In this article we will discus about:- 1. Introduction to Bacillus Cereus 2. The Organism of Bacillus Cereus and its Characteristics 3. Pathogenesis and Clinical Features of Bacillus Cereus 4. Isolation and Identification 5. Association with Foods.
- Introduction to Bacillus Cereus
- The Organism of Bacillus Cereus and its Characteristics
- Pathogenesis and Clinical Features of Bacillus Cereus
- Isolation and Identification of Bacillus Cereus
- Bacillus Cereus Association with Foods
1. Introduction to Bacillus Cereus:
An early report associating food poisoning with Bacillus spp. was made in 1906 when Lubenau described an outbreak in a sanatorium where 300 inmates and staff developed symptoms of profuse diarrhoea, stomach cramps and vomiting. A spore forming bacillus was isolated from meatballs from the incriminated meal.
Although Lubenau named the organism Bacillus peptonificans, the properties he described resemble those of Bacillus cereus. Subsequently, aerobic spore formers were implicated in a number of outbreaks in Europe and between 1936 and 1943 they were suspected of causing 117 of 367 cases investigated by the Stockholm Board of Health.
Bacillus cereus was not conclusively established as a cause of food poisoning until 1950, after the taxonomy of the genus had been clarified. Hauge described four outbreaks in Norway involving 600 people. The food vehicle was a vanilla sauce which had been prepared a day in advance and stored at room temperature before serving. Samples of the sauce later tested contained from 2.5 x 107 to 1.1 x 108 Bacillus cereus ml-1.
This classic report and many of the early ones from Europe described an illness in which diarrhoea was the predominant symptom. It is now known that Bacillus cereus is responsible for two distinct types of foodborne illness: a relatively late- onset, ‘diarrhoeal syndrome’ and a rapid-onset, ’emetic syndrome’, first described in 1971 in the UK.
Since 1975 a number of other Bacillus species have been associated with foodborne illness. In these episodes, tests have failed to find known pathogens but food remnants and/or clinical specimens have yielded high numbers of Bacillus spp.
Far less common than outbreaks featuring Bacillus cereus, they usually feature Bacillus species belonging to the same morphological group, predominantly B. subtilis but also B. licheniformis and B. pumilus.
Overall, the reported number of cases of foodborne illness due to Bacillus spp. in the UK is much lower than those for Salmonella. Since 1987 there have been 25—30 outbreaks each year involving up to a total of 445 cases. Such statistics though, are likely to underestimate the true level far more than those for Salmonella since the data come only from outbreaks and there is no estimate of sporadic cases.
2. The Organism of Bacillus Cereus and its Characteristics:
Members of the genus Bacillus are Gram-positive, aerobic, spore forming rods, though they do, on occasion, display a Gram-negative or variable reaction. They are divided into three groups based on the morphology of the sporangium (the spore-bearing cell) and the spore. Biochemical tests are used to subdivide the morphological groups into species.
Bacillus cereus is facultatively anaerobic with large vegetative cells, typically 1.0 µm by 3.0-5.0 µm in chains. It grows over a temperature range from 8 to 55 °C, optimally around 28-35 °C, and does not have any marked tolerance for low pH (min. 5.0-6.0, depending on the acidulant) or water activity (min. ~ 0.95).
Spores are central, ellipsoidal in shape and do not cause swelling in the sporangium. As a spore former, Bacillus cereus is widely distributed in the environment and can be isolated from soil, water and vegetation.
This ubiquity means that it is also a common component of the transient gut flora in humans. The spores show a variable heat resistance; recorded D values at 95 °C in phosphate buffer range between around 1 min up to 36 min. Resistance appears to vary with serovar.
In the UK, a serotyping scheme based on the flagellar (H) antigen has been devised, based on a set of 29 agglutinating antisera raised against outbreak and non- outbreak strains isolated from foods. In about 90% of outbreaks it is possible to serotype the causative organism, although only about half of environmental isolates are typable.
There does not appear to be a strong association between the two different types of Bacillus cereus food poisoning and particular serotypes.
Some have been associated with both types of syndrome, although in a study of 200 outbreaks of the emetic syndrome from around the world, serotype 1, which possesses markedly greater heat resistance than other serotypes, was isolated from implicated foods, faeces or vomitus in 63.5% of cases.
3. Pathogenesis and Clinical Features of Bacillus Cereus:
Symptoms of the diarrhoeal syndrome resemble those of Clostridium petfringens food poisoning. The onset of illness is about 8-16 h after consumption of the food, lasts for between 12 and 24 h, and is characterized by abdominal pain, profuse watery diarrhoea and rectal tenesmus. Nausea and vomiting are less frequent.
The emetic syndrome resembles the illness caused by Staphylococcus aureus. It has a shorter incubation period than the diarrhoeal syndrome, typically 1-5 h, and nausea and vomiting, lasting between 6 and 24 h, are the dominant feature.
Both syndromes are caused by distinct enterotoxins (Table 7.3). The diarrhoeal illness toxin is a heat-labile protein with a molecular mass of around 50 kDa. It is sensitive to proteolytic enzymes such as trypsin and pepsin and is produced by the organism during the late exponential phase of growth. It remains to be clarified whether, in cases of illness, the toxin is produced in the food or in the gut.
The emetic toxin has a molecular mass of less than 5 kDa is heat resistant (stable at 126 °C for 90 min) and is resistant to proteolytic enzymes and low pH. Production of the toxin occurs in the food in the late exponential to stationary phase of growth and may be related to sporulation.
Pathogenic features of the illness caused by the other Bacillus spp. are not known. The short incubation periods recorded in outbreaks (from <1 h up to 11 h) suggest an intoxication, though no toxin has been isolated and described as yet.
4. Isolation and Identification of Bacillus Cereus:
In an outbreak of Bacillus cereus food poisoning, implicated foods will contain large numbers (> 105 g-1) of organisms so enrichment techniques are not needed. The same is true of faecal or vomitus specimens and a non-selective medium such as blood agar (sometimes with the addition of polymyxin as a selective agent to suppress Gram-negatives) is commonly used.
Bacillus cereus can be identified after 24 h incubation at 37 °C by its characteristic colonial morphology of large (3-7mm diameter), flat or slightly raised, grey-green colonies with a characteristic granular or ground-glass texture and a surrounding zone of α or β haemolysis.
To confirm the identity of a blood agar isolate or to isolate smaller numbers of Bacillus cereus from foods, a more selective diagnostic agar is necessary. Several of these have been proposed which have a number of common features.
Polymyxin/pyruvate/egg yolk / mannitol / bromothymol blue agar (PEMBA) is one widely used example. It includes polymyxin as a selective agent and where yeasts and moulds are likely to be a problem actidione may also be included.
On PEMBA, Bacillus cereus produces typical crenated colonies which retain the turquoise-blue of the pH indicator (bromothymol blue) due to their inability to ferment mannitol, they are surrounded by a zone of egg-yolk precipitation caused by lecithinase activity. Pyruvate in the medium improves the egg-yolk precipitation reaction and a low level of peptone enhances sporulation.
Colonies of Bacillus cereus can be confirmed by a microscopic procedure combining a spore stain with an intracellular lipid stain. Spores appear green in a cell with red vegetative cytoplasm and containing black lipid globules.
Biochemical confirmation can be based on an isolate’s ability to produce acid from glucose but not from mannitol, xylose and arabinose. Diarrhoeal toxin can be detected by a commercially available latex agglutination kit but toxin detection and serotyping are not usually part of routine food surveillance.
5. Bacillus Cereus Association with Foods:
The ability to produce spores resistant to factors such as drying and heat means that the food-poisoning bacilli are widely distributed in foods. In most circumstances however they are only a small part of the total flora and are not present in numbers sufficient to cause illness.
Heat processing will select for spore formers and a number of surveys have reported a higher incidence of Bacillus cereus in pasteurized and other heat-processed milks (typically 35-48% of samples positive) compared with raw milk (~ 9% positive).
In most of these cases the numbers detected were low (< 103 ml-1),but when pasteurized milk or cream are stored at inadequate chill temperatures Bacillus cereus can grow and cause the type of spoilage known as ‘sweet curdling’ or ‘bitty cream’.
Despite this, milk and dairy products are rarely associated with illness caused by Bacillus cereus, although dried milk has been implicated in outbreaks when used as an ingredient in vanilla slices and macaroni cheese. A possible explanation is that though liquid milk is an excellent growth medium for the organism, toxin production is not favoured. One study in Sweden has linked this with the low aeration in static packs of milk.
The ability of spores to resist desiccation allows their survival on dried products such as cereals and flours. In the Norwegian outbreaks, the cornflour used to thicken the vanilla sauce was implicated.
Moderate heating during preparation would not inactivate the spores and subsequent extended storage of the high-αw, sauce at ambient temperature was conducive to spore germination and outgrowth.
The emetic syndrome is particularly associated with starchy products such as rice and pasta dishes. In the UK, its association with cooked rice has been sufficiently marked for it to earn the soubriquet ‘Chinese Restaurant Syndrome’. The typical scenario is where rice is prepared in bulk, in advance.
Spores, commonly those of the more heat-resistant serotype 1, survive precooking to germinate, grow and produce the emetic toxin in the product during storage.
This would be prevented by chilling to below 8°C, but the rate of cooling in the centre of a bulk of cooked rice, even if transferred to chill storage, can be slow enough for growth and toxin production to occur. Reheating the rice prior to serving will not inactivate the emetic toxin and render the product safe.
A wider range of foods have been implicated with the diarrhoeal syndrome including meat products, soups, vegetables, puddings and sauces.
Dried herbs and spices used in food preparation can be an important source of Bacillus cereus and this has often been cited as a reason for a relatively high incidence of Bacillus cereus food poisoning in Hungary, where between 1960 and 1968 it was the third most common cause of food poisoning accounting for 15.2% of persons affected.
More recent figures suggest that its relative importance has declined somewhat but whether this is due to changes in culinary practices, improvements in hygiene, decreased contamination of spices or a statistical artefact is not known.
Meat pies and pasties are common vehicles for the other food-poisoning bacilli along with a range of processed meats and meat and rice dishes. Baked goods such as bread and crumpets have been involved in a number of B. subtilis outbreaks.
Although B. subtilis is responsible for the defect known as ropey bread where spores surviving baking degrade the loaf’s internal structure and produce a sticky slime, this does not always prevent people from eating it.
In 1988, a bakery in the Isle of Man omitted propionate from their bread in order to claim for it the virtue of being free from artificial preservatives and thereby more healthy. As a result, nine people developed nausea, vomiting, diarrhoea, headache and chills 10 min after consuming ropey bread containing more than 108 organisms gm.-1.