Detection of efflux pump activity and gene expression among ciprofloxacin-resistant Staphylococcus aureus strains
Subject Areas : Medical MicrobiologyZahra Tavakoli 1 , Hassan Sahebjamee 2 , Leila Pishkar 3 , Zohreh Alimadadi 4 , Hassan Noorbazargan 5 , Amir Mirzaie 6
1 - M.Sc., Department of Biology, Tehran North Branch, Islamic Azad University, Tehran, Iran
2 - Assistant Professor, Department of Biochemistry and Biophysics, School of Biological Sciences, Varamin-Pishva Branch, Islamic Azad University, Varamin, Iran
3 - Assistant Professor, Young Researchers and Elite Club, Islamshahr Branch, Islamic Azad University, Islamshahr, Iran.
4 - M.Sc., Department of Biology, East Tehran Branch, Islamic Azad University, Tehran, Iran
5 - Ph.D., Department of Biotechnology, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
6 - Assistant Professor, Department of Biology, Roudehen Branch, Islamic Azad University, Roudehen, Iran.
Keywords: Staphylococcus aureus, Real-time PCR, efflux pump, norA gene, norB gene,
Abstract :
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.
