International Journal of Food Microbiology, 10 (1990) 125 142 Elsevier

125

F O O D 80008

Analytical methods for Bacillus cereus and other Bacillus species Kunihiro Shinagawa Iwate University, Morioka, Iwate, Japan

Bacillus cereus can give rise to two distinct forms of foodborne disease, the emetic and the diarrhoeal syndromes. The emetic syndrome is believed to be associated with an emetic toxin pre-formed in food. Cooked rice is the most common vehicle, and the symptoms are similar to those of Staphylococcus aureus intoxication. The diarrhoeal type is caused by an enterotoxin and the symptoms generally parallel those of the Clostridium perfringens food poisoning. The heat resistance of B. cereus spores and the non-fastidious nature of the organism facilitates its survival a n d / o r growth in a wide variety of foods. This review describes analytical methods available for the isolation, identification, and enumeration of the organism, in addition to details about biological and immunological methods for toxin assay. Data are also presented concerning the incidence and epidemiology of B. cereus food poisoning around the world, and especially in Japan. Key words:

Bacillus cereus; Food intoxication: Food poisoning: Analytical methods: Enterotoxin assay: Bacillus spp.

Introduction

The organisms that comprise the genus Bacillus are large, Gram-positive, rodshaped cells that grow under either aerobic or facultatively anaerobic conditions. They are capable of forming endospores that are resistant to heat, dehydration and other physical and chemical stresses. These organisms are widely distributed in the environment (soil, vegetables, etc.) and can be isolated from a wide variety of foods (Goepfert et al., 1972; Johnson, 1984). Bacillus cereus has been established as a food poisoning agent, with Bacillus subtilis, Bacillus licheniformis and Bacillus brevis, implicated as potential food poisoning organisms (Gilbert et al., 1981: Kramer et al., 1982). Two different forms of B. cereus food poisoning are recognized; the diarrhoealsyndrome type with an incubation period of 8-16 h (mean of 10 12 h) and the emetic-syndrome type with an incubation period of 1 5 h (mean of I 3 h), following the consumption of contaminated foods. The diarrhoeal type is characterized by diarrhoea and abdominal pain, resembling Clostridium perfringens food poisoning, and the emetic type is characterized by an acute attack of nausea and

Correspondence address: Iwate University, 3-18-8 Ueda, Morioka, lwate, 020, Japan.

0168-1605/90/$03.50 ~ 1990 Elsevier Science Publishers B.V.

126

vomiting which mimics the symptoms of S t a p h y l o c o c c u s a u r e u s intoxication. Recovery from both the diarrhoeal and emetic types occurs within 12 24 h (Gilbert, 1979). The symptoms associated with the diarrhoeal and emetic forms are apparently caused by two different toxins, referred to as the 'diarrheal' toxin (enterotoxin) and emetic toxin, respectively (Spira and Goepfert, 1972; Melling et al., 1976: Melling and Capel, 1978; Turnbull et al., 1979). Considerable progress has been made in the study of B. cereus strains in relation to their involvement in food poisoning, but much research remains to be done, particularly in the identification and analysis of the associated to:fins.

Incidence of

11. c e r e u s

food poisoning

There were at least 230 outbreaks of the diarrhoeal type of B. cereus food poisoning reported world-wide between 1950 and 1976 (Turnbull, 1981). It was the third most common cause of food poisoning in Hungary (117 outbreaks) between 1960 and 1968. Other countries reporting outbreaks were Finland (50 outbreaks), the Netherlands (11 outbreaks) and Canada (9 outbreaks). A wide variety of foods, including meat and vegetable soups, cooked meet and poultry, raw and cooked vegetables, pasta, milk and ice cream, were implicated. There has been an apparent decrease in the number of outbreaks identified as B. cereus food poisoning in the last decade, primarily because isolations were made only from the foods implicated and not from patient specimens (Gilbert, 1979). At least 170 outbreaks of the emetic type of B. cereus food poisoning have been reported world-wide since 1971. More than 110 outbreaks were reported in Great Britain alone between 1971 and 1978. Cooked and fried rice were implicated in 108 of the 110 outbreaks of which 104 were related to boiled or fried rice from Chinese restaurants or 'take-away' shops (Gilbert, 1979). A total of 5141 outbreaks of food poisoning occurred in Japan between 1982 and 1986, involving 185 301 cases. Bacteria were responsible for 3740 outbreaks (153 441 cases) with 86% due to Vibrio p a r a h a e m o l y t i c u s , S t a p h y l o c o c c u s a u r e u s and S a l m o n e l l a . The etiologic agent was unknown in 927 outbreaks (29 645 cases) (Table I). There were 73 outbreaks due to B. cereus (1323 cases), with 93-95% being of the emetic type; the highest incidence occurred during the summer months (June to September). The majority of outbreaks (73%) were due to some form of cooked rice, whereas 16% were due to noodles of various types and 11% to other foods (Table ll). The type of food involved in the diarrhoeal outbreaks is less clear, although puddings, hamburgers and pre-packed lunches have been implicated.

Bacteriological examination for

B. cereus

I s o l a t i o n a n d e n u m e r a t i o n o f B. cereus (Fig. 1)

At the present time B. cereus food poisoning can be established only by the isolation and enumeration of B. cereus organisms, as methods for toxin analysis are

I

of deaths

given in brackets.

183

Unknown

* Number

638 213 109 218 27 2 13 56

Bacteria Vibrro pnruhaemolytmu Salmonella spp. Staphylococcus oureus Pathogenic Escherichiu coli Clostridium botulinurn Bacillus cereu~ Others 6318

28787 (1) 6650 2936 (1) 4 804 9359 3 88 4 947 202

769 305 109 254 30 1 18 52

1095

Incidents

35536 (12) =

Cases

Incidents

(1982-1986) 1983

in Japan

1982

food poisoning

923

of bacterial

Total

Aetiological agent

Incidents

TABLE

5136

31 125 (3) 11235 3612 (1) 4493 (2) 3355 1 250 8179

37023 (13)

Cases

177

786 384 93 205 27 4 15 58

1047

Incidents

1984

4358

28345 (13) 8 222 (1) 2107 (1) 4813 6151 44 (11) 330 6678

33084 (21)

Cases

195

877 519 82 163 34 1 17 61

1177

Incidents

1985 Cases

7128

36566 14006 2412 4968 3899 1 328 10952

(1)

(3) (1) (1)

44 102 (12)

170

670 343 75 155 28 0 10 59

899

Incidents

1986

6 705

28618 12138 2363 3885 2 141 0 327 7 764

35 556 (7)

Cases

128 TABLE II Incriminated foods in Bacillus cereus vomiting type outbreaks in Japan Incriminated foods

Number of outbreaks (%)

Persons at risk

Cooked rice Fried rice Omelette and rice Curry and rice Pre-packed lunches Rice ball "Sushi" Noodles Spaghetti "Yakisoba" Other foods h

58 (72.5) 36 4 1 10 4 3 13 (16.3) 11 2 9 (11.2)

3496 508 101 2 2409 242 234 129 68 61 607

956 (27.3) 355 82 2 381 87 49 101 (78.3) 43 58 254 (41.8)

80 (100.0)

4232

1 311 (31.0)

Total

Patients (%) a

Number of persons ill in percentage of number of persons at risk. h Roast chicken; Soybean curd "Okara"; Omelette; Cooked vegetables; Rice cake; Fried fish "'Tempura".

not yet widely available. Several selective media have been developed, in which p o l y m y x i n B ( 5 - 1 0 / ~ g / m l ) , m a n n i t o l , egg yolk a n d an indicator have been incorporated to make differentiation of B. cereus easier (Jinbo et al., 1978; Bouwer-Hertzberger a n d Mossel, 1982; Johnson, 1984). Surface p l a t i n g o n t o a selective m e d i u m such as m a n n i t o l egg-yolk p o l y m y x i n ( M Y P ) agar (Mossel et al., 1967) is c o m m o n l y used for the isolation a n d e n u m e r a t i o n of B. cereus from foods a n d clinical specimens. The M Y P agar plates, inoculated with 0.1 ml of decimal dilutions of sample homogenates, are i n c u b a t e d for 18 to 24 h at 32 to 35 ° C. The suspected B. cereus colonies are e n u m e r a t e d to determine the c o u n t per gram or per milliliter of the test sample. Three or more presumptive colonies of B. cereus are transferred to n u t r i e n t agar slants for positive identification. In cases where few organisms are suspected to be present, it may be necessary to use e n r i c h m e n t procedures to isolate the organisms. This may be d o n e with b r a i n heart i n f u s i o n broth to which polym y x i n B a n d NaC1 have been added. The isolates are identified by morphological studies based on gram a n d spore staining a n d biochemical tests such as the VP reaction, liquefaction of gelatin, starch hydrolysis, indole production, glucose ferm e n t a t i o n , a n d failure to ferment m a n n i t o l , arabinose a n d xylose (Fig. 1). Biotyping, serotyping a n d phage typing are useful epidemiological tools for distinguishing B. cereus strains. C o u n t s o f B. cereus in i n c r i m i n a t e d f o o d s a n d clinical s p e c i m e n s

The c o n t a m i n a t i o n level in i n c r i m i n a t e d foods in 22 of 29 B. cereus outbreaks in J a p a n (1973 to 1984) for which q u a n t i t a t i v e data were available, ranged from 10 5 to > 1 0 9 / g with a level of 10 6 to 1 0 9 / g for 19 (86.4%) of the 22 outbreaks (Table III). A c o u n t of 10 2 to 1 0 9 / g was observed in 74 samples of faeces from 16 outbreaks,

129

Sample

1

(Suspected foods, faeces, vomitus and swabs)

I

I Direct plating culture

Enrichment culture

Enumeration of

I BHI with I polymyxin B

10% homogenate Inoculate (0.1 ml)

[

I

Media: MYP agar KG agar PEMBA agar

B. cereus

(32-35°C, 18-24 h)

[

etc. (32-35°C, 18-24 h)

l

'

(32-35°C, 18-24 h)

I

B. cereussuspectcolony

Pure culture I I

Plating culture

Plating culture

I

(32-35°C, 18-24 h)

J

l

Grey-white colony Mannitol ( - ) Egg yolk reaction ( + )

Presumptive

B. cereus

Estimate the number of B. cereus

Non-selective: Blood agar Heart Infusion agar (3-7 mm diameter, flat or slightly granular surface, irregular edge)

Morphological test

I

Im

Confirmatory biochemical tests Motility ( + ) VP reaction ( + ) Starch hydrolysis ( + / - ) Citrate utilization (+) Nitrate reduction ( + ) Acid from: Glucose ( + ) Mannitol ( - ) Xylose ( - ) Arebinose ( - )

Gram stain: Gram ( + ) Large rod Spore stain: Central ( + ) Swelling ( - ) Toxin crystals ( - )

Identification

I

I Typing tests H-serotyping Biotyping Phage typing

J

I

I

I Enterotoxln Emetic toxin (biological and/or immunological tests)

Fig. 1. Scheme of the isolation and identification of B. c e r e u s . MYP, mannitol-egg yolk polymyxin (Mossel et al., 1967); KG, Kim and Goepfert (1971) medium; PEMBA, polymyxin pyruvate egg-yolk mannitol bromothymol blue agar (Holbrook and Anderson, 1980); BH1, brain heart infusion; VP, Voges-Proskauer.

130

TABLE iIl B. cereus

counts in incriminated foods and clinical specimens associated with food poisoning outbreaks

in Japan Counts of

Number of outbreaks of food poisoning

B. cereus

(per g) 102-10

~

Faeces

Vomitus

-

12

(16.2)

-

]6

(21.6)

(2) b (3) 2(1) 7 8 (1) 4 1

14 (18.9) 13 (17.6) 9 (12.2) 5 (6.8) 3 (4.0) 2 (2.7)

10 (27.8) 12 (33.3) 4 (11.1) 2 (5.6)

74 (100.0)

36 (100.0)

10~ 104 104 105 105-106 106-10 v 10v 10~ l0 s 109 > 109 Total

Number (%) of patients "

22 (7)

-

8

(22.2)

a Faeces were obtained from 16 outbreaks and vomitus from 10 outbreaks of B. cereus food poisoning. b B. cereus detected at this level in a particular ingredient of the incriminated food vehicle.

with 52 (70.3%) having counts of 102 to 106/g. The c o u n t in 36 samples of vomitus from 10 outbreaks ranged from 102 to 107/g (Table III). The B . c e r e u s in the faeces was f o u n d exclusively as spores since the samples were heated at 75 ° C for 20 m i n prior to i n c u b a t i o n on selective media. These findings are in agreement with those reported by G i l b e r t (1983) where in 75 episodes of B . c e r e u s food poisoning the c o n t a m i n a t i o n levels in the i n c r i m i n a t e d foods ranged from 104 to > 10~°/g, with a m e a n c o u n t of 2.5 × 10V/g. Isolation of large n u m b e r s of B . c e r e u s from the i n c r i m i n a t e d foods a n d clinical specimens is a p r i m a r y criterion for diagnosing outbreaks of B. c e r e u s food poisoning.

Biochemical

properties

o f B. c e r e u s

T h e identification of B . c e r e u s is carried out using the methods a n d interpretations of G o r d o n (1973) a n d C o w a n a n d Steel (1974). Variations in the typical biochemical behaviour of strains from various sources may be observed. However analysis of 82 strains from 21 outbreaks of the emetic type, 9 strains from 3 o u t b r e a k s of the diarrhoeal type, a n d 486 strains from wholesome foods ( u n c o o k e d a n d cooked rice, meat a n d meat products, milk a n d milk products), did not show a m a r k e d variation in biological properties, with the exception of starch hydrolysis a n d salicin f e r m e n t a t i o n (Table IV). All n i n e of the strains isolated from diarrhoeal type outbreaks hydrolyzed starch, whereas n o n e of the 82 strains isolated from emetic type outbreaks were able to do so. The isolates from wholesome foods varied in their ability to hydrolyze starch: 83% of the isolates from u n c o o k e d rice, 38% of the isolates from cooked rice (Shinagawa et al., 1979) a n d 71% (199 of 281) of the

131 TABLE IV Biochemical properties of B. cereus Test

Motility Anaerobic growth Citrate utilization (Christensen) Gelatin hydrolysis Casein hydrolysis Starch hydrolysis Indole production Nitrate reduction VP reaction Urease (Christensen) Acid from: glucose mannitol xylose arabinose maltose t rehalose glycerol sucrose lactose dulcitol inositol salicin

Results

Positive % of strains Food poisoning a (strains : 91 )

Wholesome food b (strains : 486)

+ +

100 100

100 100

+ + + +/ + + - / +

96.7 100 100 9.9 0 96.7 100 0

92.6 100 100 64.2 0 85.4 100 11.7

,~

100 0 0 0 100 100 100 90.1 0 0 0 31.9

100 0 0 0 100 100 NE ~ 78.0 0 NE NE 78.0

+ + + +/ +/-

Ninety one strains were isolated from incriminated foods and clinical specimens from 21 vomiting-type outbreaks and 3 diarrhoeal-type outbreaks. b Cooked and uncooked rice, meat and meat products, meat product additives, milk and milk products, etc. c Not examined.

i s o l a t e s f r o m o t h e r f o o d s s u c h as m e a t p r o d u c t s , r a w m e a t a n d

meat product

a d d i t i v e s w e r e a b l e t o h y d r o l y z e s t a r c h . O f t h e 199 s t a r c h - h y d r o l y s i s - p o s i t i v e s t r a i n s i s o l a t e d f r o m o t h e r f o o d s , 108 (54%) w e r e c o n s i d e r e d t o b e e n t e r o t o x i n p o s i t i v e ( S h i n a g a w a et al., 1984). T h e s e r e s u l t s i n d i c a t e a p o s s i b l e r e l a t i o n s h i p b e t w e e n starch hydrolysis and enterotoxin production. T h e B. c e r e u s s t r a i n s i s o l a t e d f r o m the d i a r r h o e a l t y p e f o o d p o i s o n i n g a n d t h o s e isolated from uncooked

rice t h a t w e r e s t a r c h - h y d r o l y s i s - p o s i t i v e w e r e less h e a t

r e s i s t a n t ( 1 0 0 ° C f o r 30 m i n o r 1 0 5 ° C f o r 5 rain) t h a n t h o s e i s o l a t e d f r o m t h e e m e t i c t y p e o f f o o d p o i s o n i n g a n d t h o s e i s o l a t e d f r o m t h e c o o k e d rice t h a t w e r e s t a r c h - h y d r o l y s i s - n e g a t i v e ( S h i n a g a w a et al., 1979). T h i s m a y b e s i g n i f i c a n t in t h e i d e n t i f i c a t i o n of B. cereus strains isolated f r o m the d i f f e r e n t types of food p o i s o n ing.

132 The starch hydrolysis test is performed by streaking the B. c e r e u s strains o n t o n u t r i e n t agar s u p p l e m e n t e d with 1% soluble starch, i n c u b a t i n g at 3 2 ° C for 24 h, a n d visualising the result by p o u r i n g Lugol's iodine solution o n t o the agar surface. A clear zone a r o u n d the streak of bacterial growth is considered a positive reaction. The test is considered negative if the clear zone appears only u n d e r the streak, or the zone is absent altogether.

B. c e r e u s t y p i n g

T y p i n g studies on B. c e r e u s strains from food p o i s o n i n g outbreaks provide an i m p o r t a n t c o n t r i b u t i o n to our u n d e r s t a n d i n g of the epidemiological aspects of these outbreaks. The flagellar (H) serological typing scheme has proved to be a useful epidemiological tool in the investigation of food p o i s o n i n g (Taylor a n d Gilbert, 1975; G i l b e r t a n d Parry, 1977; K r a m e r et al., 1982) i n c l u d i n g the d i s t r i b u t i o n of serotypes in foods, food additives a n d a m o n g B. c e r e u s strains from healthy carriers. A l t h o u g h 23 serotypes (H.1 to H.23) have been identified, some serotypes are more frequently implicated in outbreaks than others. Serotype 1, alone or with other types (H.8, H.12, etc.) has been associated with 70% of the emetic-type outbreaks. The serotypes most frequently associated with the diarrhoeal type outbreaks are H.1, 2, 6, 8, 10 a n d 19. Serotypes H.1, 8, 12 a n d 19 are c o m m o n l y associated with b o t h types of outbreaks (Gilbert a n d Parry, 1977; K r a m e r et al., 1982). Serotype H.1 alone or in c o m b i n a t i o n with serotypes H.5 or H.8 has been associated with

TABLE V Distribution of H-serotypes of B. cereus from the outbreaks of food poisoning in Japan a Type of food poisoning

Vomiting type

Diarrhoeal type and Miscellaneous type Total

Serotypes isolated

Number of incidents (%)

H.I H.1 + H.5 or H.8 H.3 H.5 H.8 H.12 SH-1 b Not typable

42 (65.6) 2 1 2 6 (9.4) 2 1 3 (4.7)

H.8 H.12 Not typable -

2 1 2 64 (100.0)

The sources of the results were the publications of Itoh et al. (1982) and Shinagawa et al. (1980). b Anti-SH-1 serum was prepared by using B. cereus strains from a vomiting type episode, Shinagawa et al. (1980).

133 75% of the 59 emetic type outbreaks in Japan (Shinagawa et al., 1980; Itoh et al., 1982). The B. c e r e u s strain from one emetic outbreak, serotype SH-1, was not typable with the H.1 to H.18 antisera (Taylor and Gilbert, 1975). Only 11.9% of the strains from outbreaks were nontypable (Table V). Biotyping, using a combination of biochemical properties (VP reaction, nitrate reduction, citrate utilization, urea decomposition, starch hydrolysis, fermination of sucrose and salicin Table IV) has proved to be a useful epidemiological tool (Kozasa et al., 1977; Ueda et al., 1986). However, biotyping has not been standardized for routine use and its application and interpretation is dependent on previously established individual criteria. Phage typing of B. c e r e u s strains has not been sufficiently well developed to be useful in tracing the sources of contamination in food poisoning outbreaks. Attempts have been made, however to develop a basic set of phages for use in routine laboratory typing (Major et al., 1979). A scheme developed by Iida et ai. (1985) used six phages types isolated from soil. This set of phages lysed specifically a large number of B. c e r e u s strains at one Routine Test Dilution (RTD). Biotyping along with phage typing has been used successfully in epidemiological inquiries.

Detection of B. c e r e u s toxins

The symptoms of both the diarrhoeal and emetic types of B. c e r e u s food poisoning are caused by separate toxins. The ability to detect these toxins in incriminated foods and clinical materials would be important in the laboratory diagnosis of outbreaks, but unfortunately appropriate techniques are not widely available at the present time. Other products elaborated by B. c e r e u s such as haemolysins I and II, phospholipase C, mouse lethal toxin (MLT), etc. have not found diagnostic applications. Some of the properties and biological activities of the diarrhoeal toxin have been reported (Spira and Goepfert, 1975; Turnbull et al., 1979; Turnbull, 1981; Gilbert and Kramer, 1984) including methods for its detection: the rabbit ileal loop (RIL) test (Spira and Goepfert, 1972; Kramer et al., 1982) and the vascular permeability reaction (VPR) test (Glatz et al., 1974; Kramer et al., 1982). A monkey feeding test for diarrhoeal activity was proposed by Goepfert (1974) but this test proved to be non-specific (Thompson et al., 1984). The only test method available for the emetic toxin is the monkey feeding test (Melling et al., 1976; Melling and Capel, 1978); however, this test is cumbersome and expensive. B. c e r e u s e n t e r o t o x i n Enterotoxin production B. c e r e u s enterotoxin (ET) is produced as follows; a Heart Infusion agar slant culture is inoculated into Brain Heart Infusion (BHI) broth (Difco) supplemented with 1% glucose ( B H I G ) in a 20 ml L-test tube and incubated for 12 h at 32°C. A 0.03 ml portion of the seed culture is transferred into 3 ml of the B H I G medium in a 30 ml T-test tube which is incubated at 3 2 ° C for 6 h on a reciprocal shaker at

134 9 0 - 1 0 0 c y c l e s / m i n . The culture is centrifuged (20 m i n at 12000 × g) a n d filtered through a 0.45 /~m m e m b r a n e filter. The culture filtrate is assayed for ET immediately, or within 48 h of storage at 4 ° C . A p o r t i o n of the culture filtrate is held at - 2 0 ° C until required for the V P R test, a n d the m o u s e ileal loop test. A n o t h e r p o r t i o n (100 ml culture filtrate in a 500 ml flask) is c o n c e n t r a t e d with a m m o n i u m sulphate (70% saturation). The precipitate is dissolved in distilled water, dialysed (0.005 M Tris-HC1 buffer, p H 8.6), a n d lyophilized prior to carrying out the R I L test. Biological

assay

T h e R I L test is the only specific biological test available for detecting the diarrhoeal toxin (Spira a n d Goepfert, 1972; K r a m e r et al., 1982). In this test, a 1 ml p o r t i o n of culture filtrate is injected into ileal loops (7 8 cm sections of ileum) of y o u n g r a b b i t s (1.0 to 1.5 kg). After 6 to 8 h the rabbit is sacrificed a n d the loops are e x a m i n e d for fluid a c c u m u l a t i o n . E x a m i n a t i o n in our l a b o r a t o r y of 50-fold conc e n t r a t e d culture filtrates gave the following results: 4 to 8 diarrhoeal strains p r o d u c e d fluid a c c u m u l a t i o n (1.1 to 10 m l / l o o p ) c o m p a r e d with only 1 of 21 emetic type strains (1.1 to 5.0 m l / l o o p ) (Shinagawa, et al., 1985). The V P R test (Glatz et al., 1974; T u r n b u l l et al., 1979; K r a m e r et al., 1982) has been used also to follow the p r o d u c t i o n of enterotoxin. G o o d correlation between this test a n d the R I L test has been observed ( T u r n b u l l , 1981; Spira a n d Goepfert, 1972; G l a t z et al., 1974). The V P R test is performed as follows: the test samples (0.05 ml) are injected i n t r a d e r m a l l y into the back of a y o u n g adult rabbit (1.8-2.2 kg) a n d followed 3 h later with an injection of 2% Evans blue solution (2 m l / k g of b o d y weight) into the ear vein. The a n i m a l is killed after 1 h a n d the diameters of the dark blue zones a r o u n d the sites of injection are measured. C u l t u r e filtrates of n i n e diarrhoeal strains tested gave zones m e a s u r i n g 15 to 24.9 m m a c c o m p a n i e d by o e d e m a a n d haemorrhage; 44 emetic strains were u n a b l e to elicit a significant VPR TABLE VI Vascular permeability activity of wholesome foods Source

Food poisoning: diarrhoeal type vomiting t y p e Wholesome foods

B. c e r e ~

strains isolated from 25 food poisoning outbreaks and

Starch Strains hydrolysis tested

Number of positive strains: (mm) ~ 0.0 4.9

5.0 9.9

Positive Negative

9 (2) h 50 (1)

0 21

0 23 (1)

Positive Negative

53 60

0 56

14 3

10.014.9

15.019.9

0 6

4 0

17 1

19 0

20.024.9 5 (2) 0 3 0

Haemorrhage

9 0 18 0

a Mean diameters of dark blue reaction zones (mm). b Figures in brackets indicate the numbers of reference strains also tested; reference strains: B. cereus strain B-4ac and 4433/74 from diarrhoeal-type outbreaks. B. cereus strain 4810/72 from a vomitingtype outbreak.

135 T A B L E VII The time-course of the appearance of fluid accumulation in the mouse ileal loop Enterotoxin titre (IDU) a

Incubation time (h) 2 4

6

8

10

20

0/5 b

1/9

1/19

1/4

0/4

40 50 (positive %)

3/11 (27)

4/10 (40)

14/21 (67)

5/9 (56)

5/11 (46)

400-600

6/6

4/5

7/8

4/5

3/4

~' IDU, l m m u n o diffusion unit: the minimal toxin concentration that produces a clear precipitation line against anti-enterotoxin with the microslide gel diffusion test. b N u m b e r of positive l o o p s / n u m b e r of test loops.

(blue zones less than 9.9 mm). Another 6 emetic strains gave moderate reactions (blue zones 1.0-14.9 mm). The starch-hydrolysis-positive strains isolated from wholesome foods always demonstrated higher VPR activities than did the starch-hydrolysis-negative ones (Table VI). B. cereus ET can be detected also with the mouse ileal loop (M1L) test, traditionally used for enterotoxigenic E. coli (Punyashthiti and Finkelstein, 1971) and Clostridium perfringens (Yamamoto et al., 1979). This method is practical, easy and convenient for the assay of a small numbers of samples. In view of this, we adapted it for use in the detection of enterotoxigenic B. cereus strains. The test is performed with mice (17 to 22 g) in which two segments are tied in the ileum, each being approximately 2.5 to 3.0 cm long. 0.1 ml of a culture filtrate is injected into one test loop and 0.1 ml of sterile BHIG into the other (negative control). The ratio of the fluid volume (ml) to the loop length (cm) is used as a measure of the reaction. The time required for fluid accumulation was dependent on the toxin titre, with 10 h required for a titre of 20 Immuno Diffusion Units (IDU) and 2 h for a titre of 600 IDU. Best results were obtained using 40-50 I D U with an incubation time of 6 h, 67% of the inoculated loops giving positive results (Table VII). With the higher titres (400 to 600 IDU), the inoculated animals developed severe necrosis of the intestinal mucosae (Shinagawa, 1985). Culture filtrates from 3 of 8 diarrhoeal strains induced mild fluid accumulation (reaction ratio: 0.01 to 0.04) with only one giving a strong positive reaction (reaction ratio of > 0.05). Culture filtrates from all 42 emetic type strains were negative. Culture filtrates from 6 of 53 starch-hydrolysis-positive strains isolated from wholesome foods induced fluid accumulation. Culture filtrates from none of 60 starch-hydrolysis-negative strains were fluid-accumulation-positive. Purification o f B. cereus enterotoxin and antiserum production

Several attempts have been made to purify the enterotoxin in order to produce specific antibodies to use in immunological assays. Spira and Goepfert (1975), Turnbull et al. (1979) and Ezepchuk et al. (1979) reported the purification and

136

TABLE Vlll Some biological activities and properties of a preparation of Biological acti~.,ities Vascular permeability: Mouse ileal loop: Rabbit ileal loop: Mouse lethal activity: Cytotoxicity (Vero cell): Haemolysins: Phospholipase C: Neutralization of the above activities by immune serum:

+ + + + + +

B. c e r e u s

enterotoxin

(0.05/~g/rabbit) (0.5 ~g/mouse) (50 ~ g / l o o p ) (i.v. 12 /~g/mouse)

+

Biochemical properties Resistance to Storage 20 ° C, 60 days: 4°C, 30 days: Heating 4 5 ° C , 10 rain: 5 6 ° C , 5 min: pH 6-9 < p H 3 , > p H 11: Pepsin (pH 5): Trypsin (pH 8):

+

+

+

characterization of an enterotoxic protein from culture filtrates, but conclusive proof of enterotoxin purification was lacking. Recently, Thompson et al. (1984) isolated a toxin produced by a B. cereus strain (B-4 ac) using chromatography on Amberlite CG-400, QAE-Sephadex, Sephadex G-75 and hydroxyapatite. The toxin was associated with three antigenically distinct proteins, whose activity could be neutralized by antibodies specific for two of the proteins. We have been able to purify B. cereus ET from some food poisoning diarrhoeal type strains showing high activities of VPR and fluid accumulation in the MIL and RIL tests. Purification o f e n t e r o t o x i n

The culture supernatant fluid was treated with a m m o n i u m sulphate (70% saturation) to precipitate the enterotoxin (ET). The precipitate was redissolved and chromatographed on DEAE-Sephadex A-25 and SP-Sephadex C-25, chromatofocused (elution with poly-buffer PBE-74), and gel filtered on Sephacryl S-300. The recovery of ET was 4-6% of the amount in the culture supernatant fluid, with the greatest loss in the DEAE-Sephadex step. The toxin was found to be unstable under a variety of physicochemical conditions (Table VIII). Final identification was achieved by disc electrophoresis (7.5% polyacrylamide gel, pH 8.3) and by gel

137

diffusion using antisera prepared with the purified ET. The main band obtained in each case proved to be the toxin (Shinagawa, 1985). Characterization of enterotoxin

The biological activities as well as other properties of the purified ET are listed in Table VIII. The purified ET showed increased VPR activity, produced fluid accumulation in the M I L and R I L tests and was lethal in mice. It was negative for both haemolysin and phospholipase C activities. The instability of ET under a wide variety of conditions deserves further study. Production o f rabbit anti-enterotoxin

The purified ET was emulsified with an equal volume of Freund's complete adjuvant (Difco) and injected subcutaneously into the back of male Japanese white rabbits weighing 1.5 to 2.0 kg. Injections of increasing concentrations of ET (0.5, 2.0, 4.0, 10, 20, 40, 100, 160 ~g) were made, usually at 3 day intervals. Two weeks after the last injection, a booster injection of 200 /~g of ET without adjuvant was given. The antiserum was collected 1 week after the final inoculation. The biological activities of ET such as the VPR, fluid accumulation in the M I L and RIL tests as well as the M L T were neutralized by the anti-enterotoxin serum. Immunological assay of B. cereus enterotoxin

Investigations of B. cereus food poisoning outbreaks can be improved by testing B. cereus isolates from suspect foods and clinical specimens for ET production. The biological assays, R I L and VPR, are time consuming and somewhat expensive, and in addition, are not necessarily specific. Our results using gel diffusion methods showed that all nine B. cereus strains from three diarrhoeal-type food poisoning

TABLE IX Enterotoxigenicity of foods

B. c e r e u s

Source

Starch hydrolysis

Food poisoning: diarrhoeal type vomiting type Wholesome foods

Positive Negative Positive (%) Negative (%)

strains isolated from 24 outbreaks of food poisoning and wholesome

Strains tested

9 (2) h 50 (1) 252 142

Number of ET-producing strains (-) 0 50 (1) 107 (42.5) 129 (90.8)

1:1 0 0

1:2 2 (1) 0

1:4 7 (1) 0

1:8 " 0 0

91

44

10

0

11

2

0

0

a ET-titre, enterotoxin-producing titre of B. c e r e u s strains by microslide gel diffusion test. b Reference strains: B. c e r e u s B-4ac, 4433/73, and 4810/72.

138 TABLE X Relationship between biological and immunological assays for B. cereus enterotoxin Immunological assay (ET-titre) b

Biological assay (VPR activity ") Number of positive strains (diameter: ram) 0.0 4.9

5.0 9.9

(- )

80

I :1

0

1:2 1:4 1 :8

Total

Total

10.0 14.9

15.0 19.9

20.024.9

43

3

0

0

4

24

3

0

31

0 0

1 0

5 0

16 2

1 7

23 9

0

0

0

0

0

0

80

48

32

21

8

189

126

a VPR activity, vascular permeability reaction activity. b ET-titre, enterotoxin-producing titre of B. cereus strains by microslide gel diffusion test. o u t b r e a k s p r o d u c e d ET (2 to 4 I D U ) , whereas strains from emetic type o u t b r e a k s were negative. In a d d i t i o n , 135 (53.0%) of 252 s t a r c h - h y d r o l y s i s - p o s i t i v e strains from w h o l e s o m e foods p r o d u c e d ET (1 to 4 I D U ) as well as 13 (9.2%) of 142 s t a r c h - h y d r o l y s i s - n e g a t i v e strains (1 to 2 I D U ) ( T a b l e IX). The ET a n t i b o d y reaction does not necessarily prove that the strains are enterotoxigenic, but the fact that the a n t i b o d y does neutralize the biological activities of ET, p a r t i c u l a r l y the R I L activity, justifies its use for a n a l y z i n g B. cereus strains. T h e r e l a t i o n s h i p between i m m u n o l o g i c a l a n d biological assays for ET was s t u d i e d in 189 strains isolated from 24 d i a r r h o e a l and emetic o u t b r e a k s a n d from w h o l e s o m e foods, T h e V P R test and the microslide gel diffusion technique were used for the c o m p a r i s o n s (Table X). A c c o r d i n g to these studies, 123 strains elicited little or no V P R (blue zones: less than 9.9 ram) a n d did not p r o d u c e ET as d e t e r m i n e d by the i m m u n o l o g i c a l assay, whereas 8 strains from d i a r r h o e a l outb r e a k s strongly increased the V P R activity (blue zones: 20 24.9 mm) and p r o d u c e d ET ( 2 - 4 I D U ) . F u r t h e r i m p r o v e m e n t in the assay awaits a p p l i c a t i o n of the reversed passive latex a g g l u t i n a t i o n test a n d e n z y m e linked i m m u n o s o r b e n t assay.

Food poisoning

d u e t o o t h e r Bacillus s p p .

G i l b e r t et al. (1981) and K r a m e r et al. (1982) expressed concern that B. subtilis, B. licheniformis a n d B. brevis m a y be p o t e n t i a l food p o i s o n i n g agents. Biological a n d i m m u n o l o g i c a l tests ( V P R a n d M L T ) used in the study of B. cereus p a t h o g e n e sis have been a p p l i e d to B. subtilis and B. licheniformis isolated from food p o i s o n i n g o u t b r e a k s , b u t evidence to s u p p o r t the suggestion that they are p o t e n t i a l p a t h o g e n s has yet to be found. B. thuringiensis, a n o t h e r representative of the genus Bacillus, shows b i o c h e m i c a l p r o p e r t i e s similar to those of B. cereus. The m a j o r difference b e t w e e n the o r g a n i s m s is the p r o d u c t i o n of p r o t e i n toxin crystals by B. thuringiensis. These can be d e t e c t e d b y staining techniques either as free crystals or p a r a s p o r a l

139

inclusion bodies within the exosporium. B. thuringiensis has not been implicated in outbreaks of food poisoning, but its differentiation from B. cereus is normally overlooked. Two episodes of food poisoning caused by B. subtilis were reported in Japan (Ministry of Health and Welfare, 1970, 1980). The incriminated foods were soybean curd ('tofu') and school luncheons (vegetable with mustard). In one of these outbreaks (Ministry of Health and Welfare, 1980) 580 (62.8%) of 923 students at risk became ill. The incubation period ranged from 2 25 h and the main symptoms were abdominal pain (73.2%), nausea (71.1%), vomiting (49.8%), diarrhoea (33.4%) headache (84.0%), and fever 3 7 - 3 9 ° C (mean 39.4%). In the suspected foods the number of B. subtilis was found to be 1.0 × 10S/g and in the stools of 21 (52.5%) of 40 patients it was present at a level of 107/g. In conclusion, taking into account the above comments and observations, the following guidelines for the confirmation of outbreaks of food poisoning due to B. cereus a n d / o r other Bacillus spp. can be drawn up: detection of 105 or more microorganisms per gram in incriminated foods: detection of a large number of Bacillus spp. in clinical specimens (stool and vomitus); isolates from such samples must be of the same serotype, biotype or phage type: the specific toxin produced by the isolates in incriminated food a n d / o r clinical specimens must be detected: conclusive epidemiological features must be present, i.e., symptoms, onset and inspection of the suspected food (type, place, time and nature of preparation, preservation status, etc.).

Acknowledgements I thank Professors Carlos A.M. Lopes, University of Estadual Paulista - U N E S P and Merlin S. Bergdoll, University of Wisconsin, and Dr. John M. Kramer, Central Public Health Laboratory, London, for helpful criticism and advice during the preparation of the manucript.

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