Detection of Some Staphylococcal Enterotoxin Genes in MRSA Strains Using PCR Techniques

Main Article Content

Suha M. Abed
Waad M. Raoof
Akeel H. A. Assie
Zeina S. M. Al-Hadeithi
Farooq Ibrahim

Abstract

In view of the increasing interest in the Methicillin-resistant Staphylococcus aureus (MRSA). The extracted DNA yield was observed using the phenol-chloroform method, it ranged from (1.6-1.8) and concentration ranged from 100 to 800 ng/µl. Five classical enterotoxin genes were investigated in 20 isolates using multiplex PCR method after it had been molecularly identified into methicillin resistant using mec A (which is the key genetic component of methicillin resistance) and fem A genes in a duplex PCR technique. A multiplex PCR test based on the simultaneous amplification of the five genes genes; sea 102bp, seb 164bp, sec 451bp, sed 278 bp and see 209bp was conducted to directly detect the toxin gene content. Our results had showed that most of MRSA samples harbored at least one enterotoxin gene. Multiple toxin gene combinations were also observed. Using this PCR assay we found that among the MRSA strains obtained (n=20).  The most commonly found gene was the enterotoxin A sea (n: 18, 90%), which was found alone and together with other toxin genes.

Article Details

How to Cite
Suha M. Abed, Waad M. Raoof, Akeel H. A. Assie, Zeina S. M. Al-Hadeithi, & Farooq Ibrahim. (2023). Detection of Some Staphylococcal Enterotoxin Genes in MRSA Strains Using PCR Techniques. Tikrit Journal of Pure Science, 21(3), 33–38. https://doi.org/10.25130/tjps.v21i3.992
Section
Articles

References

1. Ataee R. A., Ataee M. H., Alishiri Gh. H., Esmaeili D., Staphylococcal Enterotoxin C in Synovial Fluid of Patients With Rheumatoid Arthritis, J. of Iran Red Crescent Med. 2014,16 (10): 1-6.

2. Duquenne M., Fleurot I., Aigle M., Darrigo C., Boreze´e-Durant E., Derzelle S., Bouix M., Deperrois-Lafarge V., and Delacroix-Buchet A., Tool for Quantification of Staphylococcal Enterotoxin Gene Expression in Cheese, J. of Appl. Environ. Microbiol., 2010, 76 (5): 1367-1374.

3. Becker K., Roth R. and Peters G., Rapid and Specific Detection of Toxigenic Staphylococcus aureus: Use of Two Multiplex PCR Enzyme Immunoassays for Amplification and Hybridization of Staphylococcal Enterotoxin Genes, Exfoliative Toxin Genes, and Toxic Shock Syndrome Toxin 1 Gene, J. of Clin. Microbiol. 1998, 36 (9): 2548-2553.

4. Sharma N. K., Rees C. E. D. and Dodd Ch. E. R., Development of a Single-Reaction Multiplex PCR Toxin Typing Assay for Staphylococcus aureus Strains, J. of Appl. Environ. Microbiol. 2000, 66 (4): 1347-1353.

5. Naffa R. G., Bdour S. M., Migdadi H. M. and Shehabi A. A., Enterotoxicity and genetic variation among clinical Staphylococcus aureus isolates in Jordan, J. of Med. Microbiol. 2006, 55:183-187.

6. Pinchuk I. V., Beswick E. J. and Reyes V. E., Staphylococcal Enterotoxins, J. of Toxins 2010, 2, 2177-2197.

7. Dinges M. M., Orwin P. M. and Schlievert P. M., Exotoxins of Staphylococcus aureus, J. of Clinic. Microbiol. Rev. 2000, 13 (1): 16-34.

8. Kayser F. H., Bienz K. A., Eckert J., Zinkernagel R. M., Medical Microbiology, Thieme Stuttgart, New York, 2005, p229.

9. Blaiotta G., Ercolini D., Pennacchia C., Fusco V., Casaburi A., Pepe O. and Villani F., PCR detection of staphylococcal enterotoxin genes in Staphylococcus spp. strains isolated from meat and dairy products. Evidence for new variants of seG and seI in S. aureus AB-8802, J. of Appl. Microbiol., 2004, 97: 719-730.

10. Chiang Y-Ch., Liao W-W., Fan Ch-M., Pai W-Y, Chiou Ch-Sh. and Tsen H-Y., PCR detection of Staphylococcal enterotoxins (SEs) N, O, P, Q, R, U, and survey of SE types in Staphylococcus aureus isolates from food-poisoning cases in Taiwan, Int.J. of Food Microbiol., 2008, 121: 66-73.

11. Morandi S., Brasca M., Andrighetto C., Lombardi A. and Lodi R., Phenotypic and Genotypic Characterization of Staphylococcus aureus Strains fromItalian Dairy Products, J. of Int. Microbiol., 2009, 10: 7.

12. Ortega E., Abriouel H., Lucas R. and Gálvez A., Multiple Roles of Staphylococcus aureus Enterotoxins: Pathogenicity, Superantigenic Activity, and Correlation to Antibiotic Resistance, J. of Toxins 2010, 2, 2117-2131.

13. Onasanya A., Mignouna H.D.and Thottappilly G., Genetic fingerprinting and phylogenetic diversity of Staphylococcus aureus isolates from Nigeria, African J. of Biotechnol 2003, 2 (8): 246-250.

14. Tang Y-W. And Stratton C. W., Advanced Techniques in Diagnostic Microbiology, Springer, New York, USA, 2006.p 426,429.

15. Kareem N.H., Sabbah M. A., Ibraheem A. N., Said A.M., Development of SYBR Green Real time PCR for Identification Methicillin-Resistance Staphylococcus aureus (MRSA), J. of Biotech. Research Center, 2013, 7 (1): 67-71.

16. Mehrotra M., Wang G. and Johnson W. M., Multiplex PCR for Detection of Genes for Staphylococcus aureus Enterotoxins, Exfoliative Toxins, Toxic Shock Syndrome Toxin 1, and Methicillin Resistance, J. of Clinic. Microbiol. 2000, 38 (3):1032.

17. Demír C., Aslantas O., Duran N., Ocak S., Ozer B., Investigation of toxin genes in Staphylococcus aureus strainsisolated in Mustafa Kemal University Hospital, J. of Turk. Med Sci. 2011, 41 (2): 343-352.

18. Strommenger B., Kettlitz C., Werner G., and Witte W., Multiplex PCR Assay for Simultaneous Detection of Nine Clinically Relevant Antibiotic Resistance Genes in Staphylococcus aureus, J. of Clinic. Microbiol. 2003, 41 (9): 4089-4094.

19. Oliveira D. C. And H. de Lencastre, Multiplex PCR Strategy for Rapid Identification of Structural Types and Variants of the mec Element in Methicillin-Resistant Staphylococcus aureus, J. of Antimicrob. Agents and Chemotherap. 2002, 46 (7): 2155-2161.

20. Hameed N. M., In vitro Evaluation of the Activity of Aspergillus niger Extract on the Viability of Staphylococcus aureus and Protoscolices of Hydatid cysts, Master thsesis, College of Medicine, University of Tikrit, 1993.

21. Bhutia K. O., Singh T. S., Biswas S., Adhikari L., Evaluation of phenotypic with genotypic methods for species identification and detection of methicillin resistant in Staphylococcus aureus, International J. of Appl. and Basic Med. Research, 2012, 2 (2): 84-91.

22. Anand T., Kalaiselvan A., Gokulakrishnan K., Chandramohan S., Elambarathi P., Analysis of Genetic Variation by Methyl Orange in Rhizobium Using RAPD–PCR, Int. J. Pharm. Tech. Res. 2012, 4 (3): 1101-1109.

23. Tan S. Ch. and Yiap B. Ch., DNA, RNA, and Protein Extraction: The Past and the Present, J. of Biomed. And Biotechnol. 2009: 1-10.

24. Moore P. C. L. and Lindsay J. A, Genetic Variation among Hospital Isolates of Methicillin-Sensitive Staphylococcus aureus: Evidence for Horizontal Transfer of Virulence Genes, J. of Clinic Microbiol. 2001, 39 (8): 2760-2767.

25. Yarwood J. M., McCormick J. K., Paustian M. L., Orwin P. M., Kapur V. and Schlievert P. M., Characterization and Expression Analysis of Staphylococcus aureus Pathogenicity Island 3, Implications for The Evolution of Sphylococcal Pathogenicity Islands, J. of Biol. Chemist., 2002, 277 (15):13138-13147.

26. Karahan M., AcÇık M. N. and Çetinkaya B., Investigation of Toxin Genes by Polymerase Chain Reaction in Staphylococcus aureus Strains Isolated from Bovine Mastitis in Turkey, J. of Foodborne Path. And Dis. 2009, 6 (8): 1029-1035.

27. Jacobsson G., Invasive Staphylococcus aureus infections, Sahlgrenska Academy University of Gothenburg, Sweden, 2009.

28. Bayles K. W. and Iandolo J. J., Genetic and Molecular Analyses of the Gene Encoding Staphylococcal Enterotoxin D, J. of Bacteriol., 1989, 171 (9):4799-4806.

29. Khudor M. H., Abbas B. A. AND Idbeis H. I., Detection of Enterotoin Genes of Staphylococcus aureus Isolates From Raw Milk, Basrah J. Vet. Res. 2012, 11 (1): 245-264.

30. Oliveira L., Rodrigues A. C., Hulland C., Ruegg P. L., Enterotoxin production, enterotoxin gene distribution, and genetic diversity of Staphylococcus aureus recovered from milk of cows with subclinical mastitis, Am. J. of Vet. Res. 2011, 72 (10): 1361-1368.

31. Sareyyüpoǧlu B., Müştak H. K., Cantekin Z. and Dɨker K. S., Molecular detection of exfoliative toxin in Staphylococcus intermedius isolates from dogs with pyoderma, J. of Ankara Üniv. Vet. Fak. Derg, 2013, 60: 15-19.

32. Holochová P., Růžičková V , Dostálová L, Pantůček R, Petráš P and Doškař J, Rapid detection and differentiation of the exfoliative toxin A-producing Staphylococcus aureus strains based on ϕ ETA prophage polymorphisms, J. Diag. Micro. Bio. 2009, 10 (8): 248-252.

33. Henegariu O., Heerema N.A., Dlouhy S.R., Vance G.H. And Vogt P.H., Multiplex PCR: Critical Parameters and Step-by-Step Protocol, Bio Techniques, 1997, 23 (3): 504-511.

34. Elnifro E. M., Ashshi A. M., Cooper R. J., AND Klapper P. E., Multiplex PCR: Optimization and Application in Diagnostic Virology, J. of Clinic. Microbiol. Rev., 2000, 13 (4): 559-570.

35. Sasaki T., Tsubakishita S., Tanaka Y., Sakusabe A., Ohtsuka M., Hirotaki Sh., Kawakami T., Fukata T., and Hiramatsu K., Multiplex-PCR Method for Species Identification of Coagulase-Positive Staphylococci, J. of Clinic. Microbiol. JCM. 2010, 48 (3): 765-769.

36. Sint D.., Raso L. and Traugott M., Advances in multiplex PCR: balancing primer efficiencies and improving detection success, J. of Methods in Ecol. and Evolu., 2012, 3: 898-905.