Psychrotrophic properties, toxigenic characteristics, and PFGE profiles of Bacillus cereus isolated from different foods and spices

Propriedades psicrotróficas, características toxigênicas e perfis PFGE de Bacillus cereus isolado de diferentes alimentos e especiarias

ABSTRACT:

Bacillus cereus is an aerobic and facultatively anaerobic, spore-forming bacterium, and it is found naturally in soil and poses a risk factor for the contamination of food and foodstuffs including cereals, vegetables, spices, ready-to-eat (RTE) foods, meats, milk, and dairy products. This study determined the prevalence of B. cereus in raw poultry meat, raw cow’s milk, cheese, spices, and RTE foods in Hatay province. The study also analysed the psychrotrophic properties, toxigenic characteristics, and pulsed-field gel electrophoresis (PFGE) profiles of the isolates. The levels of contamination with B. cereus determined for cheese, raw milk, RTE foods, spices, and raw poultry meat were 16.6%, 34.2%, 42.8%, 49%, and 55.5%, respectively. B. cereus was isolated from 84 (42%) of the 200 samples analysed and the 84 isolates were verified by PCR analysis targeting the haemolysin gene specific for B. cereus. Of the total isolates, 64 (76.1%) were psychrotrophic. The toxin gene profiling of B. cereus isolates was determined by amplifying the four genes nhe, hbl, cytK, and ces. The nhe and cytK genes were most frequently detected in the isolates, while the hbl and ces genes were not found. In addition, a high genetic relationship between the isolates was detected at a 92% similarity level by PFGE analysis. In conclusion, the occurrence of both psychrotrophic and toxigenic B. cereus strains in this study indicated a potential risk for food spoilage and food poisoning.

Key words:
Bacillus cereus; food poisoning; PCR; PFGE typing; toxin gene

RESUMO:

Bacillus cereus é uma bactéria formadora de esporos aeróbica e facultativamente anaeróbica, encontrada naturalmente no solo e representa um fator de risco para a contaminação de alimentos e alimentos, incluindo cereais, vegetais, especiarias, alimentos prontos para comer (RTE), carnes, leite e laticínios. Este estudo teve como objetivo determinar a prevalência de B. cereus em carne crua de aves, leite de vaca cru, queijo, especiarias e alimentos RTE na província de Hatay. O estudo também analisou as propriedades psicrotróficas, características toxigênicas e perfis de PFGE dos isolados. Os níveis de contaminação com B. cereus determinados para queijo, leite cru, alimentos RTE, especiarias e carne de frango crua foram de 16,6%, 34,2%, 42,8%, 49% e 55,5%, respectivamente. B. cereus foi isolado de 84 (42%) das 200 amostras analisadas e os 84 isolados foram verificados por análise de PCR visando o gene da hemolisina específico para B. cereus. Do total de isolados, 64 (76,1%) eram psicrotróficos. O perfil do gene da toxina de isolados de B. cereus foi determinado pela amplificação dos quatro genes nhe, hbl, cytK e ces. Os genes nhe e cytK foram detectados com maior frequência nos isolados, enquanto os genes hbl e ces não foram encontrados. Além disso, uma alta relação genética entre os isolados foi detectada em um nível de similaridade de 92% pela análise de PFGE. Em conclusão, a ocorrência de cepas psicrotróficas e toxigênicas de B. cereus neste estudo indica um risco potencial de deterioração e intoxicação alimentar.

Palavras-chave:
Bacillus cereus; intoxicação alimentar; PCR; tipagem PFGE; gene da toxina

INTRODUCTION:

Bacillus cereus is one of the major foodborne pathogenic bacteria that causes two types of food poisoning. The first, emetic syndrome, is characterised by nausea and vomiting and occurs within a few hours of consumption of contaminated food. Emetic syndrome is caused by a toxin called cereulide, a heat-stable peptide synthesised in the food. The second, diarrhoeal syndrome, is characterised by abdominal pain and diarrhoea. Diarrhoea is associated with some enterotoxins, the haemolysin BL (HBL), the non-haemolytic enterotoxin (NHE), and the cytotoxin K (CYTK). Diarrhoeal toxins are sensitive to heat and are synthesised during the vegetative growth of Bacillus cereus in the small intestine. These enterotoxins are particularly important in the virulence mechanism of B. cereus and are encoded by the three genes hbl, nhe, and cytK, respectively. There are also reports that the enterotoxin genes are more commonly found in B. cereus strains than the emetic gene (GRANUM & LUND, 1997; STENFORS ARNESEN et al., 2008; HARIRAM & LABBE, 2015; TEWARI et al., 2015; OWUSU-KWARTENG et al., 2017; FOGELE et al., 2018; GAO et al., 2018; BERTHOLD-PLUTA et al., 2019; SÁNCHEZ-CHICA et al., 2020b). At the same time, B. cereus can cause serious extraintestinal diseases such as bacteraemia, meningitis, arthritis, and endocarditis (LIU et al., 2016; YU et al., 2020), that need to be taken into consideration as well as food intoxication.

B. cereus is generally known as a mesophilic microorganism, which has optimal growth between 35 and 40 ^oC. However, increasing numbers of psychrotrophic B. cereus strains described can grow at refrigerator temperatures and are responsible from the microbial spoilage of refrigerated foods (GRANUM & LUND, 1997; MC KILLIP, 2000; WIJNANDS et al., 2006; STENFORS ARNESEN et al., 2008; PRETORIUS & BUYS, 2021). Until now, B. cereus has been isolated from different kinds of foods and foodstuffs such as meats, milk and dairy products, fish and seafood products, rice, starch, flours, spices and herbs, vegetables, ready-to-eat (RTE) foods, powdered foods (infant formula, powdered milk), and pastry (WIJNANDS et al., 2006; PARK et al., 2009; CADIRCI et al., 2013; HARIRAM & LABBE, 2015; TEWARI et al., 2015; OWUSU-KWARTENG et al., 2017; YIBAR et al., 2017; FOGELE et al., 2018; GAO et al., 2018; GDOURA-BEN AMOR et al., 2018; OSMAN et al., 2018; BERTHOLD-PLUTA et al., 2019; ADAME-GÓMEZ et al., 2020; SÁNCHEZ-CHICA et al., 2020a; SÁNCHEZ-CHICA et al., 2020b). The diarrhoeal disease is usually associated with proteinaceous foods, while the emetic type is more commonly due to the consumption of starch-rich foods (AGATA et al., 2002; STENFORS ARNESEN et al., 2008).

Considering that food safety is an important public health concern and consumption of foods contaminated with B. cereus can cause food intoxication, we investigated the prevalence of B. cereus in different foods and spices, which are very popular, traditional and often consumed by local people in Hatay province. B. cereus isolates were further analysed for their ability to grow at 7 ^oC, and the presence of toxin genes. Besides, PFGE analysis was carried out to evaluate the genetic relatedness of isolates.

MATERIALS AND METHODS:

Samples

In Hatay, the southeastern province of Turkey, 45 raw poultry meat, 35 raw cow’s milk, 30 cheese samples, 55 spices, and 35 ready-to-eat (RTE) foods were randomly collected from December 2018 to February 2020 and transported to the laboratory under cold chain conditions.

The spice samples were dried, powdered, and unpackaged and included thyme, mint, cumin, black pepper, red pepper, ginger, and sumac. The RTE foods included: Abagannus (a kind of salad prepared from roasted eggplant, roasted paprika, roasted tomato, pomegranate juice, garlic, olive oil, and salt), humus (hummus means chickpea in Arabic, and is made from chickpeas, sesame paste, lemon juice, garlic, salt, cumin, and olive oil), Muhammara (prepared for breakfast from walnut paste, tomato paste, spices, and olive oil), haydari (a kind of appetiser made from strained yogurt, mint, and dill), and olive salad (prepared for breakfast and made from green olives without seeds, parsley, tomatoes, green onions, olive oil, pomegranate juice, and red peppers). The cheese samples included traditional cheeses such as Sürk and Carra that are frequently consumed in Hatay province. They are both produced with spices or herbs. Carra cheese is made by mixing white cheese (manufactured from raw goat’s milk or cow’s milk) and Cokelek (cheese produced by heat-treating yogurt). Black cumin or thyme is added to the cheese. Sürk cheese is also made from Cokelek and thyme, cumin, mint, cinnamon, black peppers, ginger, and red peppers are used in its production.

Reference strain

Lyophilised Bacillus cereus strain was obtained from the National Type Culture Collection Laboratory, General Directorate of Public Health, Turkey, and used as a positive control in microbiological and molecular analysis.

Isolation and identification

The conventional culture method (TALLENT et al., 2012) and PCR technique were used in the isolation and identification of B. cereus. For the selective and pre-enrichment stages, 90 mL of Tripticase Soy Broth supplemented with polymyxin B (10 mg/mL) was added to each sample and they were homogenised. Then, the mixtures were incubated for 24 h at 30 °C. A loopful of each selective broth was taken, plated onto Chromogenic Bacillus cereus Agar (Oxoid CM1036) mixed with Brilliance Bacillus cereus Selective Supplement (Oxoid SR0230), and incubated at 30 ^oC under aerobic conditions. After 24^­_48 hours of incubation, all the suspicious blue/green colonies of B. cereus were taken and stored at ^_20 ^oC for molecular and toxin gene analysis.

Molecular analysis

For the molecular identification, all the presumptive B. cereus isolates were confirmed by the PCR technique based on the detection of the haemolysin gene. Genomic DNA was extracted using a GF-1 Nucleic Acid Extraction Kit (Vivantis, Malaysia) from the overnight Brain-Heart Infusion broth (Oxoid) cultures. DNA extraction was done according to the manufacturer’s instructions. Specific primer pairs and thermal cycling conditions described by WANG et al. (1997) were used in this study. The PCR amplicons were controlled in gel electrophoresis (1.5%) carried out at 100 V for 50 min (CS-300V, England). The gels were stained with Safe View (Abm G108) and visualised with a UV transilluminator (UVP, USA).

Toxin gene analysis

The toxin genes (hbl, nhe, cytK, ces) of B. cereus isolates were detected with a multiplex PCR. In the present study, hbl, nhe, cytK, and ces genes were searched using the following primers: (hblF, 5’-GTAAATTAIGATGAICAATTTC-3’; hblR, 5’-AGAATAGGCATTCATAGATT-3’) (nheF, 5’-AAGCIGCTCTTCGIATTC-3’; nheR, 5’-ITIGTTGAAATAAGCTGTGG-3’) (cytKF, 5’-ACAGATATCGGICAAAATGC-3’; cytKR, 5’-CAAGTIACTTGACCIGTTGC-3’) (cesF, 5’-GGTGACACATTATCATATAAGGTG-3’, cesR, 5’-GTAAGCGAACCTGTCTGTAACAACA-3’) (Ella Biotech) (EHLING-SCHULZ et al., 2005; EHLING-SCHULZ et al., 2006). The reaction mixture for multiplex PCR and amplification protocol was produced according to EHLING-SCHULZ et al. (2006).

Psychrotrophic property analysis

To detect the psychrotrophic properties of B. cereus, all isolates were subcultured in Brain-Heart Infusion broth and incubated for 24 h at 30 ^oC. Then, they were streaked on Tryptone Soya Agar. Plates were placed in a refrigerated incubator and the temperature was set at 7 ^oC. Plates were checked for bacterial growth after incubation periods of 24, 48, 72, and 168 hours.

Pulsed-field gel electrophoresis (PFGE) analysis

To better understand the genetic relationship between B. cereus strains, PFGE was performed according to LIU et al. (2016) using the SmaI macrorestriction enzyme. The 84 B. cereus isolates were first subjected to a cell lysis process to extract DNA. Then, genomic DNAs of the isolates were digested with SmaI (Takara, Japan) restriction enzyme. Following enzymatic digestion, they were subjected to electrophoresis in a CHEF-DR II system (Bio-Rad, Belgium). Then, electrophoretic bands were analysed under UV using a gel documentation system (Gel Doc, Bio-Rad, Belgium). The Bionumerics 5.10 software programme was used for dendrogram analysis, and the dendrogram was created using Dice coefficients. A similarity value was selected as 85% to reveal the genetic relationship of isolates.

RESULTS AND DISCUSSION:

Prevalence and psychrotrophic properties of Bacillus cereus

In the present study, 84 B. cereus isolates were recovered from 200 samples. High prevalence of B. cereus isolates was detected in poultry meat (25/45, 55.5%), followed by spices (27/55, 49%), RTE (15/35, 42.8%), raw milk (12/35, 34.2%), and cheese (5/30, 16.6%). Therefore, 84 isolates were molecularly confirmed as B. cereus by the amplification of the haemolysin gene (Figure 1). Sixty-four (76.1%) of the isolates had psychrotrophic abilities. All the isolated strains of poultry meat (100%) and eight of 15 (53.3%), eight of 12 (66.6%), 19 of 27 (70.3%), and four of five (80%) isolates recovered from RTE, milk, spices, and cheese, respectively, were able to grow at 7 ^oC (Table 1, 2 and 3).

Figure 1
PCR analysis of B. cereus isolates and toxin genes [M: 100 bp DNA marker, 1. Positive control, 2: Negative control, 3-5: B. cereus positive isolates, 6-7: cytK positive isolates (421 bp), 8-9: nhe (766 bp) positive isolates].

Table 1
Psychrotrophic property, toxin gene, and PFGE profiles of B. cereus isolates.

Isolate	-------Source--------	-Psychrotrophic property-	------------Toxin gene-----------			-Toxin profile-		---PFGE profile---
			cytK	nhe	hbl	ces		-------SmaI--------
1	Raw poultry meat	+	+	-	-	-	t1	A1
2	Raw poultry meat	+	-	+	-	-	t2	A1
3	Raw poultry meat	+	+	-	-	-	t1	C
4	Raw poultry meat	+	+	-	-	-	t1	C
5	Raw poultry meat	+	+	-	-	-	t1	C
6	Raw poultry meat	+	-	-	-	-	t4	C
7	Raw poultry meat	+	-	+	-	-	t2	C
8	Raw poultry meat	+	+	-	-	-	t1	C
9	Raw poultry meat	+	+	+	-	-	t3	A2
10	Raw poultry meat	+	-	-	-	-	t4	A2
11	Raw poultry meat	+	-	-	-	-	t4	A2
12	Raw poultry meat	+	-	-	-	-	t4	A2
13	Raw poultry meat	+	-	-	-	-	t4	A2
14	Raw poultry meat	+	-	-	-	-	t4	C
15	Raw poultry meat	+	-	-	-	-	t4	C
16	Raw poultry meat	+	-	-	-	-	t4	C
17	Raw poultry meat	+	-	-	-	-	t4	C
18	Raw poultry meat	+	-	-	-	-	t4	C
19	Raw poultry meat	+	-	-	-	-	t4	C
20	Raw poultry meat	+	-	-	-	-	t4	C
21	Raw poultry meat	+	-	-	-	-	t4	C
22	Raw poultry meat	+	-	-	-	-	t4	C
23	Raw poultry meat	+	-	-	-	-	t4	C
24	Raw poultry meat	+	-	-	-	-	t4	C
25	Raw poultry meat	+	-	-	-	-	t4	C
26	Raw cow milk	-	-	-	-	-	t4	C
27	Raw cow milk	+	-	-	-	-	t4	C
28	Raw cow milk	-	-	-	-	-	t4	B1
29	Raw cow milk	+	-	-	-	-	t4	B1
30	Raw cow milk	+	-	-	-	-	t4	B1
31	Raw cow milk	-	-	-	-	-	t4	C
32	Raw cow milk	+	-	-	-	-	t4	C
33	Raw cow milk	+	-	-	-	-	t4	C
34	Raw cow milk	-	-	-	-	-	t4	C
35	Raw cow milk	+	-	-	-	-	t4	C
36	Raw cow milk	+	-	-	-	-	t4	C
37	Raw cow milk	+	-	-	-	-	t4	C

• PFGE: pulsed-field gel electrophoresis; RTE: ready-to-eat; t: toxin profile; t₁: cytK ⁺, nhe - ,hbl - , ces - ; t₂: cytK ^-, nhe ⁺, hbl - , ces - ; t_3: cytK ⁺, nhe ⁺, hbl - , ces - ; t₄:cytK ^-, nhe ^-, hbl ^-, ces ^-.

Table 2
Psychrotrophic property, toxin gene, and PFGE profiles of B. cereus isolates.

Isolate	--Source--	--Psychrotrophic property--	---------------Toxin gene--------------			--Toxin profile--		--PFGE profile--
			--cytk--	--nhe--	--hbl--	-ces-		------SmaI-------
38	Cheese	-	-	+	-	-	t2	C
39	Cheese	+	-	+	-	-	t2	C
40	Cheese	+	-	-	-	-	t4	C
41	Cheese	+	-	-	-	-	t4	C
42	Cheese	+	-	+	-	-	t2	C
43	Spices	-	-	-	-	-	t4	C
44	Spices	+	-	-	-	-	t4	C
45	Spices	+	-	-	-	-	t4	C
46	Spices	+	+	+	-	-	t3	C
47	Spices	+	+	-	-	-	t1	C
48	Spices	+	+	-	-	-	t1	B2
49	Spices	+	-	-	-	-	t4	C
50	Spices	+	+	+	-	-	t3	C
51	Spices	-	+	+	-	-	t3	C
52	Spices	-	+	+	-	-	t3	C
53	Spices	+	+	+	-	-	t3	C
54	Spices	+	-	-	-	-	t4	C
55	Spices	+	+	+	-	-	t3	C
56	Spices	+	-	-	-	-	t4	C
57	Spices	+	+	+	-	-	t3	C
58	Spices	-	-	+	-	-	t2	C
59	Spices	-	+	-	-	-	t1	B1
60	Spices	-	+	-	-	-	t1	B1
61	Spices	-	+	+	-	-	t3	B1
62	Spices	+	-	+	-	-	t2	B1
63	Spices	+	+	+	-	-	t3	A2
64	Spices	+	+	+	-	-	t3	A2
65	Spices	+	-	-	-	-	t4	A2
66	Spices	+	-	+	-	-	t2	A2
67	Spices	+	+	+	-	-	t3	A2
68	Spices	-	+	+	-	-	t3	A2
69	Spices	+	-	+	-	-	t2	A2
70	RTE	-	-	-	-	-	t4	C
71	RTE	-	+	+	-	-	t3	C
72	RTE	+	+	+	-	-	t3	C

• PFGE: pulsed-field gel electrophoresis; RTE: ready-to-eat; t: toxin profile; t₁: cytK ⁺, nhe - ,hbl - , ces - ; t₂: cytK ^-, nhe ⁺, hbl - , ces - ; t_3: cytK ⁺, nhe ⁺, hbl - , ces - ; t₄:cytK ^-, nhe ^-, hbl ^-, ces ^-.

Table 3
Psychrotrophic property, toxin gene, and PFGE profiles of B. cereus isolates.

Isolate	--Source--	-Psychrotrophic property-	----------------Toxin gene-----------------				-Toxin profile-	-PFGE profile-
			--cytk--	--nhe--	--hbl--	-ces-		------SmaI------
73	RTE	+	-	-	-	-	t4	C
74	RTE	+	-	+	-	-	t2	C
75	RTE	+	-	+	-	-	t2	C
76	RTE	+	-	-	-	-	t4	C
77	RTE	-	-	-	-	-	t4	C
78	RTE	-	-	-	-	-	t4	C
79	RTE	-	-	-	-	-	t4	C
80	RTE	-	-	-	-	-	t4	C
81	RTE	+	-	-	-	-	t4	C
82	RTE	+	-	-	-	-	t4	C
83	RTE	+	-	-	-	-	t4	C
84	RTE	-	-	-	-	-	t4	C

• PFGE: pulsed-field gel electrophoresis; RTE: ready-to-eat; t: toxin profile; t₁: cytK ⁺, nhe - ,hbl - , ces - ; t₂: cytK ^-, nhe ⁺, hbl - , ces - ; t_3: cytK ⁺, nhe ⁺, hbl - , ces - ; t₄:cytK ^-, nhe ^-, hbl ^-, ces ^-.

The overall prevalence of B. cereus was 42% in this study. Compared to our data, different results have previously been reported as 30.9% by TEWARI et al. (2015), 40.4% by OWUSU-KWARTENG et al. (2017), 25.3% by GDOURA-BEN AMOR et al. (2018), 27% by GAO et al. (2018), 29.4% by ADAME-GÓMEZ et al. (2020), and 11% by SÁNCHEZ-CHICA et al. (2020b) in various foods in other countries. When comparing the results with other studies by food type, the B. cereus group was isolated from chicken meat at 50% (OSMAN et al., 2018), like this study (55.5%). WIJNANDS et al. (2006) and GDOURA-BEN AMOR et al. (2018) detected B. cereus in 3.0% vof meat and meat products, and 9.4% of raw poultry meat. Interestingly, GDOURA-BEN AMOR et al. (2018) and TEWARI et al. (2015) reported high incidence levels in cooked poultry compared to raw poultry samples. They explained this finding by the germination of B. cereus spores during inappropriate storage and the inadequate cooking of food.

Given that B. cereus is a common soil bacterium, it is often reported in milk. However, large numbers are needed in milk to cause intoxication and at this stage defects in milk are easily observed. In some dairy products, it is more difficult to observe spoilage and they pose a serious risk for Bacillus cereus food poisoning (STENFORS ARNESEN et al., 2008). For raw milk and dairy products, the prevalence of B. cereus in cheeses reported in this study was very similar to that reported by SÁNCHEZ-CHICA et al. (2020a) (19.6%), whereas WIJNANDS et al. (2006) and YIBAR et al. (2017) reported a lower prevalence (10%) in milk and milk products. Contrary to the above studies, BERTHOLD-PLUTA et al. (2019) detected B. cereus contamination in 41.7% of cheese samples.

In the studies of YIBAR et al. (2017), GAO et al. (2018), and BERTHOLD-PLUTA et al. (2019), the prevalence of B. cereus in pasteurised milk was reported as 30%, 27%, and 26%, respectively. This may be considered as higher contamination for pasteurised milk since heat treatment is applied to pasteurised milk. The authors suggested that low/inadequate pasteurisation or post-pasteurisation contamination could be the reason. The heat-stable spores of B. cereus are a problem in milk and dairy products as they are not inactivated during the pasteurisation (BENAHMED et al., 2020; PRETORIUS & BUYS, 2021). When comparing raw milk results, a lower incidence of 3.8% was reported by YIBAR et al. (2017). Contrarily, OWUSU-KWARTENG et al. (2017) reported a relatively higher prevalence in raw milk (47%), yogurt (35%), and cheese (39%). In addition, GUNDOGAN & AVCI (2014) found that raw milk, cheese, and ice cream samples were contaminated with B. cereus at levels of 90%, 70%, and 20%, respectively. B. cereus has also been identified in 13% of powdered milk (SÁNCHEZ-CHICA et al., 2020b), and 30% and 11% of infant formulas, respectively, in the studies of BERTHOLD-PLUTA et al. (2019), and SÁNCHEZ-CHICA et al. (2020b), and 7% of dairy desserts (CADIRCI et al., 2013).

Regarding the contamination level in spices, AKSU et al. (2000) and BERTHOLD-PLUTAet al. (2019) reported that the incidence of B. cereus in herbs and spices was 63%, with counts ranging from 2.0 to log 3.0 log cfu/g. In our study, the prevalence of B. cereus in spices was relatively high (49%) compared to the studies of GDOURA-BEN AMOR et al. (2018) and HARIRAM & LABBE (2015) with 28.8% and 31%, respectively, whereas FOGELE et al. (2018) reported a higher prevalence (76%) in spices. However, WIJNANDS et al. (2006) isolated B. cereus from 11.5% of flavourings in the Netherlands. Since B. cereus is a spore-forming and common soil microorganism, it could be difficult to eliminate this bacterium from food processing plants and the environment. In another context, PARK et al. (2009) found that 25% of cereal samples were positive for B. cereus in Korea. High contamination levels (56.7%) have also been reported in coffee samples by SOUZA & ABRANTES (2011). These results demonstrated that a higher prevalence in spices is possible due to the occurrence of this bacterium in soil and plants (GRANUM & LUND, 1997; STENFORS ARNESEN et al., 2008; BERTHOLD-PLUTA et al., 2019). As spices are often used as aromatic and flavour components in many types of foods (WIJNANDS et al. 2006; HARIRAM & LABBE 2015; FOGELE et al., 2018), this can be a potential risk for the cross-contamination of some food products, such as RTE foods, because they are not subjected to further heat treatment before consumption (BERTHOLD-PLUTA et al. 2019). In this sense, it is essential to ensure the microbial safety of spices. As other researchers have recommended (AKSU et al. 2000; FOGELE et al., 2018), we suggested sterilisation and the hygienic and adequate packaging of spices.

RTE foods such as Abagannus, humus, haydari, muhammara, and olive salad are very popular and commonly consumed in Hatay province, but they may pose a potential risk for B. cereus contamination as they are prepared without any heat treatment and consumed directly. In this study, 15 (42.8%) out of 35 RTE samples were found to contain B. cereus, consistent with other studies where B. cereus prevalence in RTE foods was reported as 48.2% by WIJNANDS et al. (2006) and 35% by YU et al. (2020). The majority (76.1%) of our isolates were psychrotrophic, which means that they can grow slightly during refrigeration storage and can cause spoilage in refrigerated foods. Unlike our results, WIJNANDS et al. (2006) determined that the minority of their isolates (4.4%) were psychrotrophic using the PCR method while most of them were mesophilic. However, ADAME-GÓMEZ et al. (2020) reported that almost all their isolates (91.3%) were psychrophilic. Also, BERTHOLD-PLUTA et al. (2019) reported that 25% of their isolates could grow at 7 ^oC. In this respect, the presence of psychrotropic B. cereus strains in various food products cannot be ignored since they have a significant effect especially on the quality and shelf life of refrigerated foods.

Detection of toxin genes by multiplex PCR

We found that 41.6% of our isolates harboured toxin genes while the remainder had none. The genes cytK and nhe were commonly detected in the isolates alone or together, but the other enterotoxin encoding gene, hbl, and emetic gene (ces) were not determined in this study (Table 1, 2 and 3).

According to the presence of toxin genes, four toxin gene profiles (t₁; toxin profile 1, t₂; toxin profile 2, t_3; toxin profile 3, and t_4; toxin profile 4) were detected in the study. Nine (10.7%), 11 (13%), 15 (17.8%), and 49 (58.3) isolates belonged to t₁ (cytK ⁺, nhe ^- , hbl ^- , ces ^- ), t₂ (cytK ^- , nhe ⁺, hbl ^- , ces ^- ), t₃ (cytK ⁺, nhe ⁺, hbl ^- , ces ^- ), and t₄ (cytK ^-, nhe ^-, hbl ^-, ces ^-), respectively. The most prevalent toxin profile among the enterotoxigenic isolates was t₃, including both cytK and nhe genes.

When reviewing studies performed in Turkey and elsewhere (PARK et al., 2009; CADIRCI et al. 2013; HARIRAM & LABBE, 2015; TEWARI et al., 2015; OWUSU-KWARTENG et al., 2017; WALKER YORK MOORE et al., 2017; YIBAR et al., 2017; FOGELE et al., 2018; OSMAN et al., 2018; ADAME-GÓMEZ et al., 2020; SÁNCHEZ-CHICA et al., 2020a; SÁNCHEZ-CHICA et al., 2020b; YU et al., 2020), enterotoxin genes were more frequently found than the emetic gene, indicating that diarrhoeal strains have a widespread distribution, consistent with this study. However, we did not identify any emetic strain since all the isolates lacked the ces gene, like the findings of HARIRAM & LABBE (2015), FOGELE et al. (2018), ADAME-GÓMEZ et al. (2020), SÁNCHEZ-CHICA et al. (2020a), and SÁNCHEZ-CHICA et al. (2020b).

On the contrary, in the studies of BERTHOLD-PLUTA et al. (2019), GAO et al. (2018), OWUSU-KWARTENG et al. (2017), and YU et al. (2020), the ces gene was detected but it was the least frequently observed toxin gene, reported in only 9%, 7%, 5%, and 1.2% of B. cereus strains, respectively. Also, WIJNANDS et al. (2006) found the gene responsible for cereulide-like toxin production in 8.2% of their isolates, not alone but in combination with the presence of cytK, nhe, and hbl genes. As mentioned previously, food matrices affect the growth and toxin production of B. cereus and emetic syndrome usually occurs from starchy food products (MCKILLIP, 2000; AGATA et al., 2002; WALKER YORK MOORE et al., 2017). Interestingly, the B. cereus strains carrying the emetic toxin gene or having the ability to produce cereulide-like toxin have been isolated from raw milk and milk products (OWUSU-KWARTENG et al., 2017), pasteurised milk (GAO et al., 2018), cooked meat (YU et al., 2020), and fish and fish products (WIJNANDS et al., 2006). However, according to AGATA et al. (2002), emetic toxin production was extremely low in such foods compared to bacterial growth.

Analysis of genetic relationship by PFGE

Using PFGE, three major patterns (A, B, C) were obtained with the macrorestriction enzyme SmaI among the 84 B. cereus strains investigated. PFGE patterns A and B were both divided into two sub-groups: A₁ and A_2, B₁ and B₂, whereas PFGE pattern C generated only one group. PFGE patterns B, A, and C consisted of eight (9.5%), 14 (16.6%), and 62 (73.8%) B. cereus strains, respectively. To compare clonality, a high genetic relationship between the isolates was detected at the 92% similarity level (Figure 2).

Figure 2
Pulsed-field gel electrophoresis (PFGE) dendrogram showing the relationship between B. cereus isolates.

Molecular-based techniques such as PFGE, multi-locus sequence typing (MLST), and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) are widely used for bacterial typing, revealing the genetic relationship or genetic diversity. When we analysed our isolates by PFGE, we observed a high genetic relationship between them, and three major PFGE pulsotype profiles were obtained. Unlike our study, LIU et al. (2016), GAO et al. (2018), PARK et al. (2009), and SÁNCHEZ-CHICA et al. (2020a) reported high numbers of pulsotypes using PFGE and ERIC-PCR typing, indicating that there was high genetic diversity among their isolates. GDOURA-BEN AMOR et al. (2018) found that 33.3% of their isolates were divided into 27 different clusters while the remainder were clustered individually. In another study, large genetic diversity was observed in Colombia among 46 B. cereus isolates recovered from powdered food samples that were grouped in seven clusters (SÁNCHEZ-CHICA et al., 2020b). In China, when YU et al. (2020) detected the genetic diversity of B. cereus isolates by MLST, 192 different sequence types were obtained, and 93 of them were new. In this respect, we can say that molecular typing methods can help us better understand the molecular epidemiology of B. cereus strains. Conversely, PFGE has been accepted as the gold standard for the discrimination and molecular characterisation of bacterial strains. In addition, using more than one restriction enzyme in PFGE analysis could increase the discriminatory power of the method, but we used only one restriction enzyme (SmaI) in the present study.

CONCLUSION:

Our results indicated that the tested foods were frequently contaminated with B. cereus and that 31% of the B. cereus strains were both psychrotrophic and enterotoxigenic, which may increase the potential risk for both food spoilage and food poisoning. In addition, most of the isolated strains show similar PFGE patterns indicating that there is a high genetic relationship between them. As a high genetic relationship was detected between our isolates from different samples, cross-contamination of samples with these bacteria may be considered. This study was conducted in a specific region where local people consume a great many of these products. The study reveals the potential sources of B. cereus and provides a scientific basis for further investigation. Of course, more studies should be carried out to better understand the virulence mechanism and molecular epidemiology of B. cereus strains.

ACKNOWLEDGEMENTS

Part of this study was presented at the VIII National/II International Veterinary Food Hygiene Congress, 24-27 October 2019, Antalya, Turkey.

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• CR-2021-0166.R2

Edited by

Editors:

Rudi Weiblen

Cristiano Ragagnin de Menezes

Publication Dates

• Publication in this collection
  11 Oct 2021
• Date of issue
  2022

History

• Received
  01 Mar 2021
• Accepted
  05 July 2021
• Reviewed
  24 Aug 2021