Prevalence of Bacillus cereus and Associated Risk Factors in Chinese-Style Fried Rice Available in the City of Colombo,Sri Lanka

Abstract

The present study aimed to assess the prevalence of Bacillus cereus and its associated risk factors in Chinese-style fried rice available in Colombo city. In 200 samples of fried rice the prevalence of B. cereus was 56%. The prevalence by variety of fried rice was chicken (20.0%), vegetable (18.0%), seafood (10.0%), egg (5.0%), mixed (2.0%), and beef (1.0%). Of analyzed samples, 28 (14%) had colony counts >10⁶ colony forming units per gram (cfu/g), the infectious dose for B. cereus food borne outbreaks. Occurrence of >10⁶ cfu/g of B. cereus were associated with storage of boiled rice at room temperature (p=0.030), >4 hours of storage at room temperature (p=0.042) and cooking frequency of more than once per dining session (p=0.017). The type of rice and the quantity boiled per day were not independent risk factors for high B. cereus counts. Majority of B. cereus isolates (53.7%) in this study were not typable. The serotypes observed included H15 (14.3%), H19 (14.3%), and H20 (10.7%). These serotypes are known to be associated with both emetic and diarrheal syndromes. All isolates were sensitive to ciprofloxacin (100%), erythromycin (100%), gentamicin (100%), chloramphenicol (100%), and amikacin (100%) whereas 100% resistance was observed for penicillin with minimal inhibitory concentration range of 32–256 μg/mL.

Introduction

B acillus cereus is ubiquitous in the environment and is able to survive and proliferate in soil, water, and vegetation. The highly resistant spores can survive heating, drying, radiation, freezing, and pasteurization (Kotiranta et al., 2000). It is able to contaminate wide variety of raw and processed food items such as cereals, dairy products, meat, fish, vegetables, herbs, spices, cooked rice, soups, and desserts (Granum, 2001).

B. cereus is responsible for two distinct types of food poisoning. The diarrheal syndrome is caused by enterotoxins released in the small intestine after ingestion of contaminated food. The emetic syndrome is caused by a heat stable peptide preformed in food (Vilas-Boas et al., 2007). The emetic syndrome is rapid in onset, occurring within 1–5 hours of ingestion and causes nausea, vomiting, and abdominal cramps. In contrast, the diarrheal syndrome is delayed in onset, occurring after 8–16 hours and is characterized by watery diarrhea, cramps, and abdominal pain. Usually for both syndromes symptoms do not persist beyond 24 hours and the illness is mild and self limiting (Lake et al., 2004). In addition to food poisoning, this organism is implicated in wound infections, bacteremia, endocarditis, and severe eye infections such as keratitis, endophthalmitis, and panophthalmitis. Other less frequent infections include postoperative meningitis, gingivitis, and periodontitis (Kotiranta et al., 2000).

Cooked rice is usually associated with the emetic syndrome of food poisoning. B. cereus has been documented worldwide in outbreaks associated with cooked rice (Little et al., 2002; Lake et al., 2004). Since 1971, contaminated Chinese-style fried rice has been responsible for more than 40 incidents of emetic type food poisoning in the United Kingdom (Taylor and Gilbert, 1975). In a risk assessment for B. cereus rice associated food poisoning outbreaks by Notermans and Batt (1998), “Chinese-style food” was the commonest food vehicle in Canada (1985–1986, 17/39 outbreaks), the United States (1988–1992, 12/21 outbreaks), and the Netherlands (1992–1994, 17/40 outbreaks). In addition, in England and Wales 12 outbreaks occurred after consumption of Chinese or Indian style takeaways during 1992 to 2000 (Little et al., 2002). Also, cooked rice associated outbreaks have been reported from Japan (Shinagawa et al., 1985; Shiota et al., 2010), Singapore (Tay et al., 1982), Scotland (Dickson and Morgan, 1995), Finland (Raevuori et al., 1976), India (Granum and Baird-Parker, 2000), and New Zealand (Lake et al., 2004). Consequently, B. cereus has gained prominence as a priority pathogen for enteric diseases task force work plan 2009–2010 drawn up by the WHO (WHO, 2009).

From 2005 to 2010, 7620 food poisoning cases have been reported in Sri Lanka out of which 600 have occurred in Colombo district (Ministry of Healthcare and Nutrition Sri Lanka, 2005). Clinical symptoms of many of these cases were similar to B. cereus emetic type food poisoning. However, most were not investigated due to the mild and self limiting nature of the illness. Also, the majority of cases were sporadic and not associated with outbreaks. In the recent past, B. cereus counts ranging from 1.0×10³ to 8.4×10⁸ have been reported in cooked rice and fried rice (Unpublished data from Food and Water Laboratory, Medical Research Institute, Sri Lanka). Sri Lanka is a popular tourist destination and local cuisine which includes fried rice is very much in demand. Therefore, ensuring food safety is a prime concern.

The predominance of B. cereus outbreaks linked to Chinese-style fried rice is associated with the common practice of boiling large quantities of rice and storing at room temperature before frying. This is done to avoid refrigeration which makes the grains to become sticky and clump together. Raw rice frequently contains heat resistant spores that survive boiling. When boiled rice is stored at room temperature for prolonged periods, these spores germinate, proliferate, and produce heat stable emetic toxin (Little et al., 2002). Vegetative cell growth is stimulated by the nutrient content of eggs and meat added to the rice (Morita and Woodburn, 1977). The organism is thus able to achieve the infectious dose of counts exceeding 10⁶ colony forming units per gram (cfu/g) of food (USFDA, 2001).

This study was aimed at assessing the prevalence of B. cereus in Chinese-style fried rice available in Colombo city, evaluating selected risk factors for significant counts (>10⁶ cfu/g), serotyping, and antibiotic sensitivity testing of isolates.

Materials and Methods

Sampling method

During a period of 1 year, 200 samples of Chinese-style fried rice were collected by systematic random cluster sampling of eateries within Colombo city. The samples were categorized according to the main ingredient added to fried rice during preparation. These were vegetables, chicken, seafood, eggs, beef or a mixture of all. The distribution of 200 samples included 70 (35%) vegetable fried rice, 59 (29.5%) chicken fried rice, 36 (18%) seafood fried rice, 18 (9%) egg fried rice, 10 (5%) beef fried rice, and 7 (3.5%) mixed fried rice. Samples were transported without freezing and analysis commenced within 3 hours.

A structured interviewer administered questionnaire was designed to elicit information on practices related to preparation and storage of fried rice. The questionnaire was pretested on an appropriate sample population. Data processing and analysis was conducted using SPSS 13 statistical software package.

Preparation of sample dilutions

Ten grams from each fried rice sample was randomly taken and homogenized in 90 mL of 0.1% Peptone Water (CM0009) (Oxoid Ltd) using a Stomacher 400 Circulator (Seward Ltd). With this initial 1:10 (10⁻¹) dilution, serial dilutions from 10⁻² to 10⁻⁷ were prepared in 0.1% peptone water (Bridson, 2006).

Presumptive B. cereus count

The medium used for isolation of B. cereus was prepared with B. cereus Selective Agar Base (CM617), B. cereus Selective Supplement/Polymyxin B (SR99), and Egg Yolk Emulsion (SR47) (Oxoid Ltd). Each batch of prepared medium was quality controlled using B. cereus ATCC 10876 as the positive control and Bacillus subtilis ATCC 6633 as the negative control. Duplicate plates of B. cereus selective agar were inoculated with 0.1 mL of 10⁻¹ to 10⁻⁷ dilutions. Each inoculum was evenly spread on the surface of the medium with a sterile glass spreading rod. Plates were incubated at 35°C for 24 hours and cursorily examined for Bacillus colonies. Thereafter, the plates were left at room temperature for 24 hours to allow colonies to develop typical morphology of B. cereus (Bridson, 2006). The characteristic colonies were crenated, ∼5 mm in diameter, peacock blue, and surrounded by a conspicuous egg yolk precipitate of the same color. All plates from the dilution series was examined with the naked eye and one set containing an estimated 15–150 typical colonies was selected for manual counting (Sri Lanka Standard: 516: Part 8, 1983; USFDA, 2001). This number expressed as cfu/g fried rice was the presumptive colony count.

Confirmed B. cereus count

From each positive sample, five peacock blue, lecithinase-positive colonies were picked and transferred to nutrient agar slants. These were incubated for 24 hours at 30°C and used for performing morphological and biochemical confirmatory tests. With Gram staining (212539, BD Difco™ BBL™ Stains), B. cereus appears as large Gram-positive bacilli in chains with ellipsoidal, central, or paracentral spores which do not swell the sporangium. The rapid confirmatory staining procedure (Holbrook and Anderson, 1980) using malachite green (90903; Fluka), Sudan Black (199664; Sigma-Aldrich), and aqueous 0.5% w/v safranin (212532; BD Difco BBL Stains) showed the characteristic morphology of pale green spores and black lipid globules in red stained cytoplasm.

The principle biochemical tests used for identification of B. cereus group were positive catalase (H3410; Sigma-Aldrich), positive Voges-Proskauer test (39484; Fluka), reduction of nitrate (72548; Fluka), hydrolysis of tyrosine (T3754; Sigma-Aldrich), growth in 0.001% w/v lysozyme broth (62970; Fluka), liquefaction of gelatin (48723; Fluka), and production of acid from glucose (49159; Fluka), xylose (X1500; Sigma-Aldrich), and arabinose (10850; Fluka). These biochemical tests were chosen from standard references for isolation and enumeration of B. cereus from food (Kramer et al., 1982; Sri Lanka Standard: 516: Part 8, 1983; USFDA, 2001). All biochemical media were quality controlled for performance with recommended positive and negative controls. For each batch of testing B. cereus ATCC 10876 and B. subtilis ATCC 6633 were used as additional controls.

However, these reactions are identical for all members of the B. cereus group namely, B. cereus, Bacillus mycoides, Bacillus thuringiensis, and Bacillus anthracis. Therefore, additional tests were performed by inoculating the isolates on Sheep Blood Agar (CM0854; Oxoid Ltd), Nutrient Agar (CM0003; Oxoid Ltd) and staining 3–4 day old cultures with warm 0.5% basic fuchsin (212545; BD Difco BBL Stains) for toxin crystals. B. cereus isolates were identified as those showing active motility, strong hemolysis, no rhizoid growth, and no protein toxin crystals (USFDA, 2001). The number of B. cereus was calculated by using the proportion of selected colonies confirmed as B. cereus and the dilution factor (Presumptive colony count×proportion confirmed as B. cereus×dilution factor×10). The confirmed colony counts were expressed as cfu/g of fried rice.

Assessment of risk factors

Occurrence of >10⁶ cfu/g of B. cereus were assessed with regard to five presumed risk factors. These included the type of rice (Basmathi, Samba, others), amount of rice boiled per day (>10, ≤10 kg), method of storage of boiled rice (steaming hot, room temperature, refrigerated), duration of storage of boiled rice before frying (≤4, >4 hours), and frequency of preparation of boiled rice per dining session (1, >1). Bivariate associations were assessed and those which were statistically significant (p-value<0.05) were included for binary logistic regression analysis.

Serotyping of B. cereus

Serotyping of B. cereus isolates detected at >10⁶ cfu/g was carried out at the Food Safety Microbiology Laboratory of PHLS Central Public Health Laboratory, United Kingdom. Actively motile strains obtained by several passages through semisolid nutrient agar were cultured in nutrient broth for 5 hours at 35°C with constant agitation. After confirming the presence of active motility by phase contrast microscopy, formalin 1% (v/v) was added to obtain antigen suspensions. Flagellar “H” antisera prepared by intravenous inoculation of rabbits was used for typing the isolates. Doubling dilutions of 0.25 mL portions of antirsera were made in agglutination trays and equal volumes of antigen suspension added. Final dilutions were 1:40 to 1:640. These were incubated at 50°C for 2 hours. Macroscopic agglutination was observed with dark field illumination (Taylor and Gilbert, 1975).

Antibiotic sensitivity testing of B. cereus isolates

For B. cereus isolates occurring at counts >10⁶ cfu/g, susceptibility to a standard panel of antibiotics was determined by comparative disk diffusion method. The antibiotics tested were ciprofloxacin (5 μg), erythromycin (15 μg), gentamicin (10 μg), penicillin (10 μg), chloramphenicol (30 μg), and amikacin (30 μg) (Oxoid Ltd). The resistance profile to penicillin was further investigated with Penicillin Etest^® (Biomerieux) minimal inhibitory concentration (MIC) method which was performed and interpreted per manufacturer's instructions and CLSI, M45-A, Guideline (CLSI, 2006).

Results

Of the 200 fried rice samples analyzed, B. cereus was detected in 112 (56.0%) whereas 44% (88) of the samples were negative for B. cereus. Among these positive samples, 28 (14%) had colony counts >10⁶ which corresponds to the infectious dose. Simultaneously 84 (42%) samples contained B. cereus colony counts ≤10⁶. For counts exceeding 10⁶ the highest prevalence was detected in chicken fried rice (23.7%). None of the beef fried rice contained B. cereus in excess of 10⁶. With regard to counts less than 10⁶, the highest prevalence of 44.4% was individually observed in both seafood and egg fried rice whereas beef fried rice recorded the lowest prevalence of 20% (Table 1).

Table 1.

Prevalence of Bacillus cereus Classified as Counts of ≤10⁶ cfu/g, >10⁶ cfu/g, and Nondetectable by Variety of Chinese-Style Fried Rice

	Not detected		≤10⁶ cfu/g		>10⁶ cfu/g		Total number analyzed from each variety
Variety	No:	%	No:	%	No:	%	No:	%
Vegetable	34	48.57	29	41.43	07	10.00	70	35.00
Chicken	19	32.20	26	44.07	14	23.73	59	29.60
Seafood	16	44.44	16	44.44	04	11.11	36	18.00
Egg	08	44.44	08	44.44	02	11.11	18	9.00
Beef	08	80.00	02	20.00	–	–	10	5.00
Mixed	03	42.86	03	42.86	01	14.29	07	3.50
Total	88	44.00^a	84	42.00	28	14.00	200	100.0

Total number of samples with Bacillus cereus detected=(200–88) 112 (56.00%).

cfu/g, colony forming units per gram.

In the assessment of risk factors for the occurrence of B. cereus in counts >10⁶ cfu/g three parameters emerged as independent risk factors (Table 2). These were storage at room temperature before frying (p=0.030), >4 hours of storage prior to frying (p=0.042), and frequency of cooking more than once (p=0.017).

Table 2.

Risk Factors for Bacillus cereus Counts >10⁶ cfu/g in Fried Rice with Adjusted Odds Ratios and 95% Confidence Intervals

Factor	Frequency %	p-value	Adjusted OR	95% CI
Step1
Frequency of cooking per dining session		0.067	2.80	0.93–8.41
Only once	07
More than once	21
Temperature of storage before frying		0.034	5.41	1.14–25.71
Room temperature	26
Storage in fridge/hot steaming	2
Time of storage before frying		0.064	4.39	0.91–21.05
≤4 hours	2
>4 hours	26
Step 2
Frequency of cooking per dining session		0.017	3.16	1.23–8.14
Only once	07
More than once	21
Temperature of storage before frying		0.030	5.54	1.18–25.97
Room temperature	26
Storage in fridge/hot steaming	2
Time of storage before frying		0.042	4.55	1.2–21.43
≤4 hours	16
>4 hours	12

The majority of B. cereus isolates present in counts exceeding 10⁶ were not typable. Amongst the typable isolates, the most prominent serotypes were H15 (14.3%) and H19 (14.3%). Serotype H20 was observed at 10.7% (Table 3). Antibiotic sensitivity demonstrated an identical pattern. All isolates were sensitive to ciprofloxacin (100%), erythromycin (100%), gentamicin (100%), chloramphenicol (100%), and amikacin (100%). Conversely, 100% resistance was observed for penicillin. This was confirmed by E-test which showed MIC to be within the range of 32–256 μg/mL for all isolates.

Table 3.

Serotypes of Bacillus cereus Isolates Obtained from Fried Rice Samples Containing >10⁶ cfu/g

Serovar “H”	Total number of each serovar	% of each serovar
Nontypable	15	53.57
15	04	14.29
19	04	14.29
20	03	10.71
Polyaglutination	01	3.57
Autoagglutination	01	3.57
Total	28	100

Serotyping was performed by using B. cereus H-specific antisera.

Discussion

In many food poisoning outbreaks due to B. cereus, cooked rice has been implicated as the source. Table 4 indicates some of the notable outbreaks reported since 1971. Almost all outbreaks were associated with symptoms of emetic syndrome. This may be attributed to the stimulatory effect of starch on growth of B. cereus and production of emetic toxin (Kramer and Gilbert, 1989).

Table 4.

Bacillus cereus Outbreaks Associated with Cooked Rice

Year	Country	Food item	Symptoms/syndrome	Bacillus cereus count (cfu/g)	Reference
1971	United Kingdom	Chinese-style fried rice	V, N, (D) Emetic Type	10⁶–10⁹	Taylor and Gilbert (1975)
1973–1982	Japan	Rice and chicken, Fried rice	V, N Emetic Type	10⁴–10⁸	Shinagawa et al. (1985)
1974–1999	Japan	Boiled rice, fried rice, sushi rice, rice gruel	V, N Emetic Type	10⁶–10⁸	Agata et al. (2002)
1976	Finland	Boiled rice	V, C, (D) Emetic type	1.7×10⁸	Raevuori et al. (1976)
1981	Singapore	Fried rice	V, C, (D), headache, fever Emetic type	Not documented	Tay et al. (1982)
1985	United States	Cooked rice	V, N, (D) Emetic Type	Not documented	CDC (1986)
1985	United States	Rice and chicken	D, C, N Diarrheal type	Not documented	Baddour et al. (1986)
1991	India	Cooked rice	V, N Emetic Type	Not documented	Granum and Baird-Parker (2000)
1993	United States	Fried rice with chicken	N, V, C, (D) Emetic Type	>10⁶	Khodr et al. (1994)
1994	United States	Cooked rice and macaroni	V, N Emetic Type	Not documented	Granum and Baird-Parker (2000)
1994	Scotland	Fried rice	V, N, C, Emetic Type	2.0×10⁷	Dickson and Morgan (1995)
1996	India	Cooked rice	V, N Emetic Type	Not documented	Granum and Baird-Parker (2000)
2000	United Kingdom	Cooked rice	V Emetic type	>10⁶	Ripabelli et al. (2000)
2000	The Netherland	Vegetarian rice	V, C Emetic type	10⁵–10⁸	The EFSA Journal (2005)
2008	Japan	Fried rice	V, N Emetic Type	Not documented	Shiota et al. (2010)

V, vomiting; N, nausea; C, cramps; D, diarrhea; (D), mild/occasional diarrhea.

This study revealed 56% of fried rice samples to be contaminated with B. cereus. This compares favorably with prevalence for fried rice reported by other investigators, Notermans and Batt (1998) 12%–86% in the Netherlands, Bryan et al. (1981) 85.7% in the United States, and Schiemann (1978) 33% in the United Kingdom. Similar high prevalence of B. cereus has been observed for boiled rice; 100% in India (Kamat et al., 1989), 92.9% (Bryan et al., 1981) and 91.7% (Harmon and Kautter, 1991) in the United States, 10%–93% in the Netherlands (Notermans and Batt, 1998), and 93.9% in the United Kingdom (Nichols et al., 1999). The colony counts observed in these studies ranged from <10² to ≥10^7. The colony counts observed in the current study were 10³ (13.0%), 10⁴ (9.0%), 10⁵ (11.5%), 10⁶ (8.5%), 10⁷ (3.5%), 10⁸ (2.5%), 10⁹ (4.5%), and 10¹⁰ (3.5%). B. cereus becomes a hazard when present in numbers exceeding 10⁶ cfu/g. Our study revealed 14% of samples to have this count which is considerably higher than 0.4% reported by Nichols et al. (1999) and 3.1% by Little et al. (2002).

The high counts of B. cereus associated with boiled rice could result from heat activation and germination of spores present in raw rice. Prevalence of B. cereus in raw rice has been reported as 38%–68% by coworkers in India (Vijayalakshmi et al., 1981), the United States (Ankolekar et al., 2009), Lebannon (Hassan and Nabbut, 1996), the United Kingdom (Blakey and Priest, 1980), and Korea (Park et al., 2009). In addition, B. cereus vegetative cells may be present in meat and seafood whereas dried foods and spices contain spores (Bryan et al., 1981; Little et al., 2002). There were clearly evident variations in prevalence of B. cereus by the variety of fried rice in our study. Therefore, it could be contended that addition of meat, sea food, herbs, and spices may have contributed to the observed variations which merits further investigation.

Studies reveal that the degree of B. cereus contamination of boiled rice was directly related to the temperature of storage and length of time the rice was kept before serving (Gilbert et al., 1974; Little et al., 2002). It is recommended that boiled rice not be stored at ambient temperature for more than four hours as this would permit resistant spores to germinate and proliferate. This findings were corroborated by our study which showed statistically significant association between B. cereus in counts >10⁶ cfu/g and storage of boiled rice at room temperature for >4 hours before preparation. Two outbreaks described by Khodr et al. (1994) and Shiota et al. (2010), where rice was stored at room temperature for a prolonged period and reheated are classic examples for time and temperature abuse mentioned above.

Bulk cooking of rice once per day and cooling at room temperature were shown to be associated with high B. cereus counts (WHO, 2000). This has been attributed to the presence of favorable growth conditions. However, the findings of present study contradicted this notion as it showed frequency of cooking more than once to be an independent risk factor for significant B. cereus counts. Possible explanations for this result could be postcooking contamination from inadequately cleaned utensils and addition of left over rice to new batches (Bryan et al., 1981). But the present study did not include examination of postcooking contamination. The type of rice and the quantity boiled per day was not a contributing factor for high B. cereus counts. These two parameters have not been previously investigated. Small sample size with predictably wide 95% confidence intervals was a limitation.

Majority of our B. cereus isolates were not typable. Untypable strains of B. cereus have been responsible for both emetic and diarrheal syndrome (Kramer and Gilbert, 1989). The present study revealed serotypes H15, H19, and H20. H19 has been associated with both emetic and diarrheal syndrome (Kramer et al., 1982). H15 and H20 have been reported from diarrheal and emetic syndromes respectively. Further, H15 has been isolated from raw rice, H19 from boiled rice, and H20 from both (Gilbert and Kramer, 1986).

The 100% sensitivity for ciprofloxacin, erythromycin, gentamicin, chloramphenicol, and amikacin in our study is similar to that observed by Stec (1990) and Meena et al. (2000), differing from that of Luna et al. (2007) where erythromycin sensitivity was 84%. The uniform resistance to penicillin with MIC ranging from 32 to 256 μg/mL was demonstrably higher than 95% reported for the US isolates (Luna et al., 2007).

Conclusions

With B. cereus prevalence of 56%, Chinese-style fried rice in Colombo city could be a hazard for foodborne outbreaks. Common practices in preparation such as storage of boiled rice at room temperature for prolonged periods and a greater frequency of cooking emerged as significant risk factors for B. cereus contamination. Therefore, food-handlers should be instructed on safe practices which include keeping boiled rice either at >60°C (hot steaming) or cooling rapidly and transferring to a refrigerator within 4 hours (WHO, 2000). Local serotypes could be associated with both emetic and diarrheal syndromes. Aminoglycosides, ciprofloxacin, and chloramphenicol are suitable therapeutic options for indigenous infections.

Footnotes

Acknowledgments

This project was supported by a grant from the Medical Research Institute, Colombo, Sri Lanka. For professional advice and assistance we gratefully acknowledge Professor Chandra Kodikara and Dr. Karven Cooray.

Disclosure Statement

No competing financial interests exist.

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