323

CUISTRIDXIM PERFRINGENS IN RELATION TO mxr PRODUCTS+ FRANK L. BRYAN Center for Disease Control U.S. Public Health Service Atlanta, Georgia

Neat and poultry products, particularly and turkey, are frequently incriminated as vehicles of Clostridium perfringens foodborne outbreaks (table 1). Such outbreaks are usually associated with foods prepared in food service establishments where large volumes of food are prepered several hours or a day or more in advance of serving and held during the intervening period at temperatures that are conducive t3 germination of spores and multiplication of vegetative cells. Typical outbreaks that have occurred in the United States are illustrated in table 2.

Factors, uncovered during epidemic investigations (and indicated in table 2), that contribute to outbreaks of C. perfringens foodborne illness include failure to properly refrigerate cogked foods, holding foods at warm (bacterial incubating) temperatures, preparing foods a day or more in advance of serving, and inadequate reheating (Bryan, 1972). A summary of contributory factors involved in 59 outbreaks is illustrated in table 3.

Before these factors can influence growth or survival of C. perfringens in meat, however, the meat must become contamlnated . Clostridium perfringens is a normal inhabitant of the intestinal tract of man and other animals. When enrichment procedures are used, it is not unusual to find all fecal specimens from people and most from animals positive for perfringens. It is also found in high numbers in soil and dust. Data C.on this subject has been reviewed by Smith and Holdeman (1968) and by Bryan (1969). Animal carcasses become contaminated from Fntestinal contents or from soil or dust; in addition, meat can become contaminated when handled by workers. Cross contamination, which is enhanced when both contaminated and uncontaminated meat is processed on the same equipment by the same people, also occurs.

As a result of such contemlnation, meat frequently harbors 2. perfringens. Several investigators have reported on the isolation of E. perfringens in various raw meat and poultry products (table 4). These data indicate that -C. perfringens can be expected to be present on raw meat fromtime to time.

* Presented at the 25th Annual Reciprocalkat Conference of the American Meat Science Association, 1972. TABU 1. MEAT AND RELATED PRODUCTS WHICH WERF: ATTRIBUTED AS BEING VEHICLES OF OUTBREAKS OF REPOEiTED (CONFIF~IVEDAND UNCONFIRMED) CLOSTRIDIUM PERFmGENS FOODBORNE ILWESS, 1967-1970

Year Food 1967 1968 1969 1970 Total

Beef 9 24 34 22 89

Veal 0 0 0 1 1

Pork 1 2 3 1 7 Chicken 3 6 4 2 15 Turkey 1 17 16 13 47

Other meat 2 1 0 2 5

She llf i sh 0 1 1 0 2

Fish 1 1 0 0 2

Other foods 2 5 12 8 27 unknown 5 2 2 5 14

Total 24 59 72 54 209 TABLE 2. REPORTS OF OUTBEiEAKS OF CIOSTRIDIUM PERFRINGENS FOODBORNE ILLNESS THAT HAVE APPEARED IN U.S. LITERATUREL

Number Contributory ill Vehicle factors Place References

> 20 Creamed chicken, Prepared a day before Restaurants chicken croquettes serving, failure to properly refrigerate cooked foods, inadequate reheating

181 Turkey Prepared a day before Train meal Hart --et al. (1960) serving, failure to properly refrigerate cooked foods, inadequate reheating

40 Meat and chili Failure to properly Picnic Dunham ( 1961) bean combination refrigerate cooked foods 800 to Creamed turkey --- State institution Kemp --et al. (1962) 900

55 Shrimp salad Prepered a day before Sorority house kJebb and Wiley (1962) serving, failure to properly refrigerate cooked foods

34 Roast beef Holding foods at warm Res taurant Hart (1962) temperatures

75 Rmst beef --e Scout dinner :

9 Smoked ham --- Family dinner Sart ( 1962 ) ( c ont hued ) Number Contributory ill Vehicle factors Place Reference

29 Turkey salad Prepared a day before Nursing home Seltzer --et al. (1s2) serving, failure to properly refrigerate cooked foods

Creamed chicken Holding foods at 55 warm Church dinner Fleming (m2) temperatures (restaurmt caterer)

155 Roast beef and Prepared a day before Welfare institution Werrin end Witte (1962) serving, failure to luncheon properly refrigerate w cooked foods, inadequate reheating zi:

350 Turkey Prepared a day before School Condit and Proctor (1963) serving, failure to properly refrigerate coDked foods, holding foods at warm temperatures, inadequate reheating

300 Lamb stew pie Leftover food, failure School Agar and Ploeger (1964) to properly refrigerate Nelson --et al. (1966) cooked foods, inadequate reheating

Prepared a day before School McCroan and Mixson (1965) serving, failure to properly refrigerate coDked fads

(Continued ) TABLE 2. (CONTINUED)

Number Contributory ill Vehicle factors Place Reference

171 Turkey a la king Prepared a day before School Herron --et al. (1965) serving, failure to properly refrigerate cooked foods, Wdequate reheating, holding foods at warm temperatures

14 0 Turkey Failure to properly Restaurant banquets Peterson --et al. (1966) refrigerate cooked (three ) foods, inadequate reheating W R) 366 Beef gravy Leftover food, failure School Evans and Preizler (1966) to properly refrigerate Helstad --et el. (1967) cooked foods

Chicken salad Prepared a day before Luncheon Goldblatt et al. (1966) 147 serving, failure to properly refrigerate cooked foods

56 Chicken salad --- Luncheon Houglum and Fleming (1967) 784 Prime rib au jus Holding foods at warm Ho te1 banquet Francis --et al. (1968) temperatures

Roast beef Contamination by Hot el banquet Schachner et al. 900 L- (1968) equipment after cooking, holding foods at warm temperatures

(Continued ) TAELE 2. (CONTINUED)

Nmber Contributory ill Vehicle factors Place Reference

$00 Mushroom gravy Holding foods at warm Military base Robinson and Meseer (1969) temperatures

76 Braised beef Prepared a day before School Armes --et al. (1%~) serving, failure to properly refrigerate cooked foods, inadequate reheating

590 Turkey with gravy Prepared a day before School Bryan --et al. (1971) serving, failure to w properly refrigerate ?A cooked foods, inadequate reheating

N300 Roast beef Holding foods at warm Fast food restaurant Bryan and Kilpatrick (1971) temperatures inadequate reheating

288 Spaghetti and Prepared two days befxe School dinner Deshaye --et al. (1972) meat sauce serving, failure to properly refrigerate cooked foods, inadequate reheating

1 Other outbreaks have been reported to national agencies responsible fw surveillance 3f foodborne diseases. 329

TABLE 3. FACTORS THAT CONTRIBUTE TO OUTBREAKS OF CLQSTRIDIUM PERFRINGENS FOODBOIWE ILLNESS^

Factor Percent

Inadequate holding temperature (room, refrigerator, warmer) 100 Failure to properly refrigerate cooked foods 78

Holding foods at warm (bacterial incubating) temperatures 46 Preparing foods a day or more before serving 44 Inadequate reheating of cooked, chilled foods 38 Use of leftover foods 10

1 Based on 59 outbreaks that were reported to the Center for Disease Control from 1961 through 1970. 330

Number of Number Percent samples positive1 positive2 Reference 5

RAW FED MEATS Beef- 50 35 54 13 Frozen bone les s 237 32 Carcasses 47 20 2 1% Imported-Gr . Brit. 134 28 Retail 47 17 Abattoirs 40 2 Steak and mince 10 6 Tripe 6 1H 1N Hamburger -- -- Veal- Carcasses 17 14 10 0 7 1 20 3 2 Frozen boneless 163 Pork- Carcasses 41 15 55 ll Retail 55 27 Abattoirs 14 3 21 10 38 36 fl 9N 1 Carcasses 27 14 23 1 17 0 Imported-Gr . Brit. 74 li3 Abattoir 19 12 Frozen boneless 163 2 Liver Market 100 26 Abattoir 100 12 Hospital 2 1 4 2 ‘1’ABI;E 4. (CONTINUED)

Number of Number Percent Food samples positive’ positive2 Reference5

POULTRY 26 15 58 7 6H 86 3N 44 1 Chicken (14) 25 10 40 Turkey 24 10 42 35 7 20 17 FISH 18 11 H 61 3N 17 14 a 58 100 1 1

MEAT, POULTFX, FISH 122 20 16 (no breakdown )

1 H = hemolytic; N = non-hemolytic. ( ) = heat-resistant strains; percentages are least sigpificant because both heat-sensitive and heat-resistant strains cause foodborne illness.

3 Without enrichment.

Enrichment method. 5 [l]Hall and Angelotti (1965); [21 Hobbs et al. (1953); [3] Hobbs and Wilson (1957); [41 Bryan and Kilpatrick (s’fii;[51 Sylvester and Green (1961); [61 McKillop (1959); E71 Messer et al. (1970); [81 Canada and (1964); [91 Bryan and McKinley (lE2x [lo] Zottola and Busta ; ill1 Christian and King (1971); [I21 Strong --et al. (1963). 332

It should also not be surprising to find C. perfringens In cooked meat and poultry products because its spore8 are present on the raw products and these cpores can survive the heat generated during ordinary cooking procedures and because recontamination from workers or from equipment that contact cooked meat is a likely possibility (Bryan and Kilpatrick, 1971). Several investigators have found C. perfringens in cooked meat and poultry products (table 5). "hue, if such-cooked products are held at temperatures that do not prevent the gruwth of C. perfringens, spores will germinate and vegetative cells will multiply; if gicn enough time, numbers sufficient to cause illness will develop.

By serotyping isolates from raw and cooked meat and from kitchen equipment, evidence shows that strains of C. perfringens present on raw meat or inserted into the meat by thermopgs survived cooking and can be found on or in cooked meat. Also, workers can be the source of the serotypes present on cooked, sliced meat (Bryan and Kilpatrick, 1971).

Several factors permit the development of large numbers of perfringens in cooked meat and poultry products: C.

1. It requires several amino acids (arginine, leucine, isoleucine, methionine, phenylalanine, threonine, valine, tryptophane, histidine, cysthe, glutamic acid, tyrosine, aspartic acid, and s3me strains serine and alanine) and several vitambs (adenine, biotin, nicoti- namide, pantothenate, pyridoxal, and some strains riboflavin and uracil) for growth (Boyd --et al., 1948; Fuchs and Bonde, 1957). Foods that do not contain these amino acids and vitamins are not suitable for its growth. These nutrients are available in meat, poultry, fish, legumes and milk. 2. It requires a high water activity, usually abwe a, 0.96 (Hobbs, 1965; Gough and Alford, 1965; Strong et e.,1970). Most meat and poultry products have aw values above this level.

3. It grows within the pH range of 5 to 9, but rapid growth occurs only between pH 5.5 to 7.5 (F'uchs and Bonde, 1957; Smith, 1963; Barnes et al., 1963). Most meat products are within this pH range. 4. It does not compete well in the presence of other microorganisms (Smith and Holdeman, 1968; Kafel and Ayres, 1969). When meat is cooked, however, most competitive and amensal fhra are killed; this gives heat-resistant C. perfringens spores an opportunity for germination and gives the resulting cells an opp3rtunity for rapid, unmolested multiplication when the temperature becomes favorable.

5. It requires a low oxidation-reduction potential (Eh) to initiate growth. 0ptim.m Eh to initiate growth is from -45 to -200 mv; the highest is from +3lto +23l mv (Read and Orr, 1943; Hanke and Bailey, 1945; Barnes and Ingram, 1956 ) . When meat c)r gravy is cmked, oxygen is driven out and these fmds become anaembic enough to support the grcrwth of -C. perfringens. 333

TABLE 5. INCIDENCE OF -C. PERF’HINGENS ‘IWE A IN PROCX;:;I~:ll FOOIX

Number of Number Percent Food samples positive’ positive2 Reference 5

PROCESSEX) MEATS 101 20 20 Requires full cooking 38 14 37 Requires light cooking 21 4 14 Requires no cooking 42 2 5 Steak and mince 15 1H 7 1N Sausage 25 4H 167 1N 4 Tripe 6 1 17 Black pudding 2 2 100 Roast (cold) 15 4H 27 2N 13 Cold meats 63 4H 6 4N 6 BBQ pork 15 2 13 Roast beef 31 9 29 Cooked chickens 46 13 H 28 7N 15 1 ( 2) Cooked f’urther processed chickens 118 3 3 Turkey 38 6 16 Wkg. 13 3 23 Turkey stock 4 2 50

PROCESSED FISH Hospital cooked 6 1 16 Kipper 93 7 59 13 227 Smoked haddock 16 7 44 14 0 0 Smoked 9 7 78 9 0 0 scampi 1 1 100 1 1 100 DMYDRATED SOUPS, , SAUCES AND SPAGHETTI 55 10 18 COMMERCIALLY PREPARED FROZEN FOODS 111 3 3

ROME PREPARED FOODS 16 5 3 2

( Continued ) 334

TABLE 5. (CONTINUED) H - hemolytic; N = non-hemolybic. ( ) = heat-resistant strains; percentages are least significant because both heat-sensitive and heat-resistant strains cause foodborne illness. 3 Without enrichment.

Enrichment method. 5 [l] Hall and Angelotti (1965); f21 McKillop (1959); Bryan and Kilpatrick (1971); [41 Christian and King (1971); [5] Lillard (1971); [61 Zottola and Busta (197;1); E71 Bryan and McKinley (1971); [81 Hobbs et al. (1965); (91 Nakamura and Kelly (1968); [lo] Strong --et al. (1963r - 33 5 6. Its heat-resistant spores can survive boiling for 1 to 6 hours (Hobbs et al., 1958; Barnes -et al., 1963; Hall -et a1,*, 1963; Hobbs, 1965; Nakamura and Converse, 196;r; Weiss and Strong, 1967; Roberts, 1968). Several investigators have observed its survival in cooked meat products Barnes et al., 1963; Woodburn and Kim, 1966; Strong and Ripp, 1967 7- High temperatures also heat shock, or activate, its spores, causing a higher yield of vegetative cells fromheated spore cultures than from unheated spore cultures (Barnes et a1., 1963 ) . 8. Its temperature range for growth has been reported between 54.6' and l22OF (Solberg and Elkind, 1970; Collee et al., 1961). In beef, however, it did not commence to grow until temperatures of 65O or 68OF were reached (Barnes et al., 1963; Hall and Angelotti, 1965). Optimum growth occurs between log0 to 11FF (Boyd --et al., 1948). A large volume of hot cooked meat, even if refrigerated, will stay within the growth range for C. perfrinaens for several hours (Bryan et al., 1971). At opt-- temperatures the generation time for C. perfringens has been reported to be as short as 8.5 minutes; Tt averaged 12 minutes (Arbuckle, 1960).

All of these factors are interrelated. If all are optimum for C. perfringens, rapid growth will occur after an initial lag. On the ocher hand, if any one factor is not optimum, the rate of multiplication will be decreased. Combinations of these factors at sub-optimal levels may prevent growth even if each factor alone at such levels would permit growth under otherwise optimum conditions.

Freezing and refrigerated storage is detrimental to vegetative cells of C. perfringens and freezing to a lesser extent, is detrimental to its spores TBarnes et al., 1963; Canada et al., 1964; Strong and Canada, 1964; Woodburn and Kim,TgG; Strong and Ripp, 1967). These organisms, however, can be found in frozen foods so freezing offers no assurance of safety (Bryan and Kilpatrick, 1971; Bryan et al., 1971). Also, large volumes of foods that have been stored in refrigerators overnight, and not adequately reheated, have frequently been vehicles of outbreaks (table 2).

A review of the factors that contribute to outbreaks (tables 2, 3), shows that the way to prevent C. perfringens foodborne illness from-meat products is to hold them at temperatures too cold or too hot to permit multiplication or not long enough to permit organisms to multiply; and to reheat chilled food, just before serving, to a temperature lethal for vegetative cells of C. perfringens (Bryan, 1969). Thus, to prevent C. perfrinaens foodborne illness, cooked meat and poultry should be hanzled in the following ways (Bryan and Kilpatrick, 1971; Bryan et al., 1971; Bryan and McKinley, 1972):

1. They should be eaten within 3 hours af'ter cooking. 2. If they are held at warm temperatures to be served later, hold in warmers or steam tables (which have air or water temperatures above 336

1409) only as long 88 the internal temperature of the food does not drop below 130OF. If the temperature drops below l3OOF, reheat the food. 3. If they are held cold to later be served cold, rapidly cool them to below 65OF, then cool to and hold at or below 45OF until served. One method of rapid cooling for meat and poultry is to slice them and pile to a depth no higher than 3 inches on cold pans, and then place in refrigerator or freezer. Another method is to cut roasts into smaller portions (such as 1 l/4-pound pieces) and place in double plastic bags which are surrounded by ice, then refrigerate. Methods of rapidly cooling stock and gravy are to immerse pans which hold them into ice or cold water baths or to mix in a mechanical mixer for an hour, then refrigerate. 4. If they ere held cold to be served hot later, the same procedures a8 described in item 3 should be used; in addition, heat the chilled meat to an internal temperature of at least 1609~and boil stock or gravy

If these precautions would be taken in all food service establishments, one of the most commonly occurring foodborne diseases--the disease that accounts for most reported cases of such illness in the United States-- would become a rareity. 337

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