اثر افزودن سرم گوساله بر بیان ژن‌های ویرولانس استافیلوکوکوس اورئوس
محورهای موضوعی : میکروب شناسی پزشکی
هادی کوهساری
1
(استادیار، گروه میکروبیولوژی، واحد آزادشهر، دانشگاه آزاد اسلامی، آزادشهر، ایران)
عزت الله قائمی
2
(استاد، گروه میکروبیولوژی، دانشگاه علوم پزشکی گلستان، گرگان، ایران)
نور امیرمظفری
3
(دانشیار، گروه میکروبیولوژی، دانشگاه علوم پزشکی ایران، تهران، ایران)
مریم صادق شش پلی
4
(دانش آموخته دکتری تخصصی پزشکی مولکولی، دانشگاه علوم پزشکی گلستان، گرگان، ایران.)
کلید واژه: بیان ژن, استافیلوکوکوس اورئوس, سرم گوساله, ژن‌های بیماریزایی,
چکیده مقاله :
سابقه و هدف: استافیلوکوکوس اورئوس یک پاتوژن فرصت‌طلب و یکی از مهم‌ترین عوامل تهدید کننده سلامت در سطح جهان است. بیان عوامل بیماری زایی باکتری ها در شرایط محیط‌های کشت آزمایشگاهی و شرایط in vivo یکسان نیست. هدف از مطالعه حاضر ارزیابی اثرات افزودن 5٪ سرم گوساله بر بیان ژن‌های ویرولانس استافیلوکوکوس اورئوس است.مواد و روش ها: میزان بیان ژن‌های agrA،RNAIII ، hla، spa و mecA در مدت رشد جدایه های‌های استافیلوکوکوس اورئوس در محیط کشت BHI براث و BHI براث غنی شده با 5 درصد سرم گوساله و در فازهای مختلف رشد تعیین شد. به این منظور منحنی رشد 5 جدایه از باکتری استافیلوکوکوس اورئوس در دو شرایط محیطی مختلف رسم شد. سپسRNA از فازهای مختلف رشد استخراج و میزان بیان ژن‌های یادشده با real-time PCR مورد آنالیز قرار گرفت.یافته ها: به طور میانگین در انتقال از فاز لگاریتمی به سکون میزان بیان ژن‌های agrA و RNAIII در محیط سرم‌دار به ترتیب 3.4 و 9.5 برابر افزایش داشت، اما سیستم agr نتوانست نقش تنظیمی خود را بر بیان ژن‌های ویرولانس به خوبی بازی کند. به این صورت که میزان بیان ژن hla با کاهش 0.81 برابری نشان داد و میزان بیان ژن‌های spa و mecA به ترتیب تنها افزایش 1.25 و1.03 برابری را داشتند.نتیجه گیری: صرف نظر از فاز رشد بیان همه ژن‌ها در محیط BHI براث حاوی سرم گوساله نسبت به محیط BHI براث افزایش نشان داد. از این رو به نظر می رسد که استافیلوکوکوس اورئوس در حضور فاکتورهای موجود در سرم گوساله توانایی القای بیان ژن‌های ویرولانس را مشابه با شرایط in vivo داشته باشد.
Background & Objectives: Staphylococcus aureus is an opportunistic pathogen and one of the most important health-threatening agents worldwide. Expression of bacterial virulence factors is not similar in laboratory medium conditions and in vivo. The aim of the present study was to evaluate the effects of adding 5% calf serum on the virulence genes expression of S. aureus. Materials & Methods: The expression levels of agrA, RNAIII, hla, spa, and mecA genes were determined during the growth of S. aureus isolates in BHI broth and BHI broth enriching with calf serum during different growth phases. Therefore, the growth curve of the five isolates of S. aureus in two different culture conditions was plotted. Subsequently, RNA was extracted from different phases of growth and the genes expression were analyzed by Real-time PCR. Results: In average, the expression levels of agrA and RNAIII from stationary phase to the exponential phase in BHI broth containing calf serum was increased 3.4 and 9.5-fold, respectively, while the agr system could not appropriately play its regulatory role in the expression of virulence genes. As a result, the expression of hla gene was decreased 0.81-fold and the expression levels of spa and mecA genes was only increased 1.25 and 1.03-fold, respectively. Conclusion: Regardless of the growth phase, the expression of the whole genes in BHI broth containing calf serum were increased in comparison to BHI broth. Consequently, it appears that S. aureus is capable to induce virulence genes expression in the presence of calf serum factors similar to conditions available in vivo.
therapeutic options of Staphylococcus aureus. Infect Dis. 2017; 22(10): 1-15.
2. Costa AR, Batistão DWF, Ribas RM, Sousa AM, Pereira MO, Botelho CM. Staphylococcus
aureus virulence factors and disease. Microbial pathogens and strategies for combating them:
science, technology and education (A.Méndez-Vilas,Ed.) Badajoz: Formatex Research Center.
2013; 1: 702-710.
3. Chen FJ, SiuL KK, Lin JC, Wang CH, Lu PL. Molecular typing and characterization of nasal
carriage and community-onset infection methicillin-susceptible Staphylococcus aureus isolates
in two Taiwan medical centers. BMC Infect Dis. 2012; 12: 343-351.
4. Wang B, Muir TW. Regulation of virulence in Staphylococcus aureus: Molecular mechanisms
and remaining puzzles. Cell Chem Biol. 2016; 23(2): 214-224.
5. Oogai Y, Matsuo M, Hashimoto M, Kato F, Sugai M, Komatsuzawa H. Expression of
virulence factors by Staphylococcus aureus grown in serum. Appl Environ Microbiol. 2011; 77
(22): 8097-8105.
6. Koohsari H, Amir Mozafari N, Ghazi-Saidi K, Khavari Nejad RA, Moradi A. Effect of blood
and sera on growth of Staphylococcus aureus in BHI media. J Pure Appl Microbiol. 2013; 7
(4): 2851-2855.
7. Qiu J, Zhang X, Luo M, Li H, Dong J, wang J, Leng B, Wang X, Feng H, Ren W, Deng X.
Subinhibitory concentrations of Perilla oil affect the expression of secreted virulence factor
genes in Staphylococcus aureus. PLoS ONE. 2011; 6(1): 1-8.
8. Loughman JA, Fritz SA, Storch GA, Hunstad DA. Virulence gene expression in human
community-acquired Staphylococcus aureus infections. J Infect Dis. 2009; 199(3): 294-301.
9. Mehrotra M, Wang G, Johnson WM. Multiplex PCR for detection of genes for Staphylococcus
aureus enterotoxins, exfoliative toxins, toxic shock syndrome toxin 1, and methicillin
MSSA isolates as compared to MRSA isolates,
which was also observed in this study, can be a
proof to the validity of this theory.
resistance. J Clin Microbiol. 2000; 38(3): 1032-1035.
10. Seidl K, Chen L, Bayer AS, Hady WA, Kreiswirth BN, Xiong YO. Relationship of agr
expression and function with virulence and vancomycin treatment outcomes in experimental
endocarditis due to methicillin-resistant Staphylococcus aureus. Antimicrob Agents
Chemother. 2011; 55(12): 5631-5639.
11. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time
quantitative PCR and the 2-∆∆CT method. Methods. 2001; 25 (4): 402-408.
12. Abu Othman A, Humphreys H, O'Neill E, Hughes DF. Differences in expression of
virulence genes amongst invasive and colonizing isolates of methicillin-resistant
Staphylococcus aureus. J Med Microbiol. 2011; 2: 259-261.
13. Regassa LB, Novick RP, Betley MJ. Glucose and nonmaintained pH decrease expression of
the accessory gene regulator (agr) in Staphylococcus aureus. Infect Immun. 1992; 60(8):
3381-3388.
14. Mullarky IK, Su C, Frieze N, Park YH, Sordillo LM. Staphylococcus aureus agr genotypes
with enterotoxin production capabilities can resist neutrophil bactericidal activity. Infect
Immun. 2001; 69: 45-51.
15. Malachowa N, Whitney AR, Kobayashi SD, Sturdevant DE, Kennedy AD, Braughton KR,
Shabb DW, Diep BA, Chambers HF, Otto M, Deleo FR. Global changes in Staphylococcus
aureus gene expression in human blood. PLoS ONE. 2011; 6(4): 1-13.
16. Rasmussen G, Monecke S, Ehricht R, Söderquist B. Prevalence of clonal complexes and
virulence genes among commensal and invasive Staphylococcus aureus isolates in sweden.
PLoS ONE. 2013: 8(10): 1-10.
17. Jiménez JN, Ocampo AM, Vanegas JM, Rodríguez EA, Garcés CG, Patiño LA, Ospina S,
Correa MM. Characterisation of virulence genes in methicillin susceptible and resistant
Staphylococcus aureus isolates from a paediatric population in a university hospital of
Medellín, Colombia. Mem Inst Oswaldo Cruz. 2011; 106(8): 980-985.
18. Koohsari H, Ghaemi EA, Amir Mozaffari N, Moradi A, Sadegh-Seshpoli M, Javid SN. The
Effect of adding blood on the virulence genes expression of Staphylococcus aureus in
exponential and stationary growth phase. Jundishapur J Microbiol. 2017; 10(6): 1-6.
19. Collins J, Rudkin J, Recker M, Pozzi C, O’Gara JP, Massey RC. Offsetting virulence and
antibiotic resistance costs by MRSA. ISME J. 2010; 4(4): 577-584.
20. Lee SM, Ender M, Adhikari R, Smith JM, Berger-Bachi B, Cook GM. Fitness cost of
staphylococcal cassette chromosome mec in methicillin-resistant Staphylococcus aureus by
way of continu-ous culture. Antimicrob Agents Chemother. 2007; 51(4): 1497-1499
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therapeutic options of Staphylococcus aureus. Infect Dis. 2017; 22(10): 1-15.
2. Costa AR, Batistão DWF, Ribas RM, Sousa AM, Pereira MO, Botelho CM. Staphylococcus
aureus virulence factors and disease. Microbial pathogens and strategies for combating them:
science, technology and education (A.Méndez-Vilas,Ed.) Badajoz: Formatex Research Center.
2013; 1: 702-710.
3. Chen FJ, SiuL KK, Lin JC, Wang CH, Lu PL. Molecular typing and characterization of nasal
carriage and community-onset infection methicillin-susceptible Staphylococcus aureus isolates
in two Taiwan medical centers. BMC Infect Dis. 2012; 12: 343-351.
4. Wang B, Muir TW. Regulation of virulence in Staphylococcus aureus: Molecular mechanisms
and remaining puzzles. Cell Chem Biol. 2016; 23(2): 214-224.
5. Oogai Y, Matsuo M, Hashimoto M, Kato F, Sugai M, Komatsuzawa H. Expression of
virulence factors by Staphylococcus aureus grown in serum. Appl Environ Microbiol. 2011; 77
(22): 8097-8105.
6. Koohsari H, Amir Mozafari N, Ghazi-Saidi K, Khavari Nejad RA, Moradi A. Effect of blood
and sera on growth of Staphylococcus aureus in BHI media. J Pure Appl Microbiol. 2013; 7
(4): 2851-2855.
7. Qiu J, Zhang X, Luo M, Li H, Dong J, wang J, Leng B, Wang X, Feng H, Ren W, Deng X.
Subinhibitory concentrations of Perilla oil affect the expression of secreted virulence factor
genes in Staphylococcus aureus. PLoS ONE. 2011; 6(1): 1-8.
8. Loughman JA, Fritz SA, Storch GA, Hunstad DA. Virulence gene expression in human
community-acquired Staphylococcus aureus infections. J Infect Dis. 2009; 199(3): 294-301.
9. Mehrotra M, Wang G, Johnson WM. Multiplex PCR for detection of genes for Staphylococcus
aureus enterotoxins, exfoliative toxins, toxic shock syndrome toxin 1, and methicillin
MSSA isolates as compared to MRSA isolates,
which was also observed in this study, can be a
proof to the validity of this theory.
resistance. J Clin Microbiol. 2000; 38(3): 1032-1035.
10. Seidl K, Chen L, Bayer AS, Hady WA, Kreiswirth BN, Xiong YO. Relationship of agr
expression and function with virulence and vancomycin treatment outcomes in experimental
endocarditis due to methicillin-resistant Staphylococcus aureus. Antimicrob Agents
Chemother. 2011; 55(12): 5631-5639.
11. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time
quantitative PCR and the 2-∆∆CT method. Methods. 2001; 25 (4): 402-408.
12. Abu Othman A, Humphreys H, O'Neill E, Hughes DF. Differences in expression of
virulence genes amongst invasive and colonizing isolates of methicillin-resistant
Staphylococcus aureus. J Med Microbiol. 2011; 2: 259-261.
13. Regassa LB, Novick RP, Betley MJ. Glucose and nonmaintained pH decrease expression of
the accessory gene regulator (agr) in Staphylococcus aureus. Infect Immun. 1992; 60(8):
3381-3388.
14. Mullarky IK, Su C, Frieze N, Park YH, Sordillo LM. Staphylococcus aureus agr genotypes
with enterotoxin production capabilities can resist neutrophil bactericidal activity. Infect
Immun. 2001; 69: 45-51.
15. Malachowa N, Whitney AR, Kobayashi SD, Sturdevant DE, Kennedy AD, Braughton KR,
Shabb DW, Diep BA, Chambers HF, Otto M, Deleo FR. Global changes in Staphylococcus
aureus gene expression in human blood. PLoS ONE. 2011; 6(4): 1-13.
16. Rasmussen G, Monecke S, Ehricht R, Söderquist B. Prevalence of clonal complexes and
virulence genes among commensal and invasive Staphylococcus aureus isolates in sweden.
PLoS ONE. 2013: 8(10): 1-10.
17. Jiménez JN, Ocampo AM, Vanegas JM, Rodríguez EA, Garcés CG, Patiño LA, Ospina S,
Correa MM. Characterisation of virulence genes in methicillin susceptible and resistant
Staphylococcus aureus isolates from a paediatric population in a university hospital of
Medellín, Colombia. Mem Inst Oswaldo Cruz. 2011; 106(8): 980-985.
18. Koohsari H, Ghaemi EA, Amir Mozaffari N, Moradi A, Sadegh-Seshpoli M, Javid SN. The
Effect of adding blood on the virulence genes expression of Staphylococcus aureus in
exponential and stationary growth phase. Jundishapur J Microbiol. 2017; 10(6): 1-6.
19. Collins J, Rudkin J, Recker M, Pozzi C, O’Gara JP, Massey RC. Offsetting virulence and
antibiotic resistance costs by MRSA. ISME J. 2010; 4(4): 577-584.
20. Lee SM, Ender M, Adhikari R, Smith JM, Berger-Bachi B, Cook GM. Fitness cost of
staphylococcal cassette chromosome mec in methicillin-resistant Staphylococcus aureus by
way of continu-ous culture. Antimicrob Agents Chemother. 2007; 51(4): 1497-1499