2 CLOSTRIDIUM PERFRINGENS ~OOD POISONINGl. c. L. DUNCAN Food Research Institute and Department of Bacteriology The University of Wisconsin, Madison 53106

A.BsTRAcr .------,59 Clostridium perfringens type A food poisoning in man is characterized by diarrhea and abdonrlnal pain. The disease usually follows ingestion of food contaminated with large numbers of C. perfringens cells. During the past 3 to 5 -No.I!I years, the role of this organism in food poisoning incidents &-A No. at Risk Downloaded from http://meridian.allenpress.com/jfp/article-pdf/33/2/35/2398195/0022-2747-33_2_35.pdf by guest on 27 September 2021 in the United States has acquired new emphasis as a result o-o No. Outbreaks of the increasing number of reported outbreaks and the alarm­ ing number of cases associated with these outbreaks. In 1968, C. perfringens was responsible for approximately 28% of the food poisoning outbreaks and 4~ of the cases, when compared with food poisoning caused by Salmonella, Staphy­ lococcus, Shigella, and Clostridium botulinum. The majority of reported outbreaks and cases resulting from C. perfringens are associated with mass feeding establish­ ments. The most common vehicles are beef and poultry products. The mode of action by which C. perfrlngens causes food poisoning symptoms is not fully understood. Control of this type of food poisoning must be concerned with pre­ vention of spore germination and!or multiplication of the vegetative cells in cooked foods.

As early as 1945, McClung described an outbreak of human food .poisoning in the United States result~ ing from the ingestion of boiled chicken contaminated with Clostridium perfringens (19). However, it was 4 not until 1959 that official epidemiological reports 2 were received of food poisoning outbreaks in which C. perfringens type A was identified as the etiologi­ 59 60 61 62 63 64 65 66 67 68 cal agent (2). Although only 4 outbreaks were re­ YEAR ported that year, belated recognition was at last Figure 1. Incidence of food poisoning caused by Clostri­ made of ,a food poisoning organism that was the cul­ dium perfringens in the United States during 1959 through prit in many food poisoning incidents in England 1968. Data are based on reports appearing in the follow­ since the early fifties. With the publication of the ing references: 1, 2, 1, 8, 9, 10, 18, 20, 21, 22, 23, 24, 25, 28 and 30. now classical paper on C. welchii (perfringens) food poisoning by Hobbs et al. in 1953 (16), it became obvious that here was an organism that seemed to has acquired new emphasis. This has resulted from present a particular hazard to the food service in­ the increasing number of outbreaks reported each dustry. All but' 2 of the 18 outbreaks of C. perfring­ year and the alarming number of cases associated ens poisoning. that occurred in the London area from with these outbreaks. This paper is an attempt to September, 1949 to January, 1952 occurred in school, look at the problem of C. perfringens food poisoning municipal, or factory canteens. In most instances, as it currently exists. large numbers of persons were involved. During the past 3 to 5 years, the role of this organ­ STATISTICS OF 0UTSBEAKS ism in food poisoning incidents in the United States The occurrence of food poisoning caused by C. perfringens during 1959. through 1968 is shown in Fig. 1. During the period from 1959 to 1964, there 1Published with the approval of the Director of the Research were relatively few reported outbreaks related to C. Division of the College of Agricultural and Life Sciences. perfringens. These few reports were probably made 2Presented at the Fifty-sixth Annual Meeting of the Inter­ national Association of Milk, Food, and Enviromnentai San­ only as the result of the foresight of health labora­ itarians, Inc., Lonisville, Kentucky, August 18-21, 1969. tories that included C. perfringens as a possibility in 36 CLOSTRIDWM PERF!uNGENS their search for a causal agent. For example, in 1961, 5 of the 6 outbreaks were reported by the state "::1 ~ of California alone (18). In fact, in that year in Cal­ EO"' 0 ~! :=!" :::!

TABLE 2. COMPARISON OF ETIOLOGICAL AGENTS CAUSING FOOD in public eating establishments; the remainder are 1 POISONING OUTBREAKS IN ENGLAND Al'<"D W ALES served in institutions (34). Perfringens food poison­ ing may occur when the organism grows in a cooked "..,"' food product prior to consumption. The organism s"' .E6 ~ "2 may multiply over a broad temperature range of -.:: .9..... """"!::..: ""' ..::;:; about 15 to 50 C (59 to 122 F) and over a pH .9~ ~~ ....."'" Year (;,)"" (Jl! -"' range of about 5.5 to 8.0 (31). Under optimum con­ """' ditions, large numbers of cells may be produced in % of Total 2 to 3 hr. Therefore, the danger lies in keeping food 1959 Outbreaks 2.1 95.7 2.2 at a temperature that is apparently either hot or cold 1960 Outbreaks 2.3 95.3 2.4 1961 Outbreaks 2.1 95.6 2.3 but is one at which growth of the organism will oc­ Outbreaks 2.4 95.5 2.1 cur. This hazard may often be present in mass feed­ 1966 Cases 20.3 65.6 4.1 ing establishments that rely on steam tables or cool­ Downloaded from http://meridian.allenpress.com/jfp/article-pdf/33/2/35/2398195/0022-2747-33_2_35.pdf by guest on 27 September 2021 Outbreaks 0.9 1967 2.4 96.8 ing tables for maintaining foods at ''safe" tempera­ Cases 28.5 62.8 5.5 tures. The hazard may be even greater if foods have been improperly cooled or inadequately reheated 1The values are the percentages of the total outbreaks or cases reported for only these 3 different organisms and are before placing on the steam or cold table. Table calculated from data appearing in references 36 and 37. 3 shows that, indeed, the great majority of perfring­ ens food poisoning incidents that are reported by TABLE 3. PLAcE OF ACQUISITION m· Clostridium perfringens health authorities occur in food service establishments FOOD POISONING OUTBREAKS IN THE UNITED STATES1 that are concerned with mass feeding. The table shows the place of acquistition of per­ Colleges Health or lnst!tu fringens food poisoning outbreaks in the United Year Home Resta.ura.nt Ba.nquet schools tlons Other States from 1959 through 1968. In addition, the total number of cases associated with each specific 1959 1 2 1 1960 2 place is indicated. The outbreaks reported for ban­ 1961 1 1 1 3 quets were meals that were usually served either in 1962 2 1 restaurants or school cafeterias, but a specific out­ 1963 1 break is listed under only one heading. The statis­ 1964 1 1965 1 1 3 2 tics show why such large numbers of persons are 1966 Not Available associated with perfringens food poisoning outbreaks. 1967 1 9 10 6 3 The majority of the outbreaks and cases were asso­ 1968 6 23 8 14 1 4 ciated with mass feeding establishments with restau­ Total rants accounting for the largest number of outbreaks Outbreaks 10 36 21 25 3 14 Total and banquets the largest number of cases. With Cases 82 1,618 4,7'82 4,089 1,181 1,867 large numbers of persons eating a common meal, it

1 TABLE 4. VEHICLES ASSOCIATED WITH Clostridium perfringens Data are based on reports appearing in the following refer­ 1 FOOD POISONING OUTBREAKS IN THE UNITED STATES ences: 1, 2, 7, 8, 9, 10, 18, 20, 21, 22, 23, 24, 25, 28, and 30. Unknown Otller or other Meat foods 37). This is higher than the maximum number of 56 Year Turkey Chicken Beef outbreaks reported in the United States in 1968. 1959 1 1 1 1 It is still not known how many outbreaks occur 1960 2 in the United States each year that involve C. per­ 1961 3 1 2 fringens. With better reporting of outbreaks and 1962 2 1 1963 1 more examination of suspect foods for this organism, 1964 1 it may be found that it surpasses both Staphylococcus 1965 4 1 2 and Salmonella as a causative agent of food-borne ill­ 1966 3 3 5 2 ness. 1967 1 3 14 3 8 1968 17 6 24 3 11 'The relationship of these statistics to the food Total 29 51 9 '2tf service industry is quite important. In the United States, it has been estimated that 400,000 food serv­ 1 Data are based on reports apJ?earlng in the. following refer- ice establishments serve about 100,000,000 meals ences: 1, 2, 7, 8, 9, 10, 18, 20, 21, 22, 23, 24, 25, 28, u n d daily; Eighty-two per cent of these meals are served 30. 38 CLOSTRIDIUM PERFRL.... GENS is easy to recognize when a food poisoning outbreak TABLE 5. COMPARISON OF 'l'IlE lNCUBATION PERIODS AND has occurred at a banquet, college or school cafe­ MAlN SYMPTOMS OF Clostridium perfringens, Salmonella, AND Staphylococcus FOOD POJSONlNG1 teria, or at an institutional or industrial cafeteria. However, with a restaurant or other commercial Clostridium establishment that serves a transient customer, food Characteristic. perf:rlngen.s Salmonella StaphylOCQccus poisoning incidents may go undetected. For this reason, the number of outbreaks occurring in restau­ Incubation 8-22 hr 12-24 hr 2-6 hr rants and caused by C. perfringens may be substan­ Duration 12-24 hr 1-14 days 6-24 hr tially higher than is now reported. The same may Diarrhea Extremely Very common Common be true for outbreaks that occur in the home. common Table 4 reports the vehicles associated with per­ Abdominal Present Present Present fringens outbreaks durL~g the same 1959-1968 pe­ pain riod. With a total of 51 outbreaks, beef was the Downloaded from http://meridian.allenpress.com/jfp/article-pdf/33/2/35/2398195/0022-2747-33_2_35.pdf by guest on 27 September 2021 Vomiting Very rare Not common Extremely most common vehicle. Poultry products (including common turkey and chicken) were next with a total of 41 outbreaks. Some of the outbreaks resulted from Fever Absent Very Absent common meat and/or gravy and/or dressing. These are the types of foods commonly served in mass feeding Prostration Common :R!ira-m Common establishments. They generally require low tempera­ early stages ture cooking, are usually served with a gravy, and 'After Gilbert, 19(39 (11). may be cooked in advance and reheated prior to servmg. Improper cooling of such foods after the initial cooking may allow growth of C. perfringens TABLE 6. INCIDENCE OF Clostridium perjringens IN FOODS spores that survived the heat treatment. Also, the No. of No. of Per cent reheating may not be sufficient to inactivate C. samples samples positive perfringens cells or spores which may be present examined positive samples and the temperature may not be maintained high Strong et al., 1963 (32) enough to prevent subsequent multiplication of the Commercially prepared frozen foods 111 3 2.7 cells during the actual serving of the food. Such Raw fruits and vegetables 52 2 3.8 mishandling of foods may be disastrous to those in Spices 60 3 5.0 charge of the food s·:rvice establishments. Aside from Home-prepared foods 165 3 1.8 Meat, poultry, and fish 122 20 16.4 the individual loss, such establishments may cuffer Hall and Angelotti, 1965 (12) from loss of public confidence and even legal action. Veal 17 14 82.0 Beef 50 35 70.0 THE ILLNESS Chicken 26 15 58.0 Lamb 21 14 52.0 Compared with the more familiar types of food Pork 41 15 37.0 poisoning, that caused by C. perfringens is less se­ Nakamura and Kelly, 1968 (27) vere and of short duration. Table 5 presents a com­ Spaghetti sauce and mixes 13 7 53.8 Sauce and gravy mixes 8 1 12.5 parison of the incubation periods and main symp~ Soup mixes 28 1 3.6 toms of C. perfringens, Salmonella, and Staphylococ­ Cheese and cheese sauce 6 1 16.7 cus food poisonings. The symptoms of perfringens poisoning usually appear after an incubation period of 8 to 22 hr, compared to 12 to 24 hr for Salmonella Although death from perfringens food poisoning is and 2 to 6 hr for Staphylococcus. The clinical illness rare, several cases have been reported in other coun­ of perfringens food poisoning is characterized pri­ tries. These usually have been associated with el­ marily by diarrhea and abdominal cramps. Most derly and/or debilitated persons. For instance, in patients have acute abdominal pain, while about 1967 two deaths resulting from perfringens food poi­ one-third are affected by nausea and headache. soning were reported in England and Wales (37). One Vomiting, which is common in Staphylococcus food who died was a woman aged 58 who also was suf­ poisoning, and fever, which is common in Salmonella fering with diabetic ketosis; the other was a patient food poisoning, rarely occur in patients suffering aged 42 who died in a hospital outbreak of perfring­ from perfringens food poisoning. In fact, the clinical ens food poisoning. Another death that occurred in and epidemiologic pattern of perfringens food poison­ the London area in 1966 was a 62-year-old woman ing is sufficiently characteristic as to be nearly diag­ who died 48 hr after eating turkey contaminated nostic. with C. perfringens (33). Although the woman was CLOSTRIDIDM I'ERFRINGENS 39 also suffering from Pott's disease at the time, the tion on the ligated loop of the rabbit intestine as a cause of death was certified as "acute enterocolitis possible experimental model for the study of per­ due to food poisoning." fringens food poisoning. It was found that about These facts indicate that even though the symp­ one-half of the type A strains tested that were isolat­ toms of perfringens food poisoning are usually less ed from food poisoning outbreaks consistently pro­ severe than those of other agents such as Salmonella, duced exudation of fluid in the intestinal loop when death can result in the elderly or debilitated. Out­ the challenge was made with cultures grown for a few breaks such as the one caused by C. perfringens that hours in a skim milk medium. In contrast, the ma­ occurred in a geriatric hospital in California in 1962 jority of the strains derived from sources other than and that involved 38 of 45 patients (20) become of food poisoning outbreaks failed to induce a compa­ even more concern when the lethal potential is real­ rable consistent response. In the rabbit, as in the ized. lamb, fluid accumulation was shown not to be caused by alpha toxin. Subsequent studies revealed that MECHANISM OF PATHOGEJ\'lCITY overt diarrhea could be experimentally produced in Downloaded from http://meridian.allenpress.com/jfp/article-pdf/33/2/35/2398195/0022-2747-33_2_35.pdf by guest on 27 September 2021 rabbits by injection of viable cells directly into the Clostridium perfringens food poisoning occurs af~ normal (not ligated) ileum, but not by oral chal­ ter ingestion of a food containing large numbers of lenge (4). Good correlation was obtained between the organism. Tills fact has been confirmed by sev­ the ability of the strains to produce fluid accumula­ eral human volunteer experiments (3, 15, 16). The tion in the ileal loops and overt diarrhea. The ability mechanism by which the illness is produced is not of a specific strain to induce diarrhea was dependent clear. However, the requirement that viable cells on both the number of cells in the challenge and the be ingestt~d for production of symptoms in humans method of preparation of cells for challenge. A chal­ suggests that the mode of action is that of infec­ lenge of approximately 1010 total cells obtained from tion. Yet, clinical symptoms themselves, such as the lack of fever, high primary attack rate, lack of sec­ a sporogenic medium and resuspended in skim milk ondary person to person spread, and lack of clinical was most consistent in producing diarrhea. Cells immunity, suggest an intoxication. obtained from an asporogenic medium usually would not produce diarrhea. Several years ago it was suggested that phosphoryl~ choline, a product resulting from the hydrolysis of Additional investigations were made of the ability lecithin in the presence of the alpha toxin of C. per­ of cell extracts and concentrated culture filtrates of fringens, was the agent responsible for both per­ various strains of C. perfringens to produce ileal fringens food poisoning and that caused by Bacillus loop fluid accumulation and overt diarrhea in rab­ cereus (29 ). Although this was an attractive sugges­ bits (5). Again, good correlation was obtained be­ tion, animal feeding studies (38) and human ingestion tween the ability of viable cells and of a toxic factor of phosphorylcholine ( Dack, personal communica­ present in cell extracts and culture filtrates to pro­ tion) failed to validate the hypothesis. duce both fluid accumulation in ileal loops and An understanding of the illness has been hindered diarrhea when injected into the normal ileum of the in part by the lack of a readily available laboratory rabbit. The toxic factor was shown to be heat la­ animal in which the food poisoning syndrome could bile, non-dialyzable, and was inactivated by pronase, be reproduced. Hauschild et al. (13) have shown but not by trypsin, lipase, or amylase. The toxic that lambs developed diarrhea, the principal symp­ factor was present in cell-free preparations when tom of food poisoning in man, after administration cells were grown in a sporogenic medium but not of C. perfringens cells either orally or intraduodenal­ when they were grown in an asporogenic medium, ly via a fistula. Onset and duration of diarrhea in which correlated with the usual inability of viable the lambs were essentially the same as in humans cells grown in an asporogenic medium to produce after experimental ingestion of the organism. It also diarrhea. This was the first published report on the was reported that immunization of lambs against repeated production of a diarrhea response in an alpha toxin prior to challenge had no effect on the experimental animal by such cell-free preparations. ability of the cells to produce diarrhea. A later re­ Further studies are necessary to determine if the port (14) showed that fluid accumulation occurred in diarrhea-producing factor active in rabbits is as­ ligated intestinal loops of lambs when C. perfringens sociated with diarrhea production by C. perfringens cells suspended in fresh medium were injected. Cul­ in cases of human food poisoning. If the agent ture supernatant fluid had no effect. Again, their active in both humans and rabbits is one and the results indicated that alpha toxin was not the factor same, the failure to obtain food poisoning symptoms responsible for fluid accumulation in the loops. in humans fed culture filtrates of C. perfringens (3) Duncan et al. (6) reported results of an investlga- may have resulted from the low concentration of the 40 CLOSTRIDIUM PERFRINGENS q.c_'tive factor present in culture filtrates or the ab­ procedure used for detection of the organism. Strong sence of the factor as a result of the particular et al. used a direct plating technique for enumera­ growth medium used for preparation of the chal­ tion, whereas Hall and Angelotti, as well as Naka­ lenge cells. mura and Kelly, used an enrichment technique and Results obtained using the rabbit as an animal therefore obtained more positive samples. The data model are not inconsistent with the idea that the of Nakamura and Kelly show that various sauce and mechanism of perfringens food poisoning may be that gravy mixes also arc contaminated with C. perfring­ of an "infection," if indeed the term infection is used ens. Some of the products tested required heating in the context indicated by Hobbs and Sutton (17). for less than 10 min. If such foods were allowed to They state that "the term 'infection' does not neces­ cool and were left at room temperature for several sarily imply the invasion of tissue with the corre­ hours before serving, they could become potential sponding host responses, but may also include the sources of food poisoning. multiplication of the organisms within the intestinal With the evident contamination of foods with C. canal without the invasion of tissue". Multiplication perfringens and with the ubiquitous nature of the or­ Downloaded from http://meridian.allenpress.com/jfp/article-pdf/33/2/35/2398195/0022-2747-33_2_35.pdf by guest on 27 September 2021 of the organisms in the intestine would allow elabora­ ganism, it would be difficult if not impossible to rely tion of the toxic factor found to be active in the rab­ on complete elimination of the organism from food bit. That the toxic factor may be detected in a as a control procedure. Since spores of some strains sporogenic medium but not in an asporogenic medi­ of the organisms are very heat resistant, it may be um is consistent with the fact that sporulation seems expected also that co.oked food may contain surviv­ to occur readily in the intestine. It is not known ing spores. Even heat sensitive spores of C. perfring­ if production of the toxic factor is associated with ens have ·been shown to survive the cooking process sporulation or if failure to obtain the factor in an (39) and indeed, the heat sensitive strains are also asporogenic medium results only from a nutritional responsible for food poisoning outbreaks (3.3). There­ .iil)balancc. fore, it is impossible to heat all foods sufficiently to inactivate all C. perfringens spores without making PREVENTIVE MEASURES the food organoleptically undesirable. Control lies Clostridium perfringens may be grouped into five in prevention of spore germination and/or multipli­ different types, A, B, C (including the type previous­ cation of the vegetative cells. This also is true of ly designated as type F), D, and E, which are separat­ foods that have become contaminated subsequent to ed on the basis of their soluble antigens or toxins. In­ cooking. dications are ·that all strains of type A and some of In general, to prevent perfringens food poisoning, the type C strains are potential food poisoning or­ foods should be either cooked and immediately eaten ganisms (17). hot, or cooled rapidly and refrigerated within 1 to The organism is widely distributed in nature. It 1.5 hr until required. Partial cooking of meat on may be found in the air, dust, soil, and waters. Al­ one day with subsequent reheating the next day so, ithas been isolated from a great variety of foods, should be avoided. If this is impossible, the food and is present in the intestinal contents of man and should be boiled or re-cooked thoroughly. A particu­ animals. Therefore, it aptly may be called a ubiq­ lar problem exists with large carcasses of poultry ·uitous microorganism. The incidence of C. per­ that during cooking may not reach an .internal temp­ fringens in American foods has been studied by sev­ erature sufficient for lethality of C. perfringens. Raw eral laboratories. Results of three of these studies foods such as meat and poultry should be kept sepa­ are presented in Table 6. These data show the in­ rate from cooked foods and special attention should cidence of C. perfringens in various foods examined be used to insure that different surfaces and equip­ by Strong et al. in Wisconsin (32), by Hall and Ange­ ment are used for processing these foods. lotti in Ohio (12), and by Nakamura and Kelly in With the long holding periods often involved dur­ Montana (27). ing the preparation and serving of foods in mass feed­ The data of Strong et al. show an incidence of C. ing establishments, it is not too surprising that such perfringens in commercial pr:epared frozen foods, establishments and their patrons frequently become raw :fruits and vegetables, spices, and home prepared victimized by this -organism. However, the prob­ foods ranging from 1.8 to 5.0%. However, in the lem of perfringens food poisoning may not reside only meat, poultry, and fish category the incidence was in the mass feeding establishment. In the past few 16.4%. The later study by Hall and Angelotti shows years there has been a great increase in the availabil­ a much higher incidence of the organism in meat and ity of convenience foods on the American market. poultry, with up to 82% .of the veal samples being Many of these are pre-cooked and are either frozen, positive. This higher incidence is a reflection of the refrigerated, or are hot. In the hands of the consum- CLOSTRIDIUM PmwroNGENS 41 er they may require only warming or a short cooking Foodborne disease in California with special reference to time before use. Another convenience food service Clostridium perfringens (welchii). Public Health Reports 77: 910-914. is that of vended foods. These are now widely used 19. McClung, L. S. 1945. Human food poisoning due to in schools, colleges, hospitals, factories, and other growth of Clostridium perfringens {C. welchii) in freshly cook­ places. Abuse of such foods, especially those con­ ed chicken. J. Bacteriol. 50:229-231. taining meat or poultry products by either the pro­ 20. Morbidity and Mortality Weekly Report. 1962. U, S. ducer or the consumer may result in an unexpected Public Health. Service, Communicable Disease Center, At­ encounter with perfringens food poisoning. lanta, Georgia. 11: 314-315. 21. Morbidity and Mortality Weekly Report. 1962. U. S, Public Health Service, Communicable Disease Center, At­ REFERENCES lanta, Georgia. 11:378. 1. Dauer, C. C. 19fH. 1960 summary of disease out­ 22. Morbidity and Mortality Weekly Report. 1963. U. S. breaks and a 10-year resume. Public Health Reports 76:915- Public Health Service, Communicable Disease Center, At­ 922. lanta, Georgia. 12:99. 2. Dauer, C. C., and D. L. Davids. 1960. 1959 summary 23. Morbidity and Mortality Weekly Report. 1963. U. S. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/33/2/35/2398195/0022-2747-33_2_35.pdf by guest on 27 September 2021 of disease outbreaks. 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