Characterization of Bacillus cereus in Dairy Products in China

Bacillus cereus is a common and ubiquitous foodborne pathogen with an increasing prevalence rate in dairy products in China. High and unmet demands for such products, particularly milk, raise the risk of B. cereus associated contamination. The presence of B. cereus and its virulence factors in dairy products may cause food poisoning and other illnesses. Thus, this review first summarizes the epidemiological characteristics and analytical assays of B. cereus from dairy products in China, providing insights into the implementation of intervention strategies. In addition, the recent achievements on the cytotoxicity and mechanisms of B. cereus are also presented to shed light on the therapeutic options for B. cereus associated infections.

Most toxins of B. cereus belong to the family of pore forming toxins (PFTs); among them, Nhe and Hbl are similar to the well-known cytolysin A (ClyA) of α-PFTs family, while CytK and hemolysins are members of β-PFTs [17,24,25]. PFTs have the capacities of altering the plasma membrane permeability Toxins 2020, 12, 454; doi:10.3390/toxins12070454 www.mdpi.com/journal/toxins of target cells, potentially leading to cell death and triggering the signaling pathways [26][27][28]. Nhe and Hbl have a similar mechanism of action; the three components of Nhe complex display the highest cytotoxicity at a ratio close to 10:10:1 for NheA, B and C [29,30]. In terms of Hbl, the ratio at L2: L1: B = 1:1:10/ 10:1:10 prompts the most rapid pore formation [20]. Recent studies also found that LITAF and CDIP1 work as the Hbl receptor [31]. The signaling pathways triggered by Nhe, as shown in Figure 1a, include the induced cell apoptosis though ASK1 and Fas-p38 MAPK mediated caspase-8 dependent pathways [32]. In addition, the most recent research suggested that Nhe and Hbl operate synergistically to activate the NLRP3 inflammasome and induce inflammation [33]. Moreover, Nhe have the concerted action with sphingomyelinase in pathogenic B. cereus to cause full virulence and formation of disease [34]. The two caspase-1 dependent inflammatory pathways triggered by Nhe include the form of inflammation initiated by IL-1β release and pyroptosis induced by the activation of GSDMD (Figure 1a). Moreover, the emetic toxin, cereulide is a K + ionophore toxin that damages the cellular membrane potential through inhibiting the synthesis of RNA [35], affecting mitochondrial function, resulting in expansion of mitochondria and formation of vacuoles in the protoplasm of sensitive cells, inducing cell apoptosis and even fulminant liver failure [36,37] (Figure 1b). Moreover, cereulide is terribly unwholesome and could accumulate in multiple organs [38]. CytK and hemolysins otherwise do harm to the target membrane, bringing cell lysis and apoptosis in macrophages [39] ( Figure 1c). hemolysins are members of β-PFTs [17,24,25]. PFTs have the capacities of altering the plasma membrane permeability of target cells, potentially leading to cell death and triggering the signaling pathways [26][27][28]. Nhe and Hbl have a similar mechanism of action; the three components of Nhe complex display the highest cytotoxicity at a ratio close to 10:10:1 for NheA, B and C [29,30]. In terms of Hbl, the ratio at L2: L1: B = 1:1:10/ 10:1:10 prompts the most rapid pore formation [20]. Recent studies also found that LITAF and CDIP1 work as the Hbl receptor [31]. The signaling pathways triggered by Nhe, as shown in Figure 1a, include the induced cell apoptosis though ASK1 and Fas-p38 MAPK mediated caspase-8 dependent pathways [32]. In addition, the most recent research suggested that Nhe and Hbl operate synergistically to activate the NLRP3 inflammasome and induce inflammation [33]. Moreover, Nhe have the concerted action with sphingomyelinase in pathogenic B. cereus to cause full virulence and formation of disease [34]. The two caspase-1 dependent inflammatory pathways triggered by Nhe include the form of inflammation initiated by IL-1β release and pyroptosis induced by the activation of GSDMD (Figure 1a). Moreover, the emetic toxin, cereulide is a K + ionophore toxin that damages the cellular membrane potential through inhibiting the synthesis of RNA [35], affecting mitochondrial function, resulting in expansion of mitochondria and formation of vacuoles in the protoplasm of sensitive cells, inducing cell apoptosis and even fulminant liver failure [36,37] (Figure 1b). Moreover, cereulide is terribly unwholesome and could accumulate in multiple organs [38]. CytK and hemolysins otherwise do harm to the target membrane, bringing cell lysis and apoptosis in macrophages [39] (Figure 1c). Besides, B. cereus can survive in the gastrointestinal tract with versatile virulence factors [40,41]. Therefore, many other infections associated with B. cereus have been reported including meningitis, brain abscess [42], cellulitis [43], endophthalmitis [44,45], pneumonia [46], endocarditis [47] and osteomyelitis [48]. More seriously, persistent B. cereus strains, which are highly detrimental pathogenic bacteria against antibiotic therapies, were also found in patients in the USA [49]. Altogether, tracing the source of B. cereus, such as in dairy products, is of particular concern.
At present, to the best of our knowledge, there is no detailed information focusing on the potential risk of B. cereus from dairy products in China, not to mention the summaries about the detection and toxicity mechanisms of Bacillus virulence factors. In the current review, we summarized the demand for dairy products in China, the prevalence and detection of B. cereus and the virulence Besides, B. cereus can survive in the gastrointestinal tract with versatile virulence factors [40,41]. Therefore, many other infections associated with B. cereus have been reported including meningitis, brain abscess [42], cellulitis [43], endophthalmitis [44,45], pneumonia [46], endocarditis [47] and osteomyelitis [48]. More seriously, persistent B. cereus strains, which are highly detrimental pathogenic bacteria against antibiotic therapies, were also found in patients in the USA [49]. Altogether, tracing the source of B. cereus, such as in dairy products, is of particular concern.
At present, to the best of our knowledge, there is no detailed information focusing on the potential risk of B. cereus from dairy products in China, not to mention the summaries about the detection and toxicity mechanisms of Bacillus virulence factors. In the current review, we summarized the demand for dairy products in China, the prevalence and detection of B. cereus and the virulence factors. We aim to give an overview of B. cereus in dairy products, which may contribute to the implementation of effective strategies to prevent and control foodborne pathogenic B. cereus in dairy products.

Dairy Products with Bacillus cereus
Dairy products are excellent nutrition for both young animals and human beings [50][51][52]. Notably, milk contains all eight kinds of essential amino acids, minerals, vitamins, and fatty acids with optimal proportions of nutrients [8,9]. To satisfy the increasing needs of human, milk associated dairy products are derived from diverse sources, which are known to fall into various categories including liquid milk, milk powder, cheese, condensed milk, milk fat and ice cream [51][52][53]. Demand for dairy products varies sharply from one region to another [54]. For instance, liquid milk had a top priority for Chinese customers in contrast to American and those from other countries, the proportions being 55% in China and 25% in America, respectively, while cheese had the smallest demand (just 13%, yellow part) in China compared with other countries (Figure 2a). In addition, dairy products in the Chinese market have been in short supply, and the export volume of these was far less than the import amount in China from the year of 2013 to 2018 ( Figure 2b). According to the phased consumption targets of the National Food and Nutrition Advisory Committee, per capita consumption of milk will reach 28kg by 2020 and 41kg by 2030 [53]. Likewise, the sales of milk in China will increase from 119.5 to 128.3 billion yuan from 2018 to 2022, with an average annual compound growth rate of 1.7%, and the size of the milk market will sustain a steady growth, as put forward by the China Business Research Institute. Consequently, the sale volume of milk in China will gradually augment, and the market prospect is considerable [55,56]. In view of the high demand for dairy products and the outbreaks of bacterial contamination in liquid milk and milk powder in China, it is urgent to carry out effective analytical tests, especially the detection of microbes during manufacturing, selling and importing of milk [12,[57][58][59]. In addition, the uncertain microbial growth rate and toxicity of microbial metabolites all threaten food safety [60][61][62][63]. As shown in Figure 2c, although preventive measures have been made to manage the contamination of dairy products in the last century [64], the outbreaks of B. cereus spp. contamination constantly arose in various milk products all over the world [61,62,[65][66][67][68]. The prevalence of B. cereus in dairy products is difficult to estimate, and food poisoning incidents caused by B. cereus still remain a thorny problem worldwide due to the high tolerance of B. cereus to various environments and strong propagation capacity of B. cereus spores [2,5,13,47,69]. The B. cereus isolates correspondingly are suspected of threatening the safety of raw milk and dairy products in China [5,13,70]. Therefore, we will next focus on the pattern of B. cereus contamination in dairy products in China.  [64]. Red marked isolates were B. cereus spp. strains.

Contamination of Bacillus cereus Isolates
B. cereus prevails in soil and dairy farms and often pollutes foods like raw milk and all dairy products [3,71]. Spores of B. cereus can primarily spread through soil and air [69]. Researches showed that 1g of soil contains 50-380,000 CFUs (colony-forming units) B. cereus spores, and 1 m 3 of air has at least 100 CFUs of B. cereus spores. Thus, the abundant B. cereus group spores in the environment are a major cause of the high prevalence rate of B. cereus [5,72]. B. cereus and its spores subsequently have a great opportunity to circularly contaminate dairy farms, human market, food supply places, and dairy products and colonize the intestinal tract of invertebrates and cause illness in humans afterwards ( Figure 3a). The Centers for Disease Control (CDC) website claimed that there were 619 confirmed outbreaks of Bacillus-related poisoning from 1998 to 2015 [2,73]. Specifically, the diarrheal illness caused by B. cereus is often related to meats, milk, vegetables and fish, while the emetic type is most possibly associated with rice products [15]. Previous studies have shown that B. cereus isolated from raw milk have the ability to remain active after pasteurization or ultra-high temperature (UHT) sterilization, which ensues in bacterial pollution in the final products [12,66,73,74]. Thus, the health hazards originated from B. cereus in milk industry in China require rapid and proper handling.  [64]. Red marked isolates were B. cereus spp. strains.

Contamination of Bacillus cereus Isolates
B. cereus prevails in soil and dairy farms and often pollutes foods like raw milk and all dairy products [3,71]. Spores of B. cereus can primarily spread through soil and air [69]. Researches showed that 1g of soil contains 50-380,000 CFUs (colony-forming units) B. cereus spores, and 1 m 3 of air has at least 100 CFUs of B. cereus spores. Thus, the abundant B. cereus group spores in the environment are a major cause of the high prevalence rate of B. cereus [5,72]. B. cereus and its spores subsequently have a great opportunity to circularly contaminate dairy farms, human market, food supply places, and dairy products and colonize the intestinal tract of invertebrates and cause illness in humans afterwards ( Figure 3a). The Centers for Disease Control (CDC) website claimed that there were 619 confirmed outbreaks of Bacillus-related poisoning from 1998 to 2015 [2,73]. Specifically, the diarrheal illness caused by B. cereus is often related to meats, milk, vegetables and fish, while the emetic type is most possibly associated with rice products [15]. Previous studies have shown that B. cereus isolated from raw milk have the ability to remain active after pasteurization or ultra-high temperature (UHT) sterilization, which ensues in bacterial pollution in the final products [12,66,73,74]. Thus, the health hazards originated from B. cereus in milk industry in China require rapid and proper handling.

Distribution of Bacillus cereus in Milk and Milk Products in China
The prevalence of B. cereus in China has distinct traits, owing to the specific market demands and physical differences. Liquid milk and milk powder are consumed by the largest part of the population in China (Figure 2a). It is quite a coincidence that a high prevalence rate of B. cereus appears in liquid milk (44%) and milk powder (26.1%) (Figure 3b). A recent report claimed that B. cereus were widely present in pasteurized milk in China, showing that 100 of B. cereus isolates are distributed in most Chinese cities including Hong Kong, Guangzhou, Shenzhen, Harbin, Ningxia, Beihai, Hai kou, etc. [14]. In general, there is a relatively lower prevalence rate of B. cereus in dairy products in southern China than in the northern region with exceptions (Table 1 and Figure 3c). For instance, infant formula in the Liaoning province (42%) was contaminated with B. cereus more seriously than that in Yunnan province (12%) ( Table 1). The regular rates of B. cereus in other cities or provinces, such as Beijing, Liaoning, Gansu, Yunnan, northeast China, were found to be 30%, 27%, 19%, 10% and 16%, respectively [13,[75][76][77]. Among these data, Yunnan province and the Northeast China indeed had a relatively low prevalence of B. cereus in powdered infant formula (PIF). Dairy products in Gansu province was polluted by B. cereus to a moderate extent (19%). The most striking data in Table 1 were observed in Beijing and Liaoning province, and the investigation conducted in major cities of China during 2011-2016 suggested that approximately 27% of the pasteurized milk contained B. cereus, and the contamination of B. cereus was 31% (11/36) in northern China and 25% (33/132) in southern China. Concerning infant formula in the Chinese market, two reports were produced in 2012-2013 and 2015, according to which 14% and 42% of formula contained B. cereus, respectively. It was also revealed that 8.2% of PIF samples in China were contaminated with B. cereus strains [78]. Overall, the regional characteristics of the prevalence of B. cereus in dairy products in China cannot be clearly defined by latitudes or longitudes. More scientific research into the epidemiological nature of B. cereus in milk is worth pursuing. From an international perspective, dairy products from African nations are more likely to be polluted by B. cereus, which is aided by the poor sanitary conditions [74]. A nationwide survey conducted in America manifested that a total of 18 (8.9%) of 202 samples were positive for presumptive B. cereus using the MPN technique (<10 to 50 CFU/mL), which cannot be directly compared with those of most other studies [79]. Remarkably, ice cream tested in Bavaria, Germany, was found to carry B. cereus with a high rate of 62.7%, and artisan cheese sold in Mexico had a rather low rate of 28.4% [80]. Nevertheless, no certain epidemiological profile can be obtained from the scanty information on dairy products, domestically and globally. Therefore, it is urgent for countries to address the prevalence of Bacillus cereus in their territory.  The China map shows the regional specificity of B. cereus contamination. Blue indicates no reliable data are found in these provinces. There are four different levels of contamination rate and the darker the color is, the rate of B. cereus is higher. In particular, the reddish, orange, yellow and pale pink color signify the rate of 50%-40%, 40%-30%, 30%-20% and 20%-5%, respectively.

Distribution of Bacillus cereus in Milk and Milk Products in China
The prevalence of B. cereus in China has distinct traits, owing to the specific market demands and physical differences. Liquid milk and milk powder are consumed by the largest part of the population in China (Figure 2a). It is quite a coincidence that a high prevalence rate of B. cereus appears in liquid milk (44%) and milk powder (26.1%) (Figure 3b). A recent report claimed that B. The China map shows the regional specificity of B. cereus contamination. Blue indicates no reliable data are found in these provinces. There are four different levels of contamination rate and the darker the color is, the rate of B. cereus is higher. In particular, the reddish, orange, yellow and pale pink color signify the rate of 50-40%, 40-30%, 30-20% and 20-5%, respectively.

Virulence Factors of Bacillus cereus and Detection Techniques
Many diseases caused by Bacillus groups, such as bovine mastitis, are associated with the virulence factors [40,89,90]. Therefore, it is necessary to further detect the virulence factors secreted from B. cereus isolates in a clinical setting. In particular, the analytical assays of virulence factors in a clinical setting would be enormously beneficial to understand the pathogenic mechanism of virulence factors of B. cereus and to develop effective treatment strategies.

Detection of Bacillus cereus Isolates
B. cereus is a ubiquitous Gram-positive, aerobic or facultative anaerobic, endospore-forming, rod-shaped bacterium [13,71]. The detection and isolation of B. cereus strains are mainly based on the colony count technique ISO 7932 [96]. B. cereus or presumptive colonies of Bacillus are counted on the varieties of Bacillus agar by spiral-plating or spread-plating techniques, most probable number (MPN) method and so on [97]. Both B. cereus cells and spores in the examined products can be counted according to the colony-forming units (CFUs) [5,98,99]. In addition, B. cereus isolates have the capacity for casein, starch and tributyrin hydrolysis as well as lactose fermentation, which inspired the invention of chromogenic medium for B. cereus [100,101]. Instead, the identification or analysis of B. cereus isolates also can use the PCR, the quantitative real-time PCR by targeting the 16S rRNA gene [102,103], groEL/gyrB genes [104] and panC gene [105][106][107], or cross-priming amplification [108] and so on.

Detection of Toxins Secreted from Bacillus cereus
PCR, RT-PCR and multiplex PCR are the major analytical techniques that are reported in research articles to identify the virulence factors by detecting toxin genes in B. cereus ( Figure 4a). As we know, the tripartite enterotoxins-Nhe complex consists of NheA, NheB and NheC that were encoded by nheA, nheB and nheC genes [19], as well as the components of Hbl-L2, L1, B were encoded by hblA, hblC and hblD genes separately [21]. Thus, Nhe and Hbl were usually recognized though PCR by targeting their toxic genes [109]. Clinically, a higher rate of nheA, B, C genes than that of hblA, hblC and hblD genes was disclosed in Bacillus samples (Table 1). Other toxin genes such as cytK and cytK-2 genes of CytK, hly gene of hemolysins or cesB gene of cereulide are also served as the main approaches to determine the positive strains of B. cereus by PCR [23,[110][111][112][113][114][115][116]. Furthermore, another detection tool, Enzyme Immunoassay (EIA), is employed for the direct inspection on the protein level of the toxic components of B. cereus by targeting specific mAbs [117,118], which are able to purify toxins such as NheB and the NheB-C complex and neutralize the cytotoxicity [21,119]. Liquid chromatography-mass spectrometry (LC-MS) and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis are often used for the rapid detection of emetic Bacillus isolates in food products, by analyzing ribosomal subunit proteins [120] or targeting the distinct molecular of cereulide [70,[121][122][123][124]. Cereulide is a cyclic molecule composed of a 36membered ring and a small heat and acid stable cyclic dodecadepsipeptide of 1165 Da, which consist of alternating ester and amide bonds and the structure [-D-O-Leu-D-Ala-L-O-Val-L-Val-]3 [23]. Besides, cereulide belongs to the surfactin-like peptides and is biosynthesized via nonribosomal peptide synthesis (NRPS) [94]. Recent reports revealed that the identification of cereulide from bacterial extracts peak at m/z 1191 with a limit of detection (LOD) of 30 ng/mL [121].
Normally, cytotoxicity tests or cell culture methods are used for evaluating the virulence of B. cereus isolates. The cytotoxicity of the complexes of diarrheal Bacillus enterotoxins accounts for over 90% of the total toxicity [109]. A study showed that Vero and primary endothelial cells (HUVEC) were most sensitive to Nhe, whereas Hep-G2, Vero and A549 cell lines were highly susceptible to Nhe and Hbl. CytK exhibited the highest toxicity on CaCo-2 cells [40], and the emetic toxin cereulide prevented cell proliferation in HepG2 cells by 2 nM [35], also causing vacuolation in HEp-2 cells [125,126]. In general, B. cereus toxins exhibited a wide cytotoxicity to those epithelial cells [127] and some toxins also acted on immune cells [39,128].

Analysis of Bacillus Toxin Detections in Dairy Products in China
B. cereus and its virulence factors are frequently present in dairy products in China [12][13][14][15]57,58]. This not only impacts the quality of dairy products but also potentially impairs human health. Since there is a huge demand for dairy products among Chinese people of all ages [50,53], once Bacillus contamination has occurred, the scope of the damage is extensive and inestimable.
Currently, there is a large number of potential toxin genes related to diarrhea in these B. cereus strains, including the genes of hcbl, nhe, cytK and enterotoxin FM (entFM), as well as potential enterotoxins hlyII and enterotoxin T (BceT) [14]. As shown in Table 2, a study conducted in 10 local Liquid chromatography-mass spectrometry (LC-MS) and matrix assisted laser desorption/ ionization-time of flight (MALDI-TOF) analysis are often used for the rapid detection of emetic Bacillus isolates in food products, by analyzing ribosomal subunit proteins [120] or targeting the distinct molecular of cereulide [70,[121][122][123][124]. Cereulide is a cyclic molecule composed of a 36-membered ring and a small heat and acid stable cyclic dodecadepsipeptide of 1165 Da, which consist of alternating ester and amide bonds and the structure [-D -O-Leu-D -Ala-L -O-Val-L -Val-] 3 [23]. Besides, cereulide belongs to the surfactin-like peptides and is biosynthesized via nonribosomal peptide synthesis (NRPS) [94]. Recent reports revealed that the identification of cereulide from bacterial extracts peak at m/z 1191 with a limit of detection (LOD) of 30 ng/mL [121].
Normally, cytotoxicity tests or cell culture methods are used for evaluating the virulence of B. cereus isolates. The cytotoxicity of the complexes of diarrheal Bacillus enterotoxins accounts for over 90% of the total toxicity [109]. A study showed that Vero and primary endothelial cells (HUVEC) were most sensitive to Nhe, whereas Hep-G2, Vero and A549 cell lines were highly susceptible to Nhe and Hbl. CytK exhibited the highest toxicity on CaCo-2 cells [40], and the emetic toxin cereulide prevented cell proliferation in HepG2 cells by 2 nM [35], also causing vacuolation in HEp-2 cells [125,126]. In general, B. cereus toxins exhibited a wide cytotoxicity to those epithelial cells [127] and some toxins also acted on immune cells [39,128].

Analysis of Bacillus Toxin Detections in Dairy Products in China
B. cereus and its virulence factors are frequently present in dairy products in China [12][13][14][15]57,58]. This not only impacts the quality of dairy products but also potentially impairs human health. Since there is a huge demand for dairy products among Chinese people of all ages [50,53], once Bacillus contamination has occurred, the scope of the damage is extensive and inestimable.
Currently, there is a large number of potential toxin genes related to diarrhea in these B. cereus strains, including the genes of hcbl, nhe, cytK and enterotoxin FM (entFM), as well as potential enterotoxins hlyII and enterotoxin T (BceT) [14]. As shown in Table 2, a study conducted in 10 local dairy farms in Beijing suggested that the nhe, hbl, and ces genes were detected at the rate of 100%, 79.3%, and 1.1%, respectively [13]. Meanwhile, a research involving 12 provinces in China showed that the average virulence gene number in powdered milk was 5.71, and no comparison of the distribution of those genes between different provinces was made [129]. It is logical to conclude that the virulence factors of B. cereus can exert influence on the quality of dairy products during their processing, transporting and selling. Still, like B. cereus, no striking feature of the distribution of their virulence factors in diverse provinces could be obtained. Similarly, high numbers of isolates carried nheA (84.1%), nheB (89.9%), nheC (84.1%), hblA (59.4%), hblC (44.9%), hblD (53.6%) and cytK (53.6%) genes in the production chain of milk in Brazil [62], which is notably bigger than the rate found in Ghana [73]. It is really challenging to sum up the rule of distribution of Bacillus toxins in dairy products in China, and this is limited by the deficient detection methods and shortage of useful data, as the notion of Bacillus contamination in dairy products only came into view in recent years. Note: FBO indicates foodborne outbreak; "-" represents no analytical tests was performed to identify specific toxin genes.
To produce a more forthright analysis of the Bacillus toxins, we collected the prevalence of the toxin genes in Bacillus samples from pasteurized milk and milk powder as they are the most popular dairy products in China [12][13][14][15]53,57,82,112] (Figure 3a). The data showed that the genes of nhe A B C were found in almost all the Bacillus isolates from pasteurized milk, while at least 80% of nhe A and approximate 100% nhe B and C in milk powder (Tables 1 and 2 and Figure 4b). More genes of hbl A C D were discovered in pasteurized milk (45%) than in milk powder (36.8%). Similarly, the positive rate of cytK genes in pasteurized milk and milk powder was 73% and 44.7%, respectively. The cesB gene was mainly found in milk powder (2.6%), and hlyII gene only grew in pasteurized milk (54%) (Figure 4b).
However, the detection techniques of B. cereus and its virulence factors are strongly limited [5,110,114], and B. cereus diagnostic method is still a field to be developed [135]. The widely used nucleic acid-based detection technology cannot accurately determine the bacterial activity and toxin expression [110,114]. Thus, the data analysis was incomplete since it is only based on the occurrence of toxin genes, while the level of protein expression and its toxic effects stay implicit. Therefore, the diversity and perfection of detection techniques of B. cereus and its virulence factors are necessary to improve the safety of dairy products in the future. Beyond that, the toxicity mechanisms of Bacillus virulence factors deserve to be illustrated, as host cells have frequently interacted with B. cereus infection [1,31,33,136,137]. Thus, comprehending the cytotoxicity of B. cereus is greatly beneficial in providing the therapeutic strategy for related illnesses caused by intaking Bacillus-contaminated dairy products. We believe that the summary of detection techniques of B. cereus will certainly be helpful for a more accurate examination and evaluation in the future.

Conclusions and Future Perspectives
The awareness of food safety in China has risen significantly with the mounting needs for high-quality foods. However, there still exist food poisoning incidents caused by bacteria like B. cereus in dairy products [5,12,13,70]. B. cereus group is an opportunistic spore-producing pathogen that causes food poisoning with symptoms of vomiting and diarrhea, exhalation of toxins that are the main culprit of damaging liver tissue and inflammatory diseases such as gastroenteritis and meningitis [16,17]. The infectious bacteria were the main focus of some widespread epidemics in history, and therefore, the safety of dairy products should not ever be ignored. In the past decades, although we have made arduous efforts to ensure food safety, the contamination of dairy products with B. cereus is still an issue in China. With regard to the average level in China, the investigation carried out from 2011 to 2016 unveiled that about 27.1% of the pasteurized milk on shelf were infested with B. cereus and also that the environments of milk production, handling and processing could introduce B. cereus into milk products. Together, these assessments implied the high prevalence of B. cereus and existence of potential hazards in contaminated pasteurized milk ( Figure 3 and Table 1).
On the other hand, Bacillus strains can also be used as human probiotics [138], and this field is gaining greater attention [90,139], as B. cereus strains or spores serve as probiotics for human use [140,141]. Some countries even utilize their connection with the dairy chain as a source to culture novel probiotic products [142]; however, the consequences could be unfavorable. In contrast to chemical drugs that quantitatively decrease or remain unchanged after metabolic process, the variation trend of the amounts of microbes like B. cereus in probiotics is undefined and could even rise exponentially.
In this review, we summarized the risk of B. cereus in dairy products in China and provided the analytical assays of B. cereus and its toxins. PCR is the most commonly used analysis method, accounting for 49% of articles on B. cereus (Figure 4a). However, the expression of genes does not completely represent the toxicity of the virulence factors. We need more comprehensive and rapid testing methods such as cytotoxicity tests or LC-MS analysis. Thus, the role in cell toxicity of the virulence factors of B. cereus is unquestionably important, and understanding the actions of B. cereus and its toxins at the cellular level would benefit the prevention of Bacillus infections. In addition, the cellular mechanism and the interaction between different virulence factors should be further studied. Recent reports showed that the persistent B. cereus interaction with host cells is even hard to control [1,49,143], and compared to other persistent bacteria, B. cereus are more dangerous due to the high transmission and viability of their spores [69,99,144]. In sum, strong emphasis should be placed on the B. cereus in dairy products to guarantee the safety of human life in China.

Conflicts of Interest:
The authors declare no conflicts of interest.