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Research Article
 

Prevalence of Virulence Determinants among Bacillus cereus Isolated from Milk Products with Potential Public Health Concern



Eman El-Shahat Abdeen, Heba Hussien, Ghada Abd Elmonsef Hadad and Walid Saad Mousa
 
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ABSTRACT

Background and objective: Bacillus cereus is described as one of the public health pathogen causing severe food poisoning outbreaks worldwide. Accurate surveillance about B. cereus in Egypt is scanty. For this reason, the current study was conducted to determine the prevalence of B. cereus and its virulence genes among milk powder and Ras-cheese products. Materials and Methods: Two hundred samples (130 and 70) from milk powder and Ras-cheese, respectively were aseptically collected and cultured onto specific media. The obtained isolates were subjected to mPCR for screening of virulence genes (nhe, cytK, pc-plc, hblD, hbI and ces) among of B. cereus isolates that obtained from milk powder and Ras-cheese. Results: The result revealed that B. cereus was recovered with 6.9 and 8.5% from milk powder and Ras-cheese, respectively. The nhe gene was detected and dominated in all isolates 100% from both products. In milk powder, pc-plc was the most prevalent gene (100%). However, cytK, hblD, hbl and ces genes were prevalent with 55.5, 33.3, 33.3 and 22.2%, respectively. Regarding to Ras-cheese, the prevalence of cytK was (83.33%) while each of hbI, hblD, pc-plc and ces genes were recovered in 50% of tested isolates. Conclusion: This result provided an important epidemiological view about the contamination rate and the most prevalent virulence genes of B. cereus in milk products in Egypt.

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  How to cite this article:

Eman El-Shahat Abdeen, Heba Hussien, Ghada Abd Elmonsef Hadad and Walid Saad Mousa, 2020. Prevalence of Virulence Determinants among Bacillus cereus Isolated from Milk Products with Potential Public Health Concern. Pakistan Journal of Biological Sciences, 23: 206-212.

DOI: 10.3923/pjbs.2020.206.212

URL: https://scialert.net/abstract/?doi=pjbs.2020.206.212
 
Copyright: © 2020. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

INTRODUCTION

Bacillus cereus was identified as a common opportunistic bacterium in young children foods1. Also it is widely distributed in water, various food products as rice, meat and milk products2. B. cereus is Gram positive, spore forming bacilli that exerts emetic and diarrheal toxins. These toxins are high resistance to temperature and pasteurization that causing a serious public health hazard to human3. B. cereus was listed as one of the common etiological agent incriminated in contamination of food products and poisoning4. Furthermore, B. thuringiensis and B. cereus were implicated as foodborne pathogens producing toxin and enzymes, which potentially possess serious illness to human consumers5. For examples, in United States about 19% of foodborne outbreaks from 1998-2009 were attributed to B. cereus, causing 2 disease forms by emetic and diarrheal toxins6. The emetic form observed clinically by nausea and vomiting7. Meanwhile, the diarrheal form exhibited abdominal cramps and watery diarrhea after ingestion of contaminated foods8. The main effect of B. cereus toxins depend on the induction of cellular membrane pores and electrolyte imbalance with severe losses of Na and Cl ions9. Furthermore, these toxins include hemolysin BL (HBL), nonhemolytic enterotoxin (NHE) and cytotoxin K (cytK), enterotoxin FM (EntFM), enterotoxin T (BceT) and hemolysin II (Hly II) were identified as critical virulence determinants in the diarrheal syndrome10. The major influence of hbl toxins were related to its composition of 2 lytic proteins, L1 and L2, that are encoded, by hblD and hblc genes respectively and a binding component B encoded by hblA gene11. In contrast, cytotoxin K was discovered in 2000 from gastrointestinal outbreaks in France which include cytK-1 and cytK-2 causing necrotic and heamolytic damage12,13. The phosphatidylcholine-specific phospholipase C and sphingomyelinase encoded by the plc and sph genes, respectively together linked to the hemolytic activity in B. cereus isolates14. Moreover, ces genes contribute a major role in the production of toxin, through control of the function of chromosomal key transcriptional regulators15. As results of lacking of important literatures about the prevalence of B. cereus in milk products in Egypt. So, this study aimed to spot the light upon the public health implications of B. cereus among milk powder and Ras-cheese in some Egyptian products. Furthermore, this study explains that B. cereus specially found in milk products still have a serious concern on health in Egypt as a foodborne pathogen and should be strengthen the public awareness, health education and hygienic measures to overcome contamination of food products. As well as applying of modern molecular tools as mPCR for detection of the substantial virulence genes of B. cereus and their role in pathogenicity. This study act as survey about the existence of B. cereus in some milk products in Egyptian markets and will help in construct a plan for prevent contamination of these products during manufacturing.

MATERIALS AND METHODS

Collection of milk products: Two hundred, samples were collected from milk powder and Ras-cheese (130 and 70 samples, respectively) from local markets in Menoufia Governorate, Egypt. The collected samples were put in sterile plastic bags and then transported rapidly as possible in an ice box to the laboratory for further isolation and identification.

Phenotypic identification of B. cereus isolates: From each sample (1 g) was transferred to 9 mL of peptone water tubes (0.1%, PH 7.0), incubated at 37°C for 24 h then a loopful from each incubated tubes were streaked on polymyxin pyruvate-egg yolk-mannitol-bromothymol blue agar (PEMBA) and Mannitol Egg Yolk Polymyxin (MYP) at 37°C for 24 h. Colonies of B. cereus appear as peacock blue and pink color surrounded by a zone of precipitation. Then, prepared film stained with Gram's stain examined microscopically for Gram positive rods. The standard biochemical tests (Nitrate reduction, starch hydrolysis, haemolysis, phenol red glucose broth) were performed as described by Sandra et al.16.

Molecular detection of B. cereus virulence genes
DNA extraction: DNA extraction from samples using commercial kits (Qiagen, Germany, GmbH) with the manufacturer’s recommendations. Briefly, 200 μL of the sample suspension was incubated with 10 μL of proteinase K and 200 μL of lysis buffer at 56°C for 10 min. Then added 200 μL of 100% ethanol to the lysate followed by sample washing and centrifugation. Finally, 100 μL of elution buffer was added to elute the nucleic acid.

Primers sequences, target genes, amplicon sizes and cycling conditions of B. cereus virulence genes as listed in Table 1.

PCR amplification: The mPCR was used for detection of ces, cytK, hbl and nhe genes and duplex PCR for pc-plc and hblD genes, all were utilized in a 50 μL reaction comprised of 25 μL of PCR Master Mix (Takara, Japan), 1 μL of each primer of 20 pmol concentrations, 13 μL of water and 8 μL of DNA template. The reaction was performed in an Applied Biosystems 2720 thermal cycler.

Table 1:
Primers sequences, target genes, amplicon sizes and cycling conditions of B. cereus virulence genes

Table 2:
Prevalence of B. cereus from milk powder and Ras-cheese
Percentage was estimated according to the number of each product (70 and 130, respectively)

Table 3:
Distribution and frequency of B. cereus virulence genes among tested isolates

Analysis of the PCR products: The PCR products were separated by electrophoresis on 1% agarose gel (Applichem, Germany, GmbH) in 1x TBE buffer at room temperature using gradients of 5V/cm. About 40 μL of the products was loaded in each gel slot. The gel was photographed by a gel documentation system (Alpha Innotech, Biometra) and the data was analyzed through computer software

RESULTS

Prevalence of B. cereus from milk powder and Ras-cheese:The bacteriological examination of 130 and 70 samples from milk powder and Ras-cheese respectively recorded that the prevalence rate of B. cereus in these products was 6.9% and 8.5%, respectively as shown in Table 2.

Distribution and frequency of B. cereus virulence genes among tested isolates: The results in Table 3 showed the distribution and frequency of B. cereus virulence genes from milk powder and Ras-cheese. The results revealed that nhe gene was the most common and prevalent gene with 100% in milk powder and Ras-cheese and successful amplified at 766 bp as illustrated in Fig. 1.

Fig. 1:
Agarose gel electrophoresis (%) of PCR products showed amplification of cytK gene at 421 bp, positive samples (2, 3, 6, 7, 9 from milk powder) and (10, 11, 12, 13 and 15 from Ras-cheese), ces gene at 1271 bp positive samples (7, 8 from milk powder) and (10, 11 and 14 from Ras-cheese), nhe gene at 766 bp positive samples at (1-9 from milk powder) and (10-15 from Ras-cheese) and hbI gene at 1091 bp positive samples (1, 7, 8 from milk powder) and (10,11 and 13 from Ras-cheese)

Fig. 2:
Agarose gel electrophoresis (%) of PCR products showed amplification of pc-plc gene detect at 411 bp positive samples (1-9 from milk powder) and ( 11, 12 and 15 from Ras-cheese), hbID gene at 148 bp positive samples (1, 7 and 8 from milk powder) and (10, 11 and 14 from Ras-cheese)

In contrast to milk powder, the pc-plc gene was detected in 100% and amplified at 411 bp as shown in Fig. 2, while, cytK, hbl, hblD and ces genes were predominate with 55.5, 33.3, 33.3 and 22.2%, respectively and amplified at 421,1091, 148 and 1271bp, respectively as shown in Fig. 1 and 2 . In contrast to the prevalence of these virulence genes in Ras-cheese, the cytK gene was prevalent with 83.33% and amplified at 421 bp as shown in Fig. 1, while hbl, hblD, pc-plc and ces virulence genes were prevalent with 50% for each gene and amplified at 148, 1091, 411 and 1271 bp, respectively as illustrated in (Fig. 1 and 2).

DISCUSSION

WHO classified B. cereus as one of the foodborne enterotoxigenic pathogens of public health that contaminated to several types of food products20. The genus bacilli includes B. cereus, is a bacterial organism responsible for intestinal and extraintestinal infections via production of endospore21. Diarrheal or emetic syndromes have been recognized in several cases of foodborne outbreaks due to B. cereus toxins contaminated to food products22. In this study the prevalence of B. cereus from milk powder and Ras-cheese in Egypt was 6.9 and 8.5%, respectively. In comparing to previous studies, much higher prevalence 42% was recorded by Rahimi et al.23 from 200 sample and (35.7%) and (38.7%) recorded by Owusu-Kwarteng et al.24 obtained from raw milk and Nunu cheese, respectively. Moreover, B. cereus was common prevalent bacteria in some milk products such as cheese, ice cream, milk powder and milk with prevalence ranged25 (33-55%). In other studies, a wide range in the prevalence of B. cereus was reported. In Norway, 33 and 22% in Finland26. Meanwhile, significant lower prevalence was reported in England and Wales (0.7%), Japan (0.8%), USA (1.3%) and Canada (2.2%)27. The discrepancy in prevalence may be attributed up to post-pasteurization contamination and/or storage temperature abuse the high incidence of B. cereus, in particular milk powder which is likely due to biofilm formation on the milk evaporators that can be a source of recurrent contamination of the final product28. Some putative virulence factors such as hemolysins, lipases, enterotoxins and proteases possess significant virulence and pathogenicity among B. cereus isolates29. The virulence determinants of B. cereus were potentially expressed by various genes, as hemolysin BL (hbl) and nonhemolytic enterotoxin (nhe)29,30. These enterotoxins genes and cytotoxin K enhanced the ability of B. cereus to cause diarrheal syndrome31. Many investigation support the contribution of several enterotoxins in increasing the B. cereus pathogenicity especially nhe gene. According to Gao et al.32 recorded that 45% of B. cereus strains harbored enterotoxigenic genes and most of isolates carried nheABC gene cluster with the incidence of other virulence genes (cytK, entFM, bceT, hlyII and cesb). In Our findings, the nhe gene was the most dominant gene in both screened products (milk powder and Ras-cheese) with 100%. The prevalence of cytK, hbl, hblD, pc-plc and ces virulence genes in milk powder were 55.5, 33.3, 33.3 and 22.2%, respectively. Meanwhile the prevalence of cytK was 83.3% in Ras-cheese as well as other screened genes hbl, hblD, pc-plc and ces were 50% for each gene. This result is quite similar to the results of Wijnands et al.33, who demonstrated that nhe was the prevalent gene in more than 97% of the isolates, while hbl and cytotoxin K were 66 and 50%, respectively. Although, the wide diversity in incidence of hbl genes within the B. cereus is depended on some factors such as suitable temperature for growth, food matrix and nutritional availability Carlin et al.34 or may be due to the effect of hemolytic enterotoxin, hbl, which encoded mainly by the hblCDA operon35. Other studies declared that both nheABC and entFM gene were the most prevalent genes in B. cereus isolates from foods and environmental sourcess18 36,37. Additionally, similar studies illustrated that incidence rate of hbl gene complex ranged from 40 and 70.6% from milk and dairy products respectively38-40. Although Hendriksen et al.10 pointed out that hbl, nhe and cytK are key virulence factors responsible for the production of enterotoxins in B. cereus isolates. Furthermore, the entFM gene encoded (enterotoxin FM) and bceT (B. cereus enterotoxin) were identified as virulence factors for B. cereus41. Regarding to the activities of pc-plc and sph genes as potential virulence factors of B. cereus toxins which comprise the cytolytic unit cereolysin AB42. In particular the hemolysis properties that produced due to effect of phosphatidylcholine-specific phospholipase C (pc-plc) and Sphingomyelinase (sph)14. As well as s a significant reduction of plcA and hblC activity may occurred due to the mutation in flhA gene and proposed the existence of other PlcR-regulated virulence determinants within the flagellar apparatus43. On explaining the role of the ces gene, ces operons that comprise cesA/cesB genes coded for the non-ribosomal peptide synthetase that plays a worthy and critical role in toxin production15. Our findings reported that ces gene was detected with 50 and 22.2% from Ras-cheese and milk powder, respectively. Although, lower prevalence 3 and 5% were reported for ces gene by Gao et al.32 and Ding et al.44, respectively. This obvious difference in this gene prevalence may be explained due to the difference in samples sources and size and hygienic standards applied at dairy products industry.

CONCLUSION

In conclusion, B. cereus is common bacterial pathogen incriminated in food poisoning outbreaks. Our results revealed B. cereus prevalence rate 9 and 6.9% in milk powder and Ras-cheese, respectively. The application of modern molecular techniques as multiplex PCR was efficiently for detection of nhe, pc-plc, cytK, ces, hbI and hbID virulence genes among B. cereus isolates from milk powder and Ras-cheese. These genes were found in higher percentage suggesting its critical role in pathogenicity and food poisoning outbreaks. In conclusion, these results provide valuable knowledge about the prevalence of B. cereus in some milk products that constitute public health importance.

SIGNIFICANCE STATEMENT

This study provided an important view insight about the prevalence of B. cereus in milk powder and Ras-cheese collected from local markets in Egypt. Also, identified most substantial putative virulence genes among the identified isolates. These results will help the researchers to identified the most virulence genes among B. cereus isolates recovered from some milk products in Egypt and suggested the possible recommendation and hygienic measures needed to applied to prevent the potential public health hazard of B. cereus contamination to milk products.

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