Genetic characterisation of food borne Bacillus cereus strains from ...

Genetic characterisation of food borne Bacillus cereus strains

International Journal of Integrative Biology

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A journal for biology beyond borders

ISSN 0973-8363

Genetic characterisation of food borne Bacillus cereus strains from milk, cheese and rice by multiplex PCR assay Nduhiu Gitahi J1,2,*, Jackson N Ombui1, Dorcas W Nduati1, Michael M Gicheru2 1

Public Health Pharmacology and Toxicology, University of Nairobi, Nairobi, Kenya 2 Zoological Sciences, Kenyatta University, Nairobi, Kenya Submitted: 4 Nov. 2008; Revised: 30 Dec. 2008; Accepted: 6 Feb. 2009

Abstract This study characterises enterotoxin genes including nheA, nheB, nheC, hblA, hblC, hblD, entFM,cytK, bceT, and ess in 51 Bacillus cereus (B. cereus) strains isolated from pasteurized milk, processed cheese and cooked rice. A colony multiplex polymerase chain reaction was used to test for presence of genes in B. cereus isolates. Of the fifty one B. cereus isolates, 12 (33.3%) were from milk, 8 (22.2%) from cheese and 31 (60.7%) from rice. The isolates were classified into 15 toxigenic groups A - O according to the genes they contained. Group G had the highest number of strains, 8(15.7%) while groups D and H had one isolate each. The emetic toxin gene sequence (ess) was found in 18% while bceT was found in 20% of rice isolates. The cytK gene was present in 4% of milk and 8% of rice isolates. The cytK gene was not found in any of the cheese isolates. All the other genes were found in isolates from all the three food types. The toxigenic profiles of isolates from milk and cheese differed from those of rice isolates, indicating different food preferences among B. cereus strains. Keywords: Bacillus cereus, Multiplex PCR, Gene profile patterns, food-borne.

INTRODUCTION Bacillus cereus is a gram positive, spore-forming motile, aerobic rod, commonly found in soil, water and foods. The organism has been isolated from various foods, including cooked rice, dairy products, eggs, meats and spices (Ombui et al., 2008; Kramer & Gilbert, 1989); and from pharmaceutical products (Garcia-Arribas et al., 1988). It causes emetic and diarrhea type food poisoning in human which are a major concern worldwide. The poisoning is due to the various enterotoxins produced by the various strains of this organism. The type of poisoning caused is determined by the kind of enterotoxin elaborated by the organism. Bacillus cereus produces different types of enterotoxins. They include two protein complexes: the hemolysin BL(HBL) (Beecher et al., 1995) and the non-hemolytic enterotoxin (NHE) (Lund and Granum, 1997) as well as three different single enterotoxic proteins, including cytotoxin K(CytK) ( Lund et al., 2000), enterotoxin FM * Corresponding author: Nduhiu Gitahi, M.Sc. Public Health Pharmacology and Toxicology, University of Nairobi, Nairobi, Kenya Email: [email protected], [email protected]

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(entFM) and enterotoxin T (bc-D-ENT). The hemolysin BL consists of binding protein B (35 kda) encoded by hblA gene (Heinrichs et al., 1993), lysing proteins L1 (36 kda) and L2 (45 kda) encoded by hblC and hblD genes respectively (Ryan et al., 1997). The nonhemolytic enterotoxin has three proteins moieties B, L1 and L2, encoded by the three genes nheA, nheB and nheC respectively (Lindback et al. 2004; Lund and Granum, 1997). The single enterotoxin protein includes enterotoxin FM that is encoded by entFM gene, Bacillus cereus enterotoxin T encoded by bceT gene (Agata, et al., 1995) and Cytotoxin K that is encoded by cytK gene (Lund et al., 2000; Hardy et al., 2001). A variant of CytK was later described and designated as CytK-2, and the original CtyK as CytK-1 (Fagerlund et al., 2004). These enterotoxic factors are proteins encoded by genes distributed on the chromosome (Okstad et al., 1999). In addition, B. cereus produces an emetic toxin which is a depsipeptide cereulide that is associated with emetic type of food poisoning. The toxin is encoded by emetic gene specific sequence and its production of emetic toxin is associated with sporulation (Mikami, et al., 1994).

IJIB, 2009, Vol. 5, No. 2, 82


Bacillus cereus strains may produce one or more types of these enterotoxins (Beecher et al., 1995). However, data on the genetic and toxigenic potential of B. cereus strains from processed foods in Kenya is not available, although B. cereus has been reported to contaminate processed dairy products. This study was aimed at determining the types of enterotoxin genes carried by B. cereus strains isolated from pasteurized milk, processed cheese and cooked rice in Kenyan market using multiplex PCR described by Yang et al., (2005). This method simultaneously detects all the B. cereus enterotoxin genes and emetic-specific sequences using bacterial colonies.

MATERIALS AND METHODS Bacterial strains 53 bacterial strains previously isolated from 108 food samples comprising 36 cheese, 36 pasteurised milk and 36 cooked rice samples from major supermarkets and hotels located in the Nairobi Central Business District (NCBD) were used. Bacillus cereus NCTC 11145 that had hblA, hblC, hblD, nheA, nheB and nheC genes and B. cereus strain BC 68 isolated in our laboratory and found to carry nheA, nheB, nheC and emetic toxin gene specific sequence (ess) were used as reference strains.

Detection of B. cereus enterotoxin genes using Multiplex PCR Assay A multiplex PCR was carried out using single colonies of all B. cereus isolated from milk, cheese and rice samples and reference strains. One pure colony was suspended in 50µl distilled water, boiled for 10min at 980C in a water bath and the suspension centrifuged for 3 minute at 15000rpm. 5μl of this DNA extract was amplified in a 25µl reaction mix containing 0.5µl each of the ten primer pairs and 12µl of supermix (Invitrogen USA) containing 0.5U of Taq polymerase and hexanucleotides. Amplification was done using 30 cycles of denaturation at 950C for 30s, annealing at 600C for 30s and extension at 720C for 45s followed by a final extension at 720C for 7min using a PCR minicycler (PTC-150 MJ Research, Inc. USA).

Agar gel electrophoresis for PCR products

DNA primers used to screen for the presence of nheA, nheB, nheC, hblA, hblC, hblD, entFM , cytK, bceT, and ess in B. cereus as described by Yang et al., (2005) and Ehling-Schulz et al., (2004). A primer pair from the 16S to 23S internal transcribed spacer was used for quality control of DNA. These primers were synthesized by Invitrogen® Co. USA.

PCR products were analyzed using 1.5% agarose gel electrophoresis in 0.5 X Tris boric ethylene diamine tetra acetic acid disodium salt (TBE) containing 0.5µg/ml ethidium bromide buffer. 22µl portions of PCR products were mixed with 4.0µl 6X gel loading dye and loaded onto gel submerged in 1 X TBE. A 100bp molecular weight marker (Invitrogen, USA) was used to determine the approximate molecular weight of PCR products. A constant voltage of 10V/cm was applied and the amplified fragments were allowed to migrate until appropriate band separation was achieved. A UV trans-illuminator (Vilber Laurmat, France) was used to visualize bands relative to the molecular weight marker and results were documented by photography using a digital Polaroid camera (PDZ 2300Z, Polaroid, China). A Chi square test was used to determine whether the toxin genes were significantly different between the B. cereus food borne strain in milk, cheese and rice samples.

Isolation of B. cereus from pasturized milk, cheeses and cooked rice

RESULTS AND DISCUSSION

DNA primers

Isolation of B. cereus from the three foods sample types was done by inoculating 0.1ml of pre-incubated sample homogenate onto the surface of BCSA plate and incubated at 350C for 24 – 48h. Plates were examined for typical B. cereus colonies after 24h and 48h. Colonies on BCSA that appeared crenated and about 5mm in diameter with a turquoise to peacock blue colour surrounded by a good egg yolk precipitate of the same colour were noted as typical B. cereus. One typical B. cereus colony was purified and confirmed as B. cereus using the rapid confirmatory staining procedure developed by Holbrook and Anderson (1980), and tested for carriage of enterotoxin genes using the described colony multiplex PCR assay.

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Distribution and profile of B. cereus strains enterotoxin genes 51 (96%) of the 53 B. cereus isolates tested positive for various profiles of the following enterotoxin genes, nheA, nheB, nheC, hblA, hblC, hblD, entFM ,cytK, bceT, and ess by colony multiplex PCR. All the isolates tested positive for the DNA quality control, using primer pair from the 16S to 23S rRNA internal transcribed spacer that was used as an internal PCR control to eliminate the false negative samples. Five genes were detected in B. cereus strain NCTC 11145 including nheC (557 bp), hblD (436 bp), hblC (386 bp), nheB (328 bp), and hblA (237). Bacillus cereus strain NCTC 11145 lacked the emetic gene specific sequence (ess), cytotoxin K (CytK), enterotoxin T (bceT), and IJIB, 2009, Vol. 5, No. 2, 83


isolates from rice. The prevalence of nheB was 19.6%, 11.8% and 59% in isolates from milk, cheese and rice. The prevalence of hblA gene was 9.8%, 11.8%, and 15.7% among isolates from milk, cheese and rice respectively. In addition 13.7%, 7.8%, and 29.4% of isolates from the respective foods had hblC gene. The prevalence of hblD gene in both milk and cheese isolates was 11.8% while the prevalence in rice isolates was 27.4%. Only isolates from rice had emetic toxin specific sequence (ess) and bceT genes with a prevalence of 17.6% and 19.6% respectively.

Figure 1: Multiplex PCR for B. cereus strains NCTC 11145 and BC 68 and some B. cereus food isolates Lanes M1 and M2: 100-bp ladder marker from two different runs; Lane 2: B. cereus strain 5C isolated from food in this study. From top to bottom are products of nheC (557 bp), hblD (436 bp), and nheB (328 bp) genes. Lane 3: B. cereus strain NTCC11145, from top to bottom are nheC (557 bp), hblD (436 bp), hblC (386 bp), nheB (328 bp), and hblA (237) genes. Lanes 4, 5 and 6: B. cereus isolates obtained from cheese, milk and rice samples in the present study respectively, the pattern was similar to that in lane 2. Lane 7: B. cereus strain BC 68; from top to bottom are products of emetic gene specific sequence (635 bp), nheA (475 bp), and nheB (328 bp); Lane 8: B. cereus isolate obtained from rice sample in the present study, from top to bottom are products of hblD (436 bp), nheB (328 bp), and hblA (237) genes. Lane 9: B. cereus food isolate. From top to bottom are products of hblD (436 bp), nheB (328 bp) genes.

18 %B. cereus 16

% of B. cereus strains

enterotoxin FM (entFM). The emetic gene specific sequence (635bp) together with nheA (475bp) and nheB (328bp) were detected in reference strain B. cereus strain BC 68 (Fig. 1, Fig. 2). These results are in agreement with those of Guinebretiere et al., (2002) who found that 63% and 36% of food borne B. cereus isolates to lack one or two of the nhe and hbl genes, respectively when tested by PCR, though all the genes were demonstrated by Southern blotting and most of the isolates tested positive for enterotoxins using immunoassay, Oxiod kit for the hbl genes and Tecra kit for nhe genes (Guinebretiere et al., 2002). Cardazzo et al., (2008) also reported 3 & 4 out of 47 new B. cereus isolates from industrial dairy product, egg product and farm dairy product without one or two of the nhe and hbl gene complex respectively. This indicates a degree of polymorphism in the hbl and nhe sequences among the food-associated B. cereus strains and may explain the inability to detect all genes by PCR in most of the strains. However, Granum (2001) found the nhe operon to be present in 100% of B. cereus isolates, while the hbl operon was present in 50% of B. cereus isolates.

The bceT gene is reported to be widely distributed among B. cereus strains, at 47% to 41 % (Agata et al., 1995; Ombui et al., 1997; Hsieh et al., 1999). The bceT gene sequence has been shown to vary among strains (Hansen and Hendriksen, 2001). The prevalence of cytK gene in isolates from milk and rice was 3.9% and 7.8% respectively. The cytK gene has previously been reported at a higher prevalence in B. cereus isolates from dairy products (Stenfors and Granum, 2001). The cytotoxin has been shown to cause severe food poisoning among consumers of foods contaminated with B. cereus strains bearing cytK gene. This may be due to the hemolytic and cytotoxic activities of this toxin (Lund et al., 2000, Hardy et al., 2001). The prevalence of entFM gene was 13.7%, 7.8% and 43.1% among the isolates from milk, cheese and rice respectively.

14 12 10 8 6 4 2 0

A

The distribution of enterotoxin genes in food borne B. cereus is given in Table 3. No association was apparent in the occurrence of non-hemolytic enterotoxin and hemolysin BL complex genes in milk, cheese and rice. The prevalence of nheA and nheC genes in isolates from both milk and cheese was 3.9%, while the prevalence of the two genes was 0% and 5.9% in International Journal of Integrative Biology © IJIB, All rights reserved

B

C

D

E

F

G

H

I

J

K

L

M

N

O

Toxigenic patterns

Figure 2: Percentage of B. cereus isolates in each of the toxigenic profiles.

Presence of nheA, nheC, hblA, CytK and hblD gene profiles between B. cereus isolates from the three types of food didn’t have any association (p>0.05), but nheB, IJIB, 2009, Vol. 5, No. 2, 84

Genetic characterisation of food borne Bacillus cereus strains Table 2: Genetic characteristics of food-borne B. cereus strains from pasteurized milk, processed cheese and cooked rice

Group

A B C D E F G H I J K L M N O

isolates

2 7 4 1 3 4 8 1 3 2 2 3 3 3 5

No. of strains in each food type Milk

Cheese

Rice

0 3 0 0 2 2 1 0 1 2 1 0 0 0 0

2 1 0 1 1 1 0 1 1 0 0 0 0 0 0

0 3 4 0 0 1 7 0 1 0 1 3 3 3 5

Genes Detected from the B. cereus Strains Isolated from the Three Food Types

nheB nheB nheB nheA nheA nheB nheB hblA nheB nheB nheB nheB nheB nheB nheB

hblA hblC hblC nheB nheC hblA hblA hblC hblC hblC hblC nheC hblC ess entFM

hblD hblD hblD nheC hblA entFM

entFM entFM bceT hblA hblD

cytK hblC

entFM hblD

ess entFM

bceT

hblD cytK hblD hblC hblD bceT

entFM hblD ess entFM

entFM

ess- emetic specific sequence. rRNA gene for 16S to 23S Internal transcribed spacer was detected in all the groups and was used as a DNA quality control.

hblC, ess, bceT, and EntFM gene profiles had an association with B. cereus isolated from the three types of food (p