall intestinal histological damage and lethality in mice. Serum analysis revealed a dose-dependent increase in serum CPE and potassium levels. CPE binding to the liver and kidney was detected, along with elevated levels of potassium in the serum. These data suggest that CPE can be absorbed from the intestine into the circulation, followed by the binding of the toxin to internal organs to induce potassium leakage, which can cause death. Finally, CPE pore complexes similar to those formed in tissue culture cells were detected in the intestine and liver, suggesting that (i) CPE actions are similarin vivoandin vitroand (ii) CPE-induced potassium release into blood may result from CPE pore formation in internal organs such as the liver.

Improved Media for Sporulation and Enterotoxin Production by Clostridium perfringens

1986 ◽  
Vol 49 (9) ◽  
pp. 706-711 ◽  
Author(s):  
STANLEY M. HARMON ◽  
DONALD A. KAUTTER
Keyword(s):  
Clostridium Perfringens ◽  
Sodium Carbonate ◽  
Food Poisoning ◽  
Latex Agglutination ◽  
Sporulation Medium ◽  
Enterotoxin Production ◽  
Heat Tolerant ◽  
Normal Controls

Sporulation of 24 strains of Clostridium perfringens isolated from stools of food poisoning patients and normal controls was improved by adding sodium carbonate to Duncan-Strong (DS) sporulation medium and replacing starch with raffinose in Taniguti's AEA medium. Viable counts of heat-tolerant spores (75°C for 20 min) were 2 to 186 times greater in modified AEA medium and 2 to 169 times greater in modified DS than in DS medium. Reversed passive latex agglutination assays revealed a corresponding increase in enterotoxin titers in supernatant fluids of the 12 enterotoxigenic strains grown in modified AEA medium and in modified DS medium.

scholarly journals Identification and Characterization of Sporulation-Dependent Promoters Upstream of the Enterotoxin Gene (cpe) of Clostridium perfringens

1998 ◽  
Vol 180 (1) ◽  
pp. 136-142 ◽  
Author(s):  
Yuling Zhao ◽  
Stephen B. Melville
Keyword(s):  
Clostridium Perfringens ◽  
Dna Sequences ◽  
Promoter Region ◽  
Food Poisoning ◽  
In Vitro Transcription ◽  
Shuttle Vectors ◽  
E Coli ◽  
Dna Template

ABSTRACT Three promoter sites (P1, P2, and P3) responsible for the sporulation-associated synthesis of Clostridium perfringensenterotoxin, a common cause of food poisoning in humans and animals, were identified. Nested and internal deletions of the cpepromoter region were made to narrow down the location of promoter elements. To measure the effects of the deletions on the expression ofcpe, translational fusions containing the promoter deletions were made with the gusA gene of Escherichia coli, which codes for β-glucuronidase; E. coli-C. perfringens shuttle vectors carrying the fusions were introduced into C. perfringens by electroporation. In addition, in vitro transcription assays were performed with the cpepromoter region as the DNA template for extracts made from sporulating cells. DNA sequences upstream of P1 were similar to consensus SigK-dependent promoters, while P2 and P3 were similar to consensus SigE-dependent promoters. SigE and SigK are sporulation-associated sigma factors known to be active in the mother cell compartment of sporulating cells of Bacillus subtilis, the same compartment in which enterotoxin is synthesized in C. perfringens.

Evaluation of a Reversed Passive Latex Agglutination Test Kit for Clostridium perfringens Enterotoxin

1986 ◽  
Vol 49 (7) ◽  
pp. 523-525 ◽  
Author(s):  
STANLEY M. HARMON ◽  
DONALD A. KAUTTER
Keyword(s):  
Clostridium Perfringens ◽  
Immunosorbent Assay ◽  
Culture Supernatant ◽  
Food Poisoning ◽  
Latex Agglutination ◽  
Agglutination Test ◽  
Enzyme Linked Immunosorbent Assay ◽  
Special Test ◽  
Clostridium Perfringens Enterotoxin ◽  
Test Kit

A reversed passive latex agglutination (RPLA) test kit for Clostridium perfringens enterotoxin (CPE) marketed by the Denka-Seiken Co., Tokyo, Japan, was evaluated by using culture supernatant fluids and extracts from feces of food poisoning patients. Nanograms of CPE were detectable with the assay and the reaction was specific, as shown by parallel activity in a double antibody enzyme-linked immunosorbent assay (ELISA). Although less sensitive, the RPLA method is easier to perform than the ELISA and counterimmunoelectrophoresis, both of which require special test reagents and equipment not generally available.

scholarly journals Induction and Characterization of Multianalyte Antibodies Against Penicillins in Egg Yolk

1997 ◽  
Vol 80 (6) ◽  
pp. 1220-1228 ◽  
Author(s):  
Peter de Leuw ◽  
Georg Kapa ◽  
Michael Petz
Keyword(s):  
Time Course ◽  
Immune Reaction ◽  
Keyhole Limpet Hemocyanin ◽  
Egg Yolk ◽  
Competitive Elisa ◽  
Enzyme Linked Immunosorbent Assay ◽  
Microtiter Plates ◽  
Polyethylene Glycol Precipitation ◽  
Different Characteristics

Abstract Antibodies against penicillins were induced in eggs of laying hens after immunization with 6-aminopeniciIlanic acid (6-APA) coupled to keyhole limpet hemocyanin (KLH). Development of the antibody titer was monitored by an indirect enzyme-linked immunosorbent assay (ELISA), with 6-APA coupled to ovalbumin as antigen for coating microtiter plates. Different characteristics (time course, affinity) of the immune reaction were observed by testing eggs of individual hens. Titer values varied between 150 and 2000. Antibodies were isolated by polyethylene glycol precipitation and affinity chromatography, using a hapten sorbent with 6-APA as ligand. Glycine buffer, pH 3.0, was used to elute the immunoglobulins. Antibody specificity was determined in a competitive ELISA with 7 penicillins and the cephalosporin cephalexin as competitors. Cross reactivities for the penicillins were between 100 and 290% (6-APA = 100%). Cephalexin cross reacted only marginally (3%).

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