1. Introduction
Bacillus cereus (Frankland and Frankland 1887) (
Bacillales, Bacillaceae) is a parthenogenic anaerobic Gram-positive bacterium. It is one of the
Bacillus species, with a short rod-like circumference, flagella, and oval-shaped budding spores usually located centrally or somewhat offset [
1]. Most of the
B. cereus exhibit different forms depending on the environment in which they are observed. They can be isolated from environmental reservoirs, such as soil, marine sediment, seawater, and plants [
2,
3]. Some
B. cereus strains have high virulence potential and are involved in various invasive and lethal infections [
4].
Bacillus cereus causes two different types of gastrointestinal disorders, namely, vomiting and diarrhea syndrome, which are caused by different types of toxins [
5]. Vomiting is caused by the vomiting toxin it produces, and diarrhea is caused by a hemolytic enterotoxin (hemolysin BL, Hbl), a non-hemolytic enterotoxin (Nhe), a cytotoxin K (CytK), and several other enterotoxins [
5,
6].
Bacillus cereus can grow rapidly under a wide range of conditions and can remain dormant under adverse environmental conditions. Thus, it can survive the heating of food and rapid multiply upon entering the body, leading to human infection [
7].
Bacillus cereus is an important foodborne pathogen that is ubiquitous in nature and can be isolated from many different foods and food ingredients [
8,
9]. In 2018, the EU had 98 foodborne illness outbreaks, with
B. cereus ranking fifth behind
Salmonella (Salmon 1885),
Campylobacter (Marshall et al. 1983),
norovirus (Kapikian 1972), and
Staphylococcus aureus (Ogston. 1882) [
10]. A study that analyzed 3654 food samples tested in connection with the occurrence of foodborne illnesses detected the presence of
B. cereus in 187 of these samples, with a contamination rate of 5% [
11]. In addition to foodborne illnesses such as diarrhea and vomiting,
B. cereus can cause various types of illnesses, including fulminant bacteremia, meningitis, pneumonia, gas gangrene infection, and endophthalmitis [
12,
13].
Bacillus cereus isolates have high genetic diversity. Conventional phenotypic identification methods distinguish between
B. cereus and its constituent members [
14]. Genome-based taxonomic criteria are increasingly being accepted for bacterial classification and species identification [
15]. With the rapid spread of whole-genome sequencing technology, the whole-genome information of
B. cereus has become widely available. Genome-wide data-based typing methods for
B. cereus provide high resolution. These include Core Genome multilocus sequence typing (cgMLST), single-nucleotide polymorphism (SNP) analysis, and a genome-wide average nucleotide identity-setting threshold-based method [
16,
17,
18]. Recent studies have found that for different taxonomic systems,
B. cereus currently includes a total of 23 published subspecies [
19].
The traceable typing of bacteria allows for the precise differentiation of bacteria, and certain differences exist in biological properties among different strains of the type within the same species, leading to various measures for their control. By studying the similarity of bacteria in an event, the type and source of the bacterial strain causing the infection can be determined, allowing for the most appropriate treatment and prevention protocols to be developed and thus prevent an outbreak. The earliest methods of bacterial trace typing include serotyping, phage typing, and high pulse-field gel electrophoresis (PFGE). However, such trace-typing methods are often accompanied by drawbacks such as cumbersome operation, low resolution, and weakly transferable results [
20]. Whole-genome-sequencing (WGS)-based bacterial typing methods have great discriminatory power and data transferability for the epidemiological surveillance and clinical treatment of bacteria. With the rapid development of sequencing technologies, the cost of WGS decreases significantly, and WGS-based strain analysis methods are extensively used for bacterial biological characterization [
21]. WGS has a higher resolution than PFGE in several strains in which horizontal transfer is known to exist. WGS-based bacterial-typing methods have begun to replace molecular methods such as PFGE as the “gold standard” for bacterial subtyping [
22]. WGS has become an important tool for investigating foodborne disease outbreaks, and some countries have incorporated WGS into their national food safety control systems. In 2019, China established a WGS-based molecular traceability network for foodborne diseases, providing a strong guarantee for the prevention and control of foodborne pathogenic bacteria [
23].
Further studies on strain WGS can provide insights into a strain’s resistance profile, virulence gene evaluation, and molecular typing characteristics. Comparative WGS analyses with routine diagnostic workups of tuberculosis mycobacteriosis have shown that WGS predicts species with 93% accuracy and offers drug sensitivity accuracy. WGS could reportedly diagnose a case of multidrug-resistant conjugate mycobacteriosis before the end of routine diagnostics, and WGS traceability showed that the analyzed samples were closely related to
Mycobacterium tuberculosis (Zopf 1883) in the United Kingdom [
24]. When PFGE and WGS are used to compare
Listeria monocytogenes (E. Murray et al. 1926) isolates, WGS found that two
L. monocytogenes strains with different geographic origins but closely related PFGE profiles significantly differed in terms of antibiotic and heavy metal resistance determinants, as well as removable genetic elements [
25]. Molecular surveillance using WGS can be an important complement to the phenotypic surveillance of antibiotic resistance. WGS can also provide insights into the genetic basis of a strain’s resistance mechanisms to antibiotics, as well as into the evolution and population dynamics of pathogens on different spatial and temporal scales [
26]. Thus, WGS has become an important tool for public health surveillance and molecular epidemiological studies of infectious diseases. It enables an accurate geographical description of transmission and the ability to monitor pathogen incidence at the genotype level. Coupled with epidemiological and environmental surveys, it can provide the ultimate solution for tracing the source of epidemic infections [
27].
4. Discussion
Six types of commercially available plant foods were sampled in eight regions of a province, and 273 food samples were obtained. A total of 73 strains of B. cereus were isolated, purified, and identified. They were subjected to molecular epidemiology studies through Illumina II whole-genome sequencing, and molecular epidemiology studies were conducted on the whole-genome sequences of the 73 strains of B. cereus by bioinformatics methods. The overall situation of B. cereus contamination in plant foods in various regions of a province is complex, with obvious differences between regions and establishments, and attention should be paid to improving the food safety protection system and strengthening the supervision, prevention, and control of seriously contaminated regions and establishments. There are many reasons for the differences, which may be due to the growth habit of plant foods, the production process, the environment of production and operation, as well as the growth characteristics of B. cereus itself.
The
B. cereus population includes many closely related species, with pathogenic harmful bacteria as well as probiotic bacteria with positive effects on human health and agricultural development, so the precise differentiation of
B. cereus subspecies is essential for public health risk assessment as well as for industrial and agricultural development.
Bacillus paranthracis is a new, recently delineated subtype that has been found in various environments [
38,
39]. Some researchers have identified five peroxidase genes that can promote the growth of lactic acid bacteria during fermentation in the genome of
B. paranthracis isolated from fermented rice bran [
40]. In the present study, 13 strains of
B. paranthracis were also detected from pickled vegetables, and these strains may have similarly positive effects on the fermentation of vegetables.
As an important foodborne pathogen,
B. cereus can cause various degrees of food poisoning. Notably, we detected
B. thuringiensis, which is widely used for agricultural pest control, in the presence of virulence genes.
Bacillus thuringiensis isolated from fermented soybeans was found to contain diarrhea-type enterotoxin genes, consistent with our results [
41]. This finding suggested that
B. thuringiensis had the same pathogenic potential, and relevant industries should be aware of the presence of such subspecies when using or regulating them to prevent the occurrence of diseases.
In this study, epidemiological investigations and traceability analyses of
B. cereus using MLST typing and cgSNP typing showed that the 73 strains of
B. cereus as a whole were widely distributed, with rich genetic diversity, and yet no correlation was found with the source of the strains. However, some researchers have analyzed 44 whole-genome sequences of
B. cereus isolates from different sources in Japan and found that
B. cereus from different regional sources are also closely related genetically [
42]. Finally, the prevalence of
B. cereus should be continuously monitored to achieve rapid epidemiological analyses of
B. cereus and effective prevention of foodborne illnesses, providing a strong guarantee for the surveillance of
B. cereus.
5. Conclusions
In this study, the prevalence of B. cereus in plant foods was investigated, and the presence of other species of bacterial contamination of the samples was found during testing. Therefore, it is necessary to identify the microbial communities of the samples, analyze the presence of other pathogenic bacteria, and study the population abundance and potential risk of pathogenicity of the samples. Due to the limited number of samples collected in this study, in order to obtain an accurate contamination rate of B. cereus in plant foods, the sampling volume may be increased appropriately for further analyses. In addition, the genomic characteristics of B. cereus subspecies are extremely similar to each other, and the GTDB typing scheme is based on multiphase genomics with gene-averaged nucleotide identity for the standardized typing scheme to classify and name the subspecies of B. cereus, which may lead to the similarity or concordance of physiological characteristics between different subspecies, thus affecting the judgement of the functional characteristics of the strains. When the virulence genes were analyzed, it was found that the characteristics of virulence genes were not obvious among different subspecies, so the physiological characteristics of the strains can be studied subsequently for different subspecies of B. cereus, which can provide a scientific basis for the classification of B. cereus. In order to ensure the effectiveness of traceability and achieve rapid traceability analysis of B. cereus, it is necessary to continuously update the content of the traceability database of B. cereus established in this study and the related bioinformatics analysis software (Version 1.0) in the server, so as to achieve the purpose of accurate traceability of B. cereus.