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Detection of the hbl complex genes in Bacillus cereus isolated from cow raw milk in northwest of Iran

    R. Alipour Banaei Saman Mahdavi

Iraqi Journal of Veterinary Sciences, 2020, Volume 34, Issue 2, Pages 459-463
10.33899/ijvs.2020.126120.1238

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Abstract

Bacillus(B) cereus is regarded as a major foodborne pathogen which is widely distributed in the nature. In addition, it plays an important role in the contamination of ready-to-eat and dairy products. B. cereus causes the two different types of food poisoning in human: the diarrheal and the emetic type. The aim of this study is detection of hbl complex genes in B. cereus isolated from cow raw milk in Northwest of Iran. In the present study, the number of the samples collected from cow raw milk were 120. All the isolates already had been identified phenotypically, and they were assessed for molecular confirmation by using the PCR method. B. cereus isolates were determined by detecting the hbl genes complex in the isolates. The result of this study showed that B. cereus were found in the raw milk samples 117 (97.5%) from the 120 samples. The frequency of the hblA, hblC, and hblD genes found in B. cereus isolates were 105 (89.7%), 102 (87.1%), and 102 (87.1%) , respectively. 99 isolates (84.6%) harboured 3 tested genes simultaneously. 12 B. cereus isolates (10.3%) lacked these genes. The results of current study showed that B. cereus isolated from raw milk have high potential in causing food poisoning and therefore the use of the procedures to reduce the bacterial contamination during the processing of dairy product is required.
Keywords:
    Bacillus cereus Raw milk hbl genes complex
Main Subjects:
  • Milk Hygiene

Detection of the hbl complex genes in Bacillus cereus isolated from cow raw milk in northwest of Iran

 

R. Alipour Banaei1 and S. Mahdavi2*

 

1 Department of Genetics, Tabriz Branch, Islamic Azad University, Tabriz, 2 Department of Microbiology, Maragheh Branch, Islamic Azad University, Maragheh, Iran, * Email: S.mahdavi@iau-maragheh.ac.ir

 

(Received September 20, 2019; Accepted February 21, 2020; Available online August 15, 2020)

 

Abstract

 

Bacillus(B) cereus is regarded as a major foodborne pathogen which is widely distributed in the nature. In addition, it plays an important role in the contamination of ready-to-eat and dairy products. B. cereus causes the two different types of food poisoning in human: the diarrheal and the emetic type. The aim of this study is detection of hbl complex genes in B. cereus isolated from cow raw milk in Northwest of Iran. In the present study, the number of the samples collected from cow raw milk were 120. All the isolates already had been identified phenotypically, and they were assessed for molecular confirmation by using the PCR method. B. cereus isolates were determined by detecting the hbl genes complex in the isolates. The result of this study showed that B. cereus were found in the raw milk samples 117 (97.5%) from the 120 samples. The frequency of the hblA, hblC, and hblD genes found in B. cereus isolates were 105 (89.7%), 102 (87.1%), and 102 (87.1%) , respectively. 99 isolates (84.6%) harboured 3 tested genes simultaneously. 12 B. cereus isolates (10.3%) lacked these genes. The results of current study showed that B. cereus isolated from raw milk have high potential in causing food poisoning and therefore the use of the procedures to reduce the bacterial contamination during the processing of dairy product is required.

 

Keywords: Bacillus cereus, raw milk, hbl genes complex

 

DOI: 10.33899/ijvs.2020.126120.1238, ©2020, College of Veterinary Medicine, University of Mosul.

This is an open access article under the CC BY 4.0 license (http://creativecommons.org/licenses/by/4.0/).

 

الکشف عن معقد الجینات hb1فی جراثیم العصویات الشمعیة المعزولة من حلیب البقر الخام

 فی شمال غرب إیران

 

رضا علیبور بانای1 و سامان مهدڤی2

 

1فرع الوراثة، قسم تبریز، تبریز، 2فرع الأحیاء المجهریة، قسم مراغة، جامعة ازدی الإسلامیة، مراغة، ایران

 

الخلاصة

 

تعتبر جرثومة العصویات الشمعیة من مسببات الامراض الرئیسیة التی تنتقل عن طریق الغذاء والمنتشرة على نطاق واسع فی الطبیعة. بالإضافة الى ذلک، فانها تلعب دورا مهما فی تلوث منتجات الالبان الجاهزة للأکل. تسبب العصیات الشمعیة نوعین مختلفین من التسمم الغذائی للإنسان: نوع الاسهال ونوع القیء. الهدف من هذه الدراسة هو الکشف عن مرکب الجینات hbl فی العصیات الشمعیة المعزولة من حلیب البقر الخام فی شمال غرب ایران. جمعت 120 عینة من حلیب الابقار الخام. تم التعرف على جمیع العزلات من خلال الصفات المظهریة، وکذلک تم تقییم العزلات للتأکید الجزیئی باستخدام طریقة تفاعل البلمرة المتسلسل. تم تحدید عزلات العصیات الشمعیة بالکشف عن مرکب الجینات hbl فی العزلات. اظهرت نتائج الدراسة العثور على العصیات الشمعیة فی 117 (97,5%) عینة من عینات الحلیب الخام من مجموع 120 عینة. تم ایجاد جینات hbl A،  hbl Cو hbl D فی العصیات الشمعیة 105 (89,7%)، 102 (87,1%) و 102 (87,1%)، على التوالی. کذلک اظهرت الدراسة ان 99 (84,6%) من العصیات الشمعیة تحتوی على ثلاث جینات، بینما اکدت الدراسة ان 12 (10,3%) من العصیات الشمعیة لا تحتوی على مرکب الجینات hbl. اظهرت نتائج الدراسة الحالیة ان العصیات الشمعیة المعزولة من الحلیب الخام لها القدرة الکبیرة فی إحداث التسمم الغذائی لذلک فان اتباع الاجراءات الصحیة المناسبة یقلل من التلوث بالجراثیم خلال مراحل تصنیع منتجات الالبان.

 

 


Introduction

 

Foodborne disease is regarded as a one of the most important disease which causes a serious problem in developed and developing countries (1). Raw milk is considered a good medium for growth and proliferation of the algae, protozoans, fungi, bacteria, and viruses because it has the most important nutrition factors. There are many types of pathogenic bacteria have been isolated from raw milk, some of these pathogenic bacteria are able to form spores and can tolerate the pasteurization conditions. B. cereus is one of the most important pathogens that tolerates the pasteurization process (2). This bacterium is usually a source of raw milk contamination and a major microbiological problem in the dairy industry. The heat resistant of the B. cereus spores is a source of contamination for milk products (3). B. cereus has many pathogenicity factors which causes diarrhea associated with production of enterotoxins such as the hemolysin BL (hbl), nonheliolateral enterotoxin (NHE), cytotoxin K, and FM enterotoxin (4). B. cereus produces the toxin in the small intestine that causes food poisoning and diarrhea (5). The hemolysin BL toxin is consisting of a three-component protein complex (6), which is formed from a sticky component (B) and two lithic components (1L and 2L), that coded the hblA, hblD, and hblC genes, respectively. The presence of the three genes are necessary for maximum activity and poisoning (7). The B. cereus infectious dose which causes the food poisoning is 104-1011 cells per 1 gram of food. The exact amount of toxin depends on the several factors, such as presence of vegetative bacterial cells, sporulated form in food, amount of produced enterotoxins, and the sensitivity of target cell population (8-10). The aim of this study was to detect the hbl complex genes in B. cereus isolated from raw cow's milk in Northwest of Iran.

 

Materials and methods

 

Samples collection

In this study, 300 cow raw milk samples collected from different regions in northwest of Iran (the period of collect the samples was from April to October in 2018). All the samples were tested by using the culture and biochemical tests to detect the characteristics of B. cereus isolates and finally, 120 B. cereus isolates were identified and they were sent to molecular identification by using PCR method.

 

Molecular detection of B. cereus and the hbl complex genes

DNA extraction of the B. cereus isolates was performed by using the DNA extraction kit (Pak Gene Yakhteh company, Iran). The quality of extracted DNA samples was evaluated by using the Nano Drop instrument and suitable samples to select for the next steps. The B. cereus specific primers used in the present study (Nano Zist Fanavaran company, Iran) (Table 1).

The PCR reaction was performed in a total volume of 20 μl containing 10×PCR buffer 2 µl, MgCl2 2 mM, dNTP 0.2mM, specific primers (0.25 µM), Taq DNA polymerase 1.5 U, and extracted DNA 4 μl using the thermal cycler (Astec, Japan). The PCR conditions for each gene are presented in the Table 2. The obtained PCR products were electrophoresed on 1.5% agarose gel (11).

 

Results

 

The result of this study decleared that B. cereus found in the 117 samples from the 120 investigated samples which were previously detected by using biochemical tests, and they were confirmed as B. cereus by using PCR reaction (Figure 1).

 

 

Table 1: Sequence of primers used for detection of B. cereus and the hbl complex genes

 

Gene

Sequence (5′-3′)

Amplicon size

Reference

Bal

F: 5′-TGCAACTGTATTAGCACAAGCT-3′

R: 5′-TACCACGAAGTTTGTTCACTACT-3′

533 bp

9

hblA

F: 5′-GTGCAGATGTTGATGCCGAT-3′

R: 5′-ATGCCACTGCGTGGACATAT-3′

320 bp

10

hblC

F: 5′-AATGGTCATCGGAACTCTAT-3′

R: 5′-CTCGCTGTTCTGCTGTTAAT-3′

750 bp

10

hblD

F: 5′-AATCAAGAGCTGTCACGAAT-3′

R: 5′-CACCAATTGACCATGCTAAT-3′

430 bp

10

                               

 

Table 2: The thermocycler programs for detection of B. cereus and hbl complex genes

 

Primer

Temperature (ºC)/Time(sec/min)

Cycle

Initial denaturation

Denaturation

Annealing

Extension

Final extension

Bal

94ºC (03:00)

94ºC (00:30)

54ºC (00:45)

72ºC (01:00)

72ºC (05:00)

35

hblA

94ºC (04:00)

94ºC (00:30)

58ºC (00:45)

72ºC (01:00)

72ºC (05:00)

35

hblC

94ºC (03:00)

94ºC (00:30)

53ºC (00:45)

72ºC (01:00)

72ºC (05:00)

35

hblD

94ºC (04:00)

94ºC (00:30)

54ºC (00:45)

72ºC (01:00)

72ºC (05:00)

35

 


The Figure 2 showed that the rate of the hblA gene found in the B. cereus isolates was 89.7% (105/117). In addition, the Figure 3 appeared that the rate of the hblC gene found in the B. cereus isolates was 87.1% (102/117). Moreover, the Figure 4 declared that the rate of the hblD gene found in the B. cereus isolates was 87.1% (102/117). Also, all the three genes were detected in the B. cereus isolates 99 (84.6%). In the other hands, 12 isolates of B. cereus (10.25%) were without studied genes (Table 3).

 

 

 

 

Figure 1. Electrophoresis of the bal gene PCR product on 1.5% agarose. Lad: ladder 50 bp; No. 1: positive control (B. cereus ATCC 11778); No. 2: negative control (double-distilled water); No. 3-6 and 8-14 and 16-20: positive B. cereus samples; No. 7 and 15: negative B. cereus sample.

 

 

 

Figure 2. Electrophoresis of the balA gene PCR product on 1.5% agarose. No. 1: ladder 50 bp; No. 2: positive control (B. cereus ATCC 11778); No. 3: negative control (double-distilled water); No. 4-15: positive B. cereus samples; No. 16-18: negative B. cereus sample.

 

 

Figure 3. Electrophoresis of the balC gene PCR product on 1.5% agarose. No. 1: ladder 50 bp; No. 2: positive control (B. cereus ATCC 11778); No. 3: negative control (double-distilled water); No. 4-15: positive B. cereus samples; No. 16-18: negative B. cereus sample.

 

 

 

Figure 4. Electrophoresis of the balD gene PCR product on 1.5% agarose. No. 1: ladder 50 bp; No. 2: positive control (B. cereus ATCC 11778); No. 3: negative control (double-distilled water); No. 4-18: positive B. cereus samples.

 

Table 3. Frequency of hbl complex genes in studied isolates

 

Genes

Isolates number

Frequency (%)

hblA

105

89.7%

hblC

102

87.1%

hblD

102

87.1%

hblA + hblC

102

87.1%

hblA + hblD

102

87.1%

hblC + hblD

99

84.6%

hblA+hblC+hblD

99

84.6%

 

Discussion

 

In present study, 120 cow raw milk samples collected from different regions in Northwest in Iran. All the B. cereus isolates were previously detected by using the phenotypic culture and the biochemical tests. After PCR reaction by using the specific primers, 117 B. cereus isolates were detected as a B. cereus, genetically. This indicates a higher accuracy of PCR method than the culture biochemical tests. The rapid methods for identify presence of enterotoxigenic B. cereus in foods is very important to ensure the foods hygiene. The culture and Biochemical tests are less accurate compared with the PCR reaction, which is more accurate and more reliable. In the present study, the frequency of hblA, hblC and hblD genes were showed 105 (89.7%), 102 (87.1%) and 102 (87.1%), respectively. In the previously study by Kim et al. (12) in South Korea reported that the prevalence of hblA and hblC genes in standard strains of B. cereus were 6.25%, and the frequency of hblD gene was 25%. In another study showed that only 12.5% of the isolates had all the three genes, simultaneously (12), which is much less than frequency of mentioned genes in present study. Deilami and Nasiri (13) reported that the frequency of the hbl complex genes in B. cereus isolated from foodstuffs in Tabriz and Zanjan restaurants was 8%, which is also much less than frequency of mentioned genes in present study. Prub et al. (14) reported that the prevalence of the hblA gene in B. cereus was 43%. Reis et al. (15) reported that 36.5% of isolated B. cereus from pasteurized, sterilized and dry milk in Brazil had the hbl complex genes. In another study, El-zamkan and Mubarak in Egypt (16), has been reported that the frequency of the hbl complex genes in B. cereus isolated from ice cream and rice-milk was 33.3% and 43.5%, respectively (16). Differences in distribution of the hbl complex genes in different B. cereus isolates in the mentioned studies probably are due to the geographical differences and the differences in ecological origin of isolated strains from milk, rice, meat, salads. Due to presence of all the three hbl complex genes simultaneously in studied B. cereus isolates in this study, the hemolysin BL enterotoxin will have its maximum activity, and these isolates will potentially be highly pathogenic, if hbl complex genes are expressed. Many factors affect the microbial quality of raw milk, which four factors considered as main sources in microbial contamination of raw milk. These resources include inside of livestock breast, exterior of livestock breast, environmental factors, and milking equipment and maintenance. Therefore, in order to provide hygienic milk and its products, health care must be respected according to Hazard Analysis and Critical Control Point (HACCP) instructions, during the production and consumption (2,17). In general, the culture method and the biochemical tests are time-consuming and less accurate than the PCR method. Using the PCR test, in addition to being quicker, has more accuracy and confidence.

 

Conclusion

 

In this study, regarding that the most of the tested Bacillus cereus isolates harboured all the three hbl genes, in the case of the expression of these genes, these isolates will have high virulence potentially.

 

Acknowledgements

 

The authors have supported all costs of the present research.

 

Conflict of interest

 

The authors declare that they have no conflict of interest.

  1. Detection of the frequency of Bacillus cereus in cow raw milk samples in northwest of Iran
  2. Determination of the frequency of hbl complex genes in these isolates.
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(2020). Detection of the hbl complex genes in Bacillus cereus isolated from cow raw milk in northwest of Iran. Iraqi Journal of Veterinary Sciences, 34(2), 459-463. doi: 10.33899/ijvs.2020.126120.1238
R. Alipour Banaei; Saman Mahdavi. "Detection of the hbl complex genes in Bacillus cereus isolated from cow raw milk in northwest of Iran". Iraqi Journal of Veterinary Sciences, 34, 2, 2020, 459-463. doi: 10.33899/ijvs.2020.126120.1238
(2020). 'Detection of the hbl complex genes in Bacillus cereus isolated from cow raw milk in northwest of Iran', Iraqi Journal of Veterinary Sciences, 34(2), pp. 459-463. doi: 10.33899/ijvs.2020.126120.1238
Detection of the hbl complex genes in Bacillus cereus isolated from cow raw milk in northwest of Iran. Iraqi Journal of Veterinary Sciences, 2020; 34(2): 459-463. doi: 10.33899/ijvs.2020.126120.1238
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  1. Mahdavi S, Sadeghi Zali MS, Farajnia S, Mehmannavaz Y, Isazadeh A. The comparison of bovine fecal and buffy coat samples for diagnosis of Johne’s disease based on PCR. Gene Cell Tissue. 2018;5:79745. Doi: 10.5812/gct.79745
  2. Moradi-Khatoonabadi Z, Maghsoudlou Y, Ezzatpanah H, Khomeiri M, Aminafshar M. Occurrence of Bacillus cereus in raw milk receiving from UF-feta cheese plants. Iran J Environ Health. 2014;6:545-556.
  3. Shaheen R, Svensson B, Andersson MA, Christiansson A, Salkinoja-Salonen M. Persistence strategies of Bacillus cereus spores isolated from dairy silo tanks. Food Microbiol. 2010;27:347-355. Doi: 10.1016/j.fm.2009.11.004
  4. Sergeev N, Distler M, Vargas M, Chizhikov V, Herold KE, Rasooly A. Microarray analysis of Bacillus cereus group virulence factors. J Microbiol Meth. 2006;65:488-502. Doi: 10.1016/j.mimet.2005.09.013
  5. Ceuppens S, Rajkovic A, Hamelink S, Van de Wiele T, Boon N, Uyttendaele M. Enterotoxin production by Bacillus cereus under gastrointestinal conditions and their immunological detection by commercially available kits. Foodborne Pathog Dis. 2012;9:1130-6. Doi: 10.1089/fpd.2012.1230
  6. Beecher DJ, Wong AC. Tripartite hemolysin BL: Isolation and characterization of two distinct homologous sets of components from a single Bacillus cereus isolate. Microbiol. 2000;146:1371-1380. doi.org/10.1099/00221287-146-6-1371.
  7. Granum PE, O’Sullivan K, Lund T. The sequence of the non-haemolytic enterotoxin operon from Bacillus cereus. FEMS Microbiol Lett. 1999;177:225-229. doi.org/10.1111/j.1574-6968.1999.tb13736.x
  8. Beecher DJ, Schoeni JL, Wong ACL. Enterotoxin activity of hemolysin BL from Bacillus cereus. Infect Immun. 1995;63:4423-4428.
  9. Chang YH, Shangkuan YH, Lin HC, Liu HW. PCR assay of the groEL gene for detection and differentiation of Bacillus cereus group cells. Appl Environ Microbiol. 2003;69:4502-10. DOI: 10.1128/AEM.69.8.4502-4510.2003.
  10. Hansen BM, Hendriksen NB. Detection of Enterotoxin Bacillus cereus and Bacillus thuringiensis Strains. Appl Environ Microbiol. 2001;67:185-189. Doi: 10.1128/AEM.67.1.185-189.2001
  11. Mahdavi S, Azizi Dehbokri M, Isazadeh A. Contamination of chicken meat with salmonella spp distributed in Mahabad city, Iran. Int J Enteric Pathog. 2018;6:65-68. Doi: 10.15171/ijep.2018.18
  12. Kim MJ, Han JK, Park JS, Lee JS, Lee SH, Cho JI. Various enterotoxin and other virulence factor genes widespread among Bacillus cereus and Bacillus thuringiensis strains. J Microbiol Biotechnol. 2015;25:872-879. DOI: 10.4014/jmb.1502.02003.
  13. Deilami KZ, Nasiri SH. Isolation of Bacillus cereus from foods and studying the cytotoxicity of them on Vero cells. Quarterly J Anim Physiol Anim Dev. 2016;9:69-77.
  14. Prub BM, Dietrich R, Nibler B, Martlbauer E, Scherer S. The hemolytic enterotoxin HBL is broadly distributed among species of the Bacillus cereus group. Appl Environ Microbiol. 1999;65:5436-42.    DOI: 10.1128/AEM.65.12.5436-5442.1999.
  15. Reis ALS, Montanhini MTM, Bittencourt JVM, Destro MT, Bersot LS. Gene detection and toxin production evaluation of hemolysin BL of Bacillus cereus isolated from milk and dairy products marketed in Brazil. Braz J Microbiol. 2013;44:1195-1198. http://dx.doi.org/ 10.1590/S1517-83822013000400024
  16. El-zamkan MA, Mubarak, AG. Detection of B. cereus and some of its virulence genes in some dairy desserts and children diarrhea. Alexandria J Vet Sci. 2017;53:28-38. Doi: 10.5455/ajvs.259758
  17. Lues J, Venter P, Van Der Westhuizen H. Enumeration of potential microbiological hazards in milk from a marginal urban settlement in central South Africa. J Food Microbiol. 2003;20:321-26. Doi: 10.1016/S0740-0020(02)00128-4

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