Azole Resistance and Detection of the ERG11 Gene in Clinical Candida albicans Isolated from Pregnant Women with Vulvovaginitis Attending Federal Medical Centre, Yenagoa, Nigeria

Main Article Content

Abdulrasheed B. Abdu
Tolulope Alade
Catherine Omotu

Abstract

Introduction: Candida albicans is one of the most important aetiological agents causing vaginal candidiasis in pregnant women. Most women will experience at least one episode during their reproductive years. Antifungal resistance is a particular problem with Candida infections. Some types of Candida are increasingly resistant to the first-line and second-line antifungal medications.

Objective: To investigate the azole susceptibility of Candida albicans (C. albicans) from pregnant vulvovaginal candidiasis patients and to detect ERG11 gene in these azole resistance isolates.

Methods: Forty-one clinical isolates of C. albicans were collected. Azole susceptibility was tested in vitro using microdilution techniques. The ERG11 genes of 27 isolates of C. albicans (All resistant to azoles) were amplified using PCR method.

Results: Of the 67 isolates recovered, 41(61.19%) were C. albicans, of which 27 (65.85%) each, and 25(60.98%) were resistant to Fluconazole, Voriconazole, and Nystatin respectively. In total, ERG11 genes were detected among 24(88.89%) of 27 C. albicans azole resistant isolates.

Conclusions: Twenty four ERG11 genes were detected among 27 azole resistant C. albicans isolates, which indicates a possible relation with the increase in resistance to azole drugs and the recurrence of vulvovaginal candidiasis.

Keywords:
Candida albicans, ERG11 gene, azole resistance, vulvovaginitis, pregnant women.

Article Details

How to Cite
Abdu, A., Alade, T., & Omotu, C. (2019). Azole Resistance and Detection of the ERG11 Gene in Clinical Candida albicans Isolated from Pregnant Women with Vulvovaginitis Attending Federal Medical Centre, Yenagoa, Nigeria. International STD Research & Reviews, 8(2), 1-11. https://doi.org/10.9734/ISRR/2019/v8i230097
Section
Original Research Article

References

Ksiezopolska E, Gabaldón T. Evolutionary emergence of drug resistance in Candida opportunistic pathogens. Genes. 2018;9 (9):461.
DOI:10.3390/genes9090461

Jyoti T, Shrayanee D, Zeeshan F, Saif H. Multidrug resistance: An emerging CRISIS. interdisciplinary perspectives on infectious diseases. 2014;7.

Article ID 541340
Available:https://doi.org/10.1155/2014/541340

Rodrigues C, Rodrigues M, Silva S, Henriques M. Candida glabrata biofilms: How far have we come? J. Fungi. 2017; 3:11.

Shapiro RS, Robbins N, Cowen LE. Regulatory circuitry governing fungal development, drug resistance, and disease. ‎Microbiol. Mol. Biol. Rev. 2011; 75:213–267.
DOI: 10.1128/MMBR.00045-10

Berkow E, Lockhart S. Fluconazole resistance in Candida species: A current perspective. Infect Drug Resist. 2017;10: 237–245.
DOI: 10.2147/IDR.S118892

Flowers SA, Colón B, Whaley SG, Schuler, MA, David RP. Contribution of clinically derived mutations in ERG11 to azole resistance in Candida albicans. Antimicrob Agents Chemother. 2015;59:450–460.
DOI: 10.1128/AAC.03470-14.

Xiang M J, Liu JY, Ni PH, Wang S, Shi C, Wei B, et al. Erg11 mutations associated with azole resistance in clinical isolates of Candida albicans. FEMS Yeast Res. 2013; 13:386–393. [PubMed] [CrossRef]
Available: 10.1111/1567-1364.12042

Morschhäuser J, Barker KS, Liu TT, Blaß-Warmuth J, Homayouni R, Rogers PD. The transcription factor Mrr1p controls expression of the MDR1 efflux pump and mediates multidrug resistance in Candida albicans. PLoS Pathog. 2007;3:1603–1616.
DOI: 10.1371/journal.ppat.0030164.

Lupetti A, Danesi R, Campa M, Del Tacca M, Kelly S. Molecular basis of resistance to azole antifungals. Trends Mol Med. 2002; 8:76–81. [PubMed]

Sardi JCO, Scorzoni L, Bernardi T, Fusco-Almeida AM, Mendes Giannini MJS. Candida species: Current epidemiology, pathogenicity, biofilm formation, natural antifungal products and new therapeutic options. J. Med. Microbiol. 2013;62:10–24. DOI: 10.1099/jmm.0.045054-0

Flowers SA, Barker KS, Berkow EL, Toner G, Chadwick SG, Gygax SE, Morschhauser J, Rogers PD. Gain-of-function mutations in UPC2 are a frequent cause of ERG11 upregulation in azole-resistant clinical isolates of Candida albicans. Eukaryot Cell. 2012;11:1289–1299.
DOI: 10.1128/EC.00215-12

Selmecki A, Gerami-Nejad M, Paulson C, Forche A, Berman J. An isochromosome confers drug resistance in vivo by amplification of two genes, ERG11 and TAC1. Mol Microbiol. 2008;68:624–641.
DOI: 10.1111/j.1365-2958.2008.06176

Selmecki A, Forche A, Berman J. Aneuploidy and isochromosome formation in drug-resistant Candida albicans. Science. 2006;313:367–370.
DOI:10.1126/science.1128242

Warrilow AG, Mullins JG, Hull CM, Parker JE, Lamb DC, Kelly DE, Kelly SL. S279 point mutations in Candida albicans sterol 14-alpha demethylase (CYP51) reduce in vitro inhibition by fluconazole. Antimicrob Agents Chemother. 2012;56:2099–2107.
DOI:10.1128/AAC.05389-11.

Kelly SL, Lamb DC, Kelly DE. Y132H substitution in Candida albicans sterol 14alpha-demethylase confers fluconazole resistance by preventing binding to haem. FEMS Microbiol Lett. 1999a;180:171–175.
DOI:10.1111/j.1574 6968.1999.tb08792.x.CrossRefPubMedWeb of ScienceGoogle Scholar

Kelly SL, Lamb DC, Loeffler J, Einsele H, Kelly DE. The G464S amino acid substitution in Candida albicans sterol 14alpha-demethylase causes fluconazole resistance in the clinic through reduced affinity. Biochem Biophys Res Commun 1999b;262:174–179. DOI:10.1006/bbrc.1999.1136.CrossRefPubMedWeb of ScienceGoogle Scholar

Marichal P, Koymans L, Willemsens S, Bellens D, Verhasselt P, Luyten W, et al. Contribution of mutations in the cytochrome P450 14alpha-demethylase (Erg 11p, Cyp 51p) to azole resistance in Candida albicans. Microbiology. Cross Ref Pub Med Web of Science Google Scholar. 1999;145:2701–2713.

Morio F, Loge C, Besse B, Hennequin C, Le Pape P. Screening for amino acid substitutions in the Candida albicans Erg11 protein of azole-susceptible and azole-resistant clinical isolates: New substitutions and a review of the literature. Diagn Microbiol Infect Dis. 2010;66(4): 373–384.

Sanglard D, Ischer F, Koymans L, Bille J. Amino acid substitutions in the cytochrome P-450 lanosterol 14alpha-demethylase (CYP51A1) from azole-resistant Candida albicans clinical isolates contribute to resistance to azole antifungal agents. Antimicrob Agents Chemother. 1998; 42:241–253.
DOI:10.1093/jac/42.2.241

McGowan K. Specimen collection, transport and processing: Mycology. In Jorgensen J, Pfaller M, Carroll K, Funke G, Landry M, Richter S, Warnock D (ed), Manual of Clinical Microbiology, Eleventh Edition. ASM Press, Washington, DC. 2015;1944-1954.
DOI: 10.1128/9781555817381.ch114

Wang B, Huang Li-Hua, Zhao Ji-Xue, Wei Man, Fang Hua, et al. ERG11 mutations associated with azole resistance in Candida albicans isolates from vulvovaginal candidosis patients. Asian Pac J Trop Biomed. 2015;5(11):909–914.

Santos MS, Souza ES, Junior RM, Talhari S, Souza JV. Identification of fungemia agents using the polymerase chain reaction and restriction fragment length restriction fragment length polymorphism analysis. Braz J Med Biol Res 2010;43(8): 712–6.

Oliveira CF, Paim TG, Reiter KC, Rieger A, D'Azevedo PA. Evaluation of four different DNA extraction methods in coagulase-negative staphylococci clinical isolates. Rev Inst Med Trop Sao Paulo. 2014;56(1), 29–33.
DOI:10.1590/S0036-46652014000100004

Vijayakumar R, Giri S, Kindo AJ. Molecular species identification of Candida from blood samples of intensive care unit patients by polymerase chain reaction – restricted fragment length polymorphism. J Lab Phys. 2012;4(1):1–4.

White TJ, Bruns TD, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ eds. PCR protocols, a guide to methods and applications. San Diego, California: Academic Press. 1990; 315-322.

Martínez M, López-Ribot JL, Kirkpatrick WR, Bachmann SP, Perea S, Ruesga MT, et al. Heterogeneous mechanisms of azole resistance in Candida albicans clinical isolates from an HIV-infected patient on continuous fluconazole therapy for oropharyngeal candidosis, J. Antimicrob. Chemother. 2002;49(3):515–524.

Clinical Laboratory Standard Institute (CLSI). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard—Third Edition — Document M27-A3. Wayne, Pa, USA: CLSI; 2008.

Monroy-Pérez E, Paniagua-Contreras G L, Rodríguez-Purata P, Vaca-Paniagua F, Vázquez-Villaseñor M, Díaz-Velásquez C, et al. High virulence and antifungal resistance in clinical strains of Candida albicans. Can J Infect Dis Med Microbiol. 2016;5930489.
DOI:10.1155/2016/5930489

Sobel JD. Vaginitis. N Engl J Med. 1997; 337(26):1896–1903. DOI:10.1056/NEJM199712253372607

El-sayed H, Hamouda A. Candida albicans causing vulvovaginitis and their clinical response to antifungal therapy. Egypt J Med Microbiol, 2007;16(1):53-62.

Al-Hedaithy S. Spectrum and proteinase production of yeasts causing vaginitis in Saudi Arabian women. Med Sci Monit. 2002;8(7):498-501.

Al-Mamari A, Al-Buryhi M, Al-Heggami MA, Al-Hag S. Identify and sensitivity to antifungal drugs of Candida species causing vaginitis isolated from vulvovaginal infected patients in Sana’a city. Der Pharma Chemica, 2014;6(1):336-342.

Alfouzan W, Dhar R, Ashkanani H, Gupta M, Rachel C, Khan ZU. Species spectrum and antifungal susceptibility profile of vaginal isolates of Candida in Kuwait. J Mycol Med. 2015;25(1):23-28.

Bello MD, Gonzalez A, Barnabé C, Larrouy G. First characterization of Candida albicans by random amplified polymorphic DNA method in Nicaragua and comparison of the diagnosis methods for vaginal candidiasis in Nicaraguan women. Mem Inst Oswaldo Cruz. 2002;97(7):985-989.

Holland J, Young M, Lee O, Lee S. Vulvovaginal carriage of yeasts other than Candida albicans species. Sex Transm Infect. 2003;79(3):249-250.

Pirotta M, Garland S. Genital Candida species detected in samples from women in Melbourne, Australia, before and after treatment with antibiotics. J Clin Microbiol. 2006;44(9):3213-3217.

Gültekin B, Yazici V, Aydin N. Distribution of Candida species in vaginal specimens and evaluation of CHROMagar Candida medium. Mikrobiyol Bul. 2005;39(3):319-324.

Pakshir K, Yazdani M, Kimiaghalam R. Etiology of vaginal candidiasis in Shiraz, Southern Iran. Res J Microbiol. 2007;2: 696-700.

Xu Y, Chen L, Li C. Susceptibility of clinical isolates of Candida species to fluconazole and detection of C. albicans ERG11 mutations. J. Antimicrob. Chemother. 2008;61(4):798-804.

Emmanuel N, Romeo O, Mebi A, Mark O, Scordino F, Bessy EI, et al. Genotyping and fluconazole susceptibility of Candida albicans strains from patients with vulvovaginal candidiasis in Jos, Nigeria. Asian Pac. J. Trop. Dis. 2012;48-50.

Babin D, Kotigadde S, Rao P, Rao TV. Clinico-mycological profile of vaginal candidiasis in a tertiary care hospital in Kerala. Int J Res Biol Sci. 2013;3(1):55-59.

Agwan V, Butola R, Madan M. Comparison of biofilm formation in clinical isolates of Candida species in a tertiary care center, North India. Indian J Pathol Microbiol. 2015;58:475-478

Deepa B, Subbannayya K, Sunil Rao P, Rao TV. Clinico-mycological profile of vaginal candidiasis in a tertiary care hospital in Kerala. ‎Int. J. Biol. Sci. 2013; 3(1):55-59.

Reddy A, Mustafa M. Phenotypic Identification of Candida species and their susceptibility profile in patients with genitourinary candidiasis. International J. Adv. Res. 2014;2(12):76-84.

Achkar JM, Fries BC. Candida infections of genitourinary tract. Clin. Microbiol. Rev. 2010;23(2):253-273.
DOI: 10.1128/CMR.00076-09

Okungbowa FI, Isikhuemhen OS, Dede AP. The distribution frequency of Candida species in the genitourinary tract among symptomatic individuals in Nigerian cities. Rev. iberoam. Micol. 2003;20(2):60-63.

Richter SS, Galask RP, Messer SA, Hollis RJ, Diekema DJ, Pfaller MA. Antifungal susceptibilities of Candida species causing vulvovaginitis and epidemiology of recurrent cases. J. Clin. Microbiol. 2005;43 (5):2155–2162.
DOI: 10.1128/JCM.43.5.2155-2162.2005

Chowdhary A, Prakash A, Sharma C, Kordalewska M, Kumar A, Sarma S, et al. A multicentre study of antifungal susceptibility patterns among 350 Candida auris isolates (2009–17) in India: Role of the ERG11 and FKS1 genes in azole and echinocandin resistance. J. Antimicrob. Chemother. 2018;73(4):891–899.
Available:https://doi.org/10.1093/jac/dkx480

Lockhart SR, Etienne KA, Vallabhaneni S, Farooqi J, Chowdhary A, Govender NP, et al. Simultaneous emergence of multidrug-resistant Candida auris on 3 continents confirmed by whole-genome sequencing and epidemiological analyses. Clin. Infect. Dis. 2017;64(15):134–140.
DOI:10.1093/cid/ciw691

Morales-López SE, Parra-Giraldo CM, Ceballos-Garzón A, Martínez HP, Rodríguez GJ, Álvarez-Moreno CA, et al. Invasive infections with multidrug-resistant yeast Candida auris, Colombia. Emerg Infect Dis. 2017;23(1):162–164.
DOI:10.3201/eid2301.161497

Schelenz S, Hagen F, Rhodes JL, Abdolrasouli A, Chowdhary A, Hall A, et al. First hospital outbreak of the globally emerging Candida auris in a European hospital. Antimicrob Resist Infect Control. 2016;5:35.
DOI:10.1186/s13756-016-0132-5

Magobo RE, Corcoran C, Seetharam S, Govender N P. Candida auris associated candidemia, South Africa. Emerg Infect Dis 2014;20(7):1250–1251. DOI:10.3201/eid2007.131765

Yang CW, Barkham TM, Chan FY, Wang Y. Prevalence of Candida species, including Candida dubliniensis, in Singapore. J. Clin. Microbiol. 2003;41(1): 472–474.
DOI:10.1128/jcm.41.1.472-474.2003

Yang YL, Cheng HH, Ho YA, Hsiao CF, Lo HJ. Fluconazole resistance rate of Candida species from different regions and hospital types in Taiwan. J Microbiol Immunol Infect. 2003;36(3):187–191.

Hazirolan G, Canton E, Sahin S, Arikan-Akdagli S. Head-to-head comparison of inhibitory and fungicidal activities of fluconazole, itraconazole, voriconazole, posaconazole, and isavuconazole against clinical isolates of Trichosporon asahii. Antimicrob. Agents Chemother. 2013;57 (10):4841–4847.
DOI:10.1128/AAC.00850-13

Choukri F, Benderdouche M, Sednaoui P. In vitro susceptibility profile of 200 recent clinical isolates of Candida spp. to topical antifungal treatments of vulvovaginal candidiasis, the imidazoles and nystatin agents. J Mycol Med. 2014;24(4):303–307.
DOI:10.1016/j.mycmed.2014.05.001 [PubMed] [CrossRef] [Google Scholar]

Fan S, Liu X, Wu C, Xu L, Li J. Vaginal nystatin versus oral fluconazole for the treatment for recurrent vulvovaginal candidiasis. Mycopathologia. 2014;179: 95–101. [PubMed] [Google Scholar]
DOI:10.1007/s11046-014-9827-4

Liu X, Li T, Wang D, Yang Y, Sun W, Liu J, et al. Synergistic antifungal effect of fluconazole combined with licofelone against resistant Candida albicans. Front Microbiol. 2017;8:2101.
DOI:10.3389/fmicb.2017.02101

Cui J, Ren B, Tong Y, Dai H, Zhang L. Synergistic combinations of antifungals and anti-virulence agents to fight against Candida albicans. Virulence. 2015;6(4): 362-371.
DOI: 10.1080/21505594.2015.103988

Pfaller MA, Jones RN, Castanheira M. Regional data analysis of Candida non-albicans strains collected in United States medical sites over a 6-year period, 2006-2011. Mycoses. 2014;57:602–11.
DOI: 10.1111/myc.12206
[PubMed] [Google Scholar]

Hamad M, Kazandji N, Awadallah S, Allam H. Prevalence and epidemiological characteristics of vaginal candidiasis in the UAE. Mycoses. 2014;57:184–90.
DOI: 10.1111/myc.12141
[PubMed] [Google Scholar]

Whaley SG, Berkow EL, Rybak JM, Nishimoto AT, Barker KS, Rogers PD. Azole antifungal resistance in Candida albicans and emerging non-albicans Candida species. Front Microbiol. 2017; 7:2173.
DOI: 10.3389/fmicb.2016.02173

Alvarez-Rueda N, Fleury A, Logé C, et al. The amino acid substitution N136Y in Candida albicans sterol 14 α-demethylase is involved in fluconazole resistance. Med Mycol. 2016;54(7):764–775.
[PubMed] [Google Scholar]

Manastir L, Ergon MC, Yücesoy M. Investigation of mutations in Erg11 gene of fluconazole resistant Candida albicans isolates from Turkish hospitals. Mycoses. 2011;54(2):99–104.
DOI: 10.1111/j.1439-0507.2009.01766.x.
[PubMed] [CrossRef] [Google Scholar]

Heilmann C, Schneider S, Barker KS, Rogers PD, Morschhäuser J. An A643T mutation in the transcription factor Upc2p causes constitutive ERG11 upregulation and increased fluconazole resistance in Candida albicans. Antimicrob Agents Chemother. 2010;54(1):353–359.

Cannon RD, Lamping E, Holmes AR, Niimi K, Tanabe K, Niimi M, et al. Candida albicans drug resistance another way to cope with stress. 2007;153(10):3211-3217.