2020, Scienceline Publication

JWPR

Poultry Research

J. World Poult. Res. 10(2S): 292-298, June 14, 2020

Journal of World’^s

Research Paper, PII: S2322455X2000035-10

License: CC BY 4.0

DOI: https://dx.doi.org/10.36380/jwpr.2020.35

Detection of Virulence Genes in Bacillus cereus isolated from Meat

Products Using PCR

Ashraf A. Abd El Tawab¹*, Fatma I. El-Hofy¹, Nahla, A.Abou El Roos²and Doaa, A.El-morsy²

¹Bacteriology, Immunology and Mycology Department Faculty of Veterinary Medicine, Banha University, Egypt.

²Animal Health research institute, Shibin El Kom Branch, Egypt.

*Corresponding author’s Email: Ashrafabdeltwab@yahoo.com; ORCID: 0000-0002-1221-3340

Received: 21 Feb. 2020

Accepted: 30 Mar. 2020

ABSTRACT

Bacillus cereus is an opportunistic pathogen that can cause food poisoning in humans as a result of consuming foods

containing toxins or bacteria. In this study, the incidence of B. cereus and its virulence genes in meat products was

investigated. Isolation of B. cereus was performed using selective PEMBA media and confirmed by morphological

and biochemical tests and Vitek2 compact system. The incidence of B. cereus strains in beef and chicken meat

products was 28%. The incidence of Bacillus cereus in frozen rice kofta, frozen kobiba-shami, chicken pane, and

chicken nuggets was 16%, 24%, 28%, and 44%, respectively. Moreover, the result of multiplex PCR of virulence

genes of groEL gene (533bp), Hbl gene (1091 bp), Nhe gene (766 bp) and Cytk gene (421bp) indicated that groEl

gene, Nhe gene, Cytck gene was found in 100% of B. cereus isolated from different meat products, while Hbl gene

was detected in 10% of isolates. The results demonstrate that meat products represent a threat to public health through

the transmission of Bacillus cereus.

Key words: Bacillus cereus, Beef meat, Chicken meat, PCR, Virulence genes, VITEK2

INTRODUCTION

components, including hemolysins, phospholipases, and

proteases (Beecher, 2001) nevertheless, the accurate role

of some toxins is still unclear. The emetic and the

diarrheal syndromes are still the foremost concerns for the

public health apprehension and the full appreciative of

their pathogenesis is imperative. These syndromes are

mainly revealed via the release of two core toxins, a heat-

labile diarrheal enterotoxin, and heat-stable emetic

enterotoxin (Stenfors et al., 2008).

The diarrheal syndrome revealed via the release of

one or three diarrheal enterotoxins: the tripartite toxins

hemolysin BL (HBL) and non-hemolytic enterotoxin

(Nhe), the two forms of cytotoxin K (cytK-1 and cytK-2)

The genus Bacillus includes harmless environmental and

pathogenic species. The B. cereus group is known as

pathogens or opportunistic pathogens to humans (Logan,

2012). The B. cereus is associated with food poisoning as

a result of the consumption of food containing pre-formed

toxins or bacteria producing toxins in the human gut

(McKillip, 2000 ).

Genus Bacillus are Gram-positive rods able to

produce endospores resistant to unfavorable external

conditions (Logan and Devos, 2009) that can be

distinguished

from

other

spore-formers

(Sporolactobacillus, Clostridium, Desulfotomaculum,

Sporosarcina, and Thermoactinomyces) due to their

aerobic character (strict or facultative), rod-shaped cells

and catalase production (Slepecky and Hemphill, 2006).

Schedule identification of B. cereus is generally combined

with isolation on selective media, illuminating of motility,

hemolysis pattern on blood agar, and acidification of

glucose (Stenfors et al., 2008).

and possibly enterotoxin

and enterotoxin FM

(Moravek et al., 2006). HBL is a three-component toxin,

that is encoded by hblD and hblC genes respectively, and a

binding component B encoded by hblA gene. The presence

of all three components is important for the activity of

toxin (Lindback and Granum, 2006 ).

The objective of this study was to conduct

bacteriological and molecular studies on B. cereus isolated

from frozen rice kofta, frozen kobiba-shami, chicken pane,

and chicken nuggets.

The pathogenesis of B. cereus-induced food

poisoning is mostly still unclear. The microorganism

transmits an expansive number of potentially toxic

To cite this paper: Abd El Tawab AA, El-Hofy FI, Abou El Roos NA and El-morsy DA (2020). Detection of Virulence Genes in Bacillus cereus isolated from Meat Products Using

PCR. J. World Poult. Res., 10 (2S): 292-298. DOI: https://dx.doi.org/10.36380/jwpr.2020.35

292

J. World Poult. Res., 10(2S): 292-298, 2020

MATERIALS AND METHODS

PCR detection of virulence Bacillus cereus isolates

DNA extraction

Collection of Samples

The isolates of B. cereus isolates from different meat

products were grown in 5ꢀmL nutrient broth with shaking

for 18ꢀh at 30ꢀ°C and collected at 5,000ꢀg for 5ꢀmin.

QIAamp DNA Mini Kit was used for genomic DNA

extraction and purification. PCR was achieved to detect

groEl gene and three enterotoxigenic encoding endotoxins

genes Nhe, hbl and cytK genes. A positive reference strain

of B. cereus ATCC 14579 and sterile MilliQ water as a

negative control was used in PCR analysis (Ehling-Schulz

et al., 2006; Das et al., 2013 ). Table 1 provides details

about the primers used.

A total of one hundred random samples of meat

products which including frozen rice kofta, frozen kobiba-

shami, chicken pane and chicken nuggets (25 of each)

were collected from different shops, supermarkets in

different localities in Menoufia and Kalyobia

governorates. Samples conveyed to the laboratory

following aseptic and safety precautions.

Isolation and identification of Bacillus cereus

group

A stomacher was used to homogenize 10ꢀg of each

sample in 90ꢀmL of buffered peptone water (BPW) for

2ꢀmin. Heat treatment of all samples at 70ꢀ°C for 15ꢀmin

was used to eliminate vegetative cells and allow the

isolation of spores (Al- Allaf, 2011). The pasteurized

samples were immediately positioned in ice to prevent

spore germination. An amount of 100ꢀμl was spread on

Preparation of PCR master mix

occurred according to Emerald Amp GT PCR

mastermi (Takara) CodeNo.RR310A kit as shown in table

Cycling conditions of the primers during PCR

PCR conditions are shown in table 3. Gel

electrophoresis was used to analyze PCR fragments for

presence and correct size compared to positive control

(Sambrook et al., 1989). PCR runs where a negative

control displayed amplification or positive control did not

amplify were overlooked and repeated.

Polymyxin-pyruvate-Egg

yolk-Mannitol-Bromothymol

blue agar (PEMBA) media plates and incubated at 37ꢀ°C

for 24-hr both aerobically and anaerobically. The plates

were examined and the presumptive B. cereus group was

confirmed based on microscopy of Gram-stained

preparations and biochemical tests (FDA, 2015 ). A

number of colonies were randomly collected and analyzed

by cell morphology under the microscope, Gram staining,

ability to form endospores, growth in the presence of

sodium chloride, anaerobic growth, catalase and oxidase

activity, Voges-Proskauer test and growth at pH 5.7. The

ability to ferment carbohydrates, starch hydrolysis, use of

citrate as a carbon source, lecithinase activity, and growth

inhibition by lysozyme were applied (Al- Allaf, 2011 and

FDA, 2015 ).

RESULTS

Prevalence of Bacillus cereus in meat products

The prevalence of B. cereus in meat products (frozen

rice kofta, frozen kobiba-shami, chicken pane, and chicken

nuggets) was 16%, 24%, 44%, and 28%, respectively. Out

of 28 Bacillus isolates, 18 (36%) isolates obtained from

chicken product samples and 10 (20%) isolates were

recovered from beef product samples. The incidence of B.

cereus group in the different meat products shown in table

Identification of Bacillus cereus using VITEK2

BCL Card

PCR results

The result obtained using agarose gel electrophoresis

Bacterial suspensions were arranged in 3.0 mL of

sterile saline and accustomed to a McFarland standard of

1.80-2.20 using the VITEK2 DensiChek (bioMe´rieux).

BCL cards were packed automatically in the VITEK

vacuum chamber, sealed, incubated at 35.5^oc and read

automatically every 15 min for 14 hours. Data were

investigated automatically using the VITEK2 database.

of multiplex PCR of virulence genes, groEL gene (533bp),

Hbl gene (1091 bp), Nhe (766 bp) and Cytk gene (421bp)

for characterization of virulence genes of B. cereus

isolated from different meat products showed that groEL

gene (Figure 1 ), Nhe gene (Figure 3) and Cytk gene

(Figure 4) were found in 100% of tested isolates of B.

cereus and the Hbl gene was detected in 10% of tested B.

cereus isolates (Figure 4 ).

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Abd El Tawab et al., 2020

Table 1. Oligonucleotide primers sequences used in this study to detect Bacillus cereus

Length of amplified

product (base pair)

Gene

groEL

Nhe

Primer sequence

Reference

F: 5'- TGCAACTGTATTAGCACAAGC T -3'

R: 5'-TACCACGAAGTTTGTTCACTACT-3'

F: 5'-AAG CIG CTC TTC GIA TTC-3'

533

Das et al. (2013)

766

421

Ehling-Schulz et al. (2006)

R: 5'-ITI GTT GAA ATA AGC TGT GG-3'

F: 5'-ACA GAT ATC GGI CAA AAT GC-3'

R: 5'-CAA GTI ACT TGA CCI GTT GC-3'

F: 5'-GTA AAT TAI GAT GAI CAA TTTC-3'

R: 5'-AGA ATA GGC ATT CAT AGA TT-3'

cytK

Hbl

1091

F: forward, R: reverse

Table 2. PCR master mix component used for PCR reaction for detection of virulence genes of Bacillus cereus

Component

Volume

Emerald Amp GT PCR mastermix (2x premix)

PCR grade water

12.5μl

4.5μl

1μl

Forward primer(20 pmol)

Reverse primer (20 pmol)

Template DNA

1μl

6μl

Total

25μl

Table 3. Temperature and time conditions used during PCR assay

Primary

denaturation

Secondary

denaturation

Final

extension

Gene

Annealing

Extension

No. of cycles

94˚C

5 min.

94˚C

30 sec.

55˚C

40 sec.

72˚C

45 sec.

72˚C

10 min.

groEL

94˚C

5 min.

94˚C

30 sec.

49˚C

40 sec.

72˚C

1 min.

72˚C

10 min.

Nhe, hbl, cytK

Table 4. Incidence of Bacillus cereus isolated from examined meat products

Total beef

products

(n=50)

Total chicken

products

(n=50)

Kobiba–

shami (n=25)

Rice kofta

(n=25)

Chicken

pane (n=25)

Chicken

nuggets (n=25)

Products

No of positive samples

Percentage of positive samples

24%

16%

20%

44%

28%

36%

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J. World Poult. Res., 10(2S): 292-298, 2020

Figure 1. Results of PCR amplification of groEl gene of Bacillus cereus isolated from different meat and chicken products.

Neg: negative control, Pos: positive control, Lane L: 100-600 bp DNA ladder, Lane 1-10: positive samples at 533 bp

Figure 2. Results of PCR amplification of hbl gene of Bacillus cereus isolated from different meat and chicken products. Neg:

negative control, Pos: positive control, Lane L: 100-1500bp DNA ladder, Lane 2: positive sample at 1091 bp. Lane 1, 3, 4, 5,

6, 7, 8, 9, and 10: negative samples.

295

Abd El Tawab et al., 2020

Figure 3. Results of PCR amplification of Nhe gene of Bacillus cereus isolated from different meat and chicken products.

Neg: negative control, Pos: positive control, Lane L: 100-1000bp DNA ladder, Lane 1-10: positive samples at 766 bp.

Figure 4. Results of PCR for amplification of cytK gene of Bacillus cereus isolated from different meat and chicken products.

Neg: negative control, Pos: positive control, Lane L: 100-600bp DNA ladder, Lane 1-10: positive samples at 421 bp.

296

J. World Poult. Res., 10(2S): 292-298, 2020

2017 ). VITEK2 BCL Card is a highly advanced method

DISCUSSION

for the identification of B. cereus (Halket et al., 2010).

In this study, 100% of tested B. cereus isolates

harbored Nhe gene that this result is in accordance with

that presented by Anderson et al. (2001) and Ashraf et al.

(2019), while Hbl gene was found in 10% of the tested

isolates, which is similar to findings reported by Ashraf et

al. (2019). Also, 100% of tested B. cereus were found to

harbor cytk gene and this result approved with Kamelia et

al. (2018) (81.5%) and Ngamwongsatit et al. (2008). Also,

groEl gene was present in 100% of tested B. cereus which

is a valuable target for phylogenetic studies to detect the B.

cereus (Chang et al., 2003) and has already been used in

PCR assay to detect the B. cereus (Taylor et al., 2005;

Chang et al., 2003 ).

Food-borne diseases are reported to be a serious hazard to

public health all over the world. Among the organisms

responsible for causing foodborne diseases, B. cereus has

emerged as a major foodborne pathogen during the last

few decades and causes two types of illness through the

elaboration of enterotoxins (Jay, 2005).

In this study, the incidence of B. cereus in meat

products was 28%. These results were nearly similar to

that obtained by Tewari et al. (2015), who isolated B.

cereus from 35% of meat products. The results of the

present study were higher than those obtained by Ashraf et

al. (2019), who isolated B. cereus at a percentage of 11.24,

while the results were lower than those obtained by

Shimaa et al. (2018 ) who isolated B. cereus at a

percentage of 47%.

DECLARATIONS

The incidence of B. cereus in beef meat products was

20% that was lower than that obtained by Hesham et al.

(2018), where the incidence of B. cereus was 38.2%. The

incidence of B. cereus in chicken meat products was 36%

nearly similar to results obtained by Hesham et al. (2018),

who isolated B. cereus from chicken meat products at a

rate of 48%.

Acknowledgments

This study was supported by Animal Health

Research Institute (AHRI) in Doki and AHRI in Shibin El

Kom branch.

Competing interests

No competing interest exists

The incidence of B. cereus in Kobeba-shami was

24%. This result was lower than that obtained by Shimaa

et al. (2018) where the incidence of B. cereus was 52%

and Hemmat et al. (2014) who isolated B. cereus from

84% of the examined kobeba-shami samples. The

incidence of B. cereus in Rice kofta was 16%. This result

was lower than that obtained by Shimaa et al. (2018) who

isolated B. cereus at a rate of 60%. The incidence of B.

cereus in nuggets was 28%. This result was lower than

that obtained by Smith et al. (2004) who isolated B. cereus

at a percentage of 91.6%. The incidence of B. cereus in

chicken pane was 44% that was higher than that obtained

by Smith et al. (2004) who failed to isolate B. cereus from

examined chicken products.

These variations in the results were attributed to the

quality of raw materials and the hygienic state during the

preparation and processing of the product. The high

frequency of isolation of B. cereus from meat products

may be attributed to the processing of minced meat also

additives and spices added to these products, which can

increase the number of Bacillus spores. Therefore it is

important to use additives from a trustful source during the

processing of raw meat and test these additives regularly

for the presence of Bacillus spore (Shawish and Tarabees,

Authors' contributions

All authors contributed equally to this work.

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