Detection of coding genes for enterotoxins in Bacillus cereus by PCR and their products by BCET-RPLA and ELISA assay.

VYLETĚLOVA, M. , BANYKO, J.: Detection of coding genes for enterotoxins in Bacillus cereus by PCR and their products by BCET-RPLA and ELISA Assay. Acta univ. agric. et silvic. Mendel. Brun. , 2010, LVIII, No. 5, pp. 417–426 Determination of enterotoxin production, diarrhoeal and emetic gene identifi cation was studied in 41 Bacillus cereus strains isolated from raw cows’ and raw goats’ milk, pasteurized milk, dairy products during technological processing and from dairy plant equipment. Presence of enterotoxins was detected by BCET-RPLA (HBL) and ELISA immunoassay (NHE). Gene identifi cation (nheA, nheB, nheC, hblA, hblC, hblD, bceT, cytK-1, cytK-2, entFM and ces) was achieved by means of PCR. Enterotoxin HBL was detected in 32 strains, enterotoxin NHE in all 41 strains. Presence of all three genes nheA, nheB and nheC was confi rmed in 40 strains and genes hblA, hblC and hblD in 29 strains. Comparison of used methods was as follow: 1) BCET-RPLA (which detects L2 component) and PCR (positive or negative all three hblA, hblC and hblD gene detection) were identical in 30 (73%); 2) ELISA (NheA) and PCR (all three nheC, nheB and nheA gene expression) were identical in 40 (98%) cases isolated strains. milk, Bacillus cereus, genes, enterotoxins, PCR, BCET-RPLA, ELISA assay Spore-forming microorganisms are a serious problem in contamination both of raw milk or dairy products. Their ability to survive pasteurization temperatures or ultrahigh-temperature treatment (UHT) facilitates their infi ltration into the foodchain, resulting in the potential incidence of alimentary disorders. Bacillus cereus is the primary cause of alimentary intoxication due to dairy product consumption in both the Czech Republic and other countries (Vyletělova et al., 2001, 2002; Pacova et al., 2003; Stenfors et al., 2008; Bartoszewicz, 2008). Alimentary intoxications by Bacillus cereus enterotoxins in food are divided into diarrhoeal and emetic poisoning, according to toxin type. The diarrhoeal type enterotoxin is formed by heat-labile proteins which can be inactivated by heat treatment at 56 °C for 5–30 min (Murray et al., 1999). Bacillus cereus produces the following diff erent diarrhoeal ente ro to xins: protein complex (haemolytic HBL and non-haemolytic NHE enterotoxins), enterotoxic proteins (enterotoxin T), cytotoxin K and enterotoxin FM (Yang et al., 2005; Lindback et al., 2004). Sergeev et al. (2006) classifi ed enterotoxin FM as a haemolytic enterotoxin and as cytotoxin. HBL toxins are formed from three proteins: two lytic components (L2 and L1) and a binding component B. These are encoded by genes hblC, hblD and hblA (Beecher et al., 1995). All three components are required for toxic activity (Yang et al., 2005). NHE consists of three proteins: NheA, NheB and NheC which are encoded by genes nheA, nheB and nheC, respectively. All three components are also needed for maximum cytotoxic activity (Lindback et al., 2004; Yang et al., 2005). The nucleotide sequence denoted bceT has been reported to encode a single component toxin T (BceT, 41-kDa protein) which exhibits Vero cell cytotoxicity and has also been attributed to having a role in the diarrheal syndrome (Agata et al., 1995a). An additional study has indicated that the BceT enterotoxin was most likely an experimental artifact that probably could not contribute to food poisoning (Choma and Granum, 2002). CytK toxin is a pore-forming cytotoxin linked to human necrotic enteritis cases (Hardy et al., 2001). Lund et al. 418 M. Vyletělova, J. Banyko (2000) isolated CytK from Bacillus cereus strains that caused a severe food poisoning outbreak of enteritis which killed three people and they found a highly cytotoxic, necrotic and haemolytic protein of 34kDa. CytK forms two variants: CytK-1 and Cyt K-2 which are encoded by cytK-1 and cytK-2 genes, respectively (Fagerlund et al. 2004). CytK-2 is highly toxic to human intestinal cells but not as toxic as CytK-1 (Fagerlund et al., 2004; Guinebretiere et al., 2006). Enterotoxic protein FM (entFM) is a relatively unknown haemolytic enterotoxin that is encoded by the entFM gene (Asano et al., 1997). In contrast to the diarrhoeal type, the emetic toxin (cereulide) is a small, heat-stable cyclic dodecadepsipeptide (Agata et al., 1995b) which can survive a temperature of 120 °C for 90 min (Lund, 1990). This enterotoxin is synthesized by a non-ribosomal peptide synthetase, encoded by the ces gene (Ehling-Schulz et al., 2004). The primary symptom of emetic toxin is vomiting but in animal models it can cause cellular damage (Shinagawa et al., 1995) and rarely liver failure in humans (Mahler et al., 1997). Detection methods for the diarrhoeal toxin are well-known and commercially available. They include BCET-RPLA test which is specifi c to the L2 component, and ELISA-(BDE VIA) test which detects mainly the NheA protein (45-kDa) – (Granum and Lund, 1997). The emetic enterotoxins can be detected by bioassay test, based on loss of sperm moti li ty (Andersson et al., 1998; Svensson et al., 2006) or HPLC-MS chemical assay (Haggblom et al., 2002). HBL and NHE enterotoxins, in order to be active, need all three of their components (L1, L2, B and NheA, NheB, NheC, respectively). However, methods used in practice can detect only one of three enterotoxin components (BCET-RPLA – L2; ELISA BDE – NheA). The results of these methods give us information about possible entorotoxin presence but not whether the toxin is active or not. PCR methods used in this study are able to detect all genes involved but also not their activity. This study is thus focused on comparison of the BCET-RPLA and ELISA BDE immunoassays with PCR results for genes (hblC, hblD, hblA and nheA, nheB, nheC) identifi cation which encode three enterotoxin components. The aim of this study was to fi nd out, if the commonly used methods (BCETRPLA, ELISA BDE) are suffi cient enough to determine presence and activity of enterotoxins or using some additional methods (as PCR) is necessary. MATERIAL AND METHODS Bacillus cereus origin and cultivation Bacillus cereus strains were isolated from raw cows’ and raw goats’ milk, pasteurized milk, dairy products during technological processing and from dairy industry equipment (Table I). Samples 1–29 were derived from diff erent samples, whereas samples 30–41 were isolated from one raw cows’ milk sample. Bacillus strains were isolated on modifi ed MYP Agar supplemented with egg yolk emulsion and Polymyxin B sulphate (HiMedia, Mumbai, India) and cultured at 30 ± 1 oC for 48 hours (CSN EN ISO 7932, 2005). Species identifi cation was provided according to biochemical and physiological chara cte ri stics and phenotypic tests (Vyletělova et al., 2001). Enterotoxin detection Enterotoxins were detected using two im munoas say kits: HBL enterotoxins using commercial kits BCET-RPLA (B. cereus Enterotoxin Reverse Passive Latex Agglutination – Oxoid, Denka SEIKEN Ltd., Japan) and NHE using the ELISA test (BDEVIA – Bacillus Diarrhoeal Enterotoxin Visual Immunoassay) from Tecra International Pty, Ltd., Frenchs Forest, Australia. Both kits were used according to the manufacturer’s instructions.