High Genetic Diversity and Virulence Potential in Bacillus cereus sensu lato Isolated from Milk and Cheeses in Apulia Region, Southern Italy

The Bacillus cereus group includes species that act as food-borne pathogens causing diarrheal and emetic symptoms. They are widely distributed and can be found in various foods. In this study, out of 550 samples of milk and cheeses, 139 (25.3%) were found to be contaminated by B. cereus sensu lato (s.l.). One isolate per positive sample was characterized by Multilocus Sequence Typing (MLST) and for the presence of ten virulence genes. Based on MLST, all isolates were classified into 73 different sequence types (STs), of which 12 isolates were assigned to new STs. Virulence genes detection revealed that 90% and 61% of the isolates harboured the nheABC and the hblCDA gene cluster, respectively. Ninety-four percent of the isolates harboured the enterotoxin genes entS and entFM; 8% of the isolates possessed the ces gene. Thirty-eight different genetic profiles were identified, suggesting a high genetic diversity. Our study clearly shows the widespread diffusion of potentially toxigenic isolates of B. cereus s.l. in milk and cheeses in the Apulia region highlighting the need to adopt GMP and HACCP procedures along every step of the milk and cheese production chain in order to reduce the public health risk linked to the consumption of foods contaminated by B. cereus s.l.


Introduction
The Bacillus cereus group, also named B. cereus sensu lato (s.l.), includes numerous closely related species widespread in the environment [1]; among these, B. cereus is the most important species from the food safety point of view. As a matter of fact, B. cereus, an anaerobic-facultative spore-forming bacteria, is able to survive in the food production environment and subsequently is able to contaminate a wide range of foodstuff: it has been found in milk [2][3][4], dairy products [3,[5][6][7], ice cream [8], fresh vegetables [9][10][11], meat [12], spices [13], seafood [14,15], cereal and derivatives [16], and rice [17]. In addition, its capability to form biofilm and viscous heat-resistant spores makes this organism very difficult to remove from food preparation surfaces and food production environments [18,19].

Strains Characterization by MLST
The results of the isolates characterization are summarized in Table 2 and Figure 1. A total of 139 isolates were characterized by the MLST and 73 STs were assigned; 12 isolates were assigned to new STs. Twenty-two isolates were grouped into nine clonal complexes

Strains Characterization by MLST
The results of the isolates characterization are summarized in Table 2 and Figure 1. A total of 139 isolates were characterized by the MLST and 73 STs were assigned; 12 isolates were assigned to new STs. Twenty-two isolates were grouped into nine clonal complexes (CCs). The dominant STs among all positive samples were ST 1986 and ST 34, instead, ST34, ST12 and ST26 were the dominant STs in Caciocavallo, ricotta cheese and mozzarella, respectively. The new detected STs were: ST2491, ST2667, ST2660, ST2661, ST2683, ST2664, ST2669, ST2666, ST2671, ST2675, ST2670 and ST2682.

Distribution of Enterotoxin Genes
The distribution of virulence genes is reported in Table 2 and Figure 2. According to the pathogenic characteristics of B. cereus s.l. the virulence genes were divided into two categories, namely enterotoxin coding genes (hblACD, nheABC, cytK, entFM, entS) and cereulide synthetase gene (ces). Among the enterotoxin genes, the genes of the nheABC cluster were detected in 90% of the isolates and in most of them was recovered the entire nheABC gene cluster; only three isolates were lacking the entire nheABC gene cluster. The gene cluster hblCDA was found in 61% of isolates, with a frequency not much different among the three genes detected: 68% of isolates harboured the hblA gene, 64% of isolates harboured the hblC gene and 75% of isolates harboured the hblD gene. The entFM and entS genes were detected in 94% of isolates; the cytK gene was identified in 44%. The ces gene was detected in 8% of the total isolates. Based on the distribution of both, enterotoxin and emetic genes, we divided our isolates in thirty-eight (1-38) different genetic profiles ( Table 2).

Distribution of Enterotoxin Genes
The distribution of virulence genes is reported in Table 2 and Figure 2. According to the pathogenic characteristics of B. cereus s.l. the virulence genes were divided into two categories, namely enterotoxin coding genes (hblACD, nheABC, cytK, entFM, entS) and cereulide synthetase gene (ces). Among the enterotoxin genes, the genes of the nheABC cluster were detected in 90% of the isolates and in most of them was recovered the entire nheABC gene cluster; only three isolates were lacking the entire nheABC gene cluster. The gene cluster hblCDA was found in 61% of isolates, with a frequency not much different among the three genes detected: 68% of isolates harboured the hblA gene, 64% of isolates harboured the hblC gene and 75% of isolates harboured the hblD gene. The entFM and entS genes were detected in 94% of isolates; the cytK gene was identified in 44%. The ces gene was detected in 8% of the total isolates. Based on the distribution of both, enterotoxin and emetic genes, we divided our isolates in thirty-eight (1-38) different genetic profiles (Table 2).

Statistical Analysis
When we compared the STs versus toxin gene profile by Spearman correlation (Graph-Pad Prism 8.1.1), no statistical significance was shown (r = −0.01462; p = 0.87144).

Discussion
B. cereus is a ubiquitous spore-forming bacterium widespread in the environment and frequently detected in a wide range of foodstuffs, including milk and cheeses, where it can act as foodborne pathogen. Because of the ubiquitous presence of B. cereus spores, dairy products can become contaminated during milking, processing or storage stages [45,46]. As a matter of fact, during 2007-2014, dairy products were responsible for 11 outbreaks due to B. cereus registered in EU [20]. The aim of our survey was to investigate the presence of B. cereus s.l., the genetic profile and the distribution of genetic virulence markers in the isolates obtained from milk (raw and pasteurized) and traditional cheeses from the Apulia region, southern Italy. In our study we found that 25.3% of all samples were contaminated by B. cereus s.l.; to the best of our knowledge, in Italy, data on the prevalence and the genetic characterization of B. cereus in milk and dairy products are scarce, thus it is not possible to make any comparison with previous reports. Other countries reported similar results that we found here: in China [2,47], Abidjan [48] and Brazil [49], B. cereus was detected in 27% of the milk and dairy products analysed. Recently, Adame-Gomez and colleagues reported a frequency of 29.5% of B. cereus contamination in artisanal cheese made with raw milk product in Mexico [50], whereas, in Ghana [3], Slovakia [51], India [4] and Northeast China [52], B. cereus was reported with a contamination level that ranged from 14 to 55%. Notably, among the food samples considered in this survey, we identified B. cereus s.l. in pasteurized milk; considering the ability of B. cereus to form spores that are able to survive heat treatment commonly applied in milk pasteurization [53,54], this result is not surprising. Although it is not possible to ascertain if the contamination occurs before, during the pasteurization or the filling process, spore germination and vegetative cells replication is a remote possibility, given that pasteurized milk must be stored under refrigeration and also considering its short shelf-life. In addition, psychrotrophic strains of B. cereus are known, but these isolates are generally recognized as not able to produce cereulide, thus the presence of this contamination should not pose a risk in refrigerated foods [55].
We analysed 390 samples of Caciocavallo, and 21.2% of these were contaminated by B. cereus s.l.. Caciocavallo is a very appreciated traditional curd-stretching cheese which has a large distribution in southern Italy, where it is produced from raw or pasteurized bovine milk; the season can last from two to six or more months [56].
In our survey, mozzarella and ricotta (soft cheeses) resulted contaminated in 25.8% and 11.5%, respectively, among all samples considered. These soft cheeses showed the highest rate of contamination compared other samples, with a contamination rate of 43.3% for mozzarella and 35.5% for ricotta. Mozzarella is the most famous Italian curd-stretching cheese, produced from raw or pasteurized cow or water buffalo milk [56]; in fact, some mozzarella cheeses have been awarded the protected designation of origin (POD). Ricotta is a much-appreciated food which is largely employed for direct consumption or as an ingredient for bakery and pastries products. The production process of this unripened acid-heat coagulated dairy originating from the whey obtained from draining cheese curds from curdling of other cheeses [57], includes a step in which it is applied a temperature that is able to reduce the pathogenic vegetative flora [58]. However, spore-forming bacteria could survive at these steps and, if other favourable conditions occur, they could germinate and multiply causing an active infection or an intoxication. In addition, it is not possible to establish whether the contamination occurs in the pre-or post-process steps, considering the heath resistance of B. cereus spores. Our results showed for the first time the high prevalence of B. cereus s.l. in milk and dairy products from Italy; it is known that dairy products, that are subjected to a mild heat treatment and are characterized by an extended shelf life under refrigeration, could be at risk because the competitive vegetative microflora is inactivated either by heat treatment or by reduced water activity, but enables the survival of the spores of B. cereus [53]. The high prevalence identified in our study supports the potential risk due to the consumption of B. cereus-contaminated dairy products; despite the existence of B. cereus psychrotolerant isolates capable to produce emetic toxin at low temperature [59], the main management option for controlling the growth of the organism in foodstuff remains the chill chain (storage temperature between <4 • C and 7 • C) that could be maintained during the entire production until the consumers receive them [49,54].
In our study, we did not assess the quantification of the samples contamination; on the other hand, it is known that it is very difficult to estimate the number of Colony Forming Unit (CFU) of B. cereus per g of food that could represent a risk for consumers: in literature cases in which 10 3 CFU/g were responsible of diseases are reported [20], although a concentration of B. cereus above 10 5 CFU/g is considered hazardous [60][61][62]. In the EU food law, B. cereus is considered as Process Hygiene Criterion for dried infant formulae and dried dietary foods for medical purposes intended for infants below six months of age; in case of exceeding the established limits (between 50 and 500 CFU/g), the food operator must improve his hygiene production procedures and prevent recontamination. In addition, they must carry out a selection of the raw material which has been used [63].
Based on the MLST profile, our isolates showed a high genetic diversity. Indeed, 12 of 73 MLST profiles (16.4% of the studied strains) were detected for the first time in this study; whereas the remaining profiles were already present in the MLST B. cereus database and, among these, twenty-three STs were previously identified in milk and milk products [64][65][66]. The other STs were previously identified in other foods or in the environment [65,[67][68][69][70]. B. cereus isolates related with emetic food poisoning, were generally associated with ST26 [33,42,43,71,72]; in our study, among the four isolates belonging to ST26, three harboured ces gene, supporting the hypothesis that this genotype frequently harbour the potential emetic strains; however, these strains did not carry the hbl cluster gene, as was already reported [33]. Interestingly, we identified nine additional STs (ST4, ST33, ST142, ST1936, ST2031, ST2045, ST2062, ST2667 and ST2666) that may become important to define the potential emetic strains for the presence of ces gene. Moreover, we noticed that all the isolates belonging to the CC18 and CC205 harboured the same virulence profile (profile 2). Furthermore, we identified an isolate that carried the ces gene and among the enterotoxin genes we detected only the entS gene. Based on genetic profile, we supposed that the infection due to the two isolates belonging to profile 1 (ST4; ST33) could be associated to a worst-case scenario [73,74], since they harboured all the genes investigated. The genetic profiles 2 and 5 represented the largest group, which included a total of 39 and 27 isolates, respectively. The isolates that belonged to genetic profile 2, including 19 STs, harboured all the enterotoxigenic genes, suggesting their enterotoxigenic potential; whereas the isolates that belonged to genetic profile 5, that included 21 STs, harboured eight of the nine enterotoxin genes, since they did not carry the cytK gene. This finding confirms that the health risk for consumers is strongly strain dependent, owing to the various pathogenic potential [20].
B. cereus diarrhoeal disease is due to the synthesis of different enterotoxins in the small intestine [29]; according to EFSA, about all strains of B. cereus carry the non-haemolytic enterotoxin complex genes (nhe), whereas about 30-70% of the isolates harboured the hbl and cytK-2 genes [20]. In our study, nine enterotoxigenic genes were detected and the proportion of hblACD, nheABC and cytK genes in B. cereus s.l. isolates were found to be, 61%, 90% and 44%, respectively. These frequencies were similar to previous studies [2,[75][76][77], suggesting that the diarrheagenic isolates have a high distribution among foods. Moreover, we detected two other enterotoxin genes (entFM and entS), which contribute to the severity of diarrheal illness [78]. Differently, the emesis is due to the heat-stable dodecadepsipeptide cereulide, that, similarly to other reports [3,79] we observed in 8% of the isolates. A number of studies failed to detect ces genes from food isolates [77,80], probably because the emetic isolates of B. cereus belong to a specific lineage that has a different geographical circulation [81]; however, it is difficult to give a conclusive opinion about the potential pathogenicity of the isolates because it is known that a large variability in synthesis of these enterotoxins exist among the B. cereus isolates, as well as it is still unknown why isolates that show the same genetic profile show differences in the amount of toxin production [19,47]. The strain's growth and the cereulide production are due to the composition of the food and the environment in different way; Ellouze recently demonstrated that in some food matrices (i.e., cereal-based foods) the growth rate of B. cereus is smaller than in other foods (i.e., milk-based products) but the cereulide production is faster than in foods where the growth is faster [82].

Conclusions
We believe that the potential hazard associated with contaminated foods by B. cereus s.l. should not be ignored. To ensure the protection of the public health, it is therefore necessary to develop procedures to prevent contamination of raw ingredients, to implement a plan based on hazard analysis and critical control point procedures during the manufacturing process of dairy products and strictly to respect the chill chain during their commercial and home storage.
The lack of information about the acidity, salinity, water activity, moisture and the cultures used for the production of the samples we analysed, factors that affect the growth of B. cereus, represent a weakness in our survey; further studies aimed at correlating these critical factors with the growth and toxin production by B. cereus in such foods are needed [44].