شناسایی جهش های نواحی جهش پذیر داغ ژن ERG11 در جدایه های مقاوم به فلوکونازول کاندیدا آلبیکنس درغرب مازندران
محورهای موضوعی : میکروب شناسی مولکولیمعصومه مجدی 1 , زینب خزائی کوهپر 2 , ایت الله نصرالهی عمران 3
1 - دانشجوی کارشناسی ارشد، گروه زیست شناسی سلولی و مولکولی، دانشکده علوم زیستی، واحد تنکابن، دانشگاه آزاد اسلامی، تنکابن
2 - استادیار، گروه زیست شناسی سلولی و مولکولی، دانشکده علوم زیستی، واحد تنکابن، دانشگاه آزاد اسلامی، تنکابن
3 - دانشیار، گروه قارچ شناسی پزشکی، واحد تنکابن، دانشگاه آزاد اسلامی، تنکابن
کلید واژه: کاندیدا آلبیکنس, فلوکونازول, ERG11, جهش V488I, جهش D504V,
چکیده مقاله :
سابقه و هدف: امروزه مصرف گسترده فلوکونازول باعث مقاومت در سویه های کاندیدا آلبیکنس شده است. تغییرات ساختاری Erg11p در نتیجه جهش در ژن ERG11 یکی از مکانیسم های مقاومت آزولی است. این مطالعه با هدف بررسی جهش های ژن ERG11 در جدایه های کاندیدا آلبیکنس مقاوم به فلوکونازول در غرب استان مازندران انجام شد.مواد و روش ها: در این مطالعه مقطعی توصیفی، نمونه های بالینی از مخاط واژینال 120 زن در بیمارستان های غرب مازندران به دست آمد. جدایه های کاندیدا آلبیکنس با استفاده از روش های استاندارد مانند لوله زایا و کشت در محیط کروم آگار تعیین هویت شدند. مقاومت و حساسیت جدایه ها نسبت به فلوکونازول به کمک روش های کربی بوئر و براث ماکرودایلوشن ارزیابی گردید. سپس جهش های ژن ERG11 در جدایه های بالینی به روش PCR و توالی یابی در مقایسه با جدایه PTCC 5027 (ATCC10231) تعیین شد.یافته ها: از 45 جدایه کاندیدا آلبیکنس،40 جدایه مقاوم و 5 جدایه حساس به فلوکونازول بودند. MIC فلوکونازول µg/ml ≤ 64 تعیین شد. آنالیز PCR- توالی یابی آشکار کرد که 18 جدایه مقاوم به فلوکونازول شش جهش بدمعنی (Y257H، E266D، V404I، D421N، V488I و D504V) را در ژن ERG11 داشتند.نتیجه گیری: جهش های شناسایی شده در این مطالعه ممکن است با کاهش تمایل فلوکونازول به ERG11p در ایجاد مقاومت به فلوکونازول در جدایه های کاندیدا آلبیکنس در غرب استان مازندران نقش داشته باشند.
Background & Objectives: Nowadays, widespread use of fluconazole has resulted in resistance in Candida albicans strains. The conformational changes of Erg11p due to mutations in the ERG11 gene is one of the mechanisms resulting in azole resistance. The aim of our study was to investigate ERG11 gene mutations in fluconazole-resistant isolates of C. albicans in the west of Mazandaran province.Materials & Methods: In this descriptive cross-sectional study, clinical specimens were obtained from vaginal mucosa of 120 women in hospitals of the west of Mazandaran province. C. albicans isolates were identified by standard methods such as germ tubes and CHROMEagar medium culture. The fluconazole resistance and susceptibility of the isolates were evaluated by Kirby Bauer and broth micro-dilution methods. Then, ERG11 gene mutations in resistant isolates were determined by PCR-sequencing method as compared with PTCC5027 (ATCC10231) reference strain.Results: Out of 45 C. albicans isolates, 40 isolates were resistant and 5 isolates were susceptible to fluconazole. The MIC of fluconazole was determined as ≥ 64 µg/ml. PCR-sequencing analysis revealed that 18 fluconazole-resistant isolates have six missense mutations (Y257H, E266D, V404I, D421N, V488I, and D504V) in the ERG11 gene.Conclusion: The identified mutations in this study may play role in developing fluconazole resistance in C. albicans isolates in the west of Mazandaran province by decreasing fluconazole affinity to ERG11p.
increased efflux pump activity as a resistance mechanism in azole-resistant vaginal
Candida albicans isolates. Antimicrob Agents Chemother. 2016; 60(10): 5858-66.
2. Mayer FL, Wilson D, Hube B. Candida albicans pathogenicity mechanisms. Virulence. 2013; 4
(2): 119-128.
3. Xu Y, Sheng F, Zhao J, Chen L, Li C. ERG11 mutations and expression of resistance genes in
fluconazole-resistant Candida albicans isolates. Arch Microbiol. 2015; 197(9): 1087-1093.
4. Sanglard D, Ischer F, Parkinson T, Falconer D, Bille J. Candida albicans mutations in the
ergosterol biosynthetic pathway and resistance to several antifungal agents. Antimicrob Agents
Chemother. 2003; 47(8): 2404-2412.
5. Balabandi S, Khazaei -Koohpar Z, Ranji N. Correlation between ERG11 gene mutations and
fluconazole resistance in Candida albicans strains isolates isolated in from Rasht in 2015-2016
years. Arak Univ Med Sci J. 2017; 20(7): 13-22. [In Persian]
6. Flowers SA, Colon B, Whaley SG, Schuler MA, Rogers PD. Contribution of clinically derived
mutations in ERG11 to azole resistance in Candida albicans. Antimicrob Agents Chemother.
2015; 59(1): 450-60.
7. Eftekhari AD, Anvari M, Ranji N. Investigation of ERG11 gene mutations in fluconazole
resistant Candida albicans isolated from a number of Rasht hospitals. Pharm Res. 2015;
18(3): 98-107.
8. Trnovsky J, Merz W, Della-Latta P, Wu F, Arendrup MC, Stender H. Rapid and accurate
identification of Candida albicans isolates by use of PNA FISH Flow. J Clin Microbiol. 2008;
46(4): 1537-1540.
9. Cockerill F, Patel J, Alder J, Bradford P, Dudley M, Eliopoulos G. Performance standards for
antimicrobial susceptibility testing: twenty-third informational supplement; M100-S23:
Clinical & Laboratory Standards Institute; 2013.
10. Lee PY, Costumbrado J, Hsu CY, Kim YH. Agarose gel electrophoresis for the separation of
DNA fragments. J Vis Exp. 2012; pii: 3923. doi: 10.3791/3923.
11. Xu Y, Chen L, Li C. Susceptibility of clinical isolates of Candida species to fluconazole and
detection of Candida albicans ERG11 mutations. J Antimicrob Chemother. 2008; 61(4):
798-804.
12. Ranji N, Rahbar Takrami S. Role of mexZ gene in ciprofloxacin resistance in Pseudomonas
aeruginosa isolates in Guilan province. Urmia Med J. 2017; 27(1): 902-913. [In Persian]
13. Mohammad-Alipour Z, Asadpour L, Ranji N. Fluoroquinolone resistance and mutation in gyrA
gene in clinical isolates of Klebsiella pnemoniae. Iran J Med Microbiol. 2016; 10(5): 31-37.
14. Teymuri M, Mamishi S, Pourakbari B, Mahmoudi S, Ashtiani MT, Sadeghi RH. Investigation
of ERG11 gene expression among fluconazole-resistant Candida albicans: first report from an
Iranian referral paediatric hospital. Br J Biomed Sci. 2015; 72(1): 28-31.
15. Farahbakhsh E, Yadegari M, Rajabi Bazl M, Taghizadeh Armaki M. Evaluation of
susceptibility of strains of Candida albicans isolated from AIDS patients to fluconazole and
determination of CDR2 resistance gene in resistant strains by RT-PCR method. Armaghane
danesh. 2011; 16(3): 201-210. [In Persian]
16. Mohammadi-Ghalehbin B, Javanpour Heravi H, Arzanlou M, Sarvi M. Prevalence and
antibiotic resistance pattern of Candida spp. isolated from pregnant women referred to health
centers in Ardabil, Iran. J Ardabil Uni Med Sci. 2017; 16(4): 409-421. [In Persian]
17. Xiang MJ, Liu JY, Ni PH, Wang S, Shi C, Wei B. Erg11 mutations associated with azole
resistance in clinical isolates of Candida albicans. FEMS Yeast Res. 2013; 13(4): 386-393.
18. Wang B, Huang LH, Zhao JX, Wei M, Fang H, Wang DY. ERG11 mutations associated with
azole resistance in Candida albicans isolates from vulvovaginal candidosis patients. Asian Pac J
Trop Biomed. 2015; 5(11): 909-914.
19. White TC, Holleman S, Dy F, Mirels LF, Stevens DA. Resistance mechanisms in clinical
isolates of Candida albicans. Antimicrob Agents Chemother. 2002; 46(6): 1704-1713.
20. Rosana Y, Yasmon A, Lestari DC. Overexpression and mutation as a genetic mechanism of
fluconazole resistance in Candida albicans isolated from human immunodeficiency virus
patients in Indonesia. J Med Microbiol. 2015; 64(9): 1046-1052.
21. Goldman GH, da Silva Ferreira ME, dos Reis Marques E, Savoldi M, Perlin D, Park S.
Evaluation of fluconazole resistance mechanisms in Candida albicans clinical isolates from
HIV-infected patients in Brazil. Diag Microbiol Infect Dis. 2004; 50(1): 25-32.
22. Manastir L, Ergon MC, Yucesoy M. Investigation of mutations in Erg11 gene of fluconazole
resistant Candida albicans isolates from Turkish hospitals. Mycoses. 2011; 54(2): 99-104.
23. Maebashi K, Kudoh M, Nishiyama Y, Makimura K, Kamai Y, Uchida K. Proliferation of
intracellular structure corresponding to reduced affinity of fluconazole for cytochrome P-450 in
two low-susceptibility strains of Candida albicans isolated from a Japanese AIDS patient.
Microbiol Immunol. 2003; 47(2): 117-124.
24. Lee MK, Williams LE, Warnock DW, Arthington-Skaggs BA. Drug resistance genes and
trailing growth in Candida albicans isolates. J Antimicrob Chemother. 2004; 53(2): 217-224.
25. Feng LJ, Wan Z, Wang XH, Li RY, Liu W. Relationship between antifungal resistance of
fluconazole resistant Candida albicans and mutations in ERG11 gene. Chinese Med J. 2010;
123(5): 544-548.
26. Hu L, Du X, Li T, Song Y, Zai S, Hu X. Genetic and phenotypic characterization of
Candida albicans strains isolated from infectious disease patients in Shanghai. J Med
Microbiol. 2015; 64(Pt 1): 74-83.
27. Wang H, Kong F, Sorrell TC, Wang B, McNicholas P, Pantarat N. Rapid detection of ERG11
gene mutations in clinical Candida albicans isolates with reduced susceptibility to
fluconazole by rolling circle amplification and DNA sequencing. BMC Microbiol. 2009; 9:167.
28. Marichal P, Koymans L, Willemsens S, Bellens D, Verhasselt P, Luyten W. Contribution of
mutations in the cytochrome P450 14alpha-demethylase (Erg11p, Cyp51p) to azole
resistance in Candida albicans. Microbiol. 1999; 145 ( Pt 10): 2701-2713.
29. Strzelczyk JK, Slemp-Migiel A, Rother M, Golabek K, Wiczkowski A. Nucleotide
substitutions in the Candida albicans ERG11 gene of azole-susceptible and azole-resistant
clinical isolates. Acta Biochim Pol. 2013; 60(4): 547-552
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increased efflux pump activity as a resistance mechanism in azole-resistant vaginal
Candida albicans isolates. Antimicrob Agents Chemother. 2016; 60(10): 5858-66.
2. Mayer FL, Wilson D, Hube B. Candida albicans pathogenicity mechanisms. Virulence. 2013; 4
(2): 119-128.
3. Xu Y, Sheng F, Zhao J, Chen L, Li C. ERG11 mutations and expression of resistance genes in
fluconazole-resistant Candida albicans isolates. Arch Microbiol. 2015; 197(9): 1087-1093.
4. Sanglard D, Ischer F, Parkinson T, Falconer D, Bille J. Candida albicans mutations in the
ergosterol biosynthetic pathway and resistance to several antifungal agents. Antimicrob Agents
Chemother. 2003; 47(8): 2404-2412.
5. Balabandi S, Khazaei -Koohpar Z, Ranji N. Correlation between ERG11 gene mutations and
fluconazole resistance in Candida albicans strains isolates isolated in from Rasht in 2015-2016
years. Arak Univ Med Sci J. 2017; 20(7): 13-22. [In Persian]
6. Flowers SA, Colon B, Whaley SG, Schuler MA, Rogers PD. Contribution of clinically derived
mutations in ERG11 to azole resistance in Candida albicans. Antimicrob Agents Chemother.
2015; 59(1): 450-60.
7. Eftekhari AD, Anvari M, Ranji N. Investigation of ERG11 gene mutations in fluconazole
resistant Candida albicans isolated from a number of Rasht hospitals. Pharm Res. 2015;
18(3): 98-107.
8. Trnovsky J, Merz W, Della-Latta P, Wu F, Arendrup MC, Stender H. Rapid and accurate
identification of Candida albicans isolates by use of PNA FISH Flow. J Clin Microbiol. 2008;
46(4): 1537-1540.
9. Cockerill F, Patel J, Alder J, Bradford P, Dudley M, Eliopoulos G. Performance standards for
antimicrobial susceptibility testing: twenty-third informational supplement; M100-S23:
Clinical & Laboratory Standards Institute; 2013.
10. Lee PY, Costumbrado J, Hsu CY, Kim YH. Agarose gel electrophoresis for the separation of
DNA fragments. J Vis Exp. 2012; pii: 3923. doi: 10.3791/3923.
11. Xu Y, Chen L, Li C. Susceptibility of clinical isolates of Candida species to fluconazole and
detection of Candida albicans ERG11 mutations. J Antimicrob Chemother. 2008; 61(4):
798-804.
12. Ranji N, Rahbar Takrami S. Role of mexZ gene in ciprofloxacin resistance in Pseudomonas
aeruginosa isolates in Guilan province. Urmia Med J. 2017; 27(1): 902-913. [In Persian]
13. Mohammad-Alipour Z, Asadpour L, Ranji N. Fluoroquinolone resistance and mutation in gyrA
gene in clinical isolates of Klebsiella pnemoniae. Iran J Med Microbiol. 2016; 10(5): 31-37.
14. Teymuri M, Mamishi S, Pourakbari B, Mahmoudi S, Ashtiani MT, Sadeghi RH. Investigation
of ERG11 gene expression among fluconazole-resistant Candida albicans: first report from an
Iranian referral paediatric hospital. Br J Biomed Sci. 2015; 72(1): 28-31.
15. Farahbakhsh E, Yadegari M, Rajabi Bazl M, Taghizadeh Armaki M. Evaluation of
susceptibility of strains of Candida albicans isolated from AIDS patients to fluconazole and
determination of CDR2 resistance gene in resistant strains by RT-PCR method. Armaghane
danesh. 2011; 16(3): 201-210. [In Persian]
16. Mohammadi-Ghalehbin B, Javanpour Heravi H, Arzanlou M, Sarvi M. Prevalence and
antibiotic resistance pattern of Candida spp. isolated from pregnant women referred to health
centers in Ardabil, Iran. J Ardabil Uni Med Sci. 2017; 16(4): 409-421. [In Persian]
17. Xiang MJ, Liu JY, Ni PH, Wang S, Shi C, Wei B. Erg11 mutations associated with azole
resistance in clinical isolates of Candida albicans. FEMS Yeast Res. 2013; 13(4): 386-393.
18. Wang B, Huang LH, Zhao JX, Wei M, Fang H, Wang DY. ERG11 mutations associated with
azole resistance in Candida albicans isolates from vulvovaginal candidosis patients. Asian Pac J
Trop Biomed. 2015; 5(11): 909-914.
19. White TC, Holleman S, Dy F, Mirels LF, Stevens DA. Resistance mechanisms in clinical
isolates of Candida albicans. Antimicrob Agents Chemother. 2002; 46(6): 1704-1713.
20. Rosana Y, Yasmon A, Lestari DC. Overexpression and mutation as a genetic mechanism of
fluconazole resistance in Candida albicans isolated from human immunodeficiency virus
patients in Indonesia. J Med Microbiol. 2015; 64(9): 1046-1052.
21. Goldman GH, da Silva Ferreira ME, dos Reis Marques E, Savoldi M, Perlin D, Park S.
Evaluation of fluconazole resistance mechanisms in Candida albicans clinical isolates from
HIV-infected patients in Brazil. Diag Microbiol Infect Dis. 2004; 50(1): 25-32.
22. Manastir L, Ergon MC, Yucesoy M. Investigation of mutations in Erg11 gene of fluconazole
resistant Candida albicans isolates from Turkish hospitals. Mycoses. 2011; 54(2): 99-104.
23. Maebashi K, Kudoh M, Nishiyama Y, Makimura K, Kamai Y, Uchida K. Proliferation of
intracellular structure corresponding to reduced affinity of fluconazole for cytochrome P-450 in
two low-susceptibility strains of Candida albicans isolated from a Japanese AIDS patient.
Microbiol Immunol. 2003; 47(2): 117-124.
24. Lee MK, Williams LE, Warnock DW, Arthington-Skaggs BA. Drug resistance genes and
trailing growth in Candida albicans isolates. J Antimicrob Chemother. 2004; 53(2): 217-224.
25. Feng LJ, Wan Z, Wang XH, Li RY, Liu W. Relationship between antifungal resistance of
fluconazole resistant Candida albicans and mutations in ERG11 gene. Chinese Med J. 2010;
123(5): 544-548.
26. Hu L, Du X, Li T, Song Y, Zai S, Hu X. Genetic and phenotypic characterization of
Candida albicans strains isolated from infectious disease patients in Shanghai. J Med
Microbiol. 2015; 64(Pt 1): 74-83.
27. Wang H, Kong F, Sorrell TC, Wang B, McNicholas P, Pantarat N. Rapid detection of ERG11
gene mutations in clinical Candida albicans isolates with reduced susceptibility to
fluconazole by rolling circle amplification and DNA sequencing. BMC Microbiol. 2009; 9:167.
28. Marichal P, Koymans L, Willemsens S, Bellens D, Verhasselt P, Luyten W. Contribution of
mutations in the cytochrome P450 14alpha-demethylase (Erg11p, Cyp51p) to azole
resistance in Candida albicans. Microbiol. 1999; 145 ( Pt 10): 2701-2713.
29. Strzelczyk JK, Slemp-Migiel A, Rother M, Golabek K, Wiczkowski A. Nucleotide
substitutions in the Candida albicans ERG11 gene of azole-susceptible and azole-resistant
clinical isolates. Acta Biochim Pol. 2013; 60(4): 547-552