تشخیص موتاسیون های نقطه ای مرتبط با مقاومت به سیپروفلوکساسین در جدایه های سودوموناس آئروجینوسا استان گیلان
محورهای موضوعی : میکروب شناسی مولکولی
نجمه رنجی
1
(استادیار، باشگاه پژوهشگران جوان و نخبگان ، واحد رشت، دانشگاه آزاد اسلامی، رشت، ایران)
فاطمه اسدی رحمانی
2
(کارشناس ارشد، دانشکده علوم پایه، واحد لاهیجان، دانشگاه آزاد اسلامی، لاهیجان، ایران)
سیده شیده پورخلیلی
3
(کارشناس ارشد، دانشکده علوم پایه، واحد رشت، دانشگاه آزاد اسلامی، رشت، ایران)
کلید واژه: سیپروفلوکساسین, سودوموناس آئروجینوسا, ژن parC, ژن gyrB, ژن nfxB,
چکیده مقاله :
سابقه و هدف: سودوموناس آئروجینوسا یک پاتوژن فرصت طلب به ویژه در بیماران با نقص ایمنی است. مقاومت دارویی در سودوموناس آئروجینوسا به واسطه مکانیسم های مختلفی مانند جهش در زیرواحدهای توپوایزومرازها و تنظیم کننده های منفی سیستم های پمپ افلاکس ایجاد می شود. هدف از این مطالعه ارزیابی جهش های نقطه ای ژن های gyrB، parC و nfxB در جدایه های سودوموناس آئروجینوسا مقاوم به سیپروفلوکساسین استان گیلان بود.مواد و روش ها: این پژوهش یه صورت مقطعی—توصیفی بر روی 200 سویه جمع آوری شده از بیمارستان ها و آزمایشگاه های رشت و لاهیجان انجام شد. با آزمون های بیوشیمیایی تعیین هویت جدایه ها انجام شد. حساسیت آنتی بیوتیکی به کمک روش های کربی-بائر و MIC تعیین گردید. برای ارزیابی جهش ها در ژن های gyrB، parC و nfxB در جدایه های مقاوم به سیپروفلوکساسین از روش PCR و تعیین توالی استفاده شد.یافته ها: از 69 جدایه سودوموناس آئروجینوسا، 26 سویه مقاوم به سیپروفلوکساسین بودند. MIC سیپروفلوکساسین در جدایه های مقاوم بین µg/ml 32-1024 تعیین گردید. با آنالیز توالی یابی مشخص گردید که بعضی از جدایه های مقاوم، جهش های بدمعنی در ژن های gyrB، parC و nfxB داشتند. جهش های N368S، I424L، L464I، E468D، M520L و I524V در ژن gyrB اولین موارد گزارش شده در ایران می باشند.نتیجه گیری: به نظر می رسد که جهش های گزارش شده در ژن های gyrB و parC روی تمایل توپوایزومرازها به سیپروفلوکساسین در جدایه های مقاوم تأثیر بگذارند. همچنین، جهش در ژن nfxB ممکن است موجب افزایش بیان پمپ افلاکس MexCD-OprJ و مقاومت به سیپروفلوکساسین گردد.
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.
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.
_||_
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.