Perfringens Vegetative Cells Research Articles

HEAT RESISTANCE OF CLOSTRIDIUM PERFRINGENS VEGETATIVE CELLS AS AFFECTED BY PRIOR HEAT SHOCK1

ABSTRACT This study quantified the heat resistance and the effects of a heat shock on the subsequent heat resistance of 10 strains of Clostridium perfringens. Beef gravy samples inoculated with vegetative cells of the pathogen were subjected to sublethal heating at 48C for 10 min, and then heated to a final temperature of 58C using a submerged‐coil heating apparatus. Heat‐treated samples were spiral plated on Shahidi‐Ferguson Perfringens agar to determine surviving bacterial population. No correlation between the heat resistance and the origin of the C. perfringens could be established due to significant variations in the heat resistance among strains. Inactivation kinetics of both heat‐shocked and nonheat‐shocked samples exhibited log‐linear decline in the number of surviving cells with time. D‐values at 58C for C. perfringens vegetative cells ranged from 1.21 to 1.60 min. Heat shocking allowed the organism to survive longer and the increase in heat resistance was as high as 1.5 fold. Also, heat shock resulted in the overexpression of proteins exhibiting epitopic and size similarity to E. coli GroEL and B. subtilis small acid soluble proteins. Increased heat resistance due to heat shock must be considered while designing cooking/reheating regimes that ensure safety of ready‐to‐eat foods contaminated with high numbers of C. perfringens vegetative cells.

The anaerobic pathogen Clostridium perfringens can escape the phagosome of macrophages under aerobic conditions

Clostridium perfringens is the most common cause of gas gangrene (clostridial myonecrosis), a disease that begins when ischaemic tissues become contaminated with C. perfringens vegetative cells or spores. An aerotolerant anaerobe, C. perfringens quickly multiplies in ischaemic tissues and spreads to healthy areas, leading to a high level of morbidity and mortality. As a species, the bacterium can synthesize 13 different toxins, and these are thought to be the major virulence factors of the disease. However, we present evidence here that C. perfringens can also persist inside macrophages, under aerobic conditions, by escaping the phagosome into the cytoplasm. C. perfringens was not killed by the cells of a clone (J774-33) of the macrophage-like murine cell line J774A.1 under aerobic or anaerobic conditions, whereas the non-pathogenic bacterium Bacillus subtilis was killed by J774-33 cells under both conditions. Electron microscopy images showed that C. perfringens cells were intact and resided mostly in the cytoplasm of J774-33 cells, whereas B. subtilis was in the phagosome. Immunofluorescence microscopy showed that intracellular C. perfringens bacteria failed to co-localize with the late endosome-lysosomal marker glycoprotein LAMP-1, whereas B. subtilis did co-localize with LAMP-1. C. perfringens also appeared to escape the phagosome of both activated and unactivated mouse peritoneal macrophages, but not as efficiently as was seen with the J774-33 cell line. In addition, cytochalasin D was used to show that phagocytosis of C. perfringens was dependent on actin polymerization and that the bacteria attach to J774-33 cells at distinct areas of the cell membrane. We propose that the ability to escape the phagosome and persist inside macrophages is an important factor in the early stages of a gangrene infection, when bacterial numbers are low and phagocytic cells are present.

Rapid Identification ofClostridium perfringensin Animal Feedstuffs

Clostridium perfringensis a common contaminant of grains and meals used for animal feeding and its presence in feedstuffs has been implicated in outbreaks of foodborne poisoning in farm animals. In order to evaluate a new rapid procedure forC. perfringensisolation and identification, we examined qualitatively 120 duplicate samples of feedstuffs used for farm animal and poultry feeding, using the Lactose-Sulfite broth (LS) proposed for rapidC. perfringensdetection and the conventional Cooked Meat Medium (CMM). The results suggest that LS medium is fairly successful in the detection ofC. perfringensvegetative cells and spores, despite the presence of the bacterial and fungal flora normally found in animal feedstuffs.

Interactive Effects of Temperature, Initial pH, Sodium Chloride, and Sodium Pyrophosphate on the Growth Kinetics of Clostridium perfringens

The interactive effects of temperature (12 to 42°C), initial pH (5.5 to 7), sodium chloride (0 to 3%) and sodium pyrophosphate concentrations (0 to 0.3%) on the growth in Trypticase-peptone-glucose-yeast extract broth of a three-strain mixture of Clostridium perfringens vegetative cells were determined. The number of viable C. perfringens cells was determined at appropriate intervals by plating on tryptose-sulfite-cycloserine agar. Growth data were analyzed by the Gompertz equation; the gompertz B and M parameters were then used to calculate lag-phase duration, exponential growth rate, generation time, and maximum population-density values. The data indicated that the growth kinetics of C. perfringens were dependent on the interaction of the four variables, particularly in regard to exponential growth rates and lag-phase durations. Cubic models based on the natural logarithm transformation of lag-phase duration and generation time were evaluated and appeared to adequately fit the data. The data suggest that sodium pyrophosphate can have significant bacteriostatic activity against C. perfringens and may provide processed meats with a degree of protection against this microorganism, particularly if employed in conjunction with a combination of acidic pH, high salt concentrations, and adequate refrigeration.

Post‐packaging Pasteurization Reduces CIostridiur perfringens and Other Bacteria in Precooked Vacuum‐packaged Beef Loin Chunks

ABSTRACTPrecooked beef loin chunks were vacuum packaged after inoculation with Clostridium perfringens vegetative cells or spores. The control group was uninoculated. Chunks were either nonpasteurized or pasteurized, and stored at 4°C for up to 85 days. Pasteurization reduced populations of vegetative cells (VC) and vegetative cells from spores (VCS) of C. perfringens on beef chunks throughout 85 days of refrigerated storage. Without pasteurization, constant populations of VC and VCS were maintained for 28 and 65 days, respectively. Indige‐nous microflora populations of nonpasteurized beef chunks increased for up to 28 days and remained high thereafter. Pasteurization prevented the microflora from exceeding populations of 200 log10/cm2 (surface) or mL (broth) for 85 days of storage. Pasteurization increased the shelf‐life of precooked beef loin chunks.

A 48 kilodalton enterotoxin-related protein from Clostridium perfringens vegetative and sporulating cells

Sporulating and vegetative cell extracts of enterotoxin-positive and enterotoxin-negative strains of Clostridium perfringens were examined by Western blotting using enterotoxin antiserum. A 48-kDa enterotoxin-related protein was found in vegetative and sporulating cell extracts of both toxin types. The results question previous reports about the ability of vegetative cells and presumptive enterotoxin-negative strains of this organism to produce enterotoxin, a protein with a molecular weight of 34 kDa.

Extraction, purification and characterization of a peptidoglycan hydrolase from vegetative cells of Clostridium perfringens type A.

A peptidoglycan hydrolase (PGH), which causes the germination and lysis of the coat-stripped spores of Clostridium perfringens, was extracted from perfringens vegetative cells. The enzyme was partially purified and showed an MW of 125, 000 and a pI of 7.6. It was sensitive to a variety of monovalent and divalent cations and was inhibited by sulfhydryl-active agents. The sites of PGH action, the cell wall and the spore-cortex, were determined by the release of free amino groups from cell wall fragments and the release of soluble fluorescent products from fluorescamine-labelled cortical fragments of the C. perfringens spores. The enzyme appears distinct from the extracellular initiation protein produced by C. perfringens. PGH may be an autolysin with corticolytic activity.

Growth and Survival of Clostridium perfringens in Rare Beef Prepared in a Water Bath

The low-temperature, long-time cooking of beef, using either a water bath or a conventional oven, resulted in partial inactivation of Clostridium perfringens vegetative cells. Beef roasts were cooked in a water bath for process times calculated to inactivate low and high levels of C. perfringens vegetative cells. Cooking beef in a water bath to an internal temperature of 60 C and holding for at least 12 min, as required by the USDA, reduced a population of C. perfringens by approximately 3 log cycles. To decrease the risk of subsequent outgrowth of C. perfringens, roasts (⩽ 1.5 kg) may be subjected to a process calculated for a 12-log reduction in population, which would include holding times of 2.3 h or longer at an internal temperature of 60 C. Recommendations are given for cooking and cooling roasts to minimize microbiological problems.

Effect of Freezing on Incidence and Levels of Clostridium perfringens in Mechanically Deboned Chicken Meat

Paucity of information about the effect of plant practices on microbiological condition of comminuted chicken meat prompted this investigation. Commercially deboned chicken backs and necks were analyzed for levels of aerobic organisms, incidence and levels of Clostridium perfringens vegetative cells and spores before and after 4—6 weeks at —23° C. Initially vegetative cells were isolated more frequently than spores. Frozen storage significantly reduced incidence and levels of vegetative cells and spores but did not affect levels of aerobic organisms. After frozen storage (a common industrial practice), with good food handling practices, C. perfringens should pose no undue hazard when comminuted chicken meat is incorporated into other food products.

SURVIVAL OF Clostridium perfringens IN REFRIGERATED AND FROZEN MEAT AND POULTRY ITEMS

ABSTRACTRefrigerated and frozen meats and poultry products from a retail source were examined for incidence of Clostridium perfringens vegetative cells and spores. Foods included raw ground beef, beef and pork liver, and precooked frozen beef and poultry items. Inoculated samples were also frozen and stored for various periods up to 42 days at ‐29°C. Survival of C. perfringens spores in frozen food samples and in a bacteriological medium was high, but vegetative cells were virtually eliminated. Thawing at low temperature for 2 days caused further reduction of vegetative cells, but not of spores. Approximately 50% of 32 retail frozen precooked food samples were positive for vegetative cells, and about 15% demonstrated spores of C. perfringens.

Inhibition of Clostridium perfringens by heated combinations of nitrite, sulfur, and ferrous or ferric ions.

Heating mixtures of sodium nitrite, cysteine, and either ferrous sulfate or ferric chloride at 121 C for 20 min at pH 6.5 or 6.3 produced a potent inhibitor of Clostridium perfringens vegetative cells and spores when added to previously heat-sterilized fluid thioglycolate medium. When the mixtures containing FeSO4 at pH 5.2 or FeCl3 at pH 2.7 were heated, the inhibitory effect was not produced. These responses seem to eliminate the possibility that cysteine nitrosothiol is the agent responsible for the heated-nitrite inhibition known as the Perigo effect. The variable pH responses also cast doubt upon the role of the black Roussin salt as the agent of the Perigo effect.

RESPONSE OF CLOSTRIDIUM PERFRINGENS SPORES AND VEGETATIVE CELLS TO TEMPERATURE VARIATION.

The vegetative cells and spores of four strains of Clostridium perfringens were examined to determine the effect of lowered and elevated temperatures. Spores were produced by following the method of Ellner, and vegetative cells were obtained from thioglycolate cultures. After exposure to freezing or refrigeration temperatures (-17.7 and 7.1 C, respectively), only small numbers of the vegetative cells were recovered. After similar treatment, 16 to 58% of the spores were recovered. Essentially no vegetative cells and few spores survived holding at 80 C for 10 min. Although all strains were isolated from food, only one strain of the four studied had its origin in a food-poisoning outbreak, and it had been carried on laboratory media for approximately 10 years.