Pathogenicity, Toxin Production, Control and Detection of Bacillus cereus

Abstract

Bacillus cereus is a toxin-producing, endospore-forming, facultative bacterium ubiquitous in the environment. It has been associated with numerous foodborne illness outbreaks and is found in a variety of foods including grains, produce and processed foods. When present in high numbers, B. cereus produces toxins leading to foodborne illness. Although disease is usually self-limiting and resolves with a short time, illness can result in complications. Moreover, B. cereus is resistant to many antimicrobials which can make treatment difficult in scenarios where more extensive treatment is required. Current control methods are limited, and detection of this pathogen in food is often difficult due to its genetic similarity to Bacillus anthracis and Bacillus thuringiensis. Given this, more research is required to identify better process controls to reduce contamination of food with this ubiquitous organism, and develop better methods for detection.

1. Introduction

Bacillus cereus (sensu lato) is a member of the Bacillus genus which currently includes Bacillus mycoides, Bacillus pseudomycoides, Bacillus weihenstephanensis, Bacillus anthracis, Bacillus thuringiensis, B. cereus sensu stricto (usually called B. cereus), B. cytotoxicus and B. toyonensis. Newly identified species have been isolated from marine sediments and other environments and added to this group: B. paranthracis, B. pacificus, B. tropicus, B. albus, B. mobilis, B. luti, B. proteolyticus, B. nitratireducens and B. paramycoides, with some other strains that under evaluation [1]. B. cereus is very closely related to B. anthracis and B. thuringiensis, and it has been proposed that B. anthracis is a lineage of B. cereus [2]. There is significant research interest in B. cereus and B. anthracis due to the pathogenicity of both species and there have been reported cases of B. cereus causing anthrax-like diseases [3, 4].

Bacillus cereus is a toxin-producing, endospore-forming, gram-positive, and facultative bacterium which is ubiquitous in the environment, and often found associated with soil, growing plants, and in the intestinal tract of animals [5, 6, 7, 8]. Its ability to form spores allows this organism to persist in the environment regardless of environmental conditions that are not favorable to growth [9]. Regarding food products, this bacterium has been isolated from air in a cow shed [10], rice, spices, milk and dairy products, vegetables, meat, farinaceous foods, desserts, and cakes [9].

This organism is a major problem to the food industry because it is difficult to eliminate from food products due to its ability to survive in different environments despite environmental stresses which would normally inhibit bacterial survival [11], spore resistance to heat, dehydration, acid, and other physical stresses, and an inability to be destroyed through pasteurization and other sanitation procedures [5, 12]. It is estimated that (1.4–12%) of all food poisoning in the world is related to B. cereus [13]. There are two types of food poisoning, emetic syndrome and diarrheal syndrome, which have been described. However, this organism is known to cause a variety of other clinical infections such as local infections, such as in burns, traumatic wounds, and the eye, bacteremia and septicemia, central nervous system infection (meningitis, abscesses, and shunt-associated infections), respiratory infections, endocarditis and pericarditis, endophthalmitis, periodontitis, and osteomyelitis [12, 14, 15].

Emetic syndrome is caused by a cereulide which is a heat-stable peptide toxin. Diarrheal syndrome is caused by a host of other enterotoxins produced by the organism including nonhemolytic enterotoxin (NHE), enterotoxin FM (EntFM), Hemolysin BL (HBL), and cytotoxin K (Cyt K) [14, 16]. Hemolysin BL has consisted of three different components; one bending component B (encoded by hblA gene) and two lytic components L1 and L2 (encoded by hblC and hblD genes) [12]. Nausea and are symptoms of emetic syndrome, which usually occurs 1–5 hours after ingestion of contaminated food [16]. Diarrheal syndrome results in abdominal pain and diarrheal 8 to 16 hours post-ingestion [5, 14]. These illnesses are generally short-lived and self-resolve within 24 hours [17].

Bacillus cereus is underreported as a food-borne pathogen. First, symptoms of disease are usually mild and transitory. Second, since it is ubiquitous in the environment, there is not a direct linkage of animals to human infection, such as Escherichia coli or Salmonella, thus it is often ignored. Third, emetic strains are not detected using standard methods for B. cereus such as the use of mannitol-egg yolk-polymyxin agar (MYP). Finally, the food sources that are involved in food poisoning outbreaks contain different B. cereus strains [18]. Nevertheless, there have been multiple outbreaks caused by B. cereus. Between 1998 and 2000 B. cereus was the cause of 60% of foodborne outbreaks in mass catering facilities, and it was the most frequently isolated microorganism from commercial kitchen samples [5]. In 2014, 287 outbreaks of food poisoning were due to B. cereus resulting in 3073 cases, and in 2015, 291 outbreaks resulting in 3131 cases were reported in European Member States [17]. An investigation found that B. cereus was responsible for 1689 food poisoning cases at mass-catering services in the European Union from 2000 to 2013 [17]. Another study provided strong evidence that B. cereus caused 564 foodborne illness outbreaks in France from 2007 to 2014 [19]. Bennett et al. reported that 235 food-borne outbreaks, involving 2050 illnesses and 17 cases hospitalizations were caused by Bacillus cereus in the United States between 1998 and 2008 [20].

Given the prevalence of B. cereus in food products, its pathogenicity, and the impact of B. cereus on the worldwide foodborne illness burden, it is imperative to improve control and detection of this pathogen in food. We aim to summarize what is currently known about B. cereus, discuss current research into control and detection of this pathogen, and identify research that needs to be conducted to reduce the presence of this pathogen in food.

2. Biological properties of Bacillus cereus

Bacillus cereus is a mesophilic bacterium and is widespread in the environment. It grows at temperatures ranging from 4 to 48°C, with optimal growth temperatures of 28–35°C. One study divided B. cereus isolates into two groups dependent on their growth temperature range; a low-temperature range of 4 to 37°C and a high-temperature range 10 to 42°C [21]. It is salt tolerant to 7.5% w/w concentrations and to a pH range of 4.5 to 9.5 [21, 22, 23]. The minimum water activity for growth is 0.93 [21, 24].

Bacillus cereus cells are rod shape and measure approximately 1.0–1.2 μm in width by 3.0–5.0 μm in length. Its colonies measure 3–8 mm which appear as grayish flat colonies with a ground glass-like appearance, and often rough edges. Bacillus cereus spores have an ellipsoidal or cylindrical shape, look green in a red vegetative cytoplasm cell, and include black lipid globules in intracellular lipid stain. Spores do not cause swelling in the sporangium [24, 25]. Chemical analysis of Bacillus cereus shows that it can make acid from glucose but not from mannitol, xylose, and arabinose [22]. Its oxidase test is negative, while its motility, catalase, citrate utilization, casein hydrolysis, nitrate reduction, and Voges-Proskauer (VP) reaction, l-tyrosine reduction, and growth in 0.001% lysozyme tests are positive [24, 25].

3. Pathogenicity of Bacillus cereus

Pathogenicity of B. cereus is based on its potential to colonize and persist in the host and invade tissues [26]. This ability varies from strain to strain, which makes it hard to understand and administrate the risks associated with B. cereus [1]. Although B. cereus is known to cause other diseases, such as respiratory tract infections, nosocomial infections, endophthalmtis, and central nervous system infections, we will be focusing on its role in foodborne illness [7].

Ultimately, food poisoning from B. cereus is due to its presence in food. Its presence may initially causes changes in the texture of food and the development of off-flavors due to the production of toxins and/or reproduction of vegetative cells [22]. After consumption of contaminated food, two main types of food poisoning may occur; emetic syndrome or diarrheal syndrome. High microbial count are required before toxin production begins, thus the infective dose of B. cereus is 10⁵ to 10⁷ for diarrheal disease and 10⁵ to 10⁸ cell/g for the emetic syndrome [27, 28]. Therefore, a consensus exists regarding the dose of B. cereus which is necessary to make the food safe for consumption and should not surpass the 10³ CFU/g [22].

3.1 Diarrheal syndrome

Diarrheal food poisoning was recognized in 1948 for the first time after a food poisoning outbreak in Oslo, Norway [29]. In 1955, Hauge voluntarily conducted an experiment and ingested vanilla sauce with a high number of B. cereus which subsequently resulted in abdominal pain and diarrhea. This experiment indicated that B. cereus infection can result in diarrheal disease. Toxins are produced by the bacteria in the small intestine when growth has reached the phase between exponential and stationary phases [25]. Three different heat-labile toxins are responsible for this syndrome: hemolysin BL (HBL), non-hemolytic enterotoxin (Nhe), and cytotoxin K (CytK). As mentioned before, HBL constitutes three protein complexes, L₁, L₂, and B (encoded by genes hblD, hblC, and hblA, respectively). This enterotoxin is cytotoxic and has hemolytic ability, which is the ability to destroy red blood cells by forming transmembrane pores. Additionally, activation of adenylate cyclase enzymes is another mechanism used to cause diarrheal disease [25, 30].

Nhe is another cytotoxic enterotoxin that has three protein components, NheA, NheB, and NheC, which are encoded by the genes nheA, nheB, and nheC. NheB is a cell binding protein,, while nheC acts as a catalyst for bringing nheA and nheB together. This enterotoxin was discovered after a Norwegian outbreak that was caused by a strain that did not produce the HBL enterotoxin. It resembles HBL structurally, and both HBL and Nhe are similar in that they need all three components to be biologically active [25, 27, 30].

In contrast, Cytotoxin K (CytK) is a single protein encoded by gene cytK which is another b-barrel pore-forming toxin. About 90% of B. cereus strains may have the gene for this toxin. Similar to other b-barrel pore-forming toxins, it creates oligomers that are susceptible to boiling and resistant to sodium dodecyl sulfate (SDS). It has two forms, CytK-1 and CytK-2 which have an 89% amino acid sequence similarity. The first form is more involved in French necrotic enteritis outbreaks [25, 30], for which fatal cases have been reported from ref. [31]. Each of these single enterotoxins could cause diarrheal symptoms individually [25].

The enterotoxins are produced in a temperature range of (10–43°C) with an optimum of 32°C. Toxins are susceptible to heat, rapidly degraded at pH 3, and hydrolyzed by proteolytic enzymes of the digestive tract. This shows that diarrheal syndrome is caused by ingestion of cells or spores rather than of produced toxins, however, in young people, both preformed toxin and ingestion of cells can cause diarrheal food poisoning because those people do not have enough stomach acid to destroy the toxins [25]. This syndrome results in a mild and watery diarrhea disease that is similar to the diarrheal syndrome caused by Clostridium perfringens. Most individuals suffering from diarrheal disease recover within 24 h without any treatment [1, 30].

3.2 Emetic syndrome

Emetic syndrome is also caused by B. cereus, is more acute than diarrheal syndrome, and has a shorter incubation time. Consumption of toxin results in vomiting, nausea, and abdominal cramps 1 to 6 h after ingestion of contaminated food. When cell populations reach 10⁵–10⁸ cells per gram sufficient toxin is produced in the food for causing this syndrome [30]. The main cause of this syndrome is cereulide toxin [1]. Some experiments relying on monkey feeding showed that the causative dose of cereulide toxin is 70 μg for this syndrome [25]. Cereulide is not eliminated by cooking or digestive processes since it is both heat and acid stable [18].

This is a nonribosomal peptide that is encoded by the 24-kb cereulide synthetase of genes (ces). Cereulide acts as a potassium ionophore and decomposes the transmembrane potential which results in mitochondrial inactivation and human natural killer cell inhibition by stimulating swelling and respiration. It can also causes degeneration of hepatocytes in higher dose [27]. This syndrome occurs when the pre-formed toxin binds to 5-HT₃ receptors and stimulates the vomiting center of the brain in a way similar to Staphylococcus enterotoxins. Because of its similarity to Staphylococcus enterotoxins, it is very hard to distinguish the symptoms [25].

Cereulide is produced in the stationary growth phase of B. cereus in food at a temperature range of 12–37°C and wth an ideal temperature of 12–15°C. [25]. Cereulide production is produced in foods with a_w > 0.953 and pH of >5.6, under aerobic conditions, when more than 10⁶ CFU of B. cereus emetic strains are present in each gram of food [27].

The prevalence of emetic strains of B. cereus is high in nonrandom food and clinical samples (32.8%), compared to the overall prevalence in B. cereus isolates. Still, 4.7% of ice cream, 1.6% of fish products, 11.0% of ready-to-eat foods, 3.9% of other food samples not implicated in food poisoning, 1.7% of soil samples, 1.5% of cow milk, 1.2% of cow bedding, and 3.9% of farm rinsing water samples contained B. cereus emetic strain isolates [18].

4. Toxins of Bacillus cereus

B. cereus produces different virulence factors including phospholipases, hemolysins, and enterotoxins [32]. One emetic toxin and three enterotoxins are the causative agent for the associated food poisonings [33]. The Emetic toxin is produced in food stuff and three enterotoxins that are responsible for diarrheal syndrome are produced in intestine [34].

4.1 Emetic toxin (cereulide)

In 1976, Melling et al. proposed for the first time that a different toxin is associated with emetic syndrome. They proposed this by feeding Rhesus monkey with two different isolated strains: one from a diarrheal associated outbreak and another one from a vomiting associated outbreaks. The first strain caused fluid accumulation while the second one did not. This indicated that a different toxin is involved in vomiting symptoms [35]. Then, Agata et al. [36], identified cereulide as the emetic toxin factor [36].

Cereulide is a ring-structured peptide (dodecadepsipeptide) which is constituted of three repeat sequences of four amino acids and/or oxy-acids. Its molecular weight is 1.2 kDa and its composition is [d-O-leu-d-ala-l-O-val-l-val] [25, 27]. This potassium binding depsipeptide is structurally similar to the ionophore valinomycin. Its lipophilic properties, as well as heat and pH resistance, are due to its structure which is characterized by peptide and ester bonds. It is not eliminated by bactofugation or filtration, nor by heat treatments (even at 121°C for 2 hours) in food processing. Also, pepsin and trypsin in the stomach do not have a lethal effect on it. Therefore, it is very important to control cereulide production. Some food additives such as polyphosphates could be effective for this purpose, while other food ingredients motivate its production [33]. It has seven different isoforms named isocereulide A-G [37], which have different cytotoxicity. Isocereulide-A, the highly cytotoxic isocereulide [37], has 10-fold cytotoxicity compared to wild cereulide type, while isocereulide-B has no cytotoxicity. This difference is due to the different membrane activity of the isocereulide [33].

Foodborne disease caused by cereulide results in heavy episodes of vomiting, while recently more severe intoxications show rhabdomyolysis, liver damage, and serious multi-organ failures. This is because of its high ionophoric activity [33]. A 17 year old boy died after consuming spaghetti with pesto that was prepared 4 days earlier. Within 2 days of having the meal, the boy developed fulminant liver failure and rhabdomyolysis which resulted in his death [38].

It is also reported that cereulide can co-occur with other microbial toxins in cereal-based foods such as mycotoxins [33]. A research by Agata et al. [39], quantified the production of B. cereus emetic toxin (cereulide) in various foods. In 13 out of 14 food samples that were involved in emetic type food-poisoning, cereulide was identified at a range of 0.01 to 1.28 μg per 1 g food. An emetic producing strain of B. cereus (B. cereus NC7401), was identified as the causative agent [39].

Considering B. cereus is ubiquitous in the environment, the emetic toxin is not produced in large amount in foods. A research study evaluated 271 samples of soil, animal feces, raw and processed vegetables, concluded that while B. cereus is common in the environment, the incidence of emetic strains of B. cereus is rare [40].

Carlin et al. [41] evaluated 100 strains of B. cereus to see if emetic toxin producing strains have different characteristics than non-emetic toxin-producing strains. Among them, 17 strains were emetic toxin (cereulide) producing and 83 strains non-emetic toxin-producing. The minimum growth temperature of emetic toxin-producing strains is 10°C and maximum of 48°C, while 11% of non-emetic toxin-producing strains grow at 4°C and 47% of them at 7°C and only 39% of them can grow at 48°C. Spores from emetic toxin-producing strains have higher resistance at temperatures of 90°C and lower germination rate at 7°C than other strains. This study indicates that emetic toxin-producing strains have different growth characteristics than other strains, which is useful for B. cereus risk assessment [41]. Another investigation showed that all cereulide producing strains contain the H-1 serovar phenotype, while non-emetic toxin producing group does not show this phenotype. The H-1 serovar group is enable to hydrolyze starch, thus the emetic toxin-producing strains cannot hydrolyze starch. This may help to control outbreaks of emetic type syndrome caused by B. cereus [42].

4.2 Enterotoxins

In the late 1970s and early 1980s researcher started to isolate enterotoxins. An unstable, and heat-labile protein, which was inactivated in 30 min at 56°C, was isolated for the first time as an enterotoxin protein. Then it was reported that diarrheal activity of B. cereus is caused by a complex of two or three protein enterotoxin [12]. Three different enterotoxins have been identified that are produced by B. cereus. Studies show B. cereus produces one single-component enterotoxin and two different three-component enterotoxins [6].

4.2.1 Hemolytic BL (Hbl)

Hbl is an eneterotoxin that was originally highly purified from B. cereus F837/ 76. [12]. It is a three-component enterotoxin containing two-lytic components, L₂, L₁, and binding (B) compound, which is encoded by the Hbl operon by hblC, hblD, and hblA respectively. Two different Hbl operons exist in some B. cereus strains, with the most common one containing a fourth gene, hblB. This gene encodes the Hbl bind compound precursor HblB’. The molecular weights of the components are 45, 36, and 35 kDa for L₂, L₁, and B, respectively [33], and 5.3 pI value [12]. Hbl has been identified in 42–73% of food poisoning strains and less frequent in non-pathogenic strains [43].

Evaluation of all three components of Hbl revealed that the proteins formed alpha-helices. Analysis of Hbl-B and L₁ contained transmembrane segments of 17 amino acids and 60 amino acids residues respectively, whereas L₂ did not contain any transmembrane domains. [12]. For biological activity of hbl, all three components are needed [43]. Hb has hemolytic activity as evidenced byring-shaped clearing zones due to hemolysis on blood agar. The hemolytic potential is based on the type of blood used in the experiment. Besides hemolytic activity, hbl increases vascular permeability in rabbit skin and is dermonecrotic. Cytotoxicity has also exhibited toxicity to Chinese hamster ovary cells and retinal tissue. In addition to fluid accumulation in the rabbit ileal loop assay, it causes pore formation in eukaryotic cell membranes [12]. Of note, hbl needs to be combined with nhe to be active biologically [43].

Heterogeneity exists in the hbl components in different strains. Schoeni and Wong [44], studied 127 B. cereus isolates and results showed that across all isolates there were only four variations of the B subunit(38, 42, 44, and 46 kDa), two different L₁ subunits (38 and 41 kDa), and three different L₂ subunits (43, 45, and 49 kDa) [44].

4.2.2 Non-hemolytic enterotoxin (Nhe)

Granum et al. [29], found that there should be one enterotoxin in addition to hemolysin BL and enterotoxin T that is responsible for diarrheal syndrome, after evaluating 321 strains of B. cereus [29]. While evaluating B. cereus strain 0075-95, isolated from a Norwegian outbreak, Nhe was purified for the first time [45]. This is also a three component open reading frame that is encoded by nheA, nheB, and nheC. Their molecular weights are 41, 39.8, and 36.5 kDa with a pI value of 5.13, 5.61, and 5.28 for nheA, nheB, and nheC respectively [12]. Also, nheA, nheB, and nheC have signal peptides of 26, 30, and 30 amino acids, mature proteins of 360, 372, and 329 amino acids, respectively [46].

Nhe protein components have similarities with each other as well as with hbl protein components. NheA has 19% similarity with nheB, and nheC, also nheB has 44% similarity with nheC. The similarities between nhe and hbl components are observed as 24% between nheA and L₂, 37% between nheB and L₁, and 25% between nheC and B [12, 46]. Some homologies also exist between nhe and hbl regarding the predicted transmembrane helices, in that hbl and nhe are a tripartite family [8, 12].

About 92 to 100% of B. cereus isolates produce nhe toxin, which is found more often than hbl production [12]. This shows that nhe is the most dominant toxin in diarrheal food poisoning, demonstrating that cytotoxicity of B. cereus is greatly related to the concentration of nhe and poorly to the concentration of hbl [47]. Studies revealed that no change in cytotoxicity was observed after inhibition of hbl and cytK in B. thuringiensis, while deletion of nhe operon in B. cereus strain ATCC 14579 reduced cytotoxicity activity [8].

The biological activities of nhe are based on the involvement of all three components of nhe. Nhe exhibits a a specific concentration of ratio of (10:10:1 for nheA:nheB:nheC) [33]. Though the mode of action of nhe is not completely clear, the disruption of the plasma membrane and pore formation in planar lipid bilayers were observed in epithelia [47]. This result shows that nhe causes cell death (cytotoxicity) via colloid osmotic lysis by forming transmembrane pores. Also, suspension assays indicate hemolytic activity of nhe toward erythrocytes [8].

4.2.3 Cytotoxin K (cytK)

This is a single protein diarrheal enterotoxin that was isolated in 1998 from B. cereus strain NVH 391/98. This strain caused a severe bloody diarrheal outbreak in France that resulted in six cases and three deaths. CytK toxin is encoded by cytK, which has two variants, cytK-1, and cytK-2, with 89% amino acid similarity [33]. In 2015, Castiaux et al. conducted a research study to determine whether cytK is a virulence factor for diarrheal syndrome. Results showed that cytK-2 is not a relevant virulence factor for this type of food poisoning [48]. Another study also declared that cytK-2 is not a good marker for the cytotoxicity of B. cereus [33].

This toxin is a member of the β-folded pore-forming group and has different health effects [49]. CytK-1 is a hemolytic, dermonecrotic, pore-forming in lipid layers, and highly toxic to the human intestinal epithelial cells. CytK-2 also proved to be hemolytic, cytotoxic, and pore-forming in lipid layers, but has about 80% lower cytotoxicity power than cytK-1 [33, 49]. Shadrin et al. [49], observed the cytK genes of some B. cereus strains and concluded that there is a distinct differentiation between cytK-1 and cytK-2. Thus, by knowing the variants of cytk in a strain, we can recognize if cytk is the main cause of extreme diarrheal disease [49]. A duplex PCR assay was developed to discriminate B. cereus strains carrying the cytK-1 gene. This was the first recognized method for identifying the strains containing this gene [50].

4.2.4 Enterotoxin FM (entFM)

This toxin is the most prevalent enterotoxin in B. cereus, and was identified from the B. cereus FM1 strain by Asano in 1997. PCR studies of 10 B. cereus isolates showed the presence of the entFM gene in all isolated strains [51, 52]. Another study revealed the presence of the entFM gene in 27 of 28 food isolates and 30 outbreaks associated with B. cereus strains [53]. In another study, the entFM gene was detected in all 616 strains of B. cereus and B. thuringiensis [54]. Therefore, because of its prevalence in approximately all B. cereus strains, it can be a target gene for assessing enterotoxigenic B. cereus isolates [51].

Although the majority of investigated strains were positive for entFM, only a small number of them could cause diarrheal toxicity. This may be due to the expression level of the entFM gene [51]. Research studies showed that entFM is related to cell wall peptidase (Cwps) which is involved in bacterial motility, shape, and biofilm formation, as well as vacuolization of macrophages as part of bacterial virulence [52].

There have been other identified enterotoxins produced by B. cereus, but little is known about their virulence and mode of action (Table 1).

Toxin Name	Toxin Type	Molecular Weight	pI	References
Cereulide	Emetic toxin	1.2 kDa	5.52	[55, 56]
Hemolytic BL; B-component; L1-component; L2-component	Enterotoxin	37–46 kDa; 35 kDa; 36 kDa; 45 kDa	5.3; 5.34; 5.33; 5.33	[12, 55]; [33, 57]; [33, 57]; [33, 57]
Non-hemolytic; nheA; nheB; nheC	Enterotoxin	36–41 kDa; 41 kDa; 39.8 kDa; 36.5 kDa	5.13; 5.61; 5.28	[55]; [12]; [12]; [12]
Cytotoxin K	Enterotoxin	34 kDa	6.1	[31, 55]

Table 1.

Molecular size and isoelectric point of toxins produced by B. cereus.

5. Antibiotic resistance of Bacillus cereus

Antibiotic resistance is a process by which microorganisms show tolerance against antibiotics that are used to treat people from diseases [58]. The growing number of antibiotic-resistant microorganisms have been reported as a major health problem in the twenty-first century. Therefore, several investigations have investigated this topic and revealed information about the antibiotic resistance potential of microorganisms [59]. Antibiotic resistance of B. cereus has been tested in different food products and reported in different studies.

Fiedler et all. In 2019, evaluated the antibiotic resistance of 147 B. cereus strains isolated from fresh vegetables including cucumbers, carrots, herbs, salad leaves, and ready-to-eat mixed salad leaves. It shows that B. cereus is highly resistant to β-lactam antibiotics such as penicillin G (PEN) and cefotaxime (CTX) (100%), ampicillin (AMP), and amoxicillin/clavulanic acid combination (AMC) (99.3%). This study showed these isolates were still is susceptible to ciprofloxacin (CIP) (99.3%), chloramphenicol (CHL) (98.6%), amikacin (AMK) (98.0%), imipenem (IPM) (93.9%), erythromycin (ERY) (91.8%), gentamicin (GEN) (88.4%), tetracycline (TET) (76.2%), and trimethoprim-sulfamethoxazole (SXT) (52.4%) [59]. Another study assessed the antibiotic resistance of 64 B. cereus strains isolated from different food products such as milk, dairy products, spices, and rice salad in Morocco. This investigation indicated that isolated strains are resistant to ampicillin (98.4%), tetracycline (90.6%), oxacillin (100%), cefepime (100%), and penicillin (100%), and were susceptible to chloramphenicol (67.2%), erythromycin (84.4%), and gentamicin (100%) [60]. These data are in concordance with other investigations in [61, 62, 63, 64, 65]. Most of these studies were performed using the Kirby-Bauer disc diffusion method for testing antibiotic resistance of the B. cereus isolates (Table 2).

Antibiotics	B. cereus reaction to antibiotics
	Susceptible	Intermediate	Resistant
Amoxicillin/Clavulanic Acid	−	−	+
Ampicillin	−	−	+
Benzyl Penicillin	−	−	+
Cefepime	−	−	+
Cefotaxime*	−	−	+
Cefoxitin	−	−	+
Cefpodoxime	−	−	+
Ceftazidime	−	−	+
Cloxacillin	−	−	+
Cotrimoxazole**	−	−	+
Cephalothin	−	−	+
Metronidazole	−	−	+
Nalidixic Acid	−	−	+
Nitrofurantoin	−	−	+
Novobiocin	−	−	+
Oxacillin	−	−	+
Penicillin G	−	−	+
Rifampicin***	−	−	+
Trimethoprim	−	−	+
Amikacin	+	−	−
Azithromycin	+	−	−
Chloramphenicol	+	−	−
Ciprofloxacin	+	−	−
Clindamycin	+	−	−
Erythromycin	+	−	−
Gentamicin	+	−	−
Imipenem	+	−	−
Kanamycin	+	−	−
Moxifloxacin	+	−	−
Telithromycin	+	−	−
Tetracycline	+	−	−
Sulfamethoxazole	+	−	−
Vancomycin	+	−	−
Ceftriaxone	−	+	−
Streptomycin	−	+	−

Table 2.

B. cereus antibiotic resistance [61, 66, 67, 68, 69].

*

Natural resistance to beta-lactams.

**

66% resistant.

***

Acquired resistance phenotype.

6. Detection methods of Bacillus cereus

Because of B. cereus’ high incidence, and its wide distribution in food and the environment, its detection is very important for recognizing pathogenic B. cereus strains and preventing food contamination and food-poisoning outbreaks [70]. Different factors are considered in developing detection techniques for B. cereus including, sensitivity, usage, and time. Therefore, to have useful techniques to detect B. cereus cells and spores from a low level of contamination, several detection methods have been developed.

6.1 Traditional methods

Agar plate-based counting (ISO 7932:2004) is a traditional method for detection and detection of B. cereus [1]. For detection and enumeration of low numbers of bacteria using the most probable number, ISO 21,871 has been used. The result of traditional methods was presented as “presumptive B. cereus” because these methods could not evaluate the toxin-producing ability of B. cereus and could not differentiate B. cereus from other Bacillus group isolates [71]. Therefore, after doing several laboratories steps, a differentiative method needed to be performed to complete the detection process and differentiate B. cereus from other Bacillus group bacteria [1]. These detection techniques were based on the use of selective media and biochemical tests, which are arduous, time-consuming, and need skilled personnel to perform [70]. Thus, novel techniques have to be developed to narrow these gaps.

6.2 Molecular methods

Since low levels of contamination might be present in food, a very sensitive method needs to be used to detect low numbers of B. cereus rapidly. The polymerase chain reaction (PCR) is a fast and sensitive method that can be used to determine the enterotoxic potential of B. cereus [72]. The PCR method was developed to identify different pathogenic bacteria. Nested PCR, Randomly Amplified Polymorphic DNA PCR (RAPD PCR), quantitative PCR (qPCR), and multiplex PCR (mPCR) [70] are different molecular techniques that have been developed. Traditional PCR uses a pair of primers to produce a nucleic acid fragment and can only be applied for a single pathogenic factor. Multiplex PCR uses more two or more pairs of primers to detect multiple pathogenic factors [73].

A research study was performed to compare multiplex PCR, enzyme immunoassay, and cell culture methods for detection of enterotoxigenic B. cereus. They assessed 176 strains of B. cereus from different sources and the results obtained from these three methods were correlated. The PCR assay was suggested as a convenient method for the detection of enterotoxigenic B. cereus isolates [74]. Another study investigated a novel method of antibiotic-based magnetic nanoprobes combined with mPCR for the detection of B. cereus as well as Staphylococcus aureus. In this study, more than one pathogen can be detected, unlike other methods that are designed for the detection of one pathogen (for detail refer to [73]). This study indicates the advantages of the PCR method when used with other methods in the detection process.

These methods are mainly based on specific gene sequences, which can result in false negatives due to improper cell disruption and nucleic acid extraction. Thus, on a small scale it is not necessarily an easy, real-time and rapid detection method for B. cereus [70], however, it has advantages compared to the traditional plating methods such as shorter time overall and higher specificity [1]. These methods are unlikely to be used by the food industry as it requires professionals well-trained in molecular techniques [1] Additionally, molecular methods experience detection interference from complex foodstuff which decreases their sensitivity [73]. Therefore, to overcome these limitations new biosensors techniques have been developed.

6.3 Biosensors methods

Over the last year, biosensors are the most reliable developed methods for the detection of pathogens [1]. This is due to their simplicity, rapidity, and high sensitivity [71]. Additionally, this technique needs a small number of samples to work [1]. Different biosensor techniques have been developed for which DNA-based [1] and electrochemical-based [71] are reported as the best biosensor methods.

Poly and monoclonal antibodies are used as diagnostic factors in biosensors as an alternative to DNA probes [1]. Enzyme-linked immunosorbent assay (ELISA) is a sensitive and convenient method for assessing the macromolecular protein, polysaccharide, and bacteria. This method is based on two antibodies, monoclonal antibody (mAb) and rabbit polyclonal antibody, and it is a rapid detection method for low numbers of cells (9 * 10² cells/mL). Immunological kits are commercially available, like the B. cereus Enterotoxin Test Kit, but they cannot be used on whole-cells. For detection of whole cells, sandwich ELISA is best for identification of B. cereus even in low concentrations [70].

The antibody-based assays have some disadvantages, such as being costly, because commercial B. cereus antibodies are expensive, have low affinity, and pH and temperature can affect their stability and binding potential [1]. To inhibit these disadvantages, bacteriophage and their proteins can be used as recognition elements instead of antibodies because they have high sensitivity, high binding affinity, and high stability to temperature and pH [75].

7. Control of Bacillus cereus

B. cereus endospores are very heat-resistant spores that survive cooking proccesses. Heat resistance increases with an increase of salinity and decreases as acidity increases [76]. Some improper food preparation methods, increase the risk of pathogens’ growth such as improper cooling methods [77]. A survey of 411 schools indicated that 78% of schools cool leftover food which is later heated and served at another meal. Only 8% of schools use blast chillers, and many cool food using improper cooling methods [78], which can allow for B. cereus to proliferate. Therefore, standard operating procedures are necessary to prevent B. cereus associated food-poisoning outbreaks.

Schneider et al. [76], reported best practices for controlling B. cereus outgrowth in food products. These recommendations were based on the National Institutes of Health (NIH), the National Institute of Allergy and Infectious Diseases (NIAID), the National Food Processors Association (NFPA), and the FDA Food Code 2013, that are effective for destroying B. cereus:

• Steaming the food in ≥145°F (63°C) under pressure, roasting, frying, and grilling methods can destroy B. cereus vegetative cells and spores.

• To inhibit the emetic toxin, foods need to be heated to 249°F (121°C) for more than 80 minutes.

• Cooking to ≥145°F (63°C) and reheating to 165°F (74°C) for 15 seconds destroy the vegetative cells. While, if the toxin has been preformed in food, it is not safe to eat.

• The quick cooling of foods after heating is the best way to prevent spores from erminating.

• For spore formation inhibition, hot foods should be kept at 135°F (57°C) and cold foods below 41°F (5°C).

• Cool or refrigerate leftovers rapidly to 41°F (5°C) or below.

Additional methods have been investigated for control of B. cereus in food. A study by Luu-Thi et al. [79] looked at the use of high pressure high temperature (HPHT) treatments for control of B. cereus. Spore inactivation was less than 1-log at 50–60°C but increased to 5-log at 100°C [79]. Their studies using the antimicoribial carvacrol in conjunction of HPHT showed reduced inactivation of spores at temperatures under 90°C and did not have an affect at 95–100°C.

Studies using combinations of radiation and antimicrobials were performed by Ayari et al. [80]. The use of carvacrol in combination of nisin enhanced radiation sensitivity and resulted in lower D₁₀ values [80]. However, repeated exposure to 1 kGy of γ-radiation resulted in an increase in radioresistance indicating that repeated exposures would be ineffective for control in food products.

Studies conducted by Yang et al. [81] looked at the use of lactic acid bacteria (LAB) starter cultures in rice fermentations as a way to control B. cereus [81]. With the decrease in pH from 6.8 to 4.0 there was a 1 log cfu/ml reduction in B. cereus. These results are consistent with results obtained by Rossland et al. [82] which showed varying results in feremented milk ranging from a 7-log reduction to as little as a 2-log reduction [82]. These results indicate that use of LAB may produce inconsistent results and may not inhibit B. cereus sufficiently to insure safety of food products.

Studies were conducted to look at the use of bactericidal activity of neutral electrolyzed water (NEW) against B. cereus inoculated onto the surface of fresh produce items (cherry tomato, miniature cucumber, carrot and parsley) and polypropylene cutting boards at ambient temperature (22°C). NEW solutions contained 60 and 120 mg/L free available chlorine (FAC). When used on cell suspensions 5 min of treatment a 2.11 to 3.03 log₁₀ CFU/mL reduction of B. cereus was observed [83]. B. cereus on inoculated produce was reduced by 2.11–2.30 and 2.41–3.16 log₁₀ CFU/g when NEW contained 120 mg/L FAC. On surfaces, after 5 mins of treatment, cell viability was reduced by 2.33 and 3.06 log₁₀ per 100 cm². These results indicated that a pre-treatment with NEW containing FAC may be a method to further investigate for pre-treatment of food products to protect against outgrowth of B. cereus.

8. Conclusion

B. cereus is an endospore-forming bacterium that is ubiquitous in the environment and can be isolated from different foods. Its spores are strongly resistant to heat, acid, and other environmental stresses. This makes contamination of B. cereus a concern as a food pathogen. Since illness with B. cereus are often a short duration and self-limiting, illnesses from B. cereus are underestimated.

Emetic and diarrheal syndromes are the two types of food-borne diseases caused by B. cereus. Cereulide toxin which is formed in food is the cause of the emetic syndrome and different enterotoxins including HBL, NHE, cytK, and entFM are the cause of the diarrheal syndrome. This bacterium is resistant to some antibiotics such as the β-lactam group and susceptible to some other antibiotics like gentamicin (GEN). It is unknown if antimicrobial resistance is increasing in B. cereus, but this needs further investigation given the number of antibiotics that B. cereus is resistant to.

Different detection methods are used for identifying pathogenic B. cereus strains to help prevent food contamination. However, there are limitations to current rapid detection of B. cereus especially given its fgenetic similarity to Bacillus anthracis, Bacillus thuringiensis, and Bacillus mycoides [84, 85]. Moreover, molecular detection methods require costly instrumentation and are not likely to be accessible by the majority of food processors. Therefore, different molecular techniques are needed to differentiate these bacteria from each other, and rapid tests that can be utilized by industry are needed.

Current control practices, as defined by FDA and other regulatory agencies are not adequate for eliminating B. cereus from food. Research into new methods to control or eliminate B. cereus from food products is desperately needed.

Conflict of interest

The authors declare no conflict of interest.