Detection of Toxigenic Bacillus cereus Strains Isolated from ...

2144 Journal of Food Protection, Vol. 77, No. 12, 2014, Pages 2144–2147 doi:10.4315/0362-028X.JFP-13-479 Copyright G, International Association for Food Protection

Research Note

Detection of Toxigenic Bacillus cereus Strains Isolated from Vegetables in Mexico City ´ N,1 IVA ´ N NATIVIDAD-BONIFACIO,1 CARLOS R. VA ´ ZQUEZ-QUIN ˜ ONES,2 KAREN A. FLORES-URBA ´ ZQUEZ-SALINAS,2 AND ELSA IRMA QUIN ˜ ONES-RAMI´REZ1* CARLOS VA 1Departamento

de Microbiologıá, Escuela Nacional de Ciencias Biolo´gicas, Instituto Politećnico Nacional, Calle Carpio y Plan de Ayala s/n, C.P. 11340, Mexico City, Mexico; and 2Departamento de Biotecnologıá, Divisioń de Ciencias Biolo´gicas y de la Salud, Universidad Autońoma Metropolitana-Iztapalapa, Avenida San Rafael Atlixco 186, Colonia Vicentina, C.P. 09340, Mexico City, Mexico MS 13-479: Received 8 November 2013/Accepted 6 July 2014

ABSTRACT Bacillus cereus can cause diarrhea and emetic syndromes after ingestion of food contaminated with it. This ability is due to the production of enterotoxins by this microorganism, these being the hemolysin BL complex, which is involved in the diarrheal syndrome, and cereulide, which is responsible for the emetic syndrome. The detection of genes associated with the production of these toxins can predict the virulence of strains isolated from contaminated food. In this paper, we analyzed 100 samples of vegetables, 25 of each kind (broccoli, coriander, carrot, and lettuce) obtained from different markets in Mexico City and its metropolitan area. B. cereus was isolated in 32, 44, 84, and 68% of the samples of broccoli, carrot, lettuce, and coriander, respectively. The hblA gene (encoding one of the three subunits of hemolysin BL) was amplified in 100% of the B. cereus isolates, and the ces gene (encoding the cereulide) could not be amplified from any of them. This is the first report of B. cereus isolation from the vegetables analyzed in this work and, also, the first report in Mexico of the isolation from vegetables of strains with potential virulence. The results should serve as evidence of the potential risk of consuming these foods without proper treatment.

The World Health Organization estimates that, each year, at least 2 billion people worldwide (approximately one-third of the world’s population) become sick from ingesting unsafe food and that 2.2 million die from these diseases (32). Bacillus cereus is one of the microorganisms involved; it can cause intestinal and extraintestinal symptoms. Among the intestinal alterations, food poisoning is characterized by diarrhea (due to various enterotoxins, such as nonhemolytic enterotoxin, the hemolysin BL [HBL] complex, cytotoxin K, enterotoxin T, and enterotoxin FM (25, 28)) and/or emetic (due to cereulide) syndromes (9, 28). The number of B. cereus cells capable of causing disease has been estimated to range between 104 and 108 cells per g of food (31). Outbreaks of diarrheal and emetic syndromes caused by this organism have been widely reported in Japan (10), the United States (2), The Netherlands (10), Thailand (6), Africa (20), South Korea (21), the United Kingdom (1, 17), Belgium (24), and Finland (26). Outbreaks caused by B. cereus have usually been associated with rice or other grains and vegetables that contain starch in their composition (14). About 95% of emetic-type food poisoning is due to the consumption of rice, macaroni, cheese, and vanilla pudding (13, 16, 28), whereas outbreaks of the diarrheal type are most often related to spices, fruits, nuts, grains, dairy * Author for correspondence. Tel: (z52 55) 57296300, Ext 62375; Fax: (z52 55) 57296007; E-mail: [email protected].

products, soups, and dry foods, although vegetables have also been involved (12, 19). The increase in the consumption of foods with a mild thermal treatment or that are minimally processed and refrigerated has favored the increase of spore-forming pathogenic bacteria, including some strains of psychrotrophic B. cereus (12, 18). Similarly, pasteurization of the final container eliminates the risks arising from vegetative pathogenic bacteria but is insufficient for the removal of spores, including those of B. cereus (24). In Mexico, there are no data available to assess the epidemiologic importance of B. cereus as the causative agent of enteric diseases, even though its possible presence in fresh vegetables could represent a health risk, especially when food is consumed that contains these vegetables with minimal processing. Thus, the aim of this work was to identify the presence of B. cereus in vegetables that are eaten fresh and to search for the hblA and ces genes, which are involved in the diarrheal and the emetic syndrome, respectively. MATERIALS AND METHODS Sampling and isolation of B. cereus. A total of 100 samples of several vegetables, including broccoli (n ~ 25), coriander (n ~ 25), carrot (n ~ 25), and lettuce (n ~ 25), obtained from various establishments in Mexico City and its surrounding metropolitan area, were analyzed. Four samples of each kind of vegetable were

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TABLE 1. Numbers of samples contaminated with Bacillus cereus (all of which showed the presence of the hblA gene) and MPN values Vegetable

Broccoli Carrot Lettuce Coriander Total

No. of positive samples/ total no. of samples (%)

8/25 11/25 21/25 17/25 57/100

(32) (44) (84) (68) (57)

MPN/g

.3–460 .3–9.2 .3–240 .3–1,100

gathered weekly (16 samples per month), between February and August 2012. For isolation, the methodology described in the Bacteriological Analytical Manual (4, 29) was followed. Series 3, 3, 3 of the most-probable-number (MPN) method were used, by weighing 50 g of each sample and placing them in a 450-ml flask containing 0.1% alkaline peptone water. From this solution, serial dilutions were made until 1023, and each dilution was transferred to 1-ml tubes containing Trypticase soy broth (Becton Dickinson France SA, Pont de Claix, France) containing polymyxin B. The tubes were incubated for 48 h at 30uC. Each tube with dense growth was cross-seeded on mannitol–egg yolk–polymyxin B (Oxoid Ltd. Basingstoke, UK) agar plates and incubated for 24 to 48 h at 30uC. Colonies morphologically characteristic of B. cereus were selected and identified using glucose–phenol red broth, nitrate broth, Voges-Proskauer broth (Becton Dickinson France SA), tyrosine agar, and lysozyme broth (Sigma-Aldrich, St. Louis, MO). The MPN per gram was determined as a measure of the concentration of B. cereus in samples, comparing the results with tables in the Bacteriological Analytical Manual (4). The reference strain B. cereus ATCC 14579 was used as the positive control. Preparation of DNA and amplification of hblA and ces genes by PCR. Genomic DNA was obtained by using the Wizard Genomic DNA purification kit (Promega, Madison, WI), following the manufacturer’s specifications. For PCR, a reaction mixture was prepared with 14.8 ml of sterile distilled water, 2.5 ml of 10| buffer (supplied with Taq polymerase), 1.3 ml of 50 mM MgCl2, 0.2 ml of deoxynucleoside triphosphates (10 mM) (Invitrogen, Carlsbad, CA), 2 ml of each primer, 1 U of Taq DNA polymerase

FIGURE 2. Agarose gel electrophoresis of PCR amplification product of the ces gene. Lane 1, lambda DNA/HindIII molecular weight marker; lane 2, positive control; lanes 3 to 6, samples. (Invitrogen), and 2 ml of a solution containing about 100 ng of DNA. Amplifications were performed on a MultiGene gradient thermal cycler, using primers for the hblA (20) and ces (11) genes. For hblA amplification, the following conditions were used: 5 min at 94uC, 30 cycles of 45 s at 94uC, 45 s at 51uC, and 1 min at 72uC, followed by one cycle of 5 min at 72uC. For ces amplification, the following conditions were used: 5 min at 94uC, 30 cycles of 45 s at 94uC, 45 s at 55uC, and 45 s at 72uC, followed by one cycle of 5 min at 72uC. A total of 6 ml of PCR product was then loaded onto an agarose gel containing ethidium bromide and visualized under UV light. DNA from the reference strains was used as positive controls; B. cereus ATCC 14579 was used for the hblA gene, and for the ces gene, the NCTC 11143 strain was used.

RESULTS B. cereus was identified in 57% of the 100 analyzed samples of different vegetables (32% of broccoli samples, 44% of carrot samples, 84% of the lettuce samples, and 68% of coriander samples). In all of them, colonial morphology and lecithinase production typical of B. cereus were observed. Table 1 shows the results of the isolation of B. cereus strains from the different samples; it is worth noting that the largest number of positive samples was obtained from lettuce. The amounts of microorganisms present ranged from .3 to 460 MPN/g in broccoli, from .3 to 9.2 MPN/g in carrots, from .3 to 240 MPN/g in lettuce, and from .3 to 1,100 MPN/g in coriander. It was possible to amplify the hblA gene in 100% of the strains tested (Table 1 and Fig. 1), although none of them was positive for the ces gene amplification (Fig. 2). DISCUSSION

FIGURE 1. Agarose gel electrophoresis of PCR amplification product of the hblA gene. Lane 1, 100-bp DNA ladder; lane 2, positive control; lanes 3 to 6, samples.

In Mexico, there are no reports on the isolation of B. cereus from the vegetable products tested in this work. Valero et al. (30) in Spain evaluated a total of 56 samples of fresh peppers, cucumbers, tomatoes, carrots, zucchini, garlic, and onions, as well as refrigerated minimally

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processed foods, finding B. cereus contamination in all of them except garlic and in a carrot and orange juice mix. In 2008 in South Korea, B. cereus was isolated in 41.7% of 36 samples of sunsik (a mixture of raw cereal powder) and in 90% of 21 rice samples (7). In 2009, the presence of B. cereus was reported in 46% of 178 samples of raw rice in the United States (2). Broccoli and carrots are often consumed with cereals such as rice, which has been associated with B. cereus food contamination. The results for the lettuce samples obtained in the present work are very similar to those reported by Altayar and Sutherland (1), who isolated this bacteria from 80% of the 25 lettuce samples they studied. Lettuce is preferably consumed in salads, which are generally not subjected to any heat treatment and remain exposed to the environment for long periods of time, which provides microorganisms (including B. cereus) with optimal conditions to develop, achieve the required infective dose, and cause disease in consumers. Regarding coriander, this is also a plant whose consumption in Mexico needs no heat treatment, and its cleaning (at the best) consists only of rinsing with drinking water. Similarly to lettuce, coriander is normally exposed to air for long periods of time. There are no reports so far regarding the isolation of B. cereus from this product. The results obtained in this study suggest that the vegetables analyzed are free of detectable emetic toxin, although a gene implicated in the diarrheal syndrome was present in 100% of the bacterial isolates. This result implies a potential risk to people who eat these foods, if the concentration of the bacteria exceeds the aforementioned infective dose. According to the literature, the presence of both genes (ces and hbl) in the same strain has a low probability. Only Chon and colleagues have described this finding, in two different works. In the first (7), they reported the presence of the ces gene in one strain (of 35 strains isolated from sunsik samples) and the genes encoding the HBL complex in 86% of the tested strains. In the second (8), the authors detected the HBL complex in 14.3% of the emetic strains analyzed. Aragon-Alegro and coworkers (3) reported the absence of the ces gene in 155 strains of B. cereus isolated from different food products in Brazil and the presence of the HBL complex in 67.7% of these strains. Ankolekar et al. (2) reported the absence of the ces gene in 83 strains isolated from rice and the presence of the hblA gene in only 47 of them. Kim et al. (15) indicated the absence of the ces gene in 120 isolates from clinical and food samples in South Korea and the presence of the HBL complex in 90% of them. Finally, Chaves et al. (5) reported the presence of B. cereus in 56.7% of coffee samples studied; of the 25 isolates, 76% possessed the HBL complex and the ces gene was not detected in any of them. The presence of the hblA gene has also been reported in strains isolated from pasteurized milk, fishery products, and fermented foods (20, 22, 23). Until this study, no reports existed worldwide reporting the search for hblA and ces genes in B. cereus isolated from horticultural products like those discussed in this article.


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