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Food Poisoning Attributed to Controversial Agents: Bacillus Cereus, Pseudomonas sp. and Faecal Streptococci
Food Poisoning Attributed to Controversial Agents: Bacillus cereus, Pseudomonas sp. and Faecal Streptococci E. M. Foster Food Research Institute and Department of Bacteriology University of Wisconsin, Madison, Wisconsin
Contents 1. 2. 3. 4. 5. 6.
Introduction Bacillus cereus Pseudomonas sp. Faecal streptococci Discussion References
Introduction Clostridium botulinum, Staphylococcus am'eus, Clostridium perfringens and the Salmonella species are well known and undisputed causes of food-borne disease. There is a sizeable group of organisms, however, whose status as food poisoning agents is controversial. All of them at one time or another have been suspected of causing illness, usually on the basis of evidenc~ such as this: (1) people become ill with symptoms of gastroenteritis after eating; (2) at some later time samples of suspect food are examined bacteriologically; (3) large numbers of the organism in question are found in one or more -of the items consumed. By itself, this evidence is not enough to satisfy the discriminating investigator. Merely finding large numbers of an organism not known to cause infection by the alimentary route or to produce a toxin that can be absorbed from the gastrointestinal tract is not sufficient basis for incriminating an organism as an agent of food-borne disease. Only if it were known that the food had been stored in a manner that would preclude bacterial growth following the meal and only if exhaustive bacteriological analysis had failed to reveal a known agent of food-borne disease would the mere presence of an organism in large numbers be anything more than suggestive. Lacking incontrovertible epidemiological evidence from actual disease outbreaks, there is no way to settle the controversy about a suspected food poisoning agent except to feed the organism in pure culture and reproduce the disease in a susceptible host. A variety of experimental animals have been used for this purpose, but direct evidence that the organism can cause illness when consumed by man is necessary for unequivocal proof. Usually it is during efforts to obtain proof that an organism's controversial status becomes established. One or more investigators observe signs of gastroenteritis after feeding a suspect organism but others are unable to confirm the findings. Thereafter, controversy exists. ~Iembership in this heterogeneous assortment of "controversial" agents is far from static. In the third edition of his book, Food Poisoning, Dack's (1956) list of "other bacteria" which were believed to cause Can. Inst. Food Sel. Teehnol. J. Vol. 6. No. 2, 1973
disease included E. coli, paracolon organisms, cloacaeaerogenes bacteria, Proteus sp., O. perfringcns and B. cereus. Apparently convinced that the enterococci could, in fact, cause food-borne illness, the author devoted an entire chapter to {(Streptococcus faecalis in Relation to Food Poisoning." Thirteen years later Riemann's book, Food-Borne Infections and Intoxications) treats O. perfrin.qens and B. cereus as established disease agents (Hobbs, 1969), but includes S. faecalis and the other enterococci among the organisms for which "proof is inconclusive" (Bryan, 1969). Other members of this "controversial" group are Proteus, Providencia, Escherichia coli, Oitrobacter, Klebsiella, Enterobacter, Pseudomonas, Bacillus subtilis, Actinomyces, phospholipase C-producing bacteria, and Oandida. Interest in and attitudes about the controversial agents seem to follow national boundaries to a considerable degree. To illustrate, Table 1 gives the causal agents of food poisoning in England and Wales, the United States and Hungary for various periods during the past decade. Clearly the salmonellae, S. aureus and O. perfringens accounted for the great majority of incidents in all three countries. Of much more interest to our present discussion are the other figures. B. cereus obviously is an important factor in Hungary, whereas Hobbs (1969) says "there have been no authentic or published accounts of food poisoning due to B. cereus or other organisms of the Bacillus group in the United Kingdom." O. botulinum, Shigella and pathogenic E. coli together have accounted for 10% of the U.S. outbreaks during the past several years but only 1% of the incidents reported in Hungary. By contrast, Hungarian authorities attributed 6% 'Of their outbreaks to Pseudomonas aeruginosa, Proteus sp. and Klebsiella sp. Neither British nor American authorities have attributed a single incident to one of these three agents in recent years. There is no doubt that ethnic differences in food habits can have enormous influence on the types of organisms causing food-borne disease. For example, the preponderance of Vibrio parcthaemolyticllS illness among food poisoning incidents in Japan likely reflects the heavy consumption of raw fish and other foods from the sea. Yet there is no escaping the conclusion that personal interests and biases of investigators greatly influence our reported statistics and thereby help perpetuate controversy. Otherwise, why should Hungary have substantial numbers of food-borne disease outbreaks from P. aeruginosa, Proteus sp. and Klebsiella sp., while the U.S. reported none? Equally significant, 126
Table 1. Relative importance of different food-borne disease agents in England and Wales, the United States and Hungary. Agent
Per cent of reported outbreaks where causal agent was identified England and Wales a
Salmonella, S. aureus, C. perfringens B. cereus Enterococci C. botulinum, Shigella sp., E. coli P. aeruginosa. Proteus sp., Klebsiella sp.
99 0 0 0 0
United States b 87
<: 1 2
10
o
HungaryC
83 8 1 1
6
a General and family outbreaks in 1966 as cited by Hobbs (1969). b Center for Disease Control annual summaries for January, 1966, through June, 1971. C Figures for 1960-68 as cited by Ormay and Novotny (1970).
American statistics show 17 outbreaks of enterococcal food poisoning in the two years, 1967 and 1968. Every single one was reported from New York Gity or otearby New Jersey. During the next two years, however, only five outbreaks were reported in the entire country; three of them were admittedly questionable and only one came from New York City. Thus, we do indeed have controversial food poisoning agents, some of which are not among our curI'ent candidates for discussion: B. cereus, Pse1tdomonas. sp. and faecal streptococci. My purpose shall be briefly to review the controversial status of each.
Bacillus cereus Goepfert et al. (1972) have provided a complete and timely review of the literature on B. cereus. Most of our information about the organism's food poisoning propensities has come from the Scandinavian countries, Hungary and The Netherlands, but reports now are beginning to appear in other countries (Midura et al., 1970; Anonymous, 1972). In many respects the food poisoning attributed to B. cereus closely resembles that caused by G. perfringens. Typically, symptoms include abdominal cramps, profuse watery diarrhea, rectal tenesmus and moderate nausea, but seldom vomiting. Onset time after eating usually is 10 to 12 hours and duration of illness is about the same. Children are more seriously affected than healthy adults. Contrary to the situation with G. perfringens, B. cereus is found in small numbers if at all in the victims' stools. Foods involved in outbreaks of disease typically contain 10 to 100 million cells 'of B. cereus per gram, but they are not overtly spoiled. A wide variety of cooked dishes have been incriminated including vanilla sauce, cornstarch puddings, sausages, various cooked meat dishes, soups, cream pastries, cooked rice and mashed potatoes. Spores of the organism are widely distributed in nature and readily grow in cooked foods of suitable composition if the temperature is favorable. Therefore, as with G. perfringens poisoning, improper food handling is the basic fault in illness caused by B. cereus. Attempts to produce B. cereus poisoning in human volunteers have given equivocal results. Although Hauge (1950, 1955) induced clearcut disease by feeding vanilla sauce containing B. cereus from an outbreak of illness, Dack and co-workers (1954) could not 127
confirm his findings with cultures from other sources. Other European workers have had indifferent success in demonstrating the food poisonil1g potential of B. cereus with human volunteers, although many have induced diarrhea and other symptoms in mice, guinea pigs, dogs and cats. Thus, B. ceretts remains a controversial organism. Epidemiological evidence strongly incriminates it in dozens of outbreaks, but uncertainty will continue until we know more about the mechanism by which it causes illness. Nygren suggested (1962) that the phospholipase C produced by B. cereus catalyzes the hydrolysis of lecithin to yield phosphorylcholine, which serves in turn as the toxic agent. This idea has not been supported in feeding trials with monkeys and a human volunteer (Dack, 1966; Weiss et al., 1966). A new hypothesis put forth by Goepfert and coworkers (1972) assumes the existence of a cell-associated enterotoxin that is formed during growth of the organism in the food and released upon lysis of the cells. Hence neither invasion of the tissue nor multiplication within the intestine need 'Occur. Characteristics of B. cereus food-borne illness which support this suggestion include: (a) the requirement for large numbers of cells to elicit response; (b) relatively rapid onset and short duration of illness; (c) lack of fever; and (d) few or no recoverable B. cereus cells in stool samples during or following illness.
Pseudomonas sp. The Gram negative pseudomonads are common inhabitants of soil, water and decomposing organic matter. One species, Pseudomonas aeruginosa, causes a variety of suppurative processes in man (Burrows, 1959) and often is found in the intestinal discharges of patients with gastroenteritis. Bryan (1969) reviewed several incidents from both D.S. and foreign literature that appeared to be related to food consumption. Shooter et al. (1969) observed a high faecal carriage rate of P. aeruginosa among hospital patients and suggested that faecal strains may be implicated as causes of sepsis in surgical patients who harbor them. The investigators isolated P. (wruginosa from 21 'Of 273 samples of food prepared in a hospital diet kitchen and found several patients to be shedding in their stools the same strains of P. aeruginosa that were found in the food. J. Inst. Can. Scl. Technol. Aliment. Vol. 6, No 2, 1973
In virulent cultures of P. aenlginosa Kubota and Liu (1971) have demonstrated a toxic protein that caused fluid accumulation in the ligated loop of a rabbit's intestine. This "enterotoxin" was inactivated by boiling and by trypsin. Virulent strains of P. aeruginosa (i.e. lethal to mice on intraperitoneal injection) tended to induce fluid accumulation, whereas non-virulent strains usually did not. From the available evidence one must suspect that certain strains of P. aeruginosa are able to produce a toxic substance which somehow can incite gastroenteritis in man. Either these strains are rare or the circumstances that lead to illness are seldom met, because P. aeruqinosa gastroenteritis is far from common. Acquisition of the organism through food appears to be possible, though not a common means of infection. One other species, Pseudomonas cocovenenans) has been shown to produce a serious and often fatal type of food poisoning in parts 'of Central Java, Indonesia (van Veen, 1966). A home-made fermented dish called "bongkrek" is prepared there from shredded coconut. Normally a Rhizopus species develops on the product but sometimes for reasons that are unknown P. cocovenenans overgrows and inhibits development of the fungus. Highly toxic product can result. When cultured on pressed coconut, but not on soybean or peanut cake, P. cocovenenans produces two toxic substances, toxoflavin and bongkrek acid. The latter, an unsaturated fatty acid, is the more toxic of the two, causing rapid and often fatal hypoglycemia in experimental animals. There seems to be no basis for controversy about the food poisoning potential of P. cocovenenans. However, we still do not know the conditions that allow the occasional development of this organism during bongkrek manufacture.
Faecal Streptococci Perhaps the most controversial of all the disputed food poisoning agents are the faecal streptococci. This group, sometimes called the Group D streps, includes the enterococci. They have been incriminated in nunumerous outbreaks involving different kinds of foods over the past 50 years. Almost invariably the evidence amounts to a demonstration of large numbers of faecal streptococci in the suspect food at some time after the incident. There is no merit to another rehashing of tIle voluminous literature on food poisoning outbreaks by the faecal streptococci. This has been done ably and thoroughly by Bryan (1969), Hartman et al. (1965), Deibel and Silliker (1963) and Shattock (1962). The typical incident goes something like this: a number of people become ill with r:.ausea, colic, diarrhea and sometimes vomiting between 2 and 36 hours after eating. Recognition of an outbreak spurs investigation and collection of samples. Meanwhile the leftover foods, which now have become suspect, may well have been held without refrigeration for two or three days before analysis. Faecal streptococci, if present, can flourish in most cooked foods and often outgrow Can. Inst. Food Sel. Teehnol. J. Vol. 6. No. 2, 1973
everything else. Thus they can be found in large numbers while other food poisoning agents may be completely overlooked. Recognizing that evidence such as this is not enough to incriminate the faecal streptococci as food poisoning agents, several investigators have fed cu!tures isolated from food poisoning outbreaks to experimental animals and human volunteers. Feeding trials with animals have been inconclusive; cats often respond but most other animals do not (Hartman et al.) 1965). Bryan (1969) states categorically that "animal experimentations tell us little, since the re· action of experimental animals may not be the same as the reaction of man." Tests with human volunteers have given conflicting results. A few investigators have reported evidence of gastroenteritis when faecal streptococci were fed, but most have not (Hartman et al.) 1965; Bryan, 1969). The most extensive trials were performed by Deibel and Silliker (1963), who fed 23 enterococcus strains to two or three human volunteers without a single indication of gastroenteritis. Ten years ago Shattock (1962) stated that "from all the evidence one is forced to conclude either that the environmental conditions for producing a pathogenic principle for faecal (Group D) streptococci are only very occasionally encountered, or that only exceptional strains are potentially enteropathogenic." That conclusion is equally valid today. Just as there is controversy over the food poisoning potential of the faecal streptococci, there is also controversy over their significance as indicators of insanitation. These organisms are indeed found in the intestinal tracts of man and animals, but they also occur in so many other places that their significance as faecal indicators is seriously restricted. They are common in meat packing establishments (Greenberg, 1965) and on the surfaces of plants (Mundt, 1963) . They occur in cheese, often by the tens to hundreds of millions per gram (Foster et al., 1942; Mattick and Shattock, 1943; Clark and Reinhold, 1966) ; they grow in fermented sausages (Niven, 1963) ; and they develop during the malting of barley (Stark, 1970). In fact, hardly any food that does not receive a terminal heating can be prepared feasibly without becoming contaminated with faecal streptococci. If the food is held above the freezing point for a reasonable length of time the organisms will increase in number (Niven, 1963). Thus, faecal streptococci effectively serve as an index of quality only for those foods which receive a substantial heat treatment during preparation (e.g. precooked frozen foods). However, to require low numbers of faecal streptococci in milk, cheese, fermented sausages, vegetables and similar products would virtually eliminate these items from our diet. As Niven (1963) has pointed out, faecal streptococci "can establish themselves and grow in or on food substances far removed from the original contaminating source. Therefore, an interpretation of the faecal streptococcal population in terms of the amount of 128
faecal contamination in the food can approach the ridiculous." In view of the widespread occurrence of faecal streptococci and with due regard for the uncertainty about their food poisoning potential, it is clear that these organisms do not deserve their unsavory reputation. Except for processed foods that have been subjected to rigorous bactericidal treatment, small numbers of faecal streptococci in a product are essentially meaningless. In fermented products the same can be said about large numbers.
Discussion Occurrence of an outbreak of food poisoning with dozens or hundreds of victims is difficult to refute. Proving the cause is another matter. No one has Jet found a satisfactory substitute for Koch's postulates in determining whether a suspect organism can, in fact, cause food-borne disease. Moreover, for conclusive proof the symptoms of disease must be induced in human volunteers, not experimental animals (Bryan, 1969). Still the matter is not simple. Organisms that have been clearly incriminated on epidemiological grounds have failed to incite disease when fed to human volunteers. Why? And why does a particular organism cause disease in some trials and not in others? Shattock (1962) alluded to this dilemma when she concluded about the Group D streptococci either that strains able to produce a pathogenic principle are exceptional, or conditions necessary for disease production are rarely met. Evidence now accumulating suggests that ways to settle some of our food poisoning controversies may soon be at hand. W'e now know that the method of preparing a challenge dose of food poisoning organisms can drastically affect their behaviOl' in a human volunteer (Strong et al.) 1971). Equally significant, the response of the rabbit ileal loop is influenced by the method of preparing the challenge. Thus we may have an explanation for past discrepancies between the results of different investigators. We may also have an animal model, the ileal loop response, that will provide a practical mechanism for studying enteropathogenicity and for identifying the actual factors involved in disease (Duncan and Strong, 19(9). Spira and Goepfert (1972) have shown the utility of the ligated ileal loop in studying food poisoning by B. cercus. No animal test will completely eliminate the need for human feeding trials but Strong et al. (1971) have shown a reasonable correlation between rabbit ileal loop response and reaction of human subjects to cells of C. per/ringens. Thus, we may now have a technique for studying B. cereus) faecal streptococci and other organisms whose food poisoning potential is unclear. Techniques worked out with C. lJer/ringens) itself once a "controversial" organism (Dack, 1956), may help resolve questions remaining about the others. Once we understand how the controversial agents cause food poisoning, if they do, it may be possible to find specific and easily measured characteristics that 129
correlate with the food poisoning property. This, then, will give us a practical tool for detecting those organisms having the potential to cause disease. It is important that the controversies surrounding B. cereus, the faecal streptococci and other questioned organisms be resolved. With the clouds now hanging over these organisms purchasers are reluctant to buy products containing them. Purchase specifications -often limit the numbers of enterococci to unreasonable and often meaninglessly low levels. Limits on the numbers of B. cereus in dehydrated potatoes have been imposed by certain purchasers. For the most part standards such as these simply represent lack of understanding of the facts. B. cereus becomes hazardious, if at all, only if the food is grossly mishandled and growth occurs. Even major differences in initial contamination levels can be quickly negated if a product is abused. Resolving the controversies may not be easy. Every negative test calls for another trial. Only a positive result is convincing. Thus a large mass of data obtained under a wide variety of test conditions will be necessary to answer the questions that exist as long as the findings are negative.
References Anonymous. 1972. Food Poisoning Associated with Bacillus cereus. Brit. Med. J., 1 (No. 5793) :189. Bryan, F. L. 1969. Infections Due to Miscellaneous Microorganisms. In Food-Borne Infections and Intoxications, H. Riemann, Ed., Academic Press, New York. Burrows, W. 1959. Textbook of Microbiology, 17th Ed., W. B. Saunders Co., Philadelphia. Center for Disease Control, U. S. Public Health Service, Atlanta, Ga., Foodborne Outbreaks, Annual Summaries for 1966, '67, '68, '69, '70 and January-June, 1971. Clark, W. S., and Reinbold, G. W. 1966. Enterococi in Young Cheddar Cheese. J. Dairy Scl., 49:1214. Dack, G. M. 1956. Food Poisoning, Rev. and Enlarged. The University of Chicago Press, Chicago. Dack, G. M. 1966. Importance of Food-borne Disease Outbreaks of Unrecognized Causes. Food Technol., 20 :1279. Dack, G. M., Sugiyama, H., Owens, F. J., and Kisner, J. B. 1954. Failure to Produce Illness in Human Volunteers Fed Bacillus cereus and Clostridium perfrlngens. J. Infect. Dis., 94:34. Deibel, R. H., and Silliker, J. H. 1963. Food-poisoning Potential of the Enterococcl. J. Bacteriol., 85 :827. Duncan, C. L., and Strong, D. H. 1969. Ileal Loop Fluid Accumulation and Production of Diarrhea in Rabbits by Cell-free Products of Clostridium perfringens. J. Bacteriol., 100:86. Foster, E. M., Garey, J. C., and Frazier, W. C. 1942. The Bacteriology of Brick Cheese. Ill. The Bacteria Involved in Ripening. J. Dairy Scl., 25 :323. Goepfert, J. M., Spira, W. M. and Kim, H. U. 1972. Bacillus cereus: Food Poisoning Organism. A Review. J. Milk & Food Technol., 35:213. Greenberg, R. A. 1965. Significance of Enterococci in Foods. Activities Report, 17 :62. Hartman, P. A., Reinbold, G. W., and Saraswat, D. S. 1965. Indicator Organisms - A Review. II. The Role of Enterococci in Food Poisoning. J. Milk and Food Technol., 28:344. Hauge, S. 1950. Matforgiftninger fremkalt av Bacillus cereus. Nordisk. Hyg. Tidskr., 31 :189. (BioI. Abstr., 25:1063. 1951.) Hauge, S. 1955. Food Poisoning Caused by Aerobic Spore-forming Bacilli. J. Appl. Bacteriol. 18 :591. Hobbs, Betty C. 1969. Clostridium perfringens and Bacillus cereus Infections. In Food-Borne Infections and Intoxications, H. Riemann, Ed., Academic Press, New York. KUbota, Y., and Liu, P. V. 1971. An Enterotoxin of Pseudomonas aeruginosa. J. Infect. Dis., 123:97. Mattlck, A. T. R., and Shattock, P. M. F. 1943. Group D Streptococci in English Hard Cheese. Mon. Bul. Emerg. Publ. Hlth. Lab. Serv. 2:73. Midura, T., Gerber, M., Wood, R., and Leonard, A. R. 1970. Outbreak of Food Poisoning Caused by Bacillus cereus. Pub. Health Reports, 85:45. Mundt, J. O. 1963. Occurrence of Enterococci on Plants in a Wild Environment. Appl. Microbiol., 11 :141. Niven, C. F. 1963. Microbial Indexes of Food Quality: Fecal Streptococci. In Microbiological Quality of Foods, Slanetz, L. N., Chichester, C. 0., Gaufin, A. R., and Ordal, Z. J., Eds., Academic Press, New York. Nygren, B. 1962. Phospholipase C-producing Bacteria and Food Poisoning. Acta Pathol. Microbiol. Scand. Suppl., 160:1. Ormay, L., and Novotny, T. 1970. Uber sogenannte unspezifische Lebensmittelvergiftungen in Ungarn. Zbl. Bakt. Abt. I. Orlg., 215:84. J. Inst. Can. Sci. Technol. Aliment. Vol. 6, No 2, 1973
Shattock, P, M. F. 1962. Enterococci. In Chemical and Biological Hazards in Foods, Ayres, J. C., Kraft, A. A., Snyder, H. E., and Walker, H. W., Eds., Iowa State University Press, Ames, Iowa. Shooter, R. A., Cooke, E. M., Gaya, H., Kumar, P., Patel, N., Parker, M. T., Thorn, B. T. and France, D. R. 1969. Food and Medicaments as Possible Sources of Hospital Strains of Pseudomonas aeruginosa. Lancet (June 21) :1227. Spira, W. M., and Goepfert, J. M. 1972. Bacillus cereus - Induced Fluid Accumulation in Rabbit Ileal Loops. Appl. Microbiol., 24: Stark, E. 1970. Fecal Streptococci on Barley and Malt Kernels and Instant Malted Milk Powder. Appl. Microbiol., 20 :200.
Can. Inst. Feod Scl. Technol. J. Vol. 6, No. 2, 1973
Strong, D. H., Duncan, C. L., and Perna, G. 1971. Clostridium perfringens Type A l"ood Poisoning. n. Response of the Rabbit Ileum as an Indication of Enteropathogenicity of Strains of Clostridium perfringens in Human Beings. Int. and Imm. 3:171. van Veen, A. G. 1966. The Bongkrek Toxins. In Biochemistry 01 Some Food-borne Microbial Toxins, R. I. Mateles and G. N. Wogan, Ed .. The M.LT. Press, Massachusetts Institute of Technology, Cambridge, Mass. Welss, K. F., Strong, D. H., and Groom, R. A. 1966. Mice and Monkeys as Assay Animals for Clostridium perfringens Food Poisoning. Appl. Microbiol., 14:479.
130
Contents 1. 2. 3. 4. 5. 6.
Introduction Bacillus cereus Pseudomonas sp. Faecal streptococci Discussion References
Introduction Clostridium botulinum, Staphylococcus am'eus, Clostridium perfringens and the Salmonella species are well known and undisputed causes of food-borne disease. There is a sizeable group of organisms, however, whose status as food poisoning agents is controversial. All of them at one time or another have been suspected of causing illness, usually on the basis of evidenc~ such as this: (1) people become ill with symptoms of gastroenteritis after eating; (2) at some later time samples of suspect food are examined bacteriologically; (3) large numbers of the organism in question are found in one or more -of the items consumed. By itself, this evidence is not enough to satisfy the discriminating investigator. Merely finding large numbers of an organism not known to cause infection by the alimentary route or to produce a toxin that can be absorbed from the gastrointestinal tract is not sufficient basis for incriminating an organism as an agent of food-borne disease. Only if it were known that the food had been stored in a manner that would preclude bacterial growth following the meal and only if exhaustive bacteriological analysis had failed to reveal a known agent of food-borne disease would the mere presence of an organism in large numbers be anything more than suggestive. Lacking incontrovertible epidemiological evidence from actual disease outbreaks, there is no way to settle the controversy about a suspected food poisoning agent except to feed the organism in pure culture and reproduce the disease in a susceptible host. A variety of experimental animals have been used for this purpose, but direct evidence that the organism can cause illness when consumed by man is necessary for unequivocal proof. Usually it is during efforts to obtain proof that an organism's controversial status becomes established. One or more investigators observe signs of gastroenteritis after feeding a suspect organism but others are unable to confirm the findings. Thereafter, controversy exists. ~Iembership in this heterogeneous assortment of "controversial" agents is far from static. In the third edition of his book, Food Poisoning, Dack's (1956) list of "other bacteria" which were believed to cause Can. Inst. Food Sel. Teehnol. J. Vol. 6. No. 2, 1973
disease included E. coli, paracolon organisms, cloacaeaerogenes bacteria, Proteus sp., O. perfringcns and B. cereus. Apparently convinced that the enterococci could, in fact, cause food-borne illness, the author devoted an entire chapter to {(Streptococcus faecalis in Relation to Food Poisoning." Thirteen years later Riemann's book, Food-Borne Infections and Intoxications) treats O. perfrin.qens and B. cereus as established disease agents (Hobbs, 1969), but includes S. faecalis and the other enterococci among the organisms for which "proof is inconclusive" (Bryan, 1969). Other members of this "controversial" group are Proteus, Providencia, Escherichia coli, Oitrobacter, Klebsiella, Enterobacter, Pseudomonas, Bacillus subtilis, Actinomyces, phospholipase C-producing bacteria, and Oandida. Interest in and attitudes about the controversial agents seem to follow national boundaries to a considerable degree. To illustrate, Table 1 gives the causal agents of food poisoning in England and Wales, the United States and Hungary for various periods during the past decade. Clearly the salmonellae, S. aureus and O. perfringens accounted for the great majority of incidents in all three countries. Of much more interest to our present discussion are the other figures. B. cereus obviously is an important factor in Hungary, whereas Hobbs (1969) says "there have been no authentic or published accounts of food poisoning due to B. cereus or other organisms of the Bacillus group in the United Kingdom." O. botulinum, Shigella and pathogenic E. coli together have accounted for 10% of the U.S. outbreaks during the past several years but only 1% of the incidents reported in Hungary. By contrast, Hungarian authorities attributed 6% 'Of their outbreaks to Pseudomonas aeruginosa, Proteus sp. and Klebsiella sp. Neither British nor American authorities have attributed a single incident to one of these three agents in recent years. There is no doubt that ethnic differences in food habits can have enormous influence on the types of organisms causing food-borne disease. For example, the preponderance of Vibrio parcthaemolyticllS illness among food poisoning incidents in Japan likely reflects the heavy consumption of raw fish and other foods from the sea. Yet there is no escaping the conclusion that personal interests and biases of investigators greatly influence our reported statistics and thereby help perpetuate controversy. Otherwise, why should Hungary have substantial numbers of food-borne disease outbreaks from P. aeruginosa, Proteus sp. and Klebsiella sp., while the U.S. reported none? Equally significant, 126
Table 1. Relative importance of different food-borne disease agents in England and Wales, the United States and Hungary. Agent
Per cent of reported outbreaks where causal agent was identified England and Wales a
Salmonella, S. aureus, C. perfringens B. cereus Enterococci C. botulinum, Shigella sp., E. coli P. aeruginosa. Proteus sp., Klebsiella sp.
99 0 0 0 0
United States b 87
<: 1 2
10
o
HungaryC
83 8 1 1
6
a General and family outbreaks in 1966 as cited by Hobbs (1969). b Center for Disease Control annual summaries for January, 1966, through June, 1971. C Figures for 1960-68 as cited by Ormay and Novotny (1970).
American statistics show 17 outbreaks of enterococcal food poisoning in the two years, 1967 and 1968. Every single one was reported from New York Gity or otearby New Jersey. During the next two years, however, only five outbreaks were reported in the entire country; three of them were admittedly questionable and only one came from New York City. Thus, we do indeed have controversial food poisoning agents, some of which are not among our curI'ent candidates for discussion: B. cereus, Pse1tdomonas. sp. and faecal streptococci. My purpose shall be briefly to review the controversial status of each.
Bacillus cereus Goepfert et al. (1972) have provided a complete and timely review of the literature on B. cereus. Most of our information about the organism's food poisoning propensities has come from the Scandinavian countries, Hungary and The Netherlands, but reports now are beginning to appear in other countries (Midura et al., 1970; Anonymous, 1972). In many respects the food poisoning attributed to B. cereus closely resembles that caused by G. perfringens. Typically, symptoms include abdominal cramps, profuse watery diarrhea, rectal tenesmus and moderate nausea, but seldom vomiting. Onset time after eating usually is 10 to 12 hours and duration of illness is about the same. Children are more seriously affected than healthy adults. Contrary to the situation with G. perfringens, B. cereus is found in small numbers if at all in the victims' stools. Foods involved in outbreaks of disease typically contain 10 to 100 million cells 'of B. cereus per gram, but they are not overtly spoiled. A wide variety of cooked dishes have been incriminated including vanilla sauce, cornstarch puddings, sausages, various cooked meat dishes, soups, cream pastries, cooked rice and mashed potatoes. Spores of the organism are widely distributed in nature and readily grow in cooked foods of suitable composition if the temperature is favorable. Therefore, as with G. perfringens poisoning, improper food handling is the basic fault in illness caused by B. cereus. Attempts to produce B. cereus poisoning in human volunteers have given equivocal results. Although Hauge (1950, 1955) induced clearcut disease by feeding vanilla sauce containing B. cereus from an outbreak of illness, Dack and co-workers (1954) could not 127
confirm his findings with cultures from other sources. Other European workers have had indifferent success in demonstrating the food poisonil1g potential of B. cereus with human volunteers, although many have induced diarrhea and other symptoms in mice, guinea pigs, dogs and cats. Thus, B. ceretts remains a controversial organism. Epidemiological evidence strongly incriminates it in dozens of outbreaks, but uncertainty will continue until we know more about the mechanism by which it causes illness. Nygren suggested (1962) that the phospholipase C produced by B. cereus catalyzes the hydrolysis of lecithin to yield phosphorylcholine, which serves in turn as the toxic agent. This idea has not been supported in feeding trials with monkeys and a human volunteer (Dack, 1966; Weiss et al., 1966). A new hypothesis put forth by Goepfert and coworkers (1972) assumes the existence of a cell-associated enterotoxin that is formed during growth of the organism in the food and released upon lysis of the cells. Hence neither invasion of the tissue nor multiplication within the intestine need 'Occur. Characteristics of B. cereus food-borne illness which support this suggestion include: (a) the requirement for large numbers of cells to elicit response; (b) relatively rapid onset and short duration of illness; (c) lack of fever; and (d) few or no recoverable B. cereus cells in stool samples during or following illness.
Pseudomonas sp. The Gram negative pseudomonads are common inhabitants of soil, water and decomposing organic matter. One species, Pseudomonas aeruginosa, causes a variety of suppurative processes in man (Burrows, 1959) and often is found in the intestinal discharges of patients with gastroenteritis. Bryan (1969) reviewed several incidents from both D.S. and foreign literature that appeared to be related to food consumption. Shooter et al. (1969) observed a high faecal carriage rate of P. aeruginosa among hospital patients and suggested that faecal strains may be implicated as causes of sepsis in surgical patients who harbor them. The investigators isolated P. (wruginosa from 21 'Of 273 samples of food prepared in a hospital diet kitchen and found several patients to be shedding in their stools the same strains of P. aeruginosa that were found in the food. J. Inst. Can. Scl. Technol. Aliment. Vol. 6, No 2, 1973
In virulent cultures of P. aenlginosa Kubota and Liu (1971) have demonstrated a toxic protein that caused fluid accumulation in the ligated loop of a rabbit's intestine. This "enterotoxin" was inactivated by boiling and by trypsin. Virulent strains of P. aeruginosa (i.e. lethal to mice on intraperitoneal injection) tended to induce fluid accumulation, whereas non-virulent strains usually did not. From the available evidence one must suspect that certain strains of P. aeruginosa are able to produce a toxic substance which somehow can incite gastroenteritis in man. Either these strains are rare or the circumstances that lead to illness are seldom met, because P. aeruqinosa gastroenteritis is far from common. Acquisition of the organism through food appears to be possible, though not a common means of infection. One other species, Pseudomonas cocovenenans) has been shown to produce a serious and often fatal type of food poisoning in parts 'of Central Java, Indonesia (van Veen, 1966). A home-made fermented dish called "bongkrek" is prepared there from shredded coconut. Normally a Rhizopus species develops on the product but sometimes for reasons that are unknown P. cocovenenans overgrows and inhibits development of the fungus. Highly toxic product can result. When cultured on pressed coconut, but not on soybean or peanut cake, P. cocovenenans produces two toxic substances, toxoflavin and bongkrek acid. The latter, an unsaturated fatty acid, is the more toxic of the two, causing rapid and often fatal hypoglycemia in experimental animals. There seems to be no basis for controversy about the food poisoning potential of P. cocovenenans. However, we still do not know the conditions that allow the occasional development of this organism during bongkrek manufacture.
Faecal Streptococci Perhaps the most controversial of all the disputed food poisoning agents are the faecal streptococci. This group, sometimes called the Group D streps, includes the enterococci. They have been incriminated in nunumerous outbreaks involving different kinds of foods over the past 50 years. Almost invariably the evidence amounts to a demonstration of large numbers of faecal streptococci in the suspect food at some time after the incident. There is no merit to another rehashing of tIle voluminous literature on food poisoning outbreaks by the faecal streptococci. This has been done ably and thoroughly by Bryan (1969), Hartman et al. (1965), Deibel and Silliker (1963) and Shattock (1962). The typical incident goes something like this: a number of people become ill with r:.ausea, colic, diarrhea and sometimes vomiting between 2 and 36 hours after eating. Recognition of an outbreak spurs investigation and collection of samples. Meanwhile the leftover foods, which now have become suspect, may well have been held without refrigeration for two or three days before analysis. Faecal streptococci, if present, can flourish in most cooked foods and often outgrow Can. Inst. Food Sel. Teehnol. J. Vol. 6. No. 2, 1973
everything else. Thus they can be found in large numbers while other food poisoning agents may be completely overlooked. Recognizing that evidence such as this is not enough to incriminate the faecal streptococci as food poisoning agents, several investigators have fed cu!tures isolated from food poisoning outbreaks to experimental animals and human volunteers. Feeding trials with animals have been inconclusive; cats often respond but most other animals do not (Hartman et al.) 1965). Bryan (1969) states categorically that "animal experimentations tell us little, since the re· action of experimental animals may not be the same as the reaction of man." Tests with human volunteers have given conflicting results. A few investigators have reported evidence of gastroenteritis when faecal streptococci were fed, but most have not (Hartman et al.) 1965; Bryan, 1969). The most extensive trials were performed by Deibel and Silliker (1963), who fed 23 enterococcus strains to two or three human volunteers without a single indication of gastroenteritis. Ten years ago Shattock (1962) stated that "from all the evidence one is forced to conclude either that the environmental conditions for producing a pathogenic principle for faecal (Group D) streptococci are only very occasionally encountered, or that only exceptional strains are potentially enteropathogenic." That conclusion is equally valid today. Just as there is controversy over the food poisoning potential of the faecal streptococci, there is also controversy over their significance as indicators of insanitation. These organisms are indeed found in the intestinal tracts of man and animals, but they also occur in so many other places that their significance as faecal indicators is seriously restricted. They are common in meat packing establishments (Greenberg, 1965) and on the surfaces of plants (Mundt, 1963) . They occur in cheese, often by the tens to hundreds of millions per gram (Foster et al., 1942; Mattick and Shattock, 1943; Clark and Reinhold, 1966) ; they grow in fermented sausages (Niven, 1963) ; and they develop during the malting of barley (Stark, 1970). In fact, hardly any food that does not receive a terminal heating can be prepared feasibly without becoming contaminated with faecal streptococci. If the food is held above the freezing point for a reasonable length of time the organisms will increase in number (Niven, 1963). Thus, faecal streptococci effectively serve as an index of quality only for those foods which receive a substantial heat treatment during preparation (e.g. precooked frozen foods). However, to require low numbers of faecal streptococci in milk, cheese, fermented sausages, vegetables and similar products would virtually eliminate these items from our diet. As Niven (1963) has pointed out, faecal streptococci "can establish themselves and grow in or on food substances far removed from the original contaminating source. Therefore, an interpretation of the faecal streptococcal population in terms of the amount of 128
faecal contamination in the food can approach the ridiculous." In view of the widespread occurrence of faecal streptococci and with due regard for the uncertainty about their food poisoning potential, it is clear that these organisms do not deserve their unsavory reputation. Except for processed foods that have been subjected to rigorous bactericidal treatment, small numbers of faecal streptococci in a product are essentially meaningless. In fermented products the same can be said about large numbers.
Discussion Occurrence of an outbreak of food poisoning with dozens or hundreds of victims is difficult to refute. Proving the cause is another matter. No one has Jet found a satisfactory substitute for Koch's postulates in determining whether a suspect organism can, in fact, cause food-borne disease. Moreover, for conclusive proof the symptoms of disease must be induced in human volunteers, not experimental animals (Bryan, 1969). Still the matter is not simple. Organisms that have been clearly incriminated on epidemiological grounds have failed to incite disease when fed to human volunteers. Why? And why does a particular organism cause disease in some trials and not in others? Shattock (1962) alluded to this dilemma when she concluded about the Group D streptococci either that strains able to produce a pathogenic principle are exceptional, or conditions necessary for disease production are rarely met. Evidence now accumulating suggests that ways to settle some of our food poisoning controversies may soon be at hand. W'e now know that the method of preparing a challenge dose of food poisoning organisms can drastically affect their behaviOl' in a human volunteer (Strong et al.) 1971). Equally significant, the response of the rabbit ileal loop is influenced by the method of preparing the challenge. Thus we may have an explanation for past discrepancies between the results of different investigators. We may also have an animal model, the ileal loop response, that will provide a practical mechanism for studying enteropathogenicity and for identifying the actual factors involved in disease (Duncan and Strong, 19(9). Spira and Goepfert (1972) have shown the utility of the ligated ileal loop in studying food poisoning by B. cercus. No animal test will completely eliminate the need for human feeding trials but Strong et al. (1971) have shown a reasonable correlation between rabbit ileal loop response and reaction of human subjects to cells of C. per/ringens. Thus, we may now have a technique for studying B. cereus) faecal streptococci and other organisms whose food poisoning potential is unclear. Techniques worked out with C. lJer/ringens) itself once a "controversial" organism (Dack, 1956), may help resolve questions remaining about the others. Once we understand how the controversial agents cause food poisoning, if they do, it may be possible to find specific and easily measured characteristics that 129
correlate with the food poisoning property. This, then, will give us a practical tool for detecting those organisms having the potential to cause disease. It is important that the controversies surrounding B. cereus, the faecal streptococci and other questioned organisms be resolved. With the clouds now hanging over these organisms purchasers are reluctant to buy products containing them. Purchase specifications -often limit the numbers of enterococci to unreasonable and often meaninglessly low levels. Limits on the numbers of B. cereus in dehydrated potatoes have been imposed by certain purchasers. For the most part standards such as these simply represent lack of understanding of the facts. B. cereus becomes hazardious, if at all, only if the food is grossly mishandled and growth occurs. Even major differences in initial contamination levels can be quickly negated if a product is abused. Resolving the controversies may not be easy. Every negative test calls for another trial. Only a positive result is convincing. Thus a large mass of data obtained under a wide variety of test conditions will be necessary to answer the questions that exist as long as the findings are negative.
References Anonymous. 1972. Food Poisoning Associated with Bacillus cereus. Brit. Med. J., 1 (No. 5793) :189. Bryan, F. L. 1969. Infections Due to Miscellaneous Microorganisms. In Food-Borne Infections and Intoxications, H. Riemann, Ed., Academic Press, New York. Burrows, W. 1959. Textbook of Microbiology, 17th Ed., W. B. Saunders Co., Philadelphia. Center for Disease Control, U. S. Public Health Service, Atlanta, Ga., Foodborne Outbreaks, Annual Summaries for 1966, '67, '68, '69, '70 and January-June, 1971. Clark, W. S., and Reinbold, G. W. 1966. Enterococi in Young Cheddar Cheese. J. Dairy Scl., 49:1214. Dack, G. M. 1956. Food Poisoning, Rev. and Enlarged. The University of Chicago Press, Chicago. Dack, G. M. 1966. Importance of Food-borne Disease Outbreaks of Unrecognized Causes. Food Technol., 20 :1279. Dack, G. M., Sugiyama, H., Owens, F. J., and Kisner, J. B. 1954. Failure to Produce Illness in Human Volunteers Fed Bacillus cereus and Clostridium perfrlngens. J. Infect. Dis., 94:34. Deibel, R. H., and Silliker, J. H. 1963. Food-poisoning Potential of the Enterococcl. J. Bacteriol., 85 :827. Duncan, C. L., and Strong, D. H. 1969. Ileal Loop Fluid Accumulation and Production of Diarrhea in Rabbits by Cell-free Products of Clostridium perfringens. J. Bacteriol., 100:86. Foster, E. M., Garey, J. C., and Frazier, W. C. 1942. The Bacteriology of Brick Cheese. Ill. The Bacteria Involved in Ripening. J. Dairy Scl., 25 :323. Goepfert, J. M., Spira, W. M. and Kim, H. U. 1972. Bacillus cereus: Food Poisoning Organism. A Review. J. Milk & Food Technol., 35:213. Greenberg, R. A. 1965. Significance of Enterococci in Foods. Activities Report, 17 :62. Hartman, P. A., Reinbold, G. W., and Saraswat, D. S. 1965. Indicator Organisms - A Review. II. The Role of Enterococci in Food Poisoning. J. Milk and Food Technol., 28:344. Hauge, S. 1950. Matforgiftninger fremkalt av Bacillus cereus. Nordisk. Hyg. Tidskr., 31 :189. (BioI. Abstr., 25:1063. 1951.) Hauge, S. 1955. Food Poisoning Caused by Aerobic Spore-forming Bacilli. J. Appl. Bacteriol. 18 :591. Hobbs, Betty C. 1969. Clostridium perfringens and Bacillus cereus Infections. In Food-Borne Infections and Intoxications, H. Riemann, Ed., Academic Press, New York. KUbota, Y., and Liu, P. V. 1971. An Enterotoxin of Pseudomonas aeruginosa. J. Infect. Dis., 123:97. Mattlck, A. T. R., and Shattock, P. M. F. 1943. Group D Streptococci in English Hard Cheese. Mon. Bul. Emerg. Publ. Hlth. Lab. Serv. 2:73. Midura, T., Gerber, M., Wood, R., and Leonard, A. R. 1970. Outbreak of Food Poisoning Caused by Bacillus cereus. Pub. Health Reports, 85:45. Mundt, J. O. 1963. Occurrence of Enterococci on Plants in a Wild Environment. Appl. Microbiol., 11 :141. Niven, C. F. 1963. Microbial Indexes of Food Quality: Fecal Streptococci. In Microbiological Quality of Foods, Slanetz, L. N., Chichester, C. 0., Gaufin, A. R., and Ordal, Z. J., Eds., Academic Press, New York. Nygren, B. 1962. Phospholipase C-producing Bacteria and Food Poisoning. Acta Pathol. Microbiol. Scand. Suppl., 160:1. Ormay, L., and Novotny, T. 1970. Uber sogenannte unspezifische Lebensmittelvergiftungen in Ungarn. Zbl. Bakt. Abt. I. Orlg., 215:84. J. Inst. Can. Sci. Technol. Aliment. Vol. 6, No 2, 1973
Shattock, P, M. F. 1962. Enterococci. In Chemical and Biological Hazards in Foods, Ayres, J. C., Kraft, A. A., Snyder, H. E., and Walker, H. W., Eds., Iowa State University Press, Ames, Iowa. Shooter, R. A., Cooke, E. M., Gaya, H., Kumar, P., Patel, N., Parker, M. T., Thorn, B. T. and France, D. R. 1969. Food and Medicaments as Possible Sources of Hospital Strains of Pseudomonas aeruginosa. Lancet (June 21) :1227. Spira, W. M., and Goepfert, J. M. 1972. Bacillus cereus - Induced Fluid Accumulation in Rabbit Ileal Loops. Appl. Microbiol., 24: Stark, E. 1970. Fecal Streptococci on Barley and Malt Kernels and Instant Malted Milk Powder. Appl. Microbiol., 20 :200.
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