CDR1, CDR2, MDR1 and ERG11 expression in azole resistant Сandida albicans isolated from HIV-infected patients in city of Moscow

Cover Page

Cite item

Full Text

Abstract

Candida fungi are common opportunistic microorganisms capable of causing infections of various localization, as well as life-threatening conditions in immunocompromised patients, such as HIV-infected individuals, oncology patients, subjects undergoing HSCT, which number has been steadily increasing in recent years. In addition, resistance to anti-fungal drugs has been spreading as well. Naturally sensitive to azoles, C. albicans possess a variety of mechanisms of acquired resistance, including efflux transporters and target protein-encoding gene amplification. This study was conducted to assess a prevalence of such mechanisms in the isolates sample obtained from HIV-infected patients in the Moscow region of the Russian Federation, characterize a relationship between these mechanisms and patterns of developing drug resistance. 18 strains of C. albicans resistant to fluconazole and voriconazole were isolated from HIV-infected patients with recurrent oropharyngeal candidiasis in the Moscow region. The expression levels of the ERG11, MDR1, CDR1, CDR2 genes involved in the formation of acquired azole resistance were measured using quantitative PCR, the –2ΔΔCT method with ACT and PMA genes as control genes and reference values of sensitive isolates. Expression levels exceeding the average values of sensitive isolates by more than 3 standard deviations were considered significantly elevated. In most of the isolates, elevated levels of CDR1 and CDR2 gene expression were found: 89% and 78%, respectively. The expression level of the MDR1 gene was increased only in 28% of cases. ERG11 expression levels were significantly elevated in 78% of the isolates. Expression levels of all resistance genes studied were significantly increased in 4 strains. In this sample of C. albicans isolates, acquired resistance is mainly associated with efflux vectors encoded by the CDR1 and CDR2 genes. Also, in most isolates, an increased expression level for the azole target protein gene — ERG11 was detected. The expression level of the efflux transporter gene MDR1 was increased in the smallest number of samples. It is also impossible to exclude a potential role of other mechanisms in developing acquired resistance, such as mutations in the ERG11 gene. It can be assumed that the identified mechanisms of resistance result from long-term, widespread, and sometimes uncontrolled use of azoles, including those in treatment and prevention of candidiasis in HIV-infected patients.

About the authors

A. D. Voropaev

I.M. Sechenov First Moscow State Medical University (Sechenov University)

Author for correspondence.
Email: advoropaev@gmail.com

PhD Student, Department of Microbiology, Virology and Immunology

Russian Federation, Moscow

D. A. Yekaterinchev

I.M. Sechenov First Moscow State Medical University (Sechenov University)

Email: ekaterinchevda@yandex.ru

PhD Student, Department of Microbiology, Virology and Immunology

Russian Federation, Moscow

Y. N. Urban

G.N. Gabrichevsky Research Institute for Epidemiology and Microbiology

Email: urbanek@mail.ru

PhD (Biology), Senior Researcher, Laboratory of Clinical Microbiology and Biotechnology

Russian Federation, Moscow

V. V. Zverev

I.M. Sechenov First Moscow State Medical University (Sechenov University)

Email: vitalyzverev@outlook.com

RAS Full Member, PhD, MD (Biology), Professor, Head of the Department of Microbiology, Virology and Immunology

Russian Federation, Moscow

Yu. V. Nesvizhsky

I.M. Sechenov First Moscow State Medical University (Sechenov University)

Email: nesviz@mail.ru

PhD, MD (Medicine), Professor, Honored Scientist of the Russian Federation, Professor of the Department of Microbiology

Russian Federation, Moscow

E. A. Voropaeva

G.N. Gabrichevsky Research Institute for Epidemiology and Microbiology

Email: voropaevaea2011@gmail.com

PhD, MD (Biology), Associate Professor, Head Researcher, Head of Medical Biotechnology Department

Russian Federation, Moscow

E. I. Likhanskaya

G.N. Gabrichevsky Research Institute for Epidemiology and Microbiology

Email: lihanskaya.ei@gmail.com

PhD (Biology), Head of the Laboratory of Microbiology and Prophylaxis of Intestinal Infections, Gabrichevsky Institute of Epidemiology and Microbiology

Russian Federation, Moscow

M. S. Afanasiev

I.M. Sechenov First Moscow State Medical University (Sechenov University)

Email: nesviz@mail.ru

PhD, MD (Medicine), Professor of the Department of Clinical Allergology and Immunology

Russian Federation, Moscow

S. S. Afanasiev

G.N. Gabrichevsky Research Institute for Epidemiology and Microbiologyor epidemiology and microbiology

Email: afanasievss409.4@bk.ru

PhD, MD (Medicine), Professor, Honored Scientist of the Russian Federation, Head Researcher

Russian Federation, Moscow

References

  1. Беженар М.Б., Плахова К.И. Механизмы развития резистентности к противогрибковым препаратам грибов рода Candida при рецидивирующем течении урогенитального кандидоза // Молекулярная генетика, микробиология и вирусология. 2020. Т. 38, № 1. С. 15–23. [Bezhenar M.B., Plakhova K.I. Mechanisms of developing antifungal drug resistance of candida spp. in recurrent urogenital candidiasis. Molekulyarnaya genetika, mikrobiologiya i virusologiya = Molecular Genetics, Microbiology and Virology, 2020, vol. 35, no. 1, pp. 15–23. (In Russ.)] doi: 10.17116/molgen20203801115
  2. Веселов А.В., Козлов Р.С. Инвазивный кандидоз: современные аспекты эпидемиологии, диагностики, терапии и профилактики у различных категорий пациентов (в вопроса и ответах) // Клиническая микробиология и антимикробная химиотерапия. 2016. Т. 18, № 2 (Приложение). С. 1–104. [Veselov A.V., Kozlov R.S. Invasive candidiasis: modern aspects of epidemiology, diagnosis, therapy, and prevention in various categories of patients. Klinicheskaya mikrobiologiya i antimikrobnaya khimioterapiya = Clinical Microbiology and Antimicrobial Chemotherapy, 2016, vol. 18, no. 2 (suppl.), pp. 1–104. (In Russ.)]
  3. Пашинина О.А., Карташова О.Л., Пашкова Т.М., Попова Л.П. Антимикотикорезистентность грибов рода Candida, выделенных из репродуктивного тракта женщин с воспалительными заболеваниями гениталий // Бюллетень Оренбургского Научного Центра УрО РАН. 2016. № 3. 9 c. [Pashinina O.A., Kartashova O.L., Pashkova T.M., Popova L.P. Antimycotic resistance of Candida fungi isolated from the reproductive tract of women with inflammatory diseases of the genitals. Byulleten’ Orenburgskogo Nauchnogo Tsentra UrO RAN = Bulletin of the Orenburg Scientific Center of the Ural Branch of the Russian Academy of Sciences, 2016, no. 3, 9 p. (In Russ.)]
  4. Araújo D., Mil-Homens D., Henriques M., Silva S. Anti-EFG1 2’-OMethylRNA oligomer inhibits Candida albicans filamentation and attenuates the candidiasis in Galleria mellonella. Mol. Ther. Nucleic Acids, 2021, vol. 27, pp. 517–523. doi: 10.1016/ j.omtn.2021.12.018
  5. Assress H.A., Selvarajan R., Nyoni H., Mamba B.B., Msagati T.A.M. Antifungal azoles and azole resistance in the environment: current status and future perspectives — a review. Reviews in Environmental Science and Bio/Technology, 2021, vol. 20, pp. 1011–1041. doi: 10.1007/s11157-021-09594-w
  6. Banerjee A., Pata J., Sharma S., Monk B.C., Falson P., Prasad R. Directed mutational strategies reveal drug binding and transport by the MDR transporters of Candida albicans. J. Fungi (Basel), 2021, vol. 7, no. 2: 68. doi: 10.3390/jof7020068
  7. Bhattacharya S., Sae-Tia S., Fries B.C. Candidiasis and mechanisms of antifungal resistance. Antibiotics, 2020, vol. 9, no. 6: 312. doi: 10.3390/antibiotics9060312
  8. Biswas C., Chen C.-A., Halliday C., Kennedy K., Playford E.G., Marriott D.J., Slavin M.A., Sorrell T.C., Sintchenko V. Identification of genetic markers of resistance to echinocandins, azoles and 5-fluorocytosine in Candida glabrata by next-generation sequencing: a feasibility study. Clin. Microbiol. Infect., vol. 23, no. 9, pp. 676.e7–676.e10. doi: 10.1016/j.cmi.2017.03.014
  9. Bongomin F., Gago S., Oladele R., Denning D. Global and multi-national prevalence of fungal diseases — estimate precision. J. Fungi (Basel), 2017, vol. 3, no. 4: 57. doi: 10.3390/jof3040057
  10. Flowers S.A., Barker K.S., Berkow E.L., Toner G., Chadwick S.G., Gygax S.E., Morschhäuser J., Rogers P.D. Gain-of-function mutations in UPC2 are a frequent cause of ERG11 upregulation in azole-resistant clinical isolates of Candida albicans. Eukaryotic Cell. 2012, vol. 11, no. 10, pp. 1289–1299. doi: 10.1128/EC.00215-12
  11. Garcia-Effron G. Molecular markers of antifungal resistance: potential uses in routine practice and future perspectives. J. Fungi (Basel), 2021, vol. 7, no. 3: 197. doi: 10.3390/jof7030197
  12. Graham D.O., Wilson R.K., Ruma Y.N., Keniya M.V., Tyndall J.D.A., Monk B.C. Structural insights into the azole resistance of the Candida albicans darlington strain using Saccharomyces cerevisiae lanosterol 14-demethylase as a surrogate. J. Fungi (Basel), 2021, vol. 7, no. 11: 897. doi: 10.3390/jof7110897
  13. Hampe I.A.I., Friedman J., Edgerton M., Morschhäuser J. An acquired mechanism of antifungal drug resistance simultaneously enables Candida albicans to escape from intrinsic host defenses. PLoS Pathog., 2017, vol. 13, no. 9: e1006655. doi: 10.1371/journal.ppat.1006655
  14. Hoving J.C., Brown G.D., Gómez B.L., Govender N.P., Limper A.H., May R.C., Meya D.B. AIDS-related mycoses: updated progress and future priorities. Trends Microbiol., 2020, vol. 28, no. 6, pp. 425–428. doi: 10.1016/j.tim.2020.01.009
  15. Kukurudz R.J., Chapel M., Wonitowy Q., Bukari A.-R.A., Sidney B., Sierhuis R., Gerstein A.C. Acquisition of cross-azole tolerance and aneuploidy in Candida albicans strains evolved to posaconazole. G3 (Bethesda), 2022, vol. 12, no. 9: jkac156 doi: 10.1093/g3journal/jkac156
  16. Lee Y., Puumala E., Robbins N., Cowen L.E. Antifungal drug resistance: molecular mechanisms in Candida albicans and beyond. Chem. Rev., 2021, vol. 121, no. 6, pp. 3390–3411. doi: 10.1021/acs.chemrev.0c00199
  17. Liu J.-Y.Y., Shi C., Wang Y., Li W.-J.J., Zhao Y., Xiang M.-J.J. Mechanisms of azole resistance in Candida albicans clinical isolates from Shanghai, China. Res. Microbiol., 2015, vol. 166, no. 3, pp. 153–161. doi: 10.1016/j.resmic.2015.02.009
  18. Livak K.J., Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 2001, vol. 25, no. 4, pp. 402–408. doi: 10.1006/meth.2001.1262
  19. Maras B., Maggiore A., Mignogna G., D’Erme M., Angiolella L. Hyperexpression of CDRs and HWP1 genes negatively impacts on Candida albicans virulence. PLoS One, 2021, vol. 16, no. 6: e0252555. doi: 10.1371/journal.pone.0252555
  20. Morio F., Pagniez F., Besse M., Oise Gay-Andrieu F., Miegeville M., Le Pape P. Deciphering azole resistance mechanisms with a focus on transcription factor-encoding genes TAC1, MRR1 and UPC2 in a set of fluconazole-resistant clinical isolates of Candida albicans. Int. J. Antimicrob. Agents, 2013, vol. 42, pp. 410–415. doi: 10.1016/j.ijantimicag.2013.07.013.
  21. Morschhäuser J. Regulation of multidrug resistance in pathogenic fungi. Fungal Genet. Biol., 2010, vol. 47, no. 2, pp. 94–106. doi: 10.1016/j.fgb.2009.08.002
  22. Moye-Rowley W.S. Linkage between genes involved in azole resistance and ergosterol biosynthesis. PLoS Pathog., 2020, vol. 16, no. 9: e1008819. doi: 10.1371/journal.ppat.1008819
  23. Niimi M., Niimi K., Tanabe K., Cannon R.D., Lamping E. Inhibitor resistant mutants give important insights into Candida albicans ABC transporter Cdr1 substrate specificity and help elucidate efflux pump inhibition. Antimicrob. Agents Chemother., 2022, vol. 66, no. 1: e0174821. doi: 10.1128/AAC.01748-21
  24. Oliveira J.M.V., Oliver J.C., Dias A.L.T., Padovan A.C.B., Caixeta E.S., Ariosa M.C.F. Detection of ERG11 Overexpression in Candida albicans isolates from environmental sources and clinical isolates treated with inhibitory and subinhibitory concentrations of fluconazole. Mycoses, 2021, vol. 64, no. 2, pp. 220–227 doi: 10.1111/myc.13208
  25. Orlandini R.K., Bepu D.A.N., Saraiva M.D.C.P., Bollela V.R., Motta A.C.F., Lourenço A.G. Are Candida albicans isolates from the oral cavity of HIV-infected patients more virulent than from non-HIV-infected patients? Systematic review and meta-analysis. Microbial Pathogenesis, 2020, vol. 149: 104477. doi: 10.1016/j.micpath.2020.104477
  26. Pankhurst C.L. Candidiasis (oropharyngeal). BMJ Clin. Evid., 2013, vol. 2013: 1304.
  27. Paul S., Moye-Rowley W.S. Multidrug resistance in fungi: regulation of transporter-encoding gene expression. Front. Physiol. Frontiers, 2014, vol. 5: 143. doi: 10.3389/fphys.2014.00143
  28. Perea S., López-Ribot J.L., Kirkpatrick W.R., McAtee R.K., Santillán R.A., Martínez M., Calabrese D., Sanglard D., Patterson T.F. Prevalence of molecular mechanisms of resistance to azole antifungal agents in candida albicans strains displaying high-level fluconazole resistance isolated from human immunodeficiency virus-infected patients. Antimicrob. Agents Chemother., 2001, vol. 45, no. 10, pp. 2676–2684. doi: 10.1128/AAC.45.10.2676-2684.2001
  29. Perlin D.S., Rautemaa-Richardson R., Alastruey-Izquierdo A. The global problem of antifungal resistance: prevalence, mechanisms, and management. Lancet Infect. Dis., 2017, vol. 17, no. 12, pp. e383–e392. doi: 10.1016/S1473-3099(17)30316-X
  30. Pfaller M.A., Carvalhaes C.G., DeVries S., Huband M.D., Castanheira M. Elderly versus nonelderly patients with invasive fungal infections: species distribution and antifungal resistance, SENTRY antifungal surveillance program 2017–2019. Diagn. Microbiol. Infect. Dis., 2022, vol. 102, no. 4: 115627. doi: 10.1016/j.diagmicrobio.2021.115627
  31. Rajadurai S.G., Maharajan M.K., Veettil S.K., Gopinath D. Comparative efficacy and safety of antifungal agents in the prophylaxis of oropharyngeal candidiasis among HIV-infected adults: a systematic review and network meta-analysis. Life (Basel), 2022, vol. 12, no. 4: 515. doi: 10.3390/life12040515
  32. Redhu A.K., Shah A.H., Prasad R. MFS transporters of Candida species and their role in clinical drug resistance. FEMS Yeast Res., 2016, vol. 16, no. 4: fow043 doi: 10.1093/femsyr/fow043
  33. Robbins N., Caplan T., Cowen L.E. Molecular evolution of antifungal drug resistance. Annu. Rev. Microbiol., 2017, vol. 71, no. 1, pp. 753–775. doi: 10.1146/annurev-micro-030117-020345
  34. Sah S.K., Hayes J.J., Rustchenko E. The role of aneuploidy in the emergence of echinocandin resistance in human fungal pathogen Candida albicans. PLoS Pathog., 2021, vol. 17, no. 5: e1009564. doi: 10.1371/journal.ppat.1009564
  35. Sanglard D., Coste A., Ferrari S. Antifungal drug resistance mechanisms in fungal pathogens from the perspective of transcriptional gene regulation. FEMS Yeast Res., 2009, vol. 9, no. 7, pp. 1029–1050. doi: 10.1111/j.1567-1364.2009.00578.x
  36. Sanguinetti M., Posteraro B., Lass-Flörl C. Antifungal drug resistance among Candida species: mechanisms and clinical impact. Mycoses, 2015, vol. 58, suppl. 2, pp. 2–13. doi: 10.1111/myc.12330
  37. Shi C., Liu J., Li W., Zhao Y., Meng L., Xiang M. Expression of fluconazole resistance-associated genes in biofilm from 23 clinical isolates of Candida albicans. Braz. J. Microbiol., 2019, vol. 50, no. 1, pp. 157–163. doi: 10.1007/s42770-018-0009-2
  38. Teo J.Q.-M., Lee S.J.-Y., Tan A.-L., Lim R.S.-M., Cai Y., Lim T.-P., Kwa A.L.-H. Molecular mechanisms of azole resistance in Candida bloodstream isolates. BMC Infect Dis., 2019, vol. 19, no. 1: 63. doi: 10.1186/s12879-019-3672-5
  39. Wakade R.S., Ristow L.C., Stamnes M.A., Kumar A., Krysan D.J. The Ndr/LATS kinase Cbk1 regulates a specific subset of Ace2 functions and suppresses the hypha-to-yeast transition in Candida albicans. mBio, 2020, vol. 11, no. 4: e01900-20. doi: 10.1128/mBio.01900-20
  40. Zhang J., Li L., Lv Q., Yan L., Wang Y., Jiang Y. The fungal CYP51s: their functions, structures, related drug resistance, and inhibitors. Front. Microbiol., 2019, vol. 10: 691. doi: 10.3389/fmicb.2019.00691

Supplementary files

Supplementary Files
Action
1. JATS XML
Download
2. Figure 1. ERG11, MDR1, CDR1, CDR2 expression levels (–2ΔΔCT)

Download (277KB)
Indexing metadata
3. Figure 2. Percentage of strains with elevated expression level of ERG11, MDR1, CDR1, CDR2

Download (28KB)
Indexing metadata

Copyright (c) 2022 Voropaev A.D., Yekaterinchev D.A., Urban Y.N., Zverev V.V., Nesvizhsky Y.V., Voropaeva E.A., Likhanskaya E.I., Afanasiev M.S., Afanasiev S.S.

Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.

Согласие на обработку персональных данных с помощью сервиса «Яндекс.Метрика»

1. Я (далее – «Пользователь» или «Субъект персональных данных»), осуществляя использование сайта https://journals.rcsi.science/ (далее – «Сайт»), подтверждая свою полную дееспособность даю согласие на обработку персональных данных с использованием средств автоматизации Оператору - федеральному государственному бюджетному учреждению «Российский центр научной информации» (РЦНИ), далее – «Оператор», расположенному по адресу: 119991, г. Москва, Ленинский просп., д.32А, со следующими условиями.

2. Категории обрабатываемых данных: файлы «cookies» (куки-файлы). Файлы «cookie» – это небольшой текстовый файл, который веб-сервер может хранить в браузере Пользователя. Данные файлы веб-сервер загружает на устройство Пользователя при посещении им Сайта. При каждом следующем посещении Пользователем Сайта «cookie» файлы отправляются на Сайт Оператора. Данные файлы позволяют Сайту распознавать устройство Пользователя. Содержимое такого файла может как относиться, так и не относиться к персональным данным, в зависимости от того, содержит ли такой файл персональные данные или содержит обезличенные технические данные.

3. Цель обработки персональных данных: анализ пользовательской активности с помощью сервиса «Яндекс.Метрика».

4. Категории субъектов персональных данных: все Пользователи Сайта, которые дали согласие на обработку файлов «cookie».

5. Способы обработки: сбор, запись, систематизация, накопление, хранение, уточнение (обновление, изменение), извлечение, использование, передача (доступ, предоставление), блокирование, удаление, уничтожение персональных данных.

6. Срок обработки и хранения: до получения от Субъекта персональных данных требования о прекращении обработки/отзыва согласия.

7. Способ отзыва: заявление об отзыве в письменном виде путём его направления на адрес электронной почты Оператора: info@rcsi.science или путем письменного обращения по юридическому адресу: 119991, г. Москва, Ленинский просп., д.32А

8. Субъект персональных данных вправе запретить своему оборудованию прием этих данных или ограничить прием этих данных. При отказе от получения таких данных или при ограничении приема данных некоторые функции Сайта могут работать некорректно. Субъект персональных данных обязуется сам настроить свое оборудование таким способом, чтобы оно обеспечивало адекватный его желаниям режим работы и уровень защиты данных файлов «cookie», Оператор не предоставляет технологических и правовых консультаций на темы подобного характера.

9. Порядок уничтожения персональных данных при достижении цели их обработки или при наступлении иных законных оснований определяется Оператором в соответствии с законодательством Российской Федерации.

10. Я согласен/согласна квалифицировать в качестве своей простой электронной подписи под настоящим Согласием и под Политикой обработки персональных данных выполнение мною следующего действия на сайте: https://journals.rcsi.science/ нажатие мною на интерфейсе с текстом: «Сайт использует сервис «Яндекс.Метрика» (который использует файлы «cookie») на элемент с текстом «Принять и продолжить».