Toxin gene pattern in Bacillus cereus group strains ... - Springer Link

Vet Res Commun (2008) 32 (Suppl 1):S323–S326 DOI 10.1007/s11259-008-9139-6 ABSTRACT

Toxin gene pattern in Bacillus cereus group strains isolated from sheep ricotta cheese E. P. L. De Santis & A. Foddai & S. Virdis & P. Marongiu & A. L. Pilo & C. Scarano

Published online: 7 August 2008 # Springer Science + Business Media B.V. 2008

Keywords B. cereus group . Sheep ricotta cheese . Toxin genes pattern . Toxin production Abbreviations BCET-RPLA Bacillus cereus enterotoxin-reversed passive latex agglutination BHI brain heart infusion ELISA enzyme-linked immunosorbent assay HBL hemolysin BL NHE non-hemolytic enterotoxin PCH phosphatidylcholine hydrolase PEMBA polymixin egg yolk mannitol bromothymol blue agar PIH phosphatidylinositol hydrase rpm revolutions per minute SM sphingomyelinase UV ultraviolet Introduction Milk and milk whey products processed at high temperatures and then stored by chilling are particularly sensitive to the health risks associated with microorganisms of the Bacillus cereus group (Heyndrickx and Scheldeman 2002). Sheep ricotta cheese is often contaminated by these spore forming microorganisms and it provides a substrate in which they can develop (Corona et al. 2002). B. cereus may cause diarrhoeic or emetic food poisoning outbreaks. Diarrhoeic syndrome is mainly due to HBL and NHE enterotoxins produced when the micro organisms multiply in the small intestine (Granum and Lund E. P. L. De Santis : A. Foddai : S. Virdis : P. Marongiu : A. L. Pilo : C. Scarano Department of Animal Biology, Faculty of Veterinary Medicine, University of Sassari, Sassari, Italy E. P. L. De Santis (*) Sezione Ispezione Alimenti di Origine Animale, Dipartimento di Biologia Animale, Facoltà di Medicina Veterinaria, Sassari, via Vienna 2/a, 07100 Sassari, Italy e-mail: [email protected]

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Vet Res Commun (2008) 32 (Suppl 1):S323–S326

1997; Hansen and Hendriksen 2001). Emetic syndrome is caused by emetic toxins or cereulide, a dodecapeptide produced when the microorganisms multiply in the food (Kramer and Gilbert 1989; Agata et al. 1996). Their pathogenic profile is also characterized by the ability to produce enterotoxin T, which shows toxic activity only in vitro (Agata et al. 1995), and cytotoxin K, which is necrotizing and hemolytic. Sporadic cases of death from food poisoning have been attributed to cytotoxin K producing strains (Lund et al. 2000). These microorganisms also produce hemolysins (cereolysins, hemolysin II, sphingomyelinases) and different phospholipases C (PIH, PCH and SM) (Granum 1994; Beecher and Wong 2000). The present work reports the molecular profile of the pathogenicity factors of “B. cereus” group strains isolated from fresh and ripened sheep ricotta-cheese. The ability of these strains to produce the hemolytic fraction L2 of HBL and the fraction A of NHE was also tested.

Materials and methods A study on toxin gene profiles was carried out on 66 Bacillus spp strains. Of these strains 36 were isolated from fresh sheep ricotta-cheese samples, and 30 from ripened ricottacheese. The samples were collected from 11 dairy plants in Sardinia (six strains from each plant). Each strain isolated from a colony showing typical characteristics on PEMBA (Oxoid, UK) (UNI EN ISO 7932, 2004) was analyzed by a preliminary microbiological test (Murray et al. 1999) and was then identified with the Vitek system (bioMérieux, France). The production of the L2 hemolytic HBL fraction and the proteic A fraction (45-kDa) was evaluated after an overnight broth (BHI, Oxoid) culture (+ 37 °C in a thermostatic bath at 120 rpm), using, respectively, RPLA (BCET-RPLA, Oxoid) and ELISA (Tecra, Australia) commercial kits, following the makers’ instructions. After extraction of the DNA (Manzano et al. 1998), PCR was used to determine the genes encoding for the following pathogenicity factors: cerA (phospholipases C), cerB (sphingomyelinases) (Schraft and Griffiths 1995), bceT (T enterotoxins) (Agata et al. 1995), cytK (k cytotoxins) (Lund et al. 2000), hbl operon (hemolytic diarroheal enterotoxins), nhe operon (hemolytic diarroheal enterotoxins) (Hansen and Hendriksen 2001) and ces, cereulide which is common in all emetic strains (Ehling-Schulz et al. 2005). The templates were visualised through a UV transilluminator (Gel-doc UV trans-illuminator, BioRad) in agarose gel (1,5%) stained with ethidium bromide (0.25 mg/ml).

Results All the strains (n=66) were identified by Vitek as follows: B. cereus (n=34), B. thuringiensis (n=29), B. mycoides (n=2) and B. licheniformis (n=1). The results of the molecular characterization were analyzed to identify the pathogenicity profile (Table 1).

Table 1 Genes encoding pathogenic factors in B. cereus group strains isolated in sheep ricotta cheese (N=66)

No %

cerA

cerB

hblA

hblC

hblD

nheC

nheA

nheB

ces

bce T

cyt K

62 (93.9)

32 (48.5)

19 (28.8)

21 (31.8)

31 (47)

47 (71.2)

44 (66.7)

37 (56)

8 (12.1)

58 (87.9)

12 (18.2)

No = positive strains


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Phospholipase C (cerA) and sphingomyelinase B (cerB): cerA was detected in 62 strains (93.9%) and cerB in 32 (48.5%). The presence of cerA indicates that these strains belong to the B. cereus group (van Netten and Kramer 1992). Hbl genes (operon hbl): the three hbl genes were identified in 17 out of 66 strains (25.8%), while in 7 strains two were detected and in 6 only one. The production of hemolytic fraction L2 was found in 30 strains (45.5%). The production of L2 was associated with the presence of hblC in 19 strains (28.8%) while the gene was not found in 11 strains. The results from the PCR (hblC) and RPLA (L2) methods were concordant in 53 strains (80.3%). Nhe genes (operon nhe): all three nhe genes were found at the same time in 22 strains (33.3%), while two genes were found in 24 strains (36.4%) and only one in 14 strains (21.1%). Subunit A of the NHE toxin was detected in 60 strains (91%). PCR (nheA) and ELISA (protein A) produced similar results in 40 strains (60.6%). In contrast, in 4 strains the presence of nheA was not associated with production of protein A and in 17 strains the production of the protein was not accompanied by detection of the corresponding gene. Emetic toxins: the ces gene was present in 8 strains (12.1%). Enterotoxin T: the bceT gene was found in 58 strains (87.9%). Cytotoxin k: the cytK gene was present in 12 strains (18.2%).

Discussion The pathogenic profile of B. cereus group strains isolated from sheep ricotta cheese was characterized by the high frequency of the bceT (87.9%), nheC (71.2%) and nheA (66.7%) genes. The last of these has been recently cited as being a major determinant of cytotoxicity in B. cereus (Svensson et al. 2007). The prevalence of ces and cytK is similar to that found by AA (van Netten and Kramer 1992). The profile of the genes encoding pathogenic factors is very variable both in all isolates and also in the strains isolated from ricotta cheese collected from the same cheese factory. The six strains that produced the proteic subunit L2, but in which hblC was not detected, were found to possess at least one of the operon hbl genes. 17 strains which produced protein A in broth culture did not harbor the nheA gene. At least one gene of the operon (nhe) was also found in these strains. The finding of production of diarroheal enterotoxins in the absence of the corresponding genes may be related to the cross-reactivity between the subunit components of the enterotoxins (Hansen and Hendriksen 2001) or to a high level of gene polymorphism in the isolated strains (Guinebretiere et al. 2002). Our results confirm that genes encoding the toxins responsible for foodborne disease are frequently found in the microorganisms of the B. cereus group. These findings, in association with the observed great variability in the genes expression, make an integrated approach to molecular, immunological and cytotoxicity test methods essential when epidemiological studies on the pathogenicity of the B. cereus strains are designed (Lotte et al. 2007). Acknowledgments This work was supported by grant from Fondazione Banco di Sardegna.

References Agata N., Ohta M., Mori M., Isobe M., 1995. A novel dodecadepsipeptide, cereulide, is an emetic toxin of Bacillus cereus. FEMS Microbiology Letters, 129(1), 17–20. Agata N., Ohta M., Mori M., 1996. Production of an emetic toxin, cereulide, is associated with a specific class of Bacillus cereus. Current Microbiology, 33, 67–69.

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Beecher D.J., and Wong A.C.L., 2000. Cooperative, synergistic and antagonistic haemolytic interactions between haemolysin BL, phosphatidylcholine phospholipase C and sphingomyelinase from Bacillus cereus. Microbiology, 146, 3033–3039. Corona A., De Santis E.P.L., Mazzette R., Virdis S., Cosseddu A.M., 2002. Caratterizzazione fenotipica e molecolare di microrganismi del “Gruppo Bacillus cereus” isolati in caseifici ovini. Atti del XII Convegno Nazionale Associazione Italiana Veterinari Igienisti, 179–184. Ehling-Schulz M., Vukov N., Schulz A., Shaheen R., Andersson M., Märtlbauer E., Scherer S., 2005. Identification and partial characterization of the nonribosomal peptide synthetase gene responsible for cereulide production in emetic Bacillus cereus. Applied and Environmental Microbiology, 71(1), 105– 713. Granum P.E., 1994. Bacillus cereus and its toxins. The Journal of Applied Bacteriology, Symposium Supplement, 76, 61S–66S. Granum P.E. and Lund T., 1997. Bacillus cereus and its food poisoning toxins. FEMS Microbiology Letters, 157, 223–228 Guinebretiere M.H., Brousolle V., Nguyen-The C., 2002. Enterotoxigenic profiles of food poisoning and food borne Bacillus cereus strains. Journal of Clinical Microbiogy, 40, 3053– 3056. Hansen B.M. and Hendriksen N.B., 2001. Detection of Enterotoxic Bacillus cereus and Bacillus thuringiensis Strains by PCR Analysis. Applied and Environmental Microbiology, 67(1), 185–189. Heyndrickx M. and Scheldeman P., 2002. Bacilli associated with spoilage in dairy products and other food. In: Applications and Systematics of Bacillus and Relatives, R.C.W. Berkeley, M. Heyndrickx, N.A. Logan and P. De Vos (Eds.). Blackwell Science: Oxford, 65–82. Kramer J.M. and Gilbert R.J., 1989. Bacillus cereus and other Bacillus species. In: Foodborne bacterial pathogens, M.P. Doyle (Ed.). Marcel Dekker Inc.: New York, 21–50. Lotte P., Arnesen S., O'Sullivan K., Granum P.E., 2007. Food poisoning potential of Bacillus cereus strains from Norwegian dairies. International Journal of Food Microbiology, 116(2), 292–296. Lund T., De Buyser M.L., Granum P.E., 2000. A new cytotoxin from Bacillus cereus that may cause necrotic enteritis. Molecular Microbiology, 38(2), 254–261. Manzano M., Cocolin L., Cantoni C., Comi G., 1998. A rapid method for the identification and partial serotyping of Listeria monocytogenes in food by PCR and restriction enzyme analysis. International Journal of Food Microbiology, 42(3), 207–212. Murray P.R., Baron E.J., Pfaller M.A., Tenover F.C., Yolken R.H., 1999. Manual of clinical microbiology, 7th edition. ASM Press: Washington. Schraft H. and Griffiths M.W., 1995. Specific oligonucleotide primers for detection of lecithinase-positive Bacillus spp. by PCR. Applied and Environmental Microbiology, 61(1), 98–102. Svensson B., Monthán A., Guinebretière M.H., Nguyen-Thé C., Christiansson A., 2007. Toxin production potential and the detection of toxin genes among strains of the Bacillus cereus group isolated along the dairy production chain. International Dairy Journal, 17(10), 1201–1208. UNI EN ISO 7932, 2004. Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of presumptive Bacillus cereus-Colony-count technique at 30 °C. van Netten P. and Kramer J.M., 1992. Media for the detection and enumeration of Bacillus cereus in foods. International Journal of Food Microbiology, 17(2), 85–99.