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Postgrad Med J: first published as 10.1136/pgmj.50.588.597 on 1 October 1974. Downloaded from Postgraduate Medical Journal (October 1974) 50, 597-602.

Clostridium welchii and cereus and intoxication BETTY C. HOBBS O.St.J., D.Sc., F.R.C.Path., Dip.Bact. Food Hygiene Laboratory, Central Public Health Laboratory, Colindale Avenue, London NW9 5HT

Summary 1948, there was a large outbreak of intestinal disease welchii type A is a common agent of food described as necroticans in Hamburg, poisoning when allowed to proliferate to large numbers Germany; it was reported in a number of papers in cooked foods, usually meat and poultry. The main (Ernst, 1948; Schutz, 1948; Marcuse and Konig, factors of importance are survival of the , 1950). The clinical and bacteriological findings were frequently found on raw products, through the clarified in papers by Zeissler and Rassfeld-Sternberg process, and possible contamination of cooked meats (1949), Oakley (1949) and Hain (1949). These workers transferred to unclean containers; subsequent germina- described a small outbreak of haemorrhagic enteritis tion ofspores and rapid multiplication ofthe vegetative in a family who had eaten tinned meat or fish;

cells during long slow cooling and non-refrigerated the outbreak was thought to be part of the much Protected by copyright. storage lead to heavy contamination. The re- larger episode. The strain of Cl. welchii isolated from sponsible is different from the soluble antigens, and its food and patients survived 1000C for 1-4 hr and formation in the intestine is associated with sporulation. produced much 3-toxin. This strain was given the Large numbers of Cl. welchii of the same sero- new type letter F; later it was described as a sub- logical types in food and faeces is the main diagnostic group (heat-resistant) of type C (Brooks, Sterne and factor. Important preventive measures are rapid cooling Warrack, 1957; Warrack, 1963; Sterne and Warrack, and cold storage to prevent growth. 1964). Hobbs et al. (1953) established Cl. welchii is an aerobic sporulating organism type A as a major cause of gastro-enteritis of a much commonly found in cereals. Outbreaks described from milder nature than that described as enteritis necro- Europe have a different aetiology with regard to food ticans by the German workers. The outbreaks re- vehicles, and symptoms from those ported in this paper were due to heat-resistant 'non- that have been reported recently in the U.K. from fried or oc-haemolytic Cl. welchii'. Later McKillop (1959), and boiled . The spores survive through cooking Hall et al. (1963), Taylor and Coetzee (1966), and procedures and grow out to cells which sporulate Sutton and Hobbs (1968) showed that the so-called http://pmj.bmj.com/ readily in the cooked food and which are assumed to 'heat-sensitive' strains, mostly P-haemolytic on horse produce toxin in the food. Large numbers of B. cereus blood agar, were also responsible for outbreaks. are found in foods causing illness and, as with Cl. Although the spores of these strains are less resistant welchii, the main preventive measure is inhibition of to heat, nevertheless they can survive cooking. growth by quick cooling and cold storage of foods Many other papers have been written on Cl. welchii cooked ahead of requirements. A comparative table of food poisoning up to the present day, when the the characteristics and clinical symptoms of Cl. means by which the organism causes symptoms is welchii and B. cereus is given. described. on September 29, 2021 by guest. Of the five types of Cl. welchii A to E differen- Clostridium welchil tiated on the proportion of soluble antigens in each, There were early reports of intestinal disturbances types A, C and D are pathogenic for man and types due to Cl. welchii in 1895 (Klein, 1895) and 1899 B, C, D and E affect animals. Type A is further (Andrewes, 1899). In 1943 Knox and MacDonald divisible into serological types; sera are obtained by described outbreaks of food poisoning in children inoculation of the whole organism into . after eating school meals. Gravy prepared in bulk on Cl. welchii type A may be isolated in small num- the previous day was heavily contaminated with bers from many foods both raw and cooked, and it anaerobic sporulating including Cl. welchii. forms a minor component of the faecal flora in most McClung (1945) in the U.S.A. was definite in his if not all healthy adults, occurring in numbers of assertion that certain food poisoning outbreaks after about 104/g of faeces. When it becomes established meals of cooked poultry were due to Cl. welchii. In in the large gut, where toxin is produced, large Postgrad Med J: first published as 10.1136/pgmj.50.588.597 on 1 October 1974. Downloaded from 598 B. C. Hobbs numbers appear in the faecal flora. Outbreaks of less than 4%/ of spores of the more heat-resistant enteritis necroticans due to the type C strains are strains grew without heating, and activation was uncommon; they have been reported from New usually detected at 75-80°C. The heat resistance, Guinea by Murrell et al. (1966a, b); Collee (1973) heat activation and rapid generation time are the describes similar enterotoxaemic diseases produced factors which predispose this organism to survival by various types of Cl. welchii in animals; the type C followed by rapid multiplication in slowly cooling strains in show evidence of bacterial adhesion to masses of meat and poultry. The organism is intestinal epithelial cells, which may explain the anaerobic, but not to a marked degree, nevertheless, more serious nature of the disease caused by type C its growth depends on anaerogenic conditions within strains in humans as well as in animals. a mass of cooked food. Large numbers (millions per The annual statistics for Cl. welchii food poisoning gram) of viable Cl. welchii cells in cooked foods is in England and Wales record 47 outbreaks and indicative of a health hazard. approximately 1534 cases in 1969 and 32 outbreaks The toxin of Cl. welchii responsible for the food and 1263 cases in 1970. The symptoms of abdominal poisoning symptoms is produced in the large intes- pain, and acute diarrhoea occur 8-24 hr after tine during sporulation. The potential for sporula- eating food, such as cooked meat and poultry, which tion in cooked foodstuffs is poor and if the toxin is has become grossly contaminated with the organism formed at all it is unlikely to be detected. In the during long slow cooling periods and storage at intestine, spores are readily formed and the toxin ambient temperatures. Roast, boiled, steamed, also. Ingestion of a large dose of Cl. welchii and and stewed meats and poultry, and pies and gravies sporulation of the organism in vivo are important are the predominant vehicles of infection, whether factors in the production and release of the entero- eaten cold or reheated. When meat is cooked in bulk, toxin. Ligated gut experiments in rabbits and lambs,

the heat gain is slow and subsequent cooling is slow. those with monkeys (Duncan and Strong, 1969; Protected by copyright. The heat drives off dissolved and the meat Hauschild, Niilo and Dorward, 1970; Duncan and maintains an anaerobic environment. The heat- Strong, 1971) and volunteer experiments in man shocked spores are activated to germinate and when (Strong, Duncan and Perna, 1971) have confirmed the temperature drops to below 50°C the out- the association between sporulation and growths of vegetative cells multiply rapidly. The production. so-called heat-sensitive strains of Cl. welchii type A, Hauschild (1973) refers to the toxin as enterotoxin, usually P-haemolytic on horse blood agar and more and he gives an excellent summary of the properties active in production of a, 0 and x , require less of the substance in his review. After precipitation heat shock for germination. Spores of these strains with ammonium sulphate the toxin may be purified present on the inside of containers may grow in by gel filtration and ion exchange chromatography. cooked meat foods poured into the containers. The purified preparation is free of nucleic acids, The spores of the organisms Cl. welchii, Cl. fatty acids, phosphatides and reducing sugars. The botulinum and B. cereus can survive in food after u.v. absorption spectrum is maximum at 278-280 cooking, they can be activated to germinate by the nm and minimum at 250 nm. By hydrolysis, 19 http://pmj.bmj.com/ heat treatment and grow at various rates in slowly amino-acids have been demonstrated, amongst which cooling food. Large numbers are assumed to be re- aspartic acid, serine, and glutamic acid are quired to initiate toxin production in the intestine predominant. The substance is probably a protein of or to produce toxin in the foodstuff before consump- mol. wt. 36,000 ± 4000 with an iso-electric point of tion. 4-3. The toxin is inactivated by pronase and B. The characteristics of Cl. welchii are important in subtilis protease but not by trypsin, chymotrypsin or relation to the food poisoning. Temperature limits papain. It is heat labile with a D,o° of 4 min. The for the growth of Cl. welchii are about 250C and MLD for mice (i.v.) is 2000/mgN and lethal doses of on September 29, 2021 by guest. 50°C and the optimum temperature for growth in toxin caused death within 20 min (Hauschild and cooked meat is between 43°C and 47°C (Collee, Hilsheimer, 1971). The enterotoxin may be isolated Knowlden and Hobbs, 1961). The generation time from the supernatant fluids of the contents of ligated can be as short as 10-12 min (Mead, 1969). Spores of intestinal loops in lambs and also from extracts of the classical strains (P-haemolytic) of Cl. welchii are cells. Intradermal injection of the enterotoxin into more sensitive to heat (D9o. 3-5 min)* than spores of guinea-pigs and rabbits causes erythema around the the 'heat-resistant' strains (D9O. 15-145 min)* using injection site within 1-2 hr. By relating the diameter the same sporulation medium (Roberts, 1968). It of the erythema to toxin concentration, the skin can was also reported by Roberts (1968) that 30-50% be used for rapid and accurate assay of the entero- of spores of the classical strains grew without heat- toxin; this test is said to be more sensitive than that ing, and heat activation was not detected. In contrast of the ligated intestinal loop. Niilo (1971) showed * (D9oo = Decimal reduction time at 90°C). that the erythema reaction in a guinea- skin was Postgrad Med J: first published as 10.1136/pgmj.50.588.597 on 1 October 1974. Downloaded from Cl. welchii and B. cereus infection and intoxication 599

preceded by increased permeability of the blood Midura et al. (1970) in the U.S.A., Pivovarov et al. capillaries around the injection site, and he suggested (1970) in the U.S.S.R., von Ludwig (1971) in that the accumulation of fluid in the intestinal lumen Germany and Fredette and Plante (1971) in Canada, might result from increased permeability of the blood all described outbreaks of food poisoning due to B. capillaries in the intestinal wall. cereus in which the incubation time and symptoms Whereas the toxin ingested by volunteers or in- were similar to those for Cl. welchii food poisoning. troduced into the stomachs of rhesus monkeys Recent reviews include those of Goepfert, Spira and produces both emesis and diarrhoea, the natural Kim (1972) and Goepfert et al. (1973). A wide variety illness in man is characterized by diarrhoea only of foods have been implicated in these outbreaks from toxin produced in the intestine. The reaction including soup, cooked meat, cooked potatoes and from the ingestion of toxin by man and monkey can vegetables, vanilla sauce, cornflour pudding, fried be neutralized with antiserum prepared against the rice and soya sauce. enterotoxin. From 1971 onwards U.K. reports have described Hauschild (1973) lists factors of similarity between several incidents of food poisoning associated with the toxins of Cl. welchii and cholerae; they are fried rice from Chinese restaurants. However, the both produced in vivo, induce fluid accumulation in incubation period and symptoms are different from ligated intestinal loops, inactivated by heat, inacti- those described hitherto. occurs about 1-6 vated by pronase but not by trypsin and are free of hr following a meal, sometimes there is diarrhoea a and reducing sugars. The mode of action may few hours later. Fried or boiled rice is the only food be similar and it is described as enhanced adeno- comm'on to those affected. Samples of cooked rice cyclase activity and inhibited phosphodiesterase showed high counts of 107-108/g of B. cereus. activity with a chloride and sodium imbalance. Neither aureus nor staphylococcal

The sequence of events in Cl. welchii food poison- enterotoxin were detected in samples of food ex- Protected by copyright. ing is described neatly by Hauschild (1973) as follows: amined carefully because of the similarity of incuba- '(a) ingestion of food with about 106-107 vegetative tion time and symptoms with staphylococcal entero- cells of Cl. welchii per gram; (b) multiplication and toxin food poisoning. sporulation of Cl. welchiii in the ; B. cereus is widely distributed in cereals and other (c) production ofenterotoxin in the sporulating cells; dried foods, and in dairy products it is a common (d) release of toxin by cell lysis; (e) accumulation of spoilage organism. Spores are readily formed in the fluid in the cell lumen caused by the enterotoxin vegetative cells; they may survive cooking procedures whose mode of action remains to be investigated; and grow out in the rice. The common fault is to and diarrhoea resulting from the excess fluid accu- allow large quantities of boiled rice to drain over- mulation'. Thus symptoms are initiated by live micro- night at ambient kitchen temperature. The following bial cells rather than preformed toxin and the causa- day the rice may be fried quickly with beaten up egg tive enterotoxin is produced in vivo. It must be which, if not freshly prepared, may also be highly emphasized that the assay of enterotoxin in food is contaminated with a miscellaneous collection of not required, what is needed is the quantitative . Often the fried rice is kept warm until http://pmj.bmj.com/ estimation of live Cl. welchii cells in incriminated customers arrive in the shop to take away portions. foods as well as in the stools of patients. The large numbers of B. cereus isolated from It was pointed out by Sutton and Hobbs (1971) samples of fried rice indicate that neither the that a large dose of the organism is required not only organism nor the toxin are destroyed in the quick to initiate growth in the intestine but also to permit passage through hot fat described as frying. survival of a fair number of organisms through the A few years ago Nygren (1962) suggested that the acid regions ofthe stomach. It appears that the meta- symptoms of both Cl. welchii and B. cereus were bolic changes set up by the sudden influx in the due to the liberation of phosphorylcholine from on September 29, 2021 by guest. intestine of enormous numbers of Cl. welchii from lecithin by the of these food encourages the indigenous Cl. welchii strains to organisms, but Stark and Duncan (1972) and grow also. Hauschild (1973) have shown that Cl. welchii food poisoning is due to an enterotoxin separate and Bacillus cereus distinct from phosphorylcholine. B. cereus as a food poisoning agent was described Similar data described by Goepfert and summar- by Hauge in 1950 and 1955; cornflour sauce pre- ized in 1973 indicate that B. cereus also produces an cooked to eat with fruit at picnics was responsible for enterotoxic substance that can be readily differen- outbreaks occurring in Norway. Christiansen, Koch tiated from . Goepfert (1973) describes and Madelung (1951) described an outbreak due to this toxic substance as 'protein in nature actively cornflour pudding. Clarenburg and Kampelmacher synthesized and secreted during logarithmic growth, (1957) in Holland, Nikodemusz (1958) in Hungary, causing fluid to accumulate in ligated ileal segments Postgrad Med J: first published as 10.1136/pgmj.50.588.597 on 1 October 1974. Downloaded from 600 B. C. Hobbs in the rabbit and inducing increased intestinal organism from faecal samples of patients is some- and overt diarrhoea upon oral administra- what irregular and may depend on the freshness of tion to rhesus monkeys'. The toxin he says is the stool sample. 'thermo-labile, susceptible to various proteolytic Work to be published by Gilbert, Stringer and and antigenic'; approximately 80% of the Peace (in press) indicates an optimum temperature B. cereus strains tested by him, presumably mostly for growth of B. cereus between 30 and 37°C. The from European sources, have shown evidence of maximum temperature for growth was thought to producing this enterotoxic substance. The work in be between 43 and 48°C, and the minimum tempera- Goepfert's laboratory suggests that there are two ture was found to fall between 10 and 15°C. antigenically distinct (not produced by The heat resistance of B. cereus spores during the same strain of B. cereus). the process of boiling is of practical concern in Among other Bacillus tested by Goepfert survival of the organism during cooking. Studies et al. (1973), B. thuringiensis was observed to carried out in 1972 (Gilbert, Stringer and Peace, give similar pathological symptoms in laboratory personal communication) suggest that whereas 300% animal models. They think it is unlikely that the of the spores of one strain of B. cereus were capable enterotoxin factors are related to the parasporal of surviving at 95°C in aqueous suspension after crystal inclusion body in the . The 20-25 min, only 0-0001 % of the spores of another enterotoxic activity of a culture filtrate of B. thurin- strain were able to germinate under the same con- giensis was neutralized by antiserum produced ditions; there was a similar pattern of survival in rice against a culture filtrate of B. cereus. allowed to stand after boiling. The growth rate of The work on B. cereus is in its early days. Explana- the two strains tested was also different. There was a tion is needed for the differences in incubation time rapid fall in counts after frying but the subsequent

and clinical symptoms between outbreaks in Eastern increase in numbers after storage at room tempera- Protected by copyright. Europe and other places and outbreaks recently ture was further evidence of the marked heat resis- associated with cooked rice dishes in the United tance and survival properties of the B. cereus spores. Kingdom (Public Health Laboratory Service, 1972, Spores of both strains of B. cereus tested showed a 1973), Australia (J. Taplin, personal communication), wide range of heat resistance and when germinated and Canada (Lefebvre et al., 1973). Furthermore, large numbers of spores rapidly appeared in the volunteer experiments in man are inevitable as final vegetative cells. In a similar way to Cl. welchii the proof of the role of the organism in food poisoning significance of B. cereus in cooked foods depends on and to fulfil Koch's postulates. The isolation of the the numbers present. Colony counts below 106/g are

TABLE 1. Characteristics in relation to food poisoning Clostridium welchii Bacillus cereus Prevalence Common Common Food, faeces, , dust Cereals, soil, dust http://pmj.bmj.com/ Growth Anaerobic growth in Aerobic growth in rice meat and poultry and other cereals Generation time 10-12 min 27 min Optimum temperature 43-470C 300C Minimum 15-200C 1-120C Maximum 500C 480C Sporulation Poor in food Good in food

Good in intestine on September 29, 2021 by guest. Heat resistance D1oo Less than 1 min to 1 min to 7-5 min 17 min Mechanism Toxin liberated in Toxin produced in food intestine Incubation 12-18 hr 2 hr (15 hr) Symptoms Diarrhoea and pain Vomiting (vomiting) (diarrhoea and pain) Dose required Large numbers of Large numbers of organisms organisms plus toxin Stool Large numbers of Variable number of organisms organisms Prevention Stop growth in food Stop growth in food (From Hobbs, 1973.) Postgrad Med J: first published as 10.1136/pgmj.50.588.597 on 1 October 1974. Downloaded from Cl. welchii and B. cereus infection and intoxication 601

probably of little significance but counts above 106/g COLLEE, J.G., KNOWLDEN, J.A. & HOBBS, B.C. (1961) Studies in foods stored at on the growth, sporulation and carriage of Clostridium cooked ambient temperatures may welchii with special reference to food poisoning strains. indicate that food poisoning could occur. The Journal of Applied Bacteriology, 24, 326. The foods responsible for outbreaks both of Cl. COLLEE, J.G. (1973) The potential enteropathogenicity of welchii and B. cereus food poisoning are invariably Clostridium welchii (Cl. perfringens) in man. 1st Interna- tional Congress for Bacteriology, Jerusalem. Abstracts, 1, those which have been prepared ahead of require- 158. ments. Their bulk and the general lack of proper DUNCAN, C.L. & STRONG, D.H. (1969) Experimental pro- facilities for cooling and refrigeration enable and duction ofdiarrhoea in rabbits with Clostridiumnperfringens. encourage the organisms to grow. Sutton, Kendall Canadian Journal of , 15, 765. and Hobbs (1972) demonstrated that of Cl. DUNCAN, C.L. & STRONG, D.H. (1971) Clostridium perfrin- spores gens type A food poisoning. I. Response of the rabbit welchii survived in large and small portions of beef ileum as an indication of the enteropathogenicity of strains cooked in conventional and moist air Rapidaire of in monkeys. Infection and ovens, whereas vegetative cells were destroyed. Immunity, 3, 167. When the cooked meats were cooled at room tem- ERNST, 0. (1948) Epidemiologische Beitrag zu Enteritis necro- ticans. Deutsche Gesundheitswesen, 3, 262. perature growth occurred, and some multiplication FREDETTE, V. & PLANTS, C. (1971) Episode montralais took place in the centre of large roasts even when d'empoisonnement alimentaire i Bacillus cereus. L'Institut they were cooled under refrigeration. Dehydrated de Microbiologie et d'Hygiene de l'Universite de Montreal, foods frequently contain bacterial spores which may 100, 1943. be added to foods at a late stage of for GOEPFERT, J.M., SPIRA, W.M., GLATZ, B.A. & KIM, H.U. cooking, (1973) Pathogenicity of Bacillus cereus. In: Proceedings of example, spices and pepper in curries and other so- the 8th International Symposium. The Microbiological called hot foods. Thus even small numbers of Cl. Safety of Food, p. 69. London: Academic Press. welchii spores in dehydrated foods may be a hazard GOEPFERT, J.M., SPIRA, W.M. & KIM, H.U. (1972) Bacillus cereus: food poisoning organism. A review. Journal of when these foods are used as ingredients. Protected by copyright. Milk and Food Technology, 35, 213. Food hygiene education should stress the facts that GOEPFERT, J.M. (1973) Pathogenicity patterns in Bacillus raw foods are frequently contaminated with food cereus food-borne disease. Ist International Congress for poisoning organisms before they even reach the Bacteriology, Jerusalem. Abstracts, 1, 140. kitchen, that bacterial spores are often present and HAIN, E. (1949) Origin of Clostridium welchii type F infection. British Medical Journal, 1, 271. that they can survive boiling and other methods of HALL, H.E., ANGELOTTI, R., LEWis, K.H. & FOTER, M.J. cooking with the exception of steam under pressure. (1963) Characteristics of Clostridium perfringens strains The outgrowth of Cl. welchii spores occurs in the associated with food and food-borne disease. The Journal absence of air inside rolled joints, poultry carcasses, of Bacteriology, 85, 1094. HAUGE, S. (1950) Matforgiftninger fremkalt au Bacillus pastry casings and at the bottom of stews; B. cereus cereus. Nordisk Hygienisk Tidskrift, 31, 189. spores will grow out under aerobic conditions in HAUGE, S. (1955) Food poisoning caused by aerobic - masses of food such as rice, cooked and left wet for forming bacilli. The Journal of Applied Bacteriology, 18, some hours at ambient temperature. Long, slow 591. HAUSCHILD, A.H.W. (1973) Food poisoning by Clostridium cooling and non-refrigerated storage provide ideal perfringens. Canadian Institute of Food Science and Tech- conditions for bacterial multiplication. A better nology, 6, 106. http://pmj.bmj.com/ understanding of these facts, provision by architects HAUSCHILD, A.H.W. & HILSHEIMER, R. (1971) Purification of facilities for rapid cooling of foods and sufficient and characteristics of the enterotoxin of Clostridium per- fringens type A. Canadian Journal of Microbiology, 17, cold storage for large bulks of food would help to 1425. prevent both Cl. welchii and B. cereus food poisoning. HAUSCHILD, A.H.W., NIILO, L. & DORWARD, W.J. (1970) Table 1 is quoted from Hobbs (1973) and shows the Response of ligated intestinal loops in lambs to an entero- relevant characteristics of the organisms Cl. welchii pathogenic factor of Clostridium perfringens type A. and B. cereus. Canadian Journal of Microbiology, 16, 339.

HOBBS, B.C. (1973) Food poisoning in England and Wales. on September 29, 2021 by guest. In: Proceedings of the 8th International Symposium. The Microbiological Safety ofFood, p. 129. London: Academic References Press. ANDREwES, F.W. (1899) On an outbreak of diarrhoea in the HOBBS, B.C., SMITH, M.E., OAKLEY, C.L., WARRACK, G.H. wards of St Bartholomew's Hospital, probably caused by & CRUICKSHANK, J.C. (1953) Clostridium welchii food infection of rice pudding with Bacillus enteritides sporo- poisoning. Journal of Hygiene, 51, 75. genes. Lancet, i, 8. KLEIN, E. (1895) Ueber einen pathogenen anaeroben Darm BROOKS, M.E., STERNE, M. & WARRACK, G.H. (1957) A re- bacillus, Bacillus enteritides sporogenes. Zentralblatt fiir assessment of the critera used for type differentiation of Bakteriologie, Parasitenkunde, Infektionskrankheiten und Clostridium perfringens. Journal of Pathology and Bacteri- Hygiene. I. Abteilung: Originale, 18, 737. ology, 74, 185. KNOX, R. & MACDONALD, E.K. (1943) Outbreaks of food CHRISTIANSEN, O., KOCH, S.O. & MADELUNG, P. (1951) Et poisoning in certain Leicester institutions. Medical Officer, udbrud af levnedsmiddelforgiftning forarsoget af Bacillus 69, 21. cereus. Nordisk Veterinarmedicin, 3, 194. LEFEBVRE, A., GREGOIRE, C.A., BRABANT, W. & TODD, E. CLARENBURG, A. & KAMPELMACHER, E.H. (1957) Bacillus (1973) Suspected Bacillus cereus food poisoning. Epidemio- cereus als oorzaak van voedselvergiftiging. Voeding, 18, 384. logical Bulletin, 17, 108. Postgrad Med J: first published as 10.1136/pgmj.50.588.597 on 1 October 1974. Downloaded from 602 B. C. Hobbs

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