Distribution of Enterotoxin- and Epsilon-Positive Clostridium perfringens Spores in U.S. Retail Spices.

The role of spices as vehicles of foodborne illness prompted an examination of bacterial spores in these products. Here, we report on the levels and characteristics of spores of Clostridium perfringens associated with 247 U.S. retail spices. Forty-three confirmed isolates from 17% of samples were obtained, present at levels ranging from 3.6 to 2,400/g. Twenty-seven (63%) of C. perfringens isolates were positive for the enterotoxin gene ( cpe). Seven random spice isolates produced enterotoxin at levels of between 4 and 16 ng/mL, compared with three outbreak (control) strains that each produced enterotoxin levels of >1,024 ng/mL. D95°C levels (1.0 to 3.3 min) of spores of four randomly selected spice isolates suggests a plasmid-localized cpe, while one had D95°C (>45 min) consistent with chromosomally located cpe. Five of the 43 isolates possessed the epsilon toxin gene ( etx, as well as cpe). Foods could easily become contaminated with spores of cpe-positive C. perfringens by the addition of spices. Because of its spore-forming ability, its rapid generation times at elevated temperatures, improper heating, cooling, and holding conditions could lead to elevated levels of C. perfringens in foods, a requirement for its implication in foodborne outbreaks.

Spore prevalence and toxigenicity of Bacillus cereus and Bacillus thuringiensis isolates from U.S. retail spices.

Recent incidents of foodborne illness associated with spices as the vehicle of transmission prompted this examination of U.S. retail spices with regard to Bacillus cereus. This study focused on the levels of aerobic-mesophilic spore-forming bacteria and B cereus spores associated with 247 retail spices purchased from five states in the United States. Samples contained a wide range of aerobic-mesophilic bacterial spore counts (< 200 to 8.3 × 10(7) CFU/g), with 19.1% of samples at levels above 10(5) CFU/g. For examples, paprika, allspice, peppercorns, and mixed spices had high levels of aerobic spores (> 10(7) CFU/g). Using a novel chromogenic agar, B. cereus and B. thuringiensis spores were isolated from 77 (31%) and 11 (4%) samples, respectively. Levels of B. cereus were <3 to 1,600 MPN/g. Eighty-eight percent of B. cereus isolates and 91% of B. thuringiensis isolates possessed at least one type of enterotoxin gene: HBL (hemolysin BL) or nonhemolytic enterotoxin (NHE). None of the 88 isolates obtained in this study possessed the emetic toxin gene (ces). Using commercially available immunological toxin detection kits, the toxigenicity of the isolates was confirmed. The NHE enterotoxin was expressed in 98% of B. cereus and 91% of B. thuringiensis isolates that possessed the responsible gene. HBL enterotoxin was detected in 87% of B. cereus and 100% of B. thuringiensis PCR-positive isolates. Fifty-two percent of B. cereus and 54% of B. thuringiensis isolates produced both enterotoxins. Ninety-seven percent of B. cereus isolates grew at 12°C, although only two isolates grew well at 9°C. The ability of these spice isolates to form spores, produce diarrheal toxins, and grow at moderately abusive temperatures makes retail spices an important potential vehicle for foodborne illness caused by B. cereus strains, in particular those that produce diarrheal toxins.

Inhibition of foodborne pathogens by pomegranate juice.

Pomegranates have health-promoting benefits because of their polyphenol constituents. Previous studies have demonstrated the antimicrobial activity of aqueous and organic extracts of pomegranate components and by-products. We sought to determine the antimicrobial activity against 40 foodborne pathogens representing eight bacterial species using juice itself. In addition, we sought to determine the synergistic antimicrobial activity between pomegranate juice and other plant products displaying antimicrobial activity. The antimicrobial activity of pomegranate juice was dependent on the test organism, which varied to highly susceptible (four Gram-positive species) to unaffected (Salmonella and Escherichia coli O157:H7). Two Gram-negative species, which were inhibited were Helicobacter pylori and Vibrio parahemolyticus. No synergistic antimicrobial activity was seen between pomegranate and either barberry, oregano, or cranberry. The antimicrobial activity of pomegranate juice is dependent on the test organism and extraction method. The sensitivity of H. pylori suggests that pomegranate juice may be an alternative or supplemental treatment for gastric ulcers caused by this organism.

Growth of enterotoxigenic Bacillus cereus on salmon (Oncorhynchus nerka).

We previously demonstrated the widespread presence of enterotoxigenic Bacillus cereus in marine foods. In view of the widespread consumption of raw fish, we sought to determine the ability of this organism to grow on the surface of wild Alaskan salmon at abusive temperatures (12, 16, and 20°C), using an isolate able to produce elevated levels of hemolysin BL enterotoxin and nonhemolytic enterotoxin. An incubation temperature of 37°C for colony formation was found to be selective for B. cereus grown on salmon held for up to 24 h at each temperature. A fivefold increase in log CFU per gram was observed after 26 and 22 h at 16 and 20°C, respectively, while a >4-log CFU/g increase occurred on salmon held at 12°C for 48 h. Generation times of 169.7, 53.5, and 45.6 min were observed at 12, 16, and 20°C. Nonhemolytic enterotoxin was detected when levels of B. cereus were in excess of 10(8) CFU/g. Nisin, at concentrations of 1 and 15 m g/g of salmon, reduced levels of B. cereus 2.5- and 25-fold, respectively. Our results indicate that fresh salmon can serve as an excellent substrate for enterotoxigenic B. cereus and that this organism can reach levels associated with foodborne illness following moderate temperature abuse.

Inhibitory potential of tea polyphenolics and influence of extraction time against Helicobacter pylori and lack of inhibition of beneficial lactic acid bacteria.

Tea polyphenolics such as catechins are known to have the potential to inhibit many bacterial pathogens. Helicobacter pylori has been identified as an etiologic agent in the development of gastric ulcer, peptic ulcer, gastritis, and many other stomach-related diseases. In this study, we investigated the effect of 9 tea extracts--3 different brands representing 4 different processed types (white, green, oolong, and black)--on the inhibition of H. pylori. Extraction times of 2 and 5 minutes were compared. Most 5-minute extracts showed H. pylori inhibition, whereas 2-minute extracts only of Choice darjeeling black and Tazo white showed inhibition. No recovery was observed after the addition of 0.5 and 5 mM proline, indicating that tea polyphenols do not inhibit H. pylori by inhibition of proline oxidation via proline dehydrogenase. Extracts that showed inhibition were further evaluated for their effect on beneficial lactic acid bacteria. None of the samples showed inhibition, suggesting that tea might be able to inhibit H. pylori without affecting the beneficial lactic acid bacteria. High-performance liquid chromatography indicated the presence of gallic acid, quercetin, caffeine, and tea catechins (including catechin, epicatechin, and epigallocatechin) in all the tea samples. Our study indicates that tea can be potentially used as a low-cost dietary support to combat H. pylori-linked gastric diseases without affecting the beneficial intestinal bacteria.

Physical characteristics of spores of food-associated isolates of the Bacillus cereus group.

All 47 food-borne isolates of Bacillus cereus sensu stricto, as well as 10 of 12 food-borne, enterotoxigenic isolates of Bacillus thuringiensis, possessed appendages. Spores were moderately to highly hydrophobic, and each had a net negative charge. These characteristics indicate that spores of food-associated B. thuringiensis and not only B. cereus sensu stricto have high potential to adhere to inert surfaces.

Survival during cooking and growth from spores of diarrheal and emetic types of Bacillus cereus in rice.

Bacillus cereus is a gram-positive, spore-forming, facultative anaerobe that is responsible for two types of gastrointestinal diseases: emesis and diarrhea. A significant difference in the D(95 degrees C)-values of spores of the emetic and the diarrheal types was initially determined. A mixture of B. cereus spores of the diarrheal type was inoculated into cooked rice. At inoculation levels of 2.5 x 10(2) spores per g of rice, cell numbers of 6.64 log were detected after 22 h at 20 degrees C and 6.81 log after 34 h at 17 degrees C, whereas at 12 degrees C the counts did not go above 4.0 log even after 48 h. When added to raw rice before cooking at inoculum levels of 10(3)/g, the number of viable spores decreased by 2 log, and a <1-log increase in cell numbers occurred after holding at 20 degrees C for 24 h. In contrast, the emetic spores survived and increased approximately 20-fold. Nonhemolytic enterotoxin was not detected in cooked rice at cell numbers of 8.0 log. Results here provide evidence that the absence of foodborne illness caused by the B. cereus diarrheal biotype with rice as the vehicle is due to the inability of their spores to survive and grow following standard heat processing procedures.

Detection of toxigenic Bacillus cereus and Bacillus thuringiensis spores in U.S. rice.

Bacillus cereus is a gram-positive, endospore forming pathogenic bacterium that is ubiquitous in the environment and is frequently associated with emetic and diarrheal types of foodborne illness. In this study, 178 samples of raw rice from retail food stores were analyzed for the presence of B. cereus spores. Spores of Bacillus species were found in 94 (52.8%) of the rice samples with an average concentration of 32.6 CFU/g (3.6-460 CFU/g for B. cereus and 3.6-23 CFU/g for Bacillus thuringiensis). Eighty three of the 94 isolates were identified as B. cereus and 11 were identified as B. thuringiensis. Bacillus mycoides (240 CFU/g) was the predominant isolate in one rice sample. Using PCR the isolates were checked for the presence of the cereulide synthetase gene (ces), the hblA and hblD genes of the hemolysin BL (HBL) complex and the nheA and nheB genes of the nonhemolytic (NHE) enterotoxin complex. The ces gene was not identified in any of the isolates. By contrast 47 (56.6%) B. cereus isolates possessed the hblA and hblD genes and 74 (89.1%) isolates possessed the nheA and nheB genes. As determined by commercial assay kits, forty four (53.0%) of the 83 B. cereus isolates produced both NHE and HBL enterotoxins whereas 78 (93.9%) were positive for either one or the other. Protein toxin crystals were detected visually in the 11 B. thuringiensis isolates. PCR analysis revealed 10 (90.9%) of those 11 isolates carried the cry gene. All the B. thuringiensis isolates were positive for NHE and HBL enterotoxins. Our results suggest that foodborne illness in the U.S. due to B. cereus with rice as the vehicle would be most likely associated with the diarrheal-type syndrome.

Stress proteins of Clostridium perfringens type A immunoreact with antiserum from rabbits infected with gas gangrene.

Various stressors were used to induce stress proteins in Clostridium perfringens. Cultures of C. perfringens FD-1041 were subjected to cold shock (28 degrees C for 1 h), acid shock (pH 4.5 for 30 min), or heat shock (50 degrees C for 30 min). Cells were lysed and protein samples were analyzed by immunoblotting with antiserum derived from rabbits suffering from gas gangrene. Eight cold shock proteins (approximate Mr 101, 82, 70, 37, 22, 12, 10 and 6 kDa) and also eight heat shock proteins (approximate Mr 101, 82, 70, 27, 22, 16, 12 and 10 kDa) were immunoreactive with the serum. No immunoreactive proteins were detected in samples subjected to acid shock proteins and purified DnaK protein was also non-immunoreactive with the serum. These immunogenic stress proteins may be important in regulating diseases caused by C. perfringens. Such proteins could be involved in cell survival mechanisms, serve as targets during infection, or play a role in recognition of the bacteria by the host.

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.

Elevation of the Heat Resistance of Vegetative Cells and Spores of Clostridium perfringens Type A by Sublethal Heat Shock.

The degree of heat resistance conferred on Clostridium perfringens by a heat shock, the kinetics of this development, and its duration were determined. A sublethal heat shock at 55°C for 30 min increased the heat tolerance of vegetative cells at least two- to threefold. The acquired tolerance was maintained for 2 h after the heat shock treatment. Heat shock applied for the first hour of incubation produced spores more tolerant to heat than the spores of the control. Acquired thermotolerance is of importance in the case of this organism because of its inherently high optimal growth temperature.

Lack of Correlation between the Enterotoxigenicity of Clostridium perfringens Isolates and Propionic Acid Formation or Amylase Activity at 46°C.

The absence in the supernatant fluid of either extracellular amylase in cultures grown at 46°C or propionic acid has been proposed for the presumptive identification of enterotoxin-positive strains of Clostridium perfringens . However we found that propionic acid was formed by several enterotoxin-positive, as well as enterotoxin-negative, strains of this organism. In addition 4 of 13 ent+ stains produced extracellular amylase during growth at 46°C. These results indicate that neither trait can be used as a reliable indicator of the enterotoxigenicity of isolates of this organism.

Enumeration and Confirmation of Clostridium perfringens.

A number of media have been proposed for the enumeration and confirmation of Clostridium perfringens in food and water. Most of these employ sulfite and iron together with selective antibiotics. This report discusses these various media and conditions for their use.

Evaluation of Plating Media for Recovery of Heated Clostridium perfringens Spores.

Four selective and eight non-selective plating media were evaluated for their ability to enumerate six strains of heat-activated and heat-injured spores of Clostridium perfringens . Trypticase-sulfite-neomycin (TSN) agar and sulfite-polymyxin-sulfadiazine (SPS) agar gave higher counts of heat-activated spores than non-selective media. In the case of heat-injured spores, wide variation in recovery was obtained depending on strain and medium. Higher counts of heat-injured spores were obtained by incubating plates at 37°C than at 45°C, although, except for one strain, no significant difference between the two temperatures was observed using heat-activated spores.