شناسایی فعالیت و بیان ژن پمپ افلاکس سویه های مقاوم به سیپروفلوکساسین استافیلوکوکوس اورئوس
محورهای موضوعی : میکروب شناسی پزشکیزهرا توکلی 1 , حسن صاحب جمعی 2 , لیلا پیشکار 3 , زهره علی مددی 4 , حسن نوربازرگان 5 , امیر میرزایی 6
1 - کارشناس ارشد، گروه زیست شناسی، واحد تهران شمال، دانشگاه آزاد اسلامی، تهران، ایران
2 - استادیار، گروه بیوشیمی و بیوفیزیک، دانشکده علوم زیستی، واحد ورامین پیشوا، دانشگاه آزاد اسلامی، ورامین، ایران
3 - استادیار، دانشگاه آزاد اسلامی، واحد اسلامشهر، باشگاه پژوهشگران جوان و نخبگان، اسلامشهر، ایران
4 - کارشناس ارشد، گروه زیست شناسی، واحد تهران شرق، دانشگاه آزاد اسلامی، تهران، ایران
5 - دکتری، گروه بیوتکنولوژی، دانشکده فناوریهای نوین پزشکی، دانشگاه علوم پزشکی شهید بهشتی، تهران، ایران
6 - استادیار، گروه زیست شناسی، واحد رودهن، دانشگاه آزاد اسلامی، رودهن، ایران
کلید واژه: استافیلوکوکوس اورئوس, پمپ افلاکس, ژن norA, ژن norB, روش real-time PCR,
چکیده مقاله :
سابقه و هدف: باکتری استافیلوکوکوس اورئوس یکی از مهمترین عوامل عفونت‌زای بیمارستانی می‌باشد. اخیرا سویه‌های استافیلوکوکوس اورئوس نسبت به آنتی بیوتیک سیپروفلوکساسین مقاوم شده‌اند و پمپ افلاکس در این مقاومت نقش مهمی را ایفا می‌کند. هدف از این مطالعه، بررسی وجود ژن های پمپ افلاکس (norA و norB)، بیان و فعایت آن در سویه‌های مقاوم به سیپروفلوکساسین استافیلوکوکوس اورئوس بود .مواد و روش ها: در این مطالعه، تعداد 250 نمونه بالینی به منظور جداسازی سویه های استافیلوکوکوس اورئوس، بررسی مقاومت آنتی بیوتیکی، وجود و بیان ژن‌های پمپ افلاکس norA و norB در آنها به ترتیب توسط روش های PCR و real-time PCR مورد مطالعه قرار گرفت. در انتها، پمپ افلاکس فعال در سویه‌های مقاوم به سیپروفلوکساسین استافیلوکوکوس اورئوس توسط روش اتیدیوم بروماید بررسی شد.یافته ها: از مجموع نمونه های مورد برررسی، 50 سویه استافیلوکوکوس اورئوس جداسازی شد که از این میان 12 جدایه (24%) مقاوم به سیپروفلوکساسین بودند. میزان شیوع ژن های norA و norB در سویه‌های مقاوم به سیپروفلوکساسین به ترتیب 100% و 83% بود. همچنین تمامی سویه‌های مقاوم به سیپروفلوکساسین دارای پمپ افلاکس فعال بودند. نتایج real-time PCR نشان داد که اختلاف معنی داری در میزان بیان پمپ های افلاکس در سویه‌های مقاوم به سیپروفلوکساسین وجود دارد. نتیجه گیری: نتایج این مطالعه نشان داد که پمپ‌های افلاکس norA و norB نقش مهمی در ایجاد مقاومت به سیپروفلوکساسین ایفا می‌کند.
Background & Objectives: Staphylococcus aureus is one of the important nosocomial infection agents. Recently, S. aureus strains have become resistant to ciprofloxacin and the efflux pump is considered as its contributor. Herein, we investigated the presence, expression, and activity of efflux pump genes (norA and norB) among ciprofloxacin-resistant S. aureus isolates. Materials & Methods: A total of 250 clinical samples were subjected to isolation of S. aureus strains. The antibiotic resistance pattern was characterized and the presence and expression level of norA and norB genes was assessed using PCR test and real-time PCR test, respectively. Finally, active efflux pumps were detected in ciprofloxacin-resistant S. aureus strains using the ethidium bromide test.Results: Among total clinical samples, 50 S. aureus strains were recovered. Of this 12 samples (24%) were resistant to ciprofloxacin. Moreover, norA and norB genes were found in 100 % and 83% of ciprofloxacin-resistant isolates, respectively. All ciprofloxacin-resistant isolates exhibited active efflux pumps. Real-time PCR results revealed that the isolates are more resistant to ciprofloxacin having a high level of efflux pump gene expression.Conclusion: The results of this study showed that norA and norB efflux pump genes play an important role in resistance to ciprofloxacin in S. aureus strains.
antigenicity, antimicrobial resistance and origin of Staphylococcus aureus isolated. Colomb
Med (Cali). 2016; 47: 15-20.
2. Hefzy EM, Hassan GM, Abd E, Reheem F. Detection of panton-valentine leukocidin-positive
methicillin-resistant Staphylococcus aureus nasal carriage among Egyptian health care
workers. Surg Infect (Larchmt). 2016; 17: 369-375.
3. Jeremić LP, Kapulica NK, Ristanović E, Josić D, Lepsanović Z. Prevalence of panton-valentine
leukocidin genes in community-associated methicillin-resistant Staphylococcus aureus in the
District of Pomoravlje. Vojnosanit Pregl. 2016; 73: 256-260.
4. Osman KM, Amer AM, Badr JM, Helmy NM, Elhelw RA, Orabi A, Bakry M, Saad AS.
Antimicrobial resistance, biofilm formation and mecA characterization of methicillinsusceptible S. aureus and Non-S. aureus of beef meat origin in Egypt. Front Microbiol. 2016;
29: 222-230.
5. Mohamed NA, Ramli S, Amin NN, Sulaiman WS, Isahak I, Jamaluddin TZ, Salleh NM.
Staphylococcus aureus carriage in selected kindergartens in Klang Valley. Med J Malaysia.
2016; 71: 62-65.
6. Firsov AA, Smirnova MV, Strukova EN, Vostrov SN, Portnoy YA, Zinner SH. Enrichment of
resistant Staphylococcus aureus at ciprofloxacin concentrations simulated within the mutant
selection window: bolus versus continuous infusion. Int J Antimicrob Agents. 2008; 32:
488-493.
7. Mustapha M, Bukar-Kolo YM, Geidam YA, Gulani IA. Phenotypic and genotypic detection of
methicillin-resistant Staphylococcus aureus in hunting dogs in Maiduguri metropolitan, Borno
State, Nigeria. Vet World 2016; 9: 501-506.
8. Poole K. Efflux pumps as antimicrobial resistance mechanisms. Ann Med. 2007; 39: 162-176.
9. Li XZ, Nikaido H. Efflux-mediated drug resistance in bacteria. Drugs. 2004; 64; 159-204.
10. Kosmidis C. B.D, Schindler P.L, Jacinto D, Patel K, Bains S.M, Seo G.W, Kaatz A.
Expression of multidrug resistance efflux pump genes in clinical and environmental isolates of
Staphylococcus aureus. Int J Antimicrob Agents. 2012; 40: 204-209.
11. Paulsen I.T, Lewis K. Microbial multidrug efflux. Horizon Scientific 2002; 3: 143-144.
12. Li XZ, Nikaido H. Efflux-mediated drug resistance in bacteria: an update. Drugs. 2009; 69:
1555-1623.
13. Soto SM. Role of efflux pumps in the antibiotic resistance of bacteria embedded in a biofilm.
Virulence. 2013; 1: 223-229.
14. De Kievit TR, Parkins MD, Gillis RJ, Srikumar R, Ceri H, Poole K, Iglewski BH, Storey DG.
Multidrug efflux pumps: expression patterns and contribution to antibiotic resistance in
Pseudomonas aeruginosa biofilms. Antimicrob Agents Chemother. 2001; 45: 1761-1770.
15. de Araújo RS, Barbosa-Filho JM, Scotti MT, Scotti L, da Cruz RM, Falcão-Silva Vdos S, de
Siqueira-Júnior JP, Mendonça-Junior FJ. Modulation of drug resistance in Staphylococcus
aureus with Coumarin derivatives. Scientifica (Cairo). 2016; 2016: 6894758.
16. Baba T, Takeuchi F, Kuroda M, Yuzawa H, Aoki K, Oguchi A, Nagai Y, Iwama N, Asano K,
Naimi T, Kuroda H, Cui L, Yamamoto K, Hiramatsu K. Genomic and virulence determinants
of high virulence community-acquired MRSA. Lancet. 2002; 359(9320): 1819-1827.
17. Clinical and laboratory standards institute (CLSI), 2006. Performance standards for
antimicrobial susceptibility testing; 16th informational supplement. CLSI, Wayne, Pa.
M100-S16, 26, no. 3. 2006.
18. Costa SS, Viveiros M, Amaral L, Couto I. Multidrug Efflux Pumps in Staphylococcus aureus:
an Update. Open Microbiol J. 2013; 7: 59-71.
19. Patel D, Kosmidis C, Seo SM, Kaatz GW. Ethidium bromide MIC screening for enhanced
efflux pump gene expression or efflux activity in Staphylococcus aureus. Antimicrob Agents
Chemother. 2010; 54(12): 5070-5073.
20. Xia J, Gao J, Tang W. Nosocomial infection and its molecular mechanisms of antibiotic
resistance. Biosci Trends. 2016; 10: 14-21.
21. Santos Costa S, Viveiros M, Rosato AE, Melo-Cristino J, Couto I. Impact of efflux in the
development of multidrug resistance phenotypes in Staphylococcus aureus. BMC Microbiol.
2015; 15: 232-230.
22. Liger F, Bouhours P, Ganem-Elbaz C, Jolivalt C, Pellet-Rostaing S, Popowycz F, Paris JM,
Lemaire M. C2 arylated benzo thiophene derivatives as Staphylococcus aureus NorA efflux
pump inhibitors. Chem Med Chem. 2016; 11: 320-330.
23. Sudhanthiramani S, Swetha CS, Bharathy S. Prevalence of antibiotic resistant Staphylococcus
aureus from raw milk samples collected from the local vendors in the region of Tirupathi,
India. Vet World. 2015; 8: 478-481.
24. Patel D, Kosmidis C, Seo SM, Kaatz GW. Ethidium bromide MIC screening for enhanced
efflux pump gene expression or efflux activity in Staphylococcus aureus. Antimicrob Agents
Chemother. 2010; 54: 5070-5073.
25. Truong-Bolduc, Strahilevitz J, Hooper D.C. NorC, a new efflux pump regulated by MgrA of
Staphylococcus aureus. Antimicrob Agents Chemother. 2006; 50: 1104-1107.
26. Sierra JM, Ruiz J, Jimenez De Anta MT, Vila J. Prevalence of two different genes encoding
NorA in 23 clinical strains of Staphylococcus aureus. J Antimicrob Chemother. 2000; 46:
145-146.
27. Ding Y, Onodera Y, Lee JC, Hooper DC. NorB, an efflux pump in Staphylococcus aureus
strain MW2, contributes to bacterial fitness in abscesses. J Bacteriol. 2008; 190: 7123-7129.
28. Saiful AJ, Mastura M, Zarizal S, Mazurah MI, Shuhaimi M, Ali AM. Efflux genes and active
efflux activity detection in Malaysian clinical isolates of methicillin-resistant Staphylococcus
aureus (MRSA). J Basic Microbiol. 2008; 48: 245-251.
29. Costa SS, Junqueira E, Palma C, Viveiros M, Melo-Cristino J, Amaral L, Couto I. Resistance
to antimicrobials mediated by efflux pumps in Staphylococcus aureus. Antibiotics (Basel).
2013; 2: 83-99.
_||_
antigenicity, antimicrobial resistance and origin of Staphylococcus aureus isolated. Colomb
Med (Cali). 2016; 47: 15-20.
2. Hefzy EM, Hassan GM, Abd E, Reheem F. Detection of panton-valentine leukocidin-positive
methicillin-resistant Staphylococcus aureus nasal carriage among Egyptian health care
workers. Surg Infect (Larchmt). 2016; 17: 369-375.
3. Jeremić LP, Kapulica NK, Ristanović E, Josić D, Lepsanović Z. Prevalence of panton-valentine
leukocidin genes in community-associated methicillin-resistant Staphylococcus aureus in the
District of Pomoravlje. Vojnosanit Pregl. 2016; 73: 256-260.
4. Osman KM, Amer AM, Badr JM, Helmy NM, Elhelw RA, Orabi A, Bakry M, Saad AS.
Antimicrobial resistance, biofilm formation and mecA characterization of methicillinsusceptible S. aureus and Non-S. aureus of beef meat origin in Egypt. Front Microbiol. 2016;
29: 222-230.
5. Mohamed NA, Ramli S, Amin NN, Sulaiman WS, Isahak I, Jamaluddin TZ, Salleh NM.
Staphylococcus aureus carriage in selected kindergartens in Klang Valley. Med J Malaysia.
2016; 71: 62-65.
6. Firsov AA, Smirnova MV, Strukova EN, Vostrov SN, Portnoy YA, Zinner SH. Enrichment of
resistant Staphylococcus aureus at ciprofloxacin concentrations simulated within the mutant
selection window: bolus versus continuous infusion. Int J Antimicrob Agents. 2008; 32:
488-493.
7. Mustapha M, Bukar-Kolo YM, Geidam YA, Gulani IA. Phenotypic and genotypic detection of
methicillin-resistant Staphylococcus aureus in hunting dogs in Maiduguri metropolitan, Borno
State, Nigeria. Vet World 2016; 9: 501-506.
8. Poole K. Efflux pumps as antimicrobial resistance mechanisms. Ann Med. 2007; 39: 162-176.
9. Li XZ, Nikaido H. Efflux-mediated drug resistance in bacteria. Drugs. 2004; 64; 159-204.
10. Kosmidis C. B.D, Schindler P.L, Jacinto D, Patel K, Bains S.M, Seo G.W, Kaatz A.
Expression of multidrug resistance efflux pump genes in clinical and environmental isolates of
Staphylococcus aureus. Int J Antimicrob Agents. 2012; 40: 204-209.
11. Paulsen I.T, Lewis K. Microbial multidrug efflux. Horizon Scientific 2002; 3: 143-144.
12. Li XZ, Nikaido H. Efflux-mediated drug resistance in bacteria: an update. Drugs. 2009; 69:
1555-1623.
13. Soto SM. Role of efflux pumps in the antibiotic resistance of bacteria embedded in a biofilm.
Virulence. 2013; 1: 223-229.
14. De Kievit TR, Parkins MD, Gillis RJ, Srikumar R, Ceri H, Poole K, Iglewski BH, Storey DG.
Multidrug efflux pumps: expression patterns and contribution to antibiotic resistance in
Pseudomonas aeruginosa biofilms. Antimicrob Agents Chemother. 2001; 45: 1761-1770.
15. de Araújo RS, Barbosa-Filho JM, Scotti MT, Scotti L, da Cruz RM, Falcão-Silva Vdos S, de
Siqueira-Júnior JP, Mendonça-Junior FJ. Modulation of drug resistance in Staphylococcus
aureus with Coumarin derivatives. Scientifica (Cairo). 2016; 2016: 6894758.
16. Baba T, Takeuchi F, Kuroda M, Yuzawa H, Aoki K, Oguchi A, Nagai Y, Iwama N, Asano K,
Naimi T, Kuroda H, Cui L, Yamamoto K, Hiramatsu K. Genomic and virulence determinants
of high virulence community-acquired MRSA. Lancet. 2002; 359(9320): 1819-1827.
17. Clinical and laboratory standards institute (CLSI), 2006. Performance standards for
antimicrobial susceptibility testing; 16th informational supplement. CLSI, Wayne, Pa.
M100-S16, 26, no. 3. 2006.
18. Costa SS, Viveiros M, Amaral L, Couto I. Multidrug Efflux Pumps in Staphylococcus aureus:
an Update. Open Microbiol J. 2013; 7: 59-71.
19. Patel D, Kosmidis C, Seo SM, Kaatz GW. Ethidium bromide MIC screening for enhanced
efflux pump gene expression or efflux activity in Staphylococcus aureus. Antimicrob Agents
Chemother. 2010; 54(12): 5070-5073.
20. Xia J, Gao J, Tang W. Nosocomial infection and its molecular mechanisms of antibiotic
resistance. Biosci Trends. 2016; 10: 14-21.
21. Santos Costa S, Viveiros M, Rosato AE, Melo-Cristino J, Couto I. Impact of efflux in the
development of multidrug resistance phenotypes in Staphylococcus aureus. BMC Microbiol.
2015; 15: 232-230.
22. Liger F, Bouhours P, Ganem-Elbaz C, Jolivalt C, Pellet-Rostaing S, Popowycz F, Paris JM,
Lemaire M. C2 arylated benzo thiophene derivatives as Staphylococcus aureus NorA efflux
pump inhibitors. Chem Med Chem. 2016; 11: 320-330.
23. Sudhanthiramani S, Swetha CS, Bharathy S. Prevalence of antibiotic resistant Staphylococcus
aureus from raw milk samples collected from the local vendors in the region of Tirupathi,
India. Vet World. 2015; 8: 478-481.
24. Patel D, Kosmidis C, Seo SM, Kaatz GW. Ethidium bromide MIC screening for enhanced
efflux pump gene expression or efflux activity in Staphylococcus aureus. Antimicrob Agents
Chemother. 2010; 54: 5070-5073.
25. Truong-Bolduc, Strahilevitz J, Hooper D.C. NorC, a new efflux pump regulated by MgrA of
Staphylococcus aureus. Antimicrob Agents Chemother. 2006; 50: 1104-1107.
26. Sierra JM, Ruiz J, Jimenez De Anta MT, Vila J. Prevalence of two different genes encoding
NorA in 23 clinical strains of Staphylococcus aureus. J Antimicrob Chemother. 2000; 46:
145-146.
27. Ding Y, Onodera Y, Lee JC, Hooper DC. NorB, an efflux pump in Staphylococcus aureus
strain MW2, contributes to bacterial fitness in abscesses. J Bacteriol. 2008; 190: 7123-7129.
28. Saiful AJ, Mastura M, Zarizal S, Mazurah MI, Shuhaimi M, Ali AM. Efflux genes and active
efflux activity detection in Malaysian clinical isolates of methicillin-resistant Staphylococcus
aureus (MRSA). J Basic Microbiol. 2008; 48: 245-251.
29. Costa SS, Junqueira E, Palma C, Viveiros M, Melo-Cristino J, Amaral L, Couto I. Resistance
to antimicrobials mediated by efflux pumps in Staphylococcus aureus. Antibiotics (Basel).
2013; 2: 83-99.