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PATHOGENS IN MILK | Bacillus cereus
PATHOGENS IN MILK Contents Bacillus cereus Brucella spp. Campylobacter spp. Clostridium spp. Coxiella burnetii Escherichia coli Enterobacteriaceae Enterobacter spp. Listeria monocytogenes Mycobacterium spp. Salmonella spp. Shigella spp. Staphylococcus aureus – Molecular Staphylococcus aureus – Dairy Yersinia enterocolitica
Bacillus cereus A Christiansson, Swedish Dairy Association, Lund, Sweden ª 2011 Elsevier Ltd. All rights reserved.
Introduction Bacillus cereus is an aerobic spore-forming bacterium, whose spores are commonly present at low levels in raw milk. In the 1960s and earlier, B. cereus was clearly a quality problem, due to coagulation (sweet curdling) of pasteurized milk and formation of flakes in cream when added to coffee (bitty cream). This was due to poorly cleansed equipment (e.g., milk cans) at the farm and in dairy factories and a lack of adequate refrigeration. Nowadays, these problems are rarely seen in countries where milk is kept at temperatures below 6 C. However, when pasteurized milk is stored at higher temperatures, B. cereus may still be a limiting factor for the keeping quality. Bacillus cereus can produce several enterotoxins causing diarrhea and vomiting. There are few dairy-related cases, but milk and cream have been incriminated in both types of illnesses.
Characteristics Morphology and Cultivation Bacillus cereus is a Gram-positive, rod-shaped, motile bacterium with peritrichous flagella. The cells tend to grow in chains but may occur singly as well. The length
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of the bacterium varies between 3 and 5 mm and the diameter is more than 1 mm. Spores are oval or cylindrical, located centrally or paracentrally/subterminally, and do not distend the cell. A typical trait of the B. cereus group is the presence of storage granules of poly- hydroxybutyrate in the cytoplasm. These are easily seen by phase contrast microscopy. Bacillus thuringiensis, B. mycoides, B. weihenstephanensis, B. pseudomycoides, and B. anthracis have similar characteristics, except that B. mycoides, B. pseudomycoides, and B. anthracis are nonmotile. The species concept within the B. cereus group (which includes the genetically very closely related entities mentioned above) is still under debate. Bacillus weihenstephanensis and B. mycoides are better adapted to growth at low temperature than the other members within the B. cereus group. If not mentioned specifically in the text, ‘B. cereus’ refers to the entire group (B. cereus sensu lato) except B. anthracis. Bacillus cereus forms colonies with typical appearance on agar media, generally with dull or frosted, grayish/whitish surface. Bacillus mycoides and B. pseudomycoides form rhizoid colonies. Widely used selective agar media for cultivation from food are mannitol egg yolk polymyxin agar (MYP) and polymyxin pyruvate egg yolk mannitol bromothymol blue agar (PEMBA). The detection of B. cereus is based on the absence of
Pathogens in Milk | Bacillus cereus
mannitol fermentation and positive egg yolk reaction (lecithinase). Bacillus cereus can also be enumerated on blood agar with polymyxin added. Colonies with clear zones of hemolysis and a very sharp margin are a useful diagnostic feature. Strains producing emetic toxin have a narrow zone of hemolysis or none at all. The zone does not enlarge upon further incubation, which is the case with nonemetic isolates. A chromogenic selective plating agar (BCM, B. cereus group plating medium) that stains colonies expressing phosphatidylinositol-specific phospholipase C turquoise blue has proven to be a good alternative to standard media. It is not possible to differentiate between the species of the B. cereus group based on colony morphology only. Physiology Bacillus cereus is a versatile microorganism with respect to growth substrates. Most strains produce proteases that can degrade casein and gelatin and enzymes for starch hydrolysis. Enzymes such as lecithinase and sphingomyelinase for degradation of phospholipids and lipase, with activity against triglycerides, can also be produced. Sweet curdling of milk is due to a protease and bitty cream due to phospholipase activity. Several carbohydrates are utilized, for example, glucose, fructose, trehalose, N-acetylglucosamine, and maltose. Others are utilized by only certain strains, for example, salicin, cellobiose, inositol, and mannose. A majority of strains do not grow on lactose. Mannitol is generally not used. Bacillus cereus is in general Voges–Proskauer (VP) positive and utilizes citrate, but not urea. Most strains can reduce nitrate. The minimum growth temperature differs among strains and is generally not lower than 5–6 C, although a few strains have been shown to grow at 4 C. Increased temperature from 6 to 9 C markedly affects the growth rate among psychrotrophic (psychrotolerant) isolates (Table 1). Some strains have temperature minima as high as 10–15 C. The optimum growth temperature is 30–37 C and the maximum growth temperature is 37–50 C. Strains producing emetic toxin do not grow below 10 C and are able to grow at 48 C. The psychrotolerant B. weihenstephanensis and B. mycoides are able to grow at 7 C or below and do not grow at 43 C. These species possess a cold-shock protein (CspA) that is detectable by PCR. Some strains that grow with a minimum temperature of 7 C and above do not have cspA and are classified as B. cereus (sensu stricto). The minimum pH for growth is 4.3–4.9 and the upper limit is 9.3. However, in the presence of organic acids, the minimum pH is higher, for example, pH 5.6 in 0.1 mol l 1 lactate. Although B. cereus grows best under aerobic conditions, anaerobic growth by fermentation of glucose or other carbohydrates or by anaerobic respiration with
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Table 1 Growth of Bacillus cereus (log cfu ml 1) in pasteurized milk at various storage temperatures Days of storage
6 C
7 C
8 C
9 C
1 2 3 4 5 6 7 8 9 10
-a 0.2 0.6 1.0 1.4
0.5 1.3 2.0 2.8 3.5
0.0 1.0 2.0 3.0 4.0 5.0 ND
1.0 2.4 3.7 5.0 ND ND ND
a Less than log 0. ND, not done. The values represent average data for pasteurized milk from 10 Swedish dairy plants in August. One milk package was collected from each plant and the milk from each package was divided aseptically into four aliquots, which were incubated in glass bottles in water baths with accurate temperature regulation (0.1 C). Original data from Christiansson A.
nitrate is possible. Bacillus cereus is able to grow in media with up to 7% NaCl if other conditions are optimum. Minimum water activity for growth is 0.92–0.95.
Spores Spores are formed on a variety of growth media under aerobic conditions, upon starvation. The presence of manganese and magnesium ions stimulates sporulation. Sporulation is a fairly lengthy and complicated process, occurring in the late logarithmic and early stationary phase of growth. Even under favorable conditions, sporulation may take up to 16–24 h to complete. Spores are never formed as a result of chilling if nutrients are available, that is, refrigeration of milk does not induce sporulation. For example, high levels of spores are not found in refrigerated pasteurized milk although the B. cereus counts may grow to 107 ml 1. On the other hand, milk diluted 1:50 with water is still a good growth medium, but nutrients will be depleted after growth and spores are formed abundantly, particularly if the milk is present in thin layers. This is relevant to the cleaning situation in a dairy plant. The spores may germinate and grow out to vegetative cells again under favorable conditions. Germination is much faster than sporulation. The germination rate is highly temperature dependent and may occur within much less than an hour at favorable temperature. In milk, it is stimulated by high-temperature, short-time (HTST) pasteurization, that is, heat treatment. The spores become activated and substances that stimulate germination may be formed as a result of heat treatment. Increased pasteurization temperature in the range of 72–85 C will lead to activation and germination of more spores. However,
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Pathogens in Milk | Bacillus cereus
initiation of growth in refrigerated milk will occur only after a lag phase of several days. The heat resistance of B. cereus spores is comparatively low. However, there is considerable variation in heat resistance among strains. Although not inactivated by HTST pasteurization, B. cereus spores are easily killed upon ultra-high temperature (UHT) treatment. Typical D-values at 100 C are in the range of 0.3–10 min. For comparison, D100 C for Bacillus stearothermophilus has been estimated to be approximately 3000 min. Strains producing emetic toxin produce spores that are among the most heat resistant within the B. cereus group. Generally, psychrotrophic strains tend to be less heat resistant than mesophilic strains, for example, with D-values of 2–9 min at 90 C. Vegetative cells are easily killed by pasteurization.
Milk-Borne Illness Bacillus cereus is a common contaminant in many food types, including milk, and a significant cause of foodborne illness worldwide. Bacillus cereus can cause diarrhea and/or vomiting when food (most often) containing large numbers of B. cereus is consumed. The symptoms are generally mild and transient, lasting no more than 24 h, generally without sequelae. Two types of outbreaks are known: diarrhealtype outbreak and emetic-type outbreak. Diarrheal-Type Outbreak The illness is characterized by a fairly long incubation period of 8–22 h. Watery diarrhea is very common, together with abdominal cramps, rectal spasms, and moderate nausea. Vomiting is rare. The duration of illness is generally 12–24 h. The delayed onset of symptoms indicates that illness is most likely due to growth of B. cereus in the small intestine, since the toxin(s) are very susceptible to inactivation by low pH and degradation by proteases. Preformed toxin in food will thus be inactivated in the stomach and ileum. Foods associated with diarrheal outbreaks generally contain high numbers of B. cereus, that is, 105–108 per gram food. Foods incriminated in diarrheal outbreaks include meat products, soups, vegetables, puddings, and milk products. Emetic-Type Outbreak The incubation period is short, that is, 0.5–5 h. The rapid onset of nausea and vomiting is due to a preformed toxin in the food. Abdominal cramps and diarrhea occur occasionally. Recovery is rapid, within 6–24 h. The level of B. cereus in incriminated food can vary between a few thousand and up to more than 5 1010 g 1, although it
is generally high. Fried and cooked rice are typical foods frequently involved, but milk-borne cases are also known. Toxins The nature of the enterotoxins produced by B. cereus has remained elusive for decades. However, during the last 15 years, the knowledge about these toxins has increased considerably. At least three types of enterotoxins capable of causing diarrhea have been identified. Two of these, hemolysin BL (HBL) and the nonhemolytic enterotoxin (NHE), are protein toxins consisting of three subunits each. All subunits are needed for full activity. Both toxins have been isolated from B. cereus strains involved in food poisoning. The third toxin, cytotoxin K (cytK), is a single protein toxin. CytK was involved in a rare foodborne outbreak, which caused the death of three persons, where the symptoms included bloody diarrhea. Additional toxins have been described but their involvement in foodborne illness is uncertain. The enterotoxin genes can be found in all species of the B. cereus group as judged by various PCR methods. Most strains are able to produce more than one toxin. Nhe genes are present in almost all strains, whereas Hbl and cytK genes can be found in approximately 50% of all strains, including strains in raw milk. However, cytK genes were not found in strains growing in pasteurized milk at refrigeration temperature. The toxin production potential (expression of the genes) varies considerably between strains and toxins. Strains involved in foodborne illness are generally more toxigenic than the average food or environmental isolate. They are often mesophilic, that is, they have a minimum growth temperature above 10 C, but food poisoning strains growing at or above 7 C are also known. However, strains belonging to B. weihenstephanensis and B. mycoides are generally less toxic than the other members of the B. cereus group. Bacillus thuringiensis and B. cereus (sensu stricto) have similar toxigenicity. From the point of food safety, there is therefore no need to differentiate between these two species as far as milk products are concerned. The toxins are heat labile and are considered to be inactivated by heating above 60 C for 5 min. PCR primers have been published for detection of all enterotoxin subunits. However, the mere presence of the genes is not sufficient to judge the pathogenicity of B. cereus. Monoclonal antibodies have been developed for all subunits of NHE and HBL and can be used for evaluation of the toxin production potential. Cytotoxicity tests using, for example, Vero cells or Caco cells can be employed to assess the overall cytotoxicity of strains. NHE seems to be the most cytotoxic toxin followed by HBL and cytK. The emetic toxin is a cyclic peptide, cereulide, which contains 12 modified amino acids and resembles the ionophore valinomycin. The molecular weight is
Pathogens in Milk | Bacillus cereus
1.2 kDa. The toxin is quite heat resistant and cannot be destroyed even by heating at 121 C for 1 h. Unlike the diarrheal toxins, the emetic toxin is encoded by a plasmid. The expression of the toxin varies strongly among strains and also depends on the composition of the food. The emetic toxin is a more serious health hazard than the diarrheal toxins and has been the cause of death in rare cases. Strains producing emetic toxin are rare in the dairy production chain (less than 1% of all isolates). Emetic strains do not grow in pasteurized milk that is kept refrigerated. A large number of cells (more than 5 log cfu g 1) are needed for toxin production. Recently, a real-time PCR method with PCR primers for detection of genes has been developed. Furthermore, a detection method for the toxin, based on the motility of boar sperm, has become available and can be used for foodstuffs. In addition to enterotoxins, several proteases, phospholipases, and hemolysins may have a role in the pathogenesis of B. cereus.
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may be at higher risk than the general population. Several factors may explain why milk-borne cases are few: Milk is generally kept at refrigeration temperature and growth of B. cereus is slow, thus the risk of exposure to high levels of bacteria is limited, although significant. In addition, sweet curdling often occurs when the product contains 106–107 B. cereus per ml of milk, with decreased risk of consumption. Psychrotrophic strains, in particular B. weihenstephanensis, will be enriched in pasteurized milk upon cold storage and these strains seem to be less toxigenic than B. cereus (sensu stricto). Psychrotrophic strains have a slower growth rate at the temperature of the human intestine (37 C; close to their maximum temperature of growth) than mesophilic strains, which grow faster. They are therefore less likely to cause food poisoning. However, temperature abuse will increase the risk of illness. Highly toxigenic (mesophilic) strains have been found in raw milk and they may be important in other products such as milk powder.
Outbreaks Related to Dairy Products Outbreaks related to dairy products are rare. Some cases are presented in Table 2. Both diarrheal and emetic symptoms have been recorded. Consumption of (refrigerated) raw milk is never associated with illness, due to the low numbers of B. cereus present. Growth to high numbers is always necessary in order to cause food poisoning. From the table, it seems that young people and elderly
Incidence in Dairy Products Vegetative B. cereus cells are found in raw milk at <10 ml 1 to a few hundred per ml. These cells are killed by pasteurization. Spores are found from <10 l 1 to a few thousand per liter milk, that is, at much lower levels. There is a marked seasonal variation in psychrotrophic
Table 2 Outbreaks of milk-borne illness caused by Bacillus cereus Product
Year
Country
People ill
Symptoms
Analytical data
Unpasteurized milk (heated and then kept at room temperature overnight) Cream, pasteurized
1972
Romania
221 school children
Diarrhea and abdominal cramps after 8–11 h
20 106 B. cereus per ml in milk. Bacillus cereus found in children’s feces
1975
England
Two 15-year-old girls
5 106 B. cereus per gram in cream
Milk, pasteurized
1981
Denmark
1-year-old boy
Vomiting after 8–10 h. One girl had diarrhea Vomiting after 1.5 h, no diarrhea
Milk powder, infant formula Human breast milk
1981
Chile
1981
India
35 neonate children Child, 6 months
Milk, pasteurized
1988
The Netherlands
42 elderly people
Ultra-high temperature milk (process failure)
1991
Japan
201 people
Diarrhea Diarrhea, occasional vomiting Nausea and vomiting after 2–14 h Vomiting 95%, average after 5 h Diarrhea 55%
2.6 106 B. cereus per ml in milk. Remaining milk was sweet curdled 1 h after consumption Bacillus cereus found in stool cultures Bacillus cereus found in breast milk 0.4 106 B. cereus per ml in milk Milk distributed at room temperature
Compiled from Christiansson A (1992) The toxicology of Bacillus cereus. International Dairy Federation Bulletin 275: 30–35; Van Netten P, van de Moosdijk A, van Hoensel P, Mossel DAA, and Perales I (1990) Psychrotrophic strains of Bacillus cereus producing enterotoxin. Journal of Applied Bacteriology 69: 73–79; Shinagawa K (1993) Serology and characterization of toxigenic Bacillus cereus. Netherlands Milk and Dairy Journal 47: 89–103; Cohen JV, Marmabio E, Lynch B, and Moreno A (1984) Bacillus cereus in food poisoning amid newborns. Revista Chilena de Pediatrica 55: 20–25.
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Pathogens in Milk | Bacillus cereus
spores, with the highest levels in summer and early autumn.
microbiological standards for powdered infant formula products, the limits for B. cereus are n = 5, c = 0, and m = 100 cfu g 1.
Milk and Cream The number of B. cereus in pasteurized milk and cream depends on the quality of the raw milk, the process hygiene at the dairy plant, the storage temperature of the product, and age of the product at the sampling time. Bacillus cereus grows slowly at temperatures below 6 C and will not be a quality problem, unless the sell-by date is set at several weeks. After 7 C storage for 7 days, the incidence of B. cereus can typically vary between 5 and 90% (winter and summer) at <10 to 105 ml 1 (including differences in dairy hygiene). When stored below 5 C, B. cereus is rarely detected unless there is a cleaning problem in the dairy plant. Fermented Milks and Cheese Bacillus cereus is rapidly inactivated in traditional yogurt manufacture as well as in the manufacture of fermented milk with lactococci. Some growth is possible within the first hours of fermentation. Multiplication in semihard cheese is likewise restricted to the first hours in the cheesemaking process. Inhibition occurs due to lactic acid at pH 5.6 but other inhibitors are also active. As the pH is lowered, vegetative B. cereus cells will die whereas spores that have not germinated may still be present. When present in these products, B. cereus seldom exceeds 100 g 1. Milk Powder Bacillus cereus is frequently found in low numbers in milk powder and infant formula. The frequency of isolation varies between 30 and 100% of samples with origin worldwide. Under certain circumstances, there may be some opportunity for growth of B. cereus in the evaporation process. Most samples contain <10 cfu g 1 but samples with more than 103 cfu g 1 have been found. These are due to hygienic problems in the factory or due to raw milk with a high degree of contamination. High levels of B. cereus in infant formula may constitute a health risk. Regulation (EC) 1771/2007 on microbiological criteria for foodstuffs in the European Union defines process hygiene criteria for presumptive B. cereus in dried infant formulae and dried dietary foods for special medical purposes intended for infants below 6 months of age. These are n (sample size) = 5, c (number of sample units giving values between m and M) = 1, acceptable limits (m) = 50 cfu g 1, and unsatisfactory limits (M) = 500 cfu g 1. In standard 1.6.1 of the Australia New Zealand Food Standards Code, which specifies
Source At the Farm Bacillus cereus is a ubiquitous microorganism. The spores are present in soil from 102 cfu g 1 and up to more than 105 cfu g 1. Consequently, food products of plant origin frequently contain B. cereus spores. Soil is an important source of contamination for milk. There is a marked seasonal variation in the spore content of raw milk, with higher levels during the pasture period, when the teats of the cow may be contaminated with soil. Dirty teats that are not cleansed before milking are an important contamination source, particularly during wet weather. Bacillus cereus is able to grow and sporulate on insufficiently cleaned milking equipment, so equipment may be a secondary source of contamination. Used bedding material and feed may also contain spores of B. cereus. In the Dairy Plant There has been considerable disagreement whether the occurrence of B. cereus in dairy products is caused by recontamination of milk at the dairy plant or by contamination at the farm. To some extent, this was due to the inability to detect the low levels of spores in raw milk, whereas B. cereus was easily detected in pasteurized milk after storage. It is now generally agreed that the original contamination occurs at the farm from soil. The seasonal variation in the occurrence of B. cereus in dairy products, kept at temperatures above 6 C, can to a large extent be explained by the increased contamination rate of the milk during the grazing period. However, additional contamination may occur from the dairy plant equipment. Since spores survive pasteurization, they will be present in the milk throughout the dairy process. Spores of B. cereus are very hydrophobic and will attach to surfaces of equipment, where they may germinate and form biofilms at sites that are difficult to clean. Several strain-typing methods (e.g., Random Amplified Polymorphic DNA-Polymerase Chain Rection analysis (RAPDPCR), Amplified Fragment Length Polymorphism analysis (AFLP), riboprinting, Repetitive element sequence polymorphism-PCR analysis (rep-PCR), and pulsed field gel electrophoresis (PFGE)) have recently been applied to strains of B. cereus. These methods demonstrate a high discriminatory power and could be helpful in finding contamination sites in dairy plants. There is a very strong diversity among strains of B. cereus in raw milk. Recontamination of milk by B. cereus has been demonstrated in silo tanks, pasteurizers, milk pipelines
Pathogens in Milk | Bacillus cereus
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The Dairy Plant
(a) (b) 96
100
(c)
At the dairy factory, cleaning and maintenance is essential. Attention must be given to proper concentrations of cleaning agents, sufficiently high cleaning temperature (at least 75 C for alkaline cleaning agents with 1–1.5% NaOH and at least 60–65 C for acid cleaning agents with 0.6–0.9% nitric acid), and proper flow rates during cleaning, since spores are difficult to remove and to kill. Bacillus cereus is considerably more resistant in a biofilm with spores than in a planktonic state. The spores are not killed by hot water disinfection, but sodium hypochlorite at pH 6–7 is effective. Regular replacement of gaskets and other rubber parts is important.
In Dairy Products
Figure 1 Examples of strain typing of Bacillus cereus isolates. (a) A milk stainless-steel pipeline with a very rough welded seam (arrow) was replaced at a dairy plant (to the left). Spores were recovered from the seam by rinsing with water and ultrasonication, collected by filtration, and then grown on blood agar plates (to the right). (b) All isolates showed the same RAPD fingerprint, which indicates that the welded seam was a source of recontamination. Similar fingerprints were found in pasteurized milk. (c) Examples of various RAPD fingerprints of strains from pasteurized milk. Lanes 1, 8, and 15 are molecular weight markers. A Christiansson, unpublished data.
with bad welding, and in packaging machines using RAPD-PCR (Figure 1). Automated ribotyping and repPCR have been used to identify surfaces of dairy equipment involved in recontamination of milk.
Control The Farm At the farm, measures to control B. cereus include careful teat cleansing before milking and proper cleaning and disinfection of the milking equipment. Since the teats become dirty with soil when the cows are outdoors during the grazing period, it is essential that they are clean before attaching the teat cups. During the indoor season, high levels of B. cereus spores may be found in used bedding material, if not replaced daily, and may contaminate the teats. The best cleansing routine includes the use of one moistened cloth per cow, followed by a dry paper towel. In addition, the milking equipment must be kept clean by careful cleaning after milking. Teat liners and other rubber material must be replaced regularly since aged rubber with cracks can harbor milk residues where B. cereus can propagate and sporulate.
The best control measure for B. cereus in pasteurized milk and cream is to keep a low storage temperature in the whole chain from the dairy plant to the customer. Below 5–6 C, growth of most strains of B. cereus is insignificant. If the temperature is higher, the sell-by date must be shortened. Suitable time/temperature combinations may be found by storage tests. Seasonal variation, occurrence of recontamination at the dairy plant as well as possible moderate temperature abuse by the customer must be taken into consideration when choosing the recommended last consumption date of the products. Milk powder is microbiologically stable and no growth of B. cereus can occur in the powder, although occurrence of contamination with B. cereus is frequent. However, milk powder is frequently used in infant formulae and in infant foods. When such powders are reconstituted, it is important that the product is consumed shortly after preparation unless it is not cooled to below 8 C. Spores of B. cereus are able to germinate rapidly at the reconstitution temperature and will grow rapidly if the product is kept at room temperature. Young children may be more susceptible to toxins that may be produced than adults.
See also: Analytical Methods: DNA-Based Assays. Biofilm Formation. Dehydrated Dairy Products: Infant Formulae; Milk Powder: Types and Manufacture. Heat Treatment of Milk: Thermization of Milk. Liquid Milk Products: Liquid Milk Products: Pasteurized Milk; Liquid Milk Products: UHT Sterilized Milks; Pasteurization of Liquid Milk Products: Principles, Public Health Aspects. Microorganisms Associated with Milk. Milking and Handling of Raw Milk: Effect of Storage and Transport on Milk Quality; Milking Hygiene. Plant and Equipment: In-place Cleaning. Psychrotrophic Bacteria: Other Psychrotrophs.
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Pathogens in Milk | Bacillus cereus
Further Reading Anonymous (2005) Opinion of the scientific panel on biological hazards on Bacillus cereus and other Bacillus spp in foodstuffs. EFSA Journal 175: 1–48. Becker H, Schaller G, von Wiese W, and Terplan G (1994) Bacillus cereus in infant foods and dried milk products. International Journal of Food Microbiology 23: 1–15. Christiansson A (1992) The toxicology of Bacillus cereus. International Dairy Federation Bulletin 275: 30–35. Cohen JV, Marmabio E, Lynch B, and Moreno A (1984) Bacillus cereus in food poisoning amid newborns. Revista Chilena de Pediatrica 55: 20–25. Fricker M, Reissbrodt R, and Ehling-Schultz M (2008) Evaluation of standard and new chromogenic selective plating media for isolation and identification of Bacillus cereus. International Journal of Food Microbiology 121: 27–34. Granum PE (2007) Bacillus cereus. In: Doyle MP and Beuchat LR (eds.) Food Microbiology: Fundamentals and Frontiers, 3rd edn., pp. 445–455. Washington, DC: ASM Press. Guinebretie`re M-H, Thompson FL, Sorokin A, et al. (2008) Ecological diversification in the Bacillus cereus group. Environmental Microbiology 10: 851–865. IDF (1992) Bacillus cereus in milk and milk products. Bulletin of the International Dairy Federation No. 275. Brussels:IDF.
Langeveld LPM and Cuperus F (1980) The relation between temperature and growth rate in pasteurized milk of different types of bacteria which are important to the deterioration of that milk. Netherlands Milk and Dairy Journal 34: 106–125. Notermans S, Dufrenne J, Teunis P, Beaumer R, te Giffel M, and Peeters Weem P (1997) A risk assessment study of Bacillus cereus present in pasteurized milk. Food Microbiology 14: 143–151. Shinagawa K (1993) Serology and characterization of toxigenic Bacillus cereus. Netherlands Milk and Dairy Journal 47: 89–103. Stenfors Arnesen LP, Fagerlund A, and Granum PE (2008) From soil to gut: Bacillus cereus and its food poisoning toxin. FEMS Microbiology Reviews 32: 579–606. Svensson B, Montha´n A, Guinebretie`re M-H, Nguyen-The´ C, and Christiansson A (2007) Toxin production potential and the detection of toxin genes among strains of the Bacillus cereus group isolated along the dairy production line. International Dairy Journal 17: 1201–1208. Van Netten P, van de Moosdijk A, van Hoensel P, Mossel DAA, and Perales I (1990) Psychrotrophic strains of Bacillus cereus producing enterotoxin. Journal of Applied Bacteriology 69: 73–79. Wijnands LM, Dufrenne JB, Zwietering MH, and van Leusden FM (2006) Spores from mesophilic Bacillus cereus strains germinate better and grow faster in simulated gastro-intestinal conditions than spores from psychrotrophic strains. International Journal of Food Microbiology 112: 120–128.
Bacillus cereus A Christiansson, Swedish Dairy Association, Lund, Sweden ª 2011 Elsevier Ltd. All rights reserved.
Introduction Bacillus cereus is an aerobic spore-forming bacterium, whose spores are commonly present at low levels in raw milk. In the 1960s and earlier, B. cereus was clearly a quality problem, due to coagulation (sweet curdling) of pasteurized milk and formation of flakes in cream when added to coffee (bitty cream). This was due to poorly cleansed equipment (e.g., milk cans) at the farm and in dairy factories and a lack of adequate refrigeration. Nowadays, these problems are rarely seen in countries where milk is kept at temperatures below 6 C. However, when pasteurized milk is stored at higher temperatures, B. cereus may still be a limiting factor for the keeping quality. Bacillus cereus can produce several enterotoxins causing diarrhea and vomiting. There are few dairy-related cases, but milk and cream have been incriminated in both types of illnesses.
Characteristics Morphology and Cultivation Bacillus cereus is a Gram-positive, rod-shaped, motile bacterium with peritrichous flagella. The cells tend to grow in chains but may occur singly as well. The length
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of the bacterium varies between 3 and 5 mm and the diameter is more than 1 mm. Spores are oval or cylindrical, located centrally or paracentrally/subterminally, and do not distend the cell. A typical trait of the B. cereus group is the presence of storage granules of poly- hydroxybutyrate in the cytoplasm. These are easily seen by phase contrast microscopy. Bacillus thuringiensis, B. mycoides, B. weihenstephanensis, B. pseudomycoides, and B. anthracis have similar characteristics, except that B. mycoides, B. pseudomycoides, and B. anthracis are nonmotile. The species concept within the B. cereus group (which includes the genetically very closely related entities mentioned above) is still under debate. Bacillus weihenstephanensis and B. mycoides are better adapted to growth at low temperature than the other members within the B. cereus group. If not mentioned specifically in the text, ‘B. cereus’ refers to the entire group (B. cereus sensu lato) except B. anthracis. Bacillus cereus forms colonies with typical appearance on agar media, generally with dull or frosted, grayish/whitish surface. Bacillus mycoides and B. pseudomycoides form rhizoid colonies. Widely used selective agar media for cultivation from food are mannitol egg yolk polymyxin agar (MYP) and polymyxin pyruvate egg yolk mannitol bromothymol blue agar (PEMBA). The detection of B. cereus is based on the absence of
Pathogens in Milk | Bacillus cereus
mannitol fermentation and positive egg yolk reaction (lecithinase). Bacillus cereus can also be enumerated on blood agar with polymyxin added. Colonies with clear zones of hemolysis and a very sharp margin are a useful diagnostic feature. Strains producing emetic toxin have a narrow zone of hemolysis or none at all. The zone does not enlarge upon further incubation, which is the case with nonemetic isolates. A chromogenic selective plating agar (BCM, B. cereus group plating medium) that stains colonies expressing phosphatidylinositol-specific phospholipase C turquoise blue has proven to be a good alternative to standard media. It is not possible to differentiate between the species of the B. cereus group based on colony morphology only. Physiology Bacillus cereus is a versatile microorganism with respect to growth substrates. Most strains produce proteases that can degrade casein and gelatin and enzymes for starch hydrolysis. Enzymes such as lecithinase and sphingomyelinase for degradation of phospholipids and lipase, with activity against triglycerides, can also be produced. Sweet curdling of milk is due to a protease and bitty cream due to phospholipase activity. Several carbohydrates are utilized, for example, glucose, fructose, trehalose, N-acetylglucosamine, and maltose. Others are utilized by only certain strains, for example, salicin, cellobiose, inositol, and mannose. A majority of strains do not grow on lactose. Mannitol is generally not used. Bacillus cereus is in general Voges–Proskauer (VP) positive and utilizes citrate, but not urea. Most strains can reduce nitrate. The minimum growth temperature differs among strains and is generally not lower than 5–6 C, although a few strains have been shown to grow at 4 C. Increased temperature from 6 to 9 C markedly affects the growth rate among psychrotrophic (psychrotolerant) isolates (Table 1). Some strains have temperature minima as high as 10–15 C. The optimum growth temperature is 30–37 C and the maximum growth temperature is 37–50 C. Strains producing emetic toxin do not grow below 10 C and are able to grow at 48 C. The psychrotolerant B. weihenstephanensis and B. mycoides are able to grow at 7 C or below and do not grow at 43 C. These species possess a cold-shock protein (CspA) that is detectable by PCR. Some strains that grow with a minimum temperature of 7 C and above do not have cspA and are classified as B. cereus (sensu stricto). The minimum pH for growth is 4.3–4.9 and the upper limit is 9.3. However, in the presence of organic acids, the minimum pH is higher, for example, pH 5.6 in 0.1 mol l 1 lactate. Although B. cereus grows best under aerobic conditions, anaerobic growth by fermentation of glucose or other carbohydrates or by anaerobic respiration with
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Table 1 Growth of Bacillus cereus (log cfu ml 1) in pasteurized milk at various storage temperatures Days of storage
6 C
7 C
8 C
9 C
1 2 3 4 5 6 7 8 9 10
-a 0.2 0.6 1.0 1.4
0.5 1.3 2.0 2.8 3.5
0.0 1.0 2.0 3.0 4.0 5.0 ND
1.0 2.4 3.7 5.0 ND ND ND
a Less than log 0. ND, not done. The values represent average data for pasteurized milk from 10 Swedish dairy plants in August. One milk package was collected from each plant and the milk from each package was divided aseptically into four aliquots, which were incubated in glass bottles in water baths with accurate temperature regulation (0.1 C). Original data from Christiansson A.
nitrate is possible. Bacillus cereus is able to grow in media with up to 7% NaCl if other conditions are optimum. Minimum water activity for growth is 0.92–0.95.
Spores Spores are formed on a variety of growth media under aerobic conditions, upon starvation. The presence of manganese and magnesium ions stimulates sporulation. Sporulation is a fairly lengthy and complicated process, occurring in the late logarithmic and early stationary phase of growth. Even under favorable conditions, sporulation may take up to 16–24 h to complete. Spores are never formed as a result of chilling if nutrients are available, that is, refrigeration of milk does not induce sporulation. For example, high levels of spores are not found in refrigerated pasteurized milk although the B. cereus counts may grow to 107 ml 1. On the other hand, milk diluted 1:50 with water is still a good growth medium, but nutrients will be depleted after growth and spores are formed abundantly, particularly if the milk is present in thin layers. This is relevant to the cleaning situation in a dairy plant. The spores may germinate and grow out to vegetative cells again under favorable conditions. Germination is much faster than sporulation. The germination rate is highly temperature dependent and may occur within much less than an hour at favorable temperature. In milk, it is stimulated by high-temperature, short-time (HTST) pasteurization, that is, heat treatment. The spores become activated and substances that stimulate germination may be formed as a result of heat treatment. Increased pasteurization temperature in the range of 72–85 C will lead to activation and germination of more spores. However,
26
Pathogens in Milk | Bacillus cereus
initiation of growth in refrigerated milk will occur only after a lag phase of several days. The heat resistance of B. cereus spores is comparatively low. However, there is considerable variation in heat resistance among strains. Although not inactivated by HTST pasteurization, B. cereus spores are easily killed upon ultra-high temperature (UHT) treatment. Typical D-values at 100 C are in the range of 0.3–10 min. For comparison, D100 C for Bacillus stearothermophilus has been estimated to be approximately 3000 min. Strains producing emetic toxin produce spores that are among the most heat resistant within the B. cereus group. Generally, psychrotrophic strains tend to be less heat resistant than mesophilic strains, for example, with D-values of 2–9 min at 90 C. Vegetative cells are easily killed by pasteurization.
Milk-Borne Illness Bacillus cereus is a common contaminant in many food types, including milk, and a significant cause of foodborne illness worldwide. Bacillus cereus can cause diarrhea and/or vomiting when food (most often) containing large numbers of B. cereus is consumed. The symptoms are generally mild and transient, lasting no more than 24 h, generally without sequelae. Two types of outbreaks are known: diarrhealtype outbreak and emetic-type outbreak. Diarrheal-Type Outbreak The illness is characterized by a fairly long incubation period of 8–22 h. Watery diarrhea is very common, together with abdominal cramps, rectal spasms, and moderate nausea. Vomiting is rare. The duration of illness is generally 12–24 h. The delayed onset of symptoms indicates that illness is most likely due to growth of B. cereus in the small intestine, since the toxin(s) are very susceptible to inactivation by low pH and degradation by proteases. Preformed toxin in food will thus be inactivated in the stomach and ileum. Foods associated with diarrheal outbreaks generally contain high numbers of B. cereus, that is, 105–108 per gram food. Foods incriminated in diarrheal outbreaks include meat products, soups, vegetables, puddings, and milk products. Emetic-Type Outbreak The incubation period is short, that is, 0.5–5 h. The rapid onset of nausea and vomiting is due to a preformed toxin in the food. Abdominal cramps and diarrhea occur occasionally. Recovery is rapid, within 6–24 h. The level of B. cereus in incriminated food can vary between a few thousand and up to more than 5 1010 g 1, although it
is generally high. Fried and cooked rice are typical foods frequently involved, but milk-borne cases are also known. Toxins The nature of the enterotoxins produced by B. cereus has remained elusive for decades. However, during the last 15 years, the knowledge about these toxins has increased considerably. At least three types of enterotoxins capable of causing diarrhea have been identified. Two of these, hemolysin BL (HBL) and the nonhemolytic enterotoxin (NHE), are protein toxins consisting of three subunits each. All subunits are needed for full activity. Both toxins have been isolated from B. cereus strains involved in food poisoning. The third toxin, cytotoxin K (cytK), is a single protein toxin. CytK was involved in a rare foodborne outbreak, which caused the death of three persons, where the symptoms included bloody diarrhea. Additional toxins have been described but their involvement in foodborne illness is uncertain. The enterotoxin genes can be found in all species of the B. cereus group as judged by various PCR methods. Most strains are able to produce more than one toxin. Nhe genes are present in almost all strains, whereas Hbl and cytK genes can be found in approximately 50% of all strains, including strains in raw milk. However, cytK genes were not found in strains growing in pasteurized milk at refrigeration temperature. The toxin production potential (expression of the genes) varies considerably between strains and toxins. Strains involved in foodborne illness are generally more toxigenic than the average food or environmental isolate. They are often mesophilic, that is, they have a minimum growth temperature above 10 C, but food poisoning strains growing at or above 7 C are also known. However, strains belonging to B. weihenstephanensis and B. mycoides are generally less toxic than the other members of the B. cereus group. Bacillus thuringiensis and B. cereus (sensu stricto) have similar toxigenicity. From the point of food safety, there is therefore no need to differentiate between these two species as far as milk products are concerned. The toxins are heat labile and are considered to be inactivated by heating above 60 C for 5 min. PCR primers have been published for detection of all enterotoxin subunits. However, the mere presence of the genes is not sufficient to judge the pathogenicity of B. cereus. Monoclonal antibodies have been developed for all subunits of NHE and HBL and can be used for evaluation of the toxin production potential. Cytotoxicity tests using, for example, Vero cells or Caco cells can be employed to assess the overall cytotoxicity of strains. NHE seems to be the most cytotoxic toxin followed by HBL and cytK. The emetic toxin is a cyclic peptide, cereulide, which contains 12 modified amino acids and resembles the ionophore valinomycin. The molecular weight is
Pathogens in Milk | Bacillus cereus
1.2 kDa. The toxin is quite heat resistant and cannot be destroyed even by heating at 121 C for 1 h. Unlike the diarrheal toxins, the emetic toxin is encoded by a plasmid. The expression of the toxin varies strongly among strains and also depends on the composition of the food. The emetic toxin is a more serious health hazard than the diarrheal toxins and has been the cause of death in rare cases. Strains producing emetic toxin are rare in the dairy production chain (less than 1% of all isolates). Emetic strains do not grow in pasteurized milk that is kept refrigerated. A large number of cells (more than 5 log cfu g 1) are needed for toxin production. Recently, a real-time PCR method with PCR primers for detection of genes has been developed. Furthermore, a detection method for the toxin, based on the motility of boar sperm, has become available and can be used for foodstuffs. In addition to enterotoxins, several proteases, phospholipases, and hemolysins may have a role in the pathogenesis of B. cereus.
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may be at higher risk than the general population. Several factors may explain why milk-borne cases are few: Milk is generally kept at refrigeration temperature and growth of B. cereus is slow, thus the risk of exposure to high levels of bacteria is limited, although significant. In addition, sweet curdling often occurs when the product contains 106–107 B. cereus per ml of milk, with decreased risk of consumption. Psychrotrophic strains, in particular B. weihenstephanensis, will be enriched in pasteurized milk upon cold storage and these strains seem to be less toxigenic than B. cereus (sensu stricto). Psychrotrophic strains have a slower growth rate at the temperature of the human intestine (37 C; close to their maximum temperature of growth) than mesophilic strains, which grow faster. They are therefore less likely to cause food poisoning. However, temperature abuse will increase the risk of illness. Highly toxigenic (mesophilic) strains have been found in raw milk and they may be important in other products such as milk powder.
Outbreaks Related to Dairy Products Outbreaks related to dairy products are rare. Some cases are presented in Table 2. Both diarrheal and emetic symptoms have been recorded. Consumption of (refrigerated) raw milk is never associated with illness, due to the low numbers of B. cereus present. Growth to high numbers is always necessary in order to cause food poisoning. From the table, it seems that young people and elderly
Incidence in Dairy Products Vegetative B. cereus cells are found in raw milk at <10 ml 1 to a few hundred per ml. These cells are killed by pasteurization. Spores are found from <10 l 1 to a few thousand per liter milk, that is, at much lower levels. There is a marked seasonal variation in psychrotrophic
Table 2 Outbreaks of milk-borne illness caused by Bacillus cereus Product
Year
Country
People ill
Symptoms
Analytical data
Unpasteurized milk (heated and then kept at room temperature overnight) Cream, pasteurized
1972
Romania
221 school children
Diarrhea and abdominal cramps after 8–11 h
20 106 B. cereus per ml in milk. Bacillus cereus found in children’s feces
1975
England
Two 15-year-old girls
5 106 B. cereus per gram in cream
Milk, pasteurized
1981
Denmark
1-year-old boy
Vomiting after 8–10 h. One girl had diarrhea Vomiting after 1.5 h, no diarrhea
Milk powder, infant formula Human breast milk
1981
Chile
1981
India
35 neonate children Child, 6 months
Milk, pasteurized
1988
The Netherlands
42 elderly people
Ultra-high temperature milk (process failure)
1991
Japan
201 people
Diarrhea Diarrhea, occasional vomiting Nausea and vomiting after 2–14 h Vomiting 95%, average after 5 h Diarrhea 55%
2.6 106 B. cereus per ml in milk. Remaining milk was sweet curdled 1 h after consumption Bacillus cereus found in stool cultures Bacillus cereus found in breast milk 0.4 106 B. cereus per ml in milk Milk distributed at room temperature
Compiled from Christiansson A (1992) The toxicology of Bacillus cereus. International Dairy Federation Bulletin 275: 30–35; Van Netten P, van de Moosdijk A, van Hoensel P, Mossel DAA, and Perales I (1990) Psychrotrophic strains of Bacillus cereus producing enterotoxin. Journal of Applied Bacteriology 69: 73–79; Shinagawa K (1993) Serology and characterization of toxigenic Bacillus cereus. Netherlands Milk and Dairy Journal 47: 89–103; Cohen JV, Marmabio E, Lynch B, and Moreno A (1984) Bacillus cereus in food poisoning amid newborns. Revista Chilena de Pediatrica 55: 20–25.
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Pathogens in Milk | Bacillus cereus
spores, with the highest levels in summer and early autumn.
microbiological standards for powdered infant formula products, the limits for B. cereus are n = 5, c = 0, and m = 100 cfu g 1.
Milk and Cream The number of B. cereus in pasteurized milk and cream depends on the quality of the raw milk, the process hygiene at the dairy plant, the storage temperature of the product, and age of the product at the sampling time. Bacillus cereus grows slowly at temperatures below 6 C and will not be a quality problem, unless the sell-by date is set at several weeks. After 7 C storage for 7 days, the incidence of B. cereus can typically vary between 5 and 90% (winter and summer) at <10 to 105 ml 1 (including differences in dairy hygiene). When stored below 5 C, B. cereus is rarely detected unless there is a cleaning problem in the dairy plant. Fermented Milks and Cheese Bacillus cereus is rapidly inactivated in traditional yogurt manufacture as well as in the manufacture of fermented milk with lactococci. Some growth is possible within the first hours of fermentation. Multiplication in semihard cheese is likewise restricted to the first hours in the cheesemaking process. Inhibition occurs due to lactic acid at pH 5.6 but other inhibitors are also active. As the pH is lowered, vegetative B. cereus cells will die whereas spores that have not germinated may still be present. When present in these products, B. cereus seldom exceeds 100 g 1. Milk Powder Bacillus cereus is frequently found in low numbers in milk powder and infant formula. The frequency of isolation varies between 30 and 100% of samples with origin worldwide. Under certain circumstances, there may be some opportunity for growth of B. cereus in the evaporation process. Most samples contain <10 cfu g 1 but samples with more than 103 cfu g 1 have been found. These are due to hygienic problems in the factory or due to raw milk with a high degree of contamination. High levels of B. cereus in infant formula may constitute a health risk. Regulation (EC) 1771/2007 on microbiological criteria for foodstuffs in the European Union defines process hygiene criteria for presumptive B. cereus in dried infant formulae and dried dietary foods for special medical purposes intended for infants below 6 months of age. These are n (sample size) = 5, c (number of sample units giving values between m and M) = 1, acceptable limits (m) = 50 cfu g 1, and unsatisfactory limits (M) = 500 cfu g 1. In standard 1.6.1 of the Australia New Zealand Food Standards Code, which specifies
Source At the Farm Bacillus cereus is a ubiquitous microorganism. The spores are present in soil from 102 cfu g 1 and up to more than 105 cfu g 1. Consequently, food products of plant origin frequently contain B. cereus spores. Soil is an important source of contamination for milk. There is a marked seasonal variation in the spore content of raw milk, with higher levels during the pasture period, when the teats of the cow may be contaminated with soil. Dirty teats that are not cleansed before milking are an important contamination source, particularly during wet weather. Bacillus cereus is able to grow and sporulate on insufficiently cleaned milking equipment, so equipment may be a secondary source of contamination. Used bedding material and feed may also contain spores of B. cereus. In the Dairy Plant There has been considerable disagreement whether the occurrence of B. cereus in dairy products is caused by recontamination of milk at the dairy plant or by contamination at the farm. To some extent, this was due to the inability to detect the low levels of spores in raw milk, whereas B. cereus was easily detected in pasteurized milk after storage. It is now generally agreed that the original contamination occurs at the farm from soil. The seasonal variation in the occurrence of B. cereus in dairy products, kept at temperatures above 6 C, can to a large extent be explained by the increased contamination rate of the milk during the grazing period. However, additional contamination may occur from the dairy plant equipment. Since spores survive pasteurization, they will be present in the milk throughout the dairy process. Spores of B. cereus are very hydrophobic and will attach to surfaces of equipment, where they may germinate and form biofilms at sites that are difficult to clean. Several strain-typing methods (e.g., Random Amplified Polymorphic DNA-Polymerase Chain Rection analysis (RAPDPCR), Amplified Fragment Length Polymorphism analysis (AFLP), riboprinting, Repetitive element sequence polymorphism-PCR analysis (rep-PCR), and pulsed field gel electrophoresis (PFGE)) have recently been applied to strains of B. cereus. These methods demonstrate a high discriminatory power and could be helpful in finding contamination sites in dairy plants. There is a very strong diversity among strains of B. cereus in raw milk. Recontamination of milk by B. cereus has been demonstrated in silo tanks, pasteurizers, milk pipelines
Pathogens in Milk | Bacillus cereus
29
The Dairy Plant
(a) (b) 96
100
(c)
At the dairy factory, cleaning and maintenance is essential. Attention must be given to proper concentrations of cleaning agents, sufficiently high cleaning temperature (at least 75 C for alkaline cleaning agents with 1–1.5% NaOH and at least 60–65 C for acid cleaning agents with 0.6–0.9% nitric acid), and proper flow rates during cleaning, since spores are difficult to remove and to kill. Bacillus cereus is considerably more resistant in a biofilm with spores than in a planktonic state. The spores are not killed by hot water disinfection, but sodium hypochlorite at pH 6–7 is effective. Regular replacement of gaskets and other rubber parts is important.
In Dairy Products
Figure 1 Examples of strain typing of Bacillus cereus isolates. (a) A milk stainless-steel pipeline with a very rough welded seam (arrow) was replaced at a dairy plant (to the left). Spores were recovered from the seam by rinsing with water and ultrasonication, collected by filtration, and then grown on blood agar plates (to the right). (b) All isolates showed the same RAPD fingerprint, which indicates that the welded seam was a source of recontamination. Similar fingerprints were found in pasteurized milk. (c) Examples of various RAPD fingerprints of strains from pasteurized milk. Lanes 1, 8, and 15 are molecular weight markers. A Christiansson, unpublished data.
with bad welding, and in packaging machines using RAPD-PCR (Figure 1). Automated ribotyping and repPCR have been used to identify surfaces of dairy equipment involved in recontamination of milk.
Control The Farm At the farm, measures to control B. cereus include careful teat cleansing before milking and proper cleaning and disinfection of the milking equipment. Since the teats become dirty with soil when the cows are outdoors during the grazing period, it is essential that they are clean before attaching the teat cups. During the indoor season, high levels of B. cereus spores may be found in used bedding material, if not replaced daily, and may contaminate the teats. The best cleansing routine includes the use of one moistened cloth per cow, followed by a dry paper towel. In addition, the milking equipment must be kept clean by careful cleaning after milking. Teat liners and other rubber material must be replaced regularly since aged rubber with cracks can harbor milk residues where B. cereus can propagate and sporulate.
The best control measure for B. cereus in pasteurized milk and cream is to keep a low storage temperature in the whole chain from the dairy plant to the customer. Below 5–6 C, growth of most strains of B. cereus is insignificant. If the temperature is higher, the sell-by date must be shortened. Suitable time/temperature combinations may be found by storage tests. Seasonal variation, occurrence of recontamination at the dairy plant as well as possible moderate temperature abuse by the customer must be taken into consideration when choosing the recommended last consumption date of the products. Milk powder is microbiologically stable and no growth of B. cereus can occur in the powder, although occurrence of contamination with B. cereus is frequent. However, milk powder is frequently used in infant formulae and in infant foods. When such powders are reconstituted, it is important that the product is consumed shortly after preparation unless it is not cooled to below 8 C. Spores of B. cereus are able to germinate rapidly at the reconstitution temperature and will grow rapidly if the product is kept at room temperature. Young children may be more susceptible to toxins that may be produced than adults.
See also: Analytical Methods: DNA-Based Assays. Biofilm Formation. Dehydrated Dairy Products: Infant Formulae; Milk Powder: Types and Manufacture. Heat Treatment of Milk: Thermization of Milk. Liquid Milk Products: Liquid Milk Products: Pasteurized Milk; Liquid Milk Products: UHT Sterilized Milks; Pasteurization of Liquid Milk Products: Principles, Public Health Aspects. Microorganisms Associated with Milk. Milking and Handling of Raw Milk: Effect of Storage and Transport on Milk Quality; Milking Hygiene. Plant and Equipment: In-place Cleaning. Psychrotrophic Bacteria: Other Psychrotrophs.
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Pathogens in Milk | Bacillus cereus
Further Reading Anonymous (2005) Opinion of the scientific panel on biological hazards on Bacillus cereus and other Bacillus spp in foodstuffs. EFSA Journal 175: 1–48. Becker H, Schaller G, von Wiese W, and Terplan G (1994) Bacillus cereus in infant foods and dried milk products. International Journal of Food Microbiology 23: 1–15. Christiansson A (1992) The toxicology of Bacillus cereus. International Dairy Federation Bulletin 275: 30–35. Cohen JV, Marmabio E, Lynch B, and Moreno A (1984) Bacillus cereus in food poisoning amid newborns. Revista Chilena de Pediatrica 55: 20–25. Fricker M, Reissbrodt R, and Ehling-Schultz M (2008) Evaluation of standard and new chromogenic selective plating media for isolation and identification of Bacillus cereus. International Journal of Food Microbiology 121: 27–34. Granum PE (2007) Bacillus cereus. In: Doyle MP and Beuchat LR (eds.) Food Microbiology: Fundamentals and Frontiers, 3rd edn., pp. 445–455. Washington, DC: ASM Press. Guinebretie`re M-H, Thompson FL, Sorokin A, et al. (2008) Ecological diversification in the Bacillus cereus group. Environmental Microbiology 10: 851–865. IDF (1992) Bacillus cereus in milk and milk products. Bulletin of the International Dairy Federation No. 275. Brussels:IDF.
Langeveld LPM and Cuperus F (1980) The relation between temperature and growth rate in pasteurized milk of different types of bacteria which are important to the deterioration of that milk. Netherlands Milk and Dairy Journal 34: 106–125. Notermans S, Dufrenne J, Teunis P, Beaumer R, te Giffel M, and Peeters Weem P (1997) A risk assessment study of Bacillus cereus present in pasteurized milk. Food Microbiology 14: 143–151. Shinagawa K (1993) Serology and characterization of toxigenic Bacillus cereus. Netherlands Milk and Dairy Journal 47: 89–103. Stenfors Arnesen LP, Fagerlund A, and Granum PE (2008) From soil to gut: Bacillus cereus and its food poisoning toxin. FEMS Microbiology Reviews 32: 579–606. Svensson B, Montha´n A, Guinebretie`re M-H, Nguyen-The´ C, and Christiansson A (2007) Toxin production potential and the detection of toxin genes among strains of the Bacillus cereus group isolated along the dairy production line. International Dairy Journal 17: 1201–1208. Van Netten P, van de Moosdijk A, van Hoensel P, Mossel DAA, and Perales I (1990) Psychrotrophic strains of Bacillus cereus producing enterotoxin. Journal of Applied Bacteriology 69: 73–79. Wijnands LM, Dufrenne JB, Zwietering MH, and van Leusden FM (2006) Spores from mesophilic Bacillus cereus strains germinate better and grow faster in simulated gastro-intestinal conditions than spores from psychrotrophic strains. International Journal of Food Microbiology 112: 120–128.