Assessment of point mutations associated with ciprofloxacin resistance in Pseudomonas aeruginosa isolates in Guilan province

Subject Areas : Molecular Microbiology

Najmeh Ranji ¹ , Fatemeh Asadi Rahmani ² , Seyedeh Shideh PourKhalili ³

1 - Assistant Professor, Young Researchers and Elite Club, Rasht branch, Islamic Azad University, Rasht, Iran.
2 - M.Sc., Faculty of Sciences, Lahijan branch, Islamic Azad University, Lahijan, Iran.
3 - M.Sc., Faculty of Sciences, Rasht branch, Islamic Azad University, Rasht, Iran.

Received: 2017-10-27 Accepted : 2017-11-30 Published : 2018-05-22

Keywords: Pseudomonas aeruginosa, Ciprofloxacin, parC gene, gyrB gene, nfxB gene,

Abstract :

Background & Objectives: Pseudomonas aeruginosa is an opportunistic pathogen especially in immunocompromised patients. Drug resistance in P. aeruginosa is caused by different mechanisms such as mutations in topoisomerases subunits and negative regulators of efflux pump systems. This study was aimed to investigate mutations in gyrB, parC, and nfxB genes in ciprofloxacin-resistant P.aeruginosa isolates in Guilan province.   Materials & Methods: In this cross-sectional study, 200 isolates were obtained from different clinical samples of Rasht and Lahijan hospitals and laboratories and subsequently identified by biochemical tests. Antibiotic susceptibility pattern was determined by Kirby Bauer disk diffusion susceptibility test and Minimum Inhibitory Concentration (MIC) test. PCR-sequencing was used to assess mutations in gyrB, parC, and nfxB genes in ciprofloxacin-resistant P. aeruginosa isolates.   Results: Out of  69 P. aeruginosa isolates, 26 isolates were ciprofloxacin-resistant. MIC of ciprofloxacin in resistant isolates was determined between 32-1024 µg/ml. PCR-sequencing analysis revealed that some resistant isolates have missense mutations in gyrB, parC, and nfxB genes. It seems that N368S, I424L, L464I, E468D, M520L, and I524V mutations in gyrB were reported for the first time in Iran.   Conclusion: It seems that reported mutations in gyrB and parC genes affect topoisomerases affinity to ciprofloxacin in resistant isolates. Furthermore, mutations in the nfxB gene may lead to overexpression of MexCD-OprJ efflux pump and ciprofloxacin resistance.  

References:

1. Odumosu BT, Adeniyi BA, Chandra R. First detection of oxa-10 extended-spectrum
   beta-lactamases and the occurrence of mexr and nfxb in clinical isolates of Pseudomonas
   aeruginosa from Nigeria. Chemother. 2016; 61(2): 87-92.
   2. Ranji N, Rahbar Takrami S. Role of mexZ gene in ciprofloxacin resistance in Pseudomonas
   aeruginosa isolates in Guilan province. J Urmia Univ Med Sci. 2017; 27(10): 902-913.
   [In Persian]
   3. Hakimi F, Ranji N, Faezi Ghasemi M. Mutations in nalC gene in ciprofloxacin resistant strains
   of Pseudomonas aeruginosa isolated from hospitals and laboratories of Guilan province in
   2014-2015 years. J Arak Uni Med Sci. 2016; 19(7): 12-21. [In Persian]
   4. Motahhary Tashi H, Ranji N. Study on oprD mutation and imipenem resistance in
   Pseudomonas aeruginosa isolates in Gilan province. J Microbial World. 2017;10(1): 26-36.
   
   [In Persian]
   5. Shakibaie MR, Jalilzadeh KA, Yamakanamardi SM. Horizontal transfer of antibiotic resistance
   genes among gram negative bacteria in sewage and lake water and influence of some
   physico-chemical parameters of water on conjugation process. J Environ Biol. 2009; 30(1):
   45-49.
   6. Ahmadpour Bijargah K, Faezi Ghasemi M, Ranji N. Mutations in parC subunit of
   topoisomerase IV in ciprofloxacin resistant isolates of Klebsiella pneumonia in Guilan
   province. J Urmia Univ Med Sci. 2017; 28(3): 223-230. [In Persian]
   7. Rahnamay Roodposhti F, Ranji N, Asadpour L. Mutations of gyrA gene in fluoroquinolone
   resistant isolates of Pseudomonas aeruginosa in Guilan province. J Mazandaran Univ Med Sci.
   2016; 26(139): 84-92. [In Persian]
   8. Vaez H, Faghri J, Isfahani BN, Moghim S, Yadegari S, Fazeli H. Efflux pump regulatory genes
   mutations in multidrug resistance Pseudomonas aeruginosa isolated from wound infections in
   Isfahan hospitals. Adv Biomed Res. 2014; 3: 1-5.
   9. Church D, Elsayed S, Reid O, Winston B, Lindsay R. Burn wound infections. Clin Microbiol
   Rev. 2006; 19(2): 403-434.
   10. Rajkumari N, John NV, Mathur P, Misra MC. Antimicrobial resistance in Pseudomonas sp.
   causing infections in Trauma patients: A 6 year experience from a south Asian country. J Glob
   Infect Dis. 2014; 6(4): 182-185.
   11. Douraghi M, Ghasemi F, Dallal MM, Rahbar M, Rahimiforoushani A. Molecular identification
   of Pseudomonas aeruginosa recovered from cystic fibrosis patients. J Prev Med Hyg. 2014; 55
   (2): 50-53.
   12. Kittinger C, Lipp M, Baumert R, Folli B, Koraimann G, Toplitsch D. Antibiotic resistance
   patterns of Pseudomonas spp. isolated from the river Danube. Front Microbiol. 2016; 7: 1-8.
   13. Ahmed Z, Saeed Khan S, Khan M. In vitro trials of some antimicrobial combinations against
   Staphylococcus aureus and Pseudomonas aeruginosa. Saudi J Biol Sci. 2013; 20(1): 79-83.
   14. Imani Fooladi A, Rostami Z, Shapouri R. Antimicrobial resistance and ESBL prevalence in
   Pseudomonas aeruginosa strains isolated from clinical specimen by phenotypic and genotypic
   methods, Zanjan, Iran. Iran J Med Microbiol. 2011: 10:189-198. [In Persian]
   15. Sadeghi A, Rahimi B, Shojapour M. Molecular detection of metallo-β-lactamase genes
   blaVIM-1, blaVIM-2, blaIMP-1, blaIMP-2 and blaSPM-1 in Pseudomonas aeruginosa isolated
   from hospitalized patients in Markazi province by duplex-PCR. Afr J Microbiol. 2012: 6(12):
   2965-2969.
   16. Nikokar I, Tishayar A, Flakiyan Z, Alijani K, Rehana-Banisaeed S, Hossinpour M.
   Antibiotic resistance and frequency of class 1 integrons among Pseudomonas aeruginosa,
   isolated from burn patients in Guilan, Iran. Iran J Microbiol. 2013; 5(1): 36-41.
   17. Goli HR, Nahaei MR, Ahangarzadeh Rezaee M, Hasani A, Samadi Kafil H, Aghazadeh M.
   Emergence of colistin resistant Pseudomonas aeruginosa at Tabriz hospitals, Iran. Iran J
   
   Microbiol. 2016; 8(1): 62-69.
   18. Kugelberg E, Löfmark S, Wretlind B, Andersson D. Reduction of the fitness burden of
   quinolone resistance in Pseudomonas aeruginosa. J Antimicrob Chemother. 2005: 55(1):
   22-30.
   19. Wang YT, Lee MF, Peng CF. Mutations in the quinolone resistance-determining regions
   associated with ciprofloxacin resistance in Pseudomonas aeruginosa isolates from Southern
   Taiwan. Biomarkers Genomic Med. 2014; 6(2): 79-83.
   20. Niga T, Ito H, Oyamada Y, Yamagishi J, Kadono M, Nishino T. Cooperation between
   alteration of DNA gyrase genes and over-expression of MexB and MexX confers high-level
   fluoroquinolone resistance in Pseudomonas aeruginosa strains isolated from a patient who
   received a liver transplant followed by treatment with fluoroquinolones. Microbiol Immunol.
   2005: 49(5): 443-446.
   21. Miyahara E, Nishie M, Takumi S, Miyanohara H, Nishi J, Yoshiie K. Environmental
   mutagens may be implicated in the emergence of drug-resistant microorganisms. FEMS
   Microbiol Lett. 2011: 317(2): 109-116.
   22. Sekiguchi J, Teruya K, Horii K, Kuroda E, Konosaki H, Mizuguchi Y. Molecular
   epidemiology of outbreaks and containment of drug-resistant Pseudomonas aeruginosa in a
   Tokyo hospital. J Infect Chemother. 2007: 13(6): 418-422.
   23. Nouri R, Ahangarzadeh Rezaee M, Hasani A, Aghazadeh M, Asgharzadeh M. The role of
   gyrA and parC mutations in fluoroquinolones-resistant Pseudomonas aeruginosa isolates from
   Iran. Braz J Microbiol. 2016; 47(4): 925-930.
   24. Khaledi A, Schniederjans M, Pohl S, Rainer R, Bodenhofer U, Xia B. Transcriptome profiling
   of antimicrobial resistance in Pseudomonas aeruginosa. Antimicrob Agents Chemother. 2016;
   60(8): 4722-4733.
   25. Nascimento APB, Ortiz MF, Martins WMBS, Morais GL, Fehlberg LCC, Almeida LGP.
   Intraclonal genome stability of the metallo-β-lactamase SPM-1-producing Pseudomonas
   aeruginosa ST277, an endemic clone disseminated in Brazilian hospitals. Front Microbiol.
   2016; 7: 1-16.
   26. Salma R, Dabboussi F, Kassaa I, Khudary R, Hamze M. gyrA and parC mutations in
   quinolone-resistant clinical isolates of Pseudomonas aeruginosa from Nini Hospital in north
   Lebanon. J Infect Chemother. 2013; 19(1): 77-81.
   27. Gorgani N, Ahlbrand S, Patterson A, Pourmand N. Detection of point mutations associated
   with antibiotic resistance in Pseudomonas aeruginosa. Int J Antimicrob Agents. 2009; 34(5):
   414-418.
   28. Jalal S, Ciofu O, Hoiby N, Gotoh N, Wretlind B. Molecular mechanisms of fluoroquinolone
   resistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients. Antimicrob Agents
   Chemother. 2000: 44(3): 710-712.
   29. Jeannot K, Elsen S, Kˆhler , Attree I, Van Delden C, PlÈsiat P. Resistance and virulence of
   
   Pseudomonas aeruginosa clinical strains overproducing the MexCD-OprJ efflux pump.
   Antimicrob Agents Chemother. 2008: 52: 2455-2462.
   30. Purssell A, Poole K. Functional characterization of the NfxB repressor of the mexCD–oprJ
   multidrug efflux operon of Pseudomonas aeruginosa. Microbiol. 2013; 159(Pt 10): 2058-2073.
   31. Vaez H, Faghri J, Isfahani B. Efflux pump regulatory genes mutations in multidrug resistance
   Pseudomonas aeruginosa isolated from wound infections in Isfahan hospitals. Adv Bjomed
   Res. 2014: 3: 1-5.
   32. Tohidpour A, Najar Peerayeh S, Najafi S. Detection of DNA gyrase mutation and multidrug
   efflux pumps hyperactivity in ciprofloxacin resistant clinical isolates of Pseudomonas
   aeruginosa. J Med Microbiol Infec Dis. 2013: 1: 1-7.
   33. Jeannot K, Elsen S, Kohler T, Attree I, van Delden C, Plesiat P. Resistance and virulence of
   Pseudomonas aeruginosa clinical strains overproducing the MexCD-OprJ efflux pump.
   Antimicrob Agents Chemother. 2008; 52(7): 2455-2462.
   34. Bruchmann S, Dotsch A, Nouri B, Chaberny IF, Haussler S. Quantitative contributions of
   target alteration and decreased drug accumulation to Pseudomonas aeruginosa fluoroquinolone
   resistance. Antimicrob Agents Chemother. 2013; 57(3): 1361-1368.