Food Bioprocess Technol (2011) 4:624–630 DOI 10.1007/s11947-009-0182-2 ORIGINAL PAPER Clostridium perfringens: A Dynamic Foodborne Pathogen Santos García & Norma Heredia Received: 17 September 2008 / Accepted: 14 January 2009 / Published online: 13 February 2009 # Springer Science + Business Media, LLC 2009 Abstract Clostridium perfringens is a spore-forming bacterium and natural inhabitant of soil and the intestinal tracts of many warm-blooded animals, including humans. The ubiquitous nature of this bacterium and its spores makes it a frequent problem for the food industry and establishments where large amounts of food are prepared. C. perfringens causes potentially lethal foodborne diseases in humans, including food poisoning and necrotic enteritis. This bacterium could be controlled properly following safety rules such as adequate heating and cooling of food during processing. Unfortunately, large C. perfringens outbreaks, sometimes with fatal outcomes are still frequently reported. This paper describes the main characteristics of C. perfringens that allow the bacterium to survive and grow in foods, and cause human disease as well as discusses strategies to control this microorganism during food processing. Keywords Food safety . Foodborne pathogens . Clostridium perfringens . Enterotoxin The Organism The genus Clostridium consists of a diverse group of bacteria that are unable to grow in the presence of oxygen and have the ability to form heat-resistant endospores Meeting Presentation: Inocuidad Alimentaria 2007, Chihuahua, Mexico, October 2007 (Food Safety 2007). S. García (*) : N. Heredia Departamento de Microbiología e Inmunología, Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León, Apdo. Postal 124-F, San Nicolás, N.L. 66451, México e-mail: santos@microbiosymas.com (Fig. 1, Heredia and Labbé 2001). C. perfringens was first described as Bacillus aerogenes in 1892 and was later called Clostridium welchii. Historically, C. perfringens has been known as the predominant cause of gas gangrene occurring in wound infections (Heredia and Labbé 2001). This bacterium is the most prolific toxin-producing species within the clostridial group. The toxins are responsible for a wide variety of human and veterinary diseases, many of which can be lethal (McClane 2007). C. perfringens cause two distinct human diseases that can be transmitted by food, one is a common form of foodborne illness, and the other is necrotic enteritis that is relatively rare. Foodborne illness caused by C. perfringens is one of the most common illnesses caused by the consumption of contaminated food (Garcia and Heredia 2009). Although the association of foodborne illness was first proposed approximately 100 years ago, it was not until the 1960s and 1970s that conclusive evidence had accumulated showing that an enterotoxin is associated with the sporulation of C. perfringens in the intestine of affected individuals (McDonel 1986). C. perfringens is a Gram-positive, anaerobic, nonmotile rod which form large, regular, round, and slightly opaque and shiny colonies on the surface of agar plates. Despite the fact that this organism is an anaerobe, it is capable of growing at Eh values of +350 mV and reducing its environment to less than −400 mV. (Brynestad and Granum 2002). C. perfringens colonies normally display a doublezone hemolysis on blood agar plates, with a clear inner theta-toxin zone and a hazy outer zone caused by alphatoxin production. The bacterium can grow between 15 °C and 50 °C, with an optimum of 37 °C to 45 °C for most strains, and with growth reported at temperatures as low as 6 °C. Generation times for enterotoxin-positive C. perfringens strains grown between 41 °C and 46 °C can be less Food Bioprocess Technol (2011) 4:624–630 Fig. 1 Sporulating cell of Clostridium perfringens than 8 min in autoclaved ground beef (Willardsen et al. 1978). The ability of C. perfringens to form heat-resistant spores and the wide temperature range in which the organism can grow are features that allow the bacterium to multiply and survive in a variety of food environments (Brynestad and Granum 2002; Heredia and Labbé 2001). Pulse field electrophoresis revealed that strain CPN50 of C. perfringens has a single circular 3.6 Mb chromosome. More than 100 restriction sites and 24 genetic loci have been located on that genome, which is slightly larger in size than that of other Gram-positive bacteria (Rood 1998). C. perfringens is classified into five types (A, B, C, D, and E) based on the production of four major toxins (alpha, beta, epsilon, and iota toxins) and hydrolytic enzymes including lecithinase, hemolysins, hyaluronidase, collagenase, DNAse, and amylase (Brynestad and Granum 2002). Molecular epidemiological surveys suggest that only a small fraction (1% to 5%) of all C. perfringens isolates, mainly belonging to type A, are capable of producing an enterotoxin responsible for food poisoning (Brynestad and Granum 2002; Rodriguez-Romo et al. 1988). In addition, it has also been reported that most, and perhaps all, C. perfringens type E that have been isolated carry silent, defective cpe (C. perfringens enterotoxin gene) sequences (Billington et al. 1998). C. perfringens are ubiquitous bacteria found in virtually all environments that have been tested, including soil, dust, in the intestinal tract of humans and other animals, in spices, and on the surfaces of vegetable products, and in other raw and processed foods (Brynestad and Granum 2002; Heredia and Labbé 2001). Recently, the frequency of C. perfringens in the normal fecal flora of healthy North 625 Americans has been investigated. About half of 43 tested subjects were colonized with the bacteria at levels of ~106 cfu/g feces, and all were type A strains; no alpha, beta2 or enterotoxin were detected in the stools of any of the donors (Carman et al. 2008). The presence of C. perfringens in each of these environments along with the longevity of the spores make C. perfringens a suitable indicator of both distant and intermittent fecal contamination (Fujioka and Shizumura 1985), therefore, C. perfringens has been used as an indicator parameter in surface water sources in Europe (Council Directive 98/83/EU). C. perfringens possess several attributes that have contributed significantly to its ability to cause foodborne illness. First, C. perfringens has a ubiquitous distribution in the natural environment, giving it ample opportunity to contaminate foods. Second, C. perfringens has the ability to form heat-resistant spores, allowing survival in a variety of environments and through processes, including the incomplete cooking of foods or improper sterilization techniques. Third, C. perfringens has the ability to grow quickly in foods, allowing the bacteria to reach the high levels that are necessary for food poisoning. Finally, C. perfringens is capable of producing an intestinally active enterotoxin (CPE) that is responsible for the characteristic gastrointestinal symptoms of C. perfringens food poisoning (Brynestad et al. 1997; McClane 2005). CPE Considerable evidence directly implicates CPE as the virulence factor responsible for the diarrhea and cramping symptoms associated with C. perfringens type A food poisoning. This toxin is a single 35-kDa polypeptide with an isoelectric point of 4.3, is heat and pH labile, is 309 amino acids in length, and has a unique mechanism of action. Specifically, CPE is produced intracellularly during the sporulation process, and it is released along with the mature spore (Heredia and Labbé 2001). The majority of the enterotoxin-positive strains that carry this gene on a plasmid and on the chromosome are pathogenic for humans, while most of the strains with episomally located cpe are pathogenic for other animals. Studies comparing the organization of the chromosomal and plasmid cpe loci of type A C. perfringens isolates have revealed that a ~3-Kb DNA region is identical in both the plasmid and chromosomal cpe locus; however, some differences have been detected (McClane 2005). Like many bacterial toxins, the cpe gene appears to be associated with mobile genetic elements. Even when chromosomally located, the cpe gene may still be associated with mobile genetic elements (McClane 2005). Several reports have indicated that when enterotoxin-positive 626 strains are transferred repeatedly without heat shocking, these can lose this enterotoxin gene (Brynestad et al. 1997). The role of CPE in the physiology of the bacterial cell remains unknown. C. perfringens Foodborne Diseases Foodborne diseases caused by C. perfringens include food poisoning, the most common illness, caused by type A strains, and necrotic enteritis, caused by type C and a few type A strains (Bos et al. 2005; Brynestad and Granum 2002). The predominant symptoms of C. perfringens food poisoning include diarrhea and severe abdominal pain; nausea is less common, and fever and vomiting are unusual. These food poisoning cases are self-limiting, and antibiotic therapy is not recommended (Heredia and Labbé 2001). In the early 1970s, CPE-positive strains of C. perfringens type A became associated with C. perfringens food poisoning, which ranks as one of the most common foodborne illness each year in many countries, including the US and UK (Adak et al. 2002). Foodborne illness is caused when food becomes contaminated with large numbers of vegetative bacterial cells (>106 CFU/g) of C. perfringens type A isolates that carry the cpe gene. Disease symptoms appear 8 to 24 h after the ingestion of contaminated food. Many of the ingested bacterial cells may die when exposed to the acidic environment of the stomach, but if the food vehicle is sufficiently contaminated, some vegetative cells will survive passage through the stomach and enter the small intestine where they multiply and sporulate (McClane 2007). CPE is then produced by these sporulating cells and is eventually released into the intestinal lumen when the sporulating cells lyse to release their endospores (McClane 2007; Heredia and Labbé 2001). The possibility of ingesting sporulating cells or preformed enterotoxin is unlikely, since studies with volunteers indicate that the amount of ingested CPE necessary to produce symptoms would require cell numbers that would impart adverse sensory qualities to such foods. This foodborne illness is typically a toxi-infection rather than intoxication, and it resolves spontaneously within the following 12 to 24 h (McClane 2007). Once the CPE is released into the small intestine, a series of events occurs. First, CPE binds to a 50-kDa protein receptor, forming a small complex of 90 kDa. Next, this small complex develops a post-binding physical change, which could represent either the insertion of CPE into the membrane, or a conformational change. This physically changed small complex and a 70-kDa membrane protein will then form a large 160-kDa complex, which will initiate a series of biochemical events that will alter the normal permeability of brush border membranes in small intestine epithelial cells (McClane 1996). This CPE-induced perme- Food Bioprocess Technol (2011) 4:624–630 ability change becomes cytotoxic and causes localized tissue damage, which leads to a breakdown in normal fluid and electrolyte transport properties and, hence, diarrhea (McClane 1996). Treatment of CPE with trypsin increases CPE activity at least twofold, suggesting a possible role for the intestinal enzyme in cases of human illness (Granum and Richardson 1991). C. perfringens food poisoning is not a reportable disease; however, in the United States, the Centers for Disease Control and Prevention (CDC) estimates that 250,000 cases of C. perfringens type A food poisoning occur annually (Linch et al. 2006). In Norway in the 1990s, this organism was registered as the most common cause of food poisoning. Similarly, the prevalence in other countries, such as Japan and the UK, is also high. Deaths due to C. perfringens are not common, but do occur in the elderly and debilitated (Brynestad and Granum 2002; Heredia and Labbé 2001). Another very serious but rare human disease is necrotic enteritis, which is due to infection with C. perfringens type A and C isolates. Here, symptoms can include diarrhea, abdominal cramps, vomiting, fever, and severe bowel necrosis, which can result in death (Bos et al. 2005; Brynestad and Granum 2002). Contamination of Food Enterotoxigenic C. perfringens is commonly found in soil, dust, in the intestinal tract of humans and other animals, in spices, on the surfaces of vegetable products, as well as in other raw and processed foods (Table 1) (Brynestad and Granum 2002; Heikinheimo 2008; Heredia and Labbé 2001). Animal carcasses and cuts of meat can become contaminated with C. perfringens from contact with soil or animal feces, or during slaughtering and processing. Many organisms that compete with C. perfringens are killed when meat and poultry are cooked, but C. perfringens spores are difficult to eliminate. C. perfringens requires more than a dozen amino acids and several vitamins for its growth, both of which are typically present in meat. Recent CDC statistics indicate that the leading food vehicles for this bacterium in the United States are meats, notably beef and poultry, and meat-containing products, such as gravies, stews, and Mexican food (Linch et al. 2006; McClane 2007). Growth in Food, Prevention, and Control C. perfringens type A food poisoning usually results from either improper cooling or temperature maintenance of food, preparation of food a day or more in advance, or inadequate reheating of food (Heredia and Labbé 2001). Food Bioprocess Technol (2011) 4:624–630 627 Table 1 Prevalence of cpe-positive Clostridium perfringens in food Number of samples studied Number of C. perfringens isolates analyzed cpe-positive C. perfringens in samples (%) Total C. perfringens in samples (%) Detection Country methoda Reference 50 0 2 16 Japan Miwa et al. 1998 10 0 0 NKb Japan Miwa et al. 1996 Pork 50 0 0 10 Japan Miwa et al. 1998 Chicken 10 50 0 0 0 12 NK 84 Japan Japan Miwa et al. 1996 Miwa et al. 1998 Sausage 75 315 NK 83 8 2.5 NK 26 Costa Rica Argentina Morera et al. 1999 Virginia et al. 2002 Hamburger 100 19 0 19 Argentina Virginia et al. 2002 Minced meat 100 24 1 24 Argentina Virginia et al. 2002 Fresh and processed retail samples Animal origin 347 17 1 5 MPNPCR Nested PCR MPNPCR PCR MPNPCR RPLA RPLA, PCR RPLA, PCR RPLA, PCR PCR USA 887 302 1.4 31 PCR USA 131 NK 0 30 PCR USA Rahmati and Labbe 2008 Wen and McClane 2004 Lin and Labbé 2003 92 94 115 1 0 14 1 3 12 Australia Australia Argentina 380 188 4 NK ND ND RPLA, PCR Dot blot Curry roux 60 Miscellaneous Animals and humans, NK animal and human food, unknown origin NK Animals and humans, animal food, unknown origin NK 616 0 8 12 NK RPLA PCR Japan USA and Canada 454 4 NK PCR USA Food Meat (raw) Meat (processed) Seafood Retail food Spices Beef Animal and nonanimal origin Diced lamb retail Ground beef Various different origins Mexico Phillips et al. 2008 Aguilera et al. 2005 Rodriguez-Romo et al. 1988 Fujisawa et al. 2001 Songer and Meer 1996 (Kokai-Kun et al. 1994) Modified and updated from Heikinheimo (2008). Detection methods: ELISA enzyme-linked immunosorbent assay, RPLA reversed passive latex agglutination, PCR polymerase chain reaction, CH colony hybridization, MPN-PCR PCR combined with the most probable number technique, RPHA reversed passive hemagglutination. b NK, not known. a Both physical and chemical treatments are used in food processing to eliminate or reduce the presence of pathogens and spoilage microorganisms. Heat-activation of spores during the cooking process would facilitate germination when temperatures become favorable for growth. Also, during cooking, Eh values drop to levels that favor subsequent multiplication of C. perfringens (Heredia and Labbé 2001). Temperatures favoring bacte- rial replication can arise during improper cooling, either keeping food at room temperature, or by refrigerating large portions, which cool slowly, or from improper holding temperatures. In these cases, bacteria commence multiplication. If these food products are served without being reheated to a temperature sufficient to kill vegetative forms of C. perfringens, illness may result (McClane 2007). 628 The specific association between C. perfringens type A chromosomal cpe isolates and food poisoning is that their spores and vegetative cells are especially heat resistant. Vegetative cells of chromosomal cpe isolates are, at 55 °C, two-fold more resistant than vegetative cells of plasmidic cpe isolates. Further, those containing the cpe gene on their chromosome produce spores that are 60-fold more resistant to heat compared with those with plasmidic cpe (McClane 2007). Although C. perfringens spores are the main source of concern in food products, vegetative cells may occasionally cause health problems in nonheat-treated foods or by recontamination of heat-treated foods. As with most foodborne pathogens, pasteurization temperatures (72 °C [161 °F]) for 5–10 min and routine well-cooking procedures readily inactivate vegetative cells of this organism (Heredia and Labbé 2001). Of far greater concern is the heat resistance of their spores that varies depending on the strain, leading to the designation of strains as being “heatresistant” or “heat-sensitive.” For example, in one study, the decimal reduction values at 95 °C (D95) for the heatresistant spores were between 17.6 and 63 min; however, in some reports, it has been as high as 200 min, compared with decimal reduction values between 1.3 and 2.8 min for the heat-sensitive spores (Ando et al. 1985). Not surprisingly, the so-called heat-resistant strains were more often associated with cases of foodborne illness. C. perfringens spores can be killed by the use of hypochlorite at a pH below 8.5 or by the use of UV light (Brynestad and Granum 2002). Improper cooling of food has been identified as an important factor associated with C. perfringens food poisoning. As cooked foods cool, they pass through the entire range of temperatures supporting the growth of the bacterium, allowing for germination and outgrowth of contaminant C. perfringens spores into vegetative cells, which can rapidly multiply to reach high numbers. Therefore, rapid cooling of cooked foods is crucial to prevent the proliferation of this pathogen (Heredia and Labbé 2001). However, unlike vegetative cells of other species, C. perfringens is unusually sensitive to refrigerated and frozen storage. At temperatures below 10 °C, no growth is observed for these bacteria; some reports have found that some strains of C. perfringens are able to grow in food maintained at 12 °C (de Joung et al. 2004). Accordingly, food samples to be tested for the presence of C. perfringens vegetative cells should be analyzed immediately, or kept refrigerated and tested as soon as possible, but never frozen. The U.S. Department of Agriculture/Food Safety Inspection Service (USDA/FSIS) draft compliance guidelines for ready-to-eat (RTE) meat and poultry products state that these products should be cooled at a rate sufficient to Food Bioprocess Technol (2011) 4:624–630 ensure that no more than a 1-log increase of C. perfringens cells takes place (USDA-FSIS 2001). These federal guidelines also state that cooling from 54.4 °C to 26.6 °C (130 °F to 80 °F) should take no longer than 1.5 h and that cooling from 26.6 °C to 4.4 °C (80 °F to 40 °F) should take no longer than 5 h. Additional guidelines allow for the cooling of certain cured cooked meats from 54.4 °C to 26.7 °C (130 °F to 80 °F) in 5 h, and from 26.7 °C to 7.2 °C (80 °F to 45 °F) in 10 h (Brynestad and Granum 2002; Heredia and Labbé 2001). Adding chemical preservatives to foods is a very common practice to reduce the microbial population, to avoid health risks, and extend the shell life of the products. In some cases, these substances have been demonstrated to be very effective in reducing or eliminating pathogenic and spoilage microorganisms. For example, Sabah et al. (2003, 2004) found that 0.5% to 4.8% sodium citrate inhibited growth of C. perfringens in cooked vacuum-packaged restructured beef that was cooled from 54.4 °C to 7.2 °C within 18 h, and that oregano oil in combination with organic acids inhibited growth of the organism during cooling of sous-vide cooked ground beef products. Juneja and Thippareddi (2004) observed that organic acid salts such as 1% sodium lactate, 1% sodium acetate, or 1% buffered sodium citrate (with or without sodium diacetate), inhibited the germination and outgrowth of C. perfringens spores during the chilling process of marinated ground turkey breast. In another study, incorporation of 0.1% carvacrol, cinnamaldehyde, thymol, and oregano oil into the beef completely inhibited C. perfringens spore germination and outgrowth during exponential cooling of cooked beef within 12 h (Juneja et al. 2006) Recently, it was shown that addition of GRAS substances, including sodium benzoate, potassium sorbate, sodium nitrite, and monosodium glutamate, to cultures of C. perfringens can influence their cold tolerance. Moreover, in some cases, these substances that would normally eliminate microorganisms increased the cold tolerance of C. perfringens, permitting cell survival at low temperatures (Limón et al. 2007). In general, food preservation techniques, either physical, chemical, or biological, can cause a variety of stresses that interfere with bacterial homeostasis to prevent growth or to kill bacteria. However, as a result of the stress response, some bacteria can survive and grow following the application of stress (Jones and Inouye 1994). Adaptation to and survival in a stress condition may be an important prerequisite to persistence in foods. This stress response has been described in C. perfringens. A specific set of seven, five, and five stress proteins have been reported as a result of heat, acid, or cold stresses, respectively (Heredia et al. 1998; Villarreal et al. 2000, 2002); cross-response between several stresses has also been reported (Limón et Food Bioprocess Technol (2011) 4:624–630 al. 2001). The implications of these findings are extremely critical. Recently, it has been demonstrated that production of exometabolites of C. perfringens during heat challenge plays an important role in heat tolerance of this bacteria (Heredia et al. 2008). 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