Two Sequential Clinical Isolates of Candida glabrata with Multidrug-Resistance to Posaconazole and Echinocandins
Abstract
:1. Introduction
2. Materials and Methods
2.1. Strains and Molecular Identification
2.2. Antifungal Susceptibility Testing
2.3. Amplification and Sequence Analysis of Resistance-Related Genes
2.4. RNA Extraction and Quantitative Real-Time Reverse-Transcription (RT)-PCR
2.5. Testing Susceptibility to FK520 with Other Agents
2.6. Statistical Analysis
2.7. Data Availability
3. Results
3.1. C. glabrata Isolates BMU10720 and BMU10722 Are Multidrug-Resistant to POS and Echinocandins
3.2. Four SNPs Exist in FKS2 Genes between C. glabrata Isolates BMU10720 and BMU10722
3.3. Contribution of Overexpression of ERG11 and CDR1 in POS-Resistance
3.3.1. Both ERG11 and CDR1 Overexpressed in BMU10720 while Only CDR1 Overexpressed in BMU10722
3.3.2. ERG11 Expression Level Can Be Down-Regulated by Cdr1 Inhibitor FK520 So as to Reverse POS-Resistance in BMU10720 and BMU10722
3.4. Contribution of Mutation and Up-Regulation of FKS1 and FKS2 in Echinocandin-Resistance
3.4.1. BMU10720 and BMU10722 Harbor S663P Substitution in FKS2 as Well as Up-Regulation of FKS1 and FKS2 Induced by CAS
3.4.2. FKS2 Expression Level Can Be Down-Regulated by FK520 So as to Partially Reverse Echinocandin-Resistance in BMU10720 and BMU10722
4. Discussion
5. Conclusions
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
- Pappas, P.G.; Lionakis, M.S.; Arendrup, M.C.; Ostrosky-Zeichner, L.; Kullberg, B.J. Invasive Candidiasis. Nat. Rev. Dis. Primers 2018, 4, 18026. [Google Scholar] [CrossRef] [PubMed]
- Alobaid, K.; Khan, Z. Epidemiologic Characteristics of Adult Candidemic Patients in a Secondary Hospital in Kuwait: A Retrospective Study. J. Mycol. Méd. 2019, 29, 35–38. [Google Scholar] [CrossRef] [PubMed]
- Kakeya, H.; Shibata, W.; Yamada, K.; Kaneko, Y. National Trends in the Japanese Distribution of Major Candida Species Causing Candidemia During 2003–2017: A Report by the Epidemiological Investigation Committee for Human Mycoses in Japan. Open Forum. Infect. Dis. 2019, 6, S142. [Google Scholar] [CrossRef]
- Perlin, D.S.; Rautemaa-Richardson, R.; Alastruey-Izquierdo, A. The Global Problem of Antifungal Resistance: Prevalence, Mechanisms, and Management. Lancet Infect. Dis. 2017, 17, e383–e392. [Google Scholar] [CrossRef]
- Hou, X.; Xiao, M.; Wang, H.; Yu, S.Y.; Zhang, G.; Zhao, Y.; Xu, Y.C. Profiling of PDR1 and MSH2 in Candida glabrata Bloodstream Isolates from a Multicenter Study in China. Antimicrob. Agents Chemother. 2018, 62, e00153-18. [Google Scholar] [CrossRef] [Green Version]
- Whaley, S.G.; Rogers, P.D. Azole Resistance in Candida glabrata. Curr. Infect. Dis. Rep. 2016, 18, 41. [Google Scholar] [CrossRef]
- Pappas, P.G.; Kauffman, C.A.; Andes, D.R.; Clancy, C.J.; Marr, K.A.; Ostrosky-Zeichner, L.; Reboli, A.C.; Schuster, M.G.; Vazquez, J.A.; Walsh, T.J.; et al. Clinical Practice Guideline for the Management of Candidiasis: 2016 Update by the Infectious Diseases Society of America. Clin. Infect. Dis 2016, 62, e1–e50. [Google Scholar] [CrossRef]
- Perlin, D.S. Mechanisms of Echinocandin Antifungal Drug Resistance. Ann. N. Y. Acad. Sci. 2015, 1354, 1–11. [Google Scholar] [CrossRef]
- Farmakiotis, D.; Tarrand, J.J.; Kontoyiannis, D.P. Drug-Resistant Candida glabrata Infection in Cancer Patients. Emerg. Infect. Dis. 2014, 20, 1833–1840. [Google Scholar] [CrossRef] [Green Version]
- Hou, X.; Xiao, M.; Chen, S.C.; Kong, F.; Wang, H.; Chu, Y.Z.; Kang, M.; Sun, Z.Y.; Hu, Z.D.; Li, R.Y.; et al. Molecular Epidemiology and Antifungal Susceptibility of Candida glabrata in China (August 2009 to July 2014): A Multi-Center Study. Front. Microbiol. 2017, 8, 880. [Google Scholar] [CrossRef]
- Song, Y.; Chen, X.; Yan, Y.; Wan, Z.; Liu, W.; Li, R. Prevalence and Antifungal Susceptibility of Pathogenic Yeasts in China: A 10-Year Retrospective Study in a Teaching Hospital. Front. Microbiol. 2020, 11, 1401. [Google Scholar] [CrossRef]
- Healey, K.R.; Perlin, D.S. Fungal Resistance to Echinocandins and the MDR Phenomenon in Candida glabrata. J. Fungi 2018, 4, 105. [Google Scholar] [CrossRef]
- Simonicova, L.; Moye-Rowley, W.S. Functional Information from Clinically-derived Drug Fesistant forms of the Candida glabrata Pdr1 Transcription Factor. PLoS Genet. 2020, 16, e1009005. [Google Scholar] [CrossRef]
- Filler, S.G.; Ferrari, S.; Ischer, F.; Calabrese, D.; Posteraro, B.; Sanguinetti, M.; Fadda, G.; Rohde, B.; Bauser, C.; Bader, O.; et al. Gain of Function Mutations in CgPDR1 of Candida glabrata Not Only Mediate Antifungal Resistance but Also Enhance Virulence. PLoS Pathog. 2009, 5, e1000268. [Google Scholar] [CrossRef] [Green Version]
- Hull, C.M.; Parker, J.E.; Bader, O.; Weig, M.; Gross, U.; Warrilow, A.G.; Kelly, D.E.; Kelly, S.L. Facultative Sterol Uptake in an Ergosterol-Deficient Clinical Isolate of Candida glabrata Harboring a Missense Mutation in ERG11 and Exhibiting Cross-Resistance to Azoles and Amphotericin B. Antimicrob. Agents Chemother. 2012, 56, 4223–4232. [Google Scholar] [CrossRef] [Green Version]
- Vanden Bossche, H.; Marichal, P.; Odds, F.C.; Le Jeune, L.; Coene, M.C. Characterization of an Azole-Resistant Candida glabrata Isolate. Antimicrob. Agents Chemother. 1992, 36, 2602–2610. [Google Scholar] [CrossRef] [Green Version]
- Redding, S.W.; Kirkpatrick, W.R.; Saville, S.; Coco, B.J.; White, W.; Fothergill, A.; Rinaldi, M.; Eng, T.; Patterson, T.F.; Lopez-Ribot, J. Multiple Patterns of Resistance to Fluconazole in Candida glabrata Isolates from a Patient with Oropharyngeal Candidiasis Receiving Head and Neck Radiation. J. Clin. Microbiol. 2003, 41, 619. [Google Scholar] [CrossRef] [Green Version]
- Marichal, P.; Vanden Bossche, H.; Odds, F.C.; Nobels, G.; Warnock, D.W.; Timmerman, V.; Van Broeckhoven, C.; Fay, S.; Mose-Larsen, P. Molecular Biological Characterization of an Azole-Resistant Candida glabrata Isolate. Antimicrob. Agents Chemother. 1997, 41, 2229–2237. [Google Scholar] [CrossRef] [Green Version]
- Neppelenbroek, K.H.; Seo, R.S.; Urban, V.M.; Silva, S.; Dovigo, L.N.; Jorge, J.H.; Campanha, N.H. Identification of Candida Species in the Clinical Laboratory: A Review of Conventional, Commercial, and Molecular Techniques. Oral. Dis. 2014, 20, 329–344. [Google Scholar] [CrossRef]
- Clinical and Laboratory Standards Institute. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts. Approved Standard-Fourth Edition. CLSI Document M27-A4. Wayne, PA, USA. 2017. Available online: https://clsi.org/standards/products/microbiology/documents/m27/ (accessed on 28 May 2021).
- Clinical and Laboratory Standards Institute. Performance Standards for Antifungal Susceptibility Testing of Yeasts, 1st ed. CLSI Document M60. Wayne, PA, USA. 2017. Available online: https://clsi.org/standards/products/microbiology/documents/m60/ (accessed on 28 May 2021).
- Clinical and Laboratory Standards Institute. Epidemiological Cutoff Values for Antifungal Susceptibility Testing, 2nd ed. CLSI Document M59. Wayne, PA, USA. 2018. Available online: https://clsi.org/standards/products/microbiology/documents/m59/ (accessed on 28 May 2021).
- Qiao, J.; Kontoyiannis, D.P.; Wan, Z.; Li, R.; Liu, W. Antifungal Activity of Statins against Aspergillus Species. Med. Mycol. 2007, 45, 589–593. [Google Scholar] [CrossRef] [Green Version]
- Li, Q.Q.; Skinner, J.; Bennett, J.E. Evaluation of Reference Genes for Real-Time Quantitative PCR Studies in Candida glabrata Following Azole Treatment. BMC Mol. Biol. 2012, 13, 22. [Google Scholar] [CrossRef] [Green Version]
- Pais, P.; California, R.; Galocha, M.; Viana, R.; Ola, M.; Cavalheiro, M.; Takahashi-Nakaguchi, A.; Chibana, H.; Butler, G.; Teixeira, M.C. Candida glabrata Transcription Factor Rpn4 Mediates Fluconazole Resistance through Regulation of Ergosterol Biosynthesis and Plasma Membrane Permeability. Antimicrob. Agents Chemother. 2020, 64, e00554-20. [Google Scholar] [CrossRef]
- Whaley, S.G.; Caudle, K.E.; Vermitsky, J.P.; Chadwick, S.G.; Toner, G.; Barker, K.S.; Gygax, S.E.; Rogers, P.D. UPC2A is Required for High-Level Azole Antifungal Resistance in Candida glabrata. Antimicrob. Agents Chemother. 2014, 58, 4543–4554. [Google Scholar] [CrossRef] [Green Version]
- Won, E.J.; Choi, M.J.; Kim, M.N.; Yong, D.; Lee, W.G.; Uh, Y.; Kim, T.S.; Byeon, S.A.; Lee, S.Y.; Kim, S.H.; et al. Fluconazole-Resistant Candida glabrata Bloodstream Isolates, South Korea, 2008–2018. Emerg. Infect. Dis. 2021, 27, 779–788. [Google Scholar] [CrossRef]
- Whaley, S.G.; Zhang, Q.; Caudle, K.E.; Rogers, P.D. Relative Contribution of the ABC Transporters Cdr1, Pdh1, and Snq2 to Azole Resistance in Candida glabrata. Antimicrob. Agents Chemother. 2018, 62, e01070-18. [Google Scholar] [CrossRef] [Green Version]
- Li, H.; Chen, Z.; Zhang, C.; Gao, Y.; Zhang, X.; Sun, S. Resistance reversal induced by a combination of fluconazole and tacrolimus (FK506) in Candida glabrata. J. Med. Microbiol. 2015, 64, 44–52. [Google Scholar] [CrossRef]
- Singh-Babak, S.D.; Babak, T.; Diezmann, S.; Hill, J.A.; Xie, J.L.; Chen, Y.L.; Poutanen, S.M.; Rennie, R.P.; Heitman, J.; Cowen, L.E. Global Analysis of the Evolution and Mechanism of Echinocandin Resistance in Candida glabrata. PLoS Pathog. 2012, 8, e1002718. [Google Scholar] [CrossRef] [Green Version]
- Erin, N.; Lehman, R.A.; Boyer, P.J.; Billingsley, M.L. In Vitro Hypoxia and Excitotoxicity in Human Brain Induce Calcineurin-Bcl-2 Interactions. Neuroscience 2003, 117, 557–565. [Google Scholar] [CrossRef]
- Katiyar, S.K.; Alastruey-Izquierdo, A.; Healey, K.R.; Johnson, M.E.; Perlin, D.S.; Edlind, T.D. Fks1 and Fks2 are Functionally Redundant but Differentially Regulated in Candida glabrata: Implications for Echinocandin Resistance. Antimicro.b Agents Chemother. 2012, 56, 6304–6309. [Google Scholar] [CrossRef] [Green Version]
- Ribeiro, M.A.; Paula, C.R. Up-regulation of ERG11 gene among fluconazole-resistant Candida albicans generated in vitro: Is there any clinical implication? Diagn. Microbiol. Infect. Dis. 2007, 57, 71–75. [Google Scholar] [CrossRef]
- Abbes, S.; Mary, C.; Sellami, H.; Michel-Nguyen, A.; Ayadi, A.; Ranque, S. Interactions between Copy Number and Expression Level of Genes Involved in Fluconazole Resistance in Candida glabrata. Front. Cell. Infect. Microbiol. 2013, 3, 74. [Google Scholar] [CrossRef] [Green Version]
- Shukla, S.; Ambudkar, S.V.; Prasad, R. Substitution of threonine-1351 in the Multidrug Transporter Cdr1p of Candida albicans Results in Hypersusceptibility to Antifungal Agents and Threonine-1351 is Essential for Synergic Effects of Calcineurin Inhibitor FK520. J. Antimicrob. Chemother. 2004, 54, 38–45. [Google Scholar] [CrossRef] [Green Version]
- Nim, S.; Rawal, M.K.; Prasad, R. FK520 Interacts with the Discrete Intrahelical Amino Acids of Multidrug Transporter Cdr1 Protein and Acts as Antagonist to Selectively Chemosensitize Azole-Resistant Clinical Isolates of Candida albicans. FEMS Yeast Res. 2014, 14, 624–632. [Google Scholar] [CrossRef] [Green Version]
- Basso, L.R., Jr.; Gast, C.E.; Mao, Y.; Wong, B. Fluconazole Transport into Candida albicans Secretory Vesicles by the Membrane Proteins Cdr1p, Cdr2p, and Mdr1p. Eukaryot. Cell 2010, 9, 960–970. [Google Scholar] [CrossRef] [Green Version]
- Hassan, Y.; Chew, S.Y.; Than, L.T.L. Candida glabrata: Pathogenicity and Resistance Mechanisms for Adaptation and Survival. J. Fungi 2021, 7, 667. [Google Scholar] [CrossRef] [PubMed]
- Alexander, B.D.; Johnson, M.D.; Pfeiffer, C.D.; Jiménez-Ortigosa, C.; Catania, J.; Booker, R.; Castanheira, M.; Messer, S.A.; Perlin, D.S.; Pfaller, M.A. Increasing Echinocandin Resistance in Candida glabrata: Clinical Failure Correlates with Presence of FKS Mutations and Elevated Minimum Inhibitory Concentrations. Clin. Infect. Dis. 2013, 56, 1724–1732. [Google Scholar] [CrossRef] [Green Version]
- Castanheira, M.; Woosley, L.N.; Messer, S.A.; Diekema, D.J.; Jones, R.N.; Pfaller, M.A. Frequency of Fks Mutations Among Candida glabrata Isolates from a 10-year Global Collection of Bloodstream Infection Isolates. Antimicrob. Agents Chemother. 2014, 58, 577–580. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Pham, C.D.; Iqbal, N.; Bolden, C.B.; Kuykendall, R.J.; Harrison, L.H.; Farley, M.M.; Schaffner, W.; Beldavs, Z.G.; Chiller, T.M.; Park, B.J.; et al. Role of FKS Mutations in Candida glabrata: MIC values, echinocandin resistance, and multidrug resistance. Antimicrob. Agents Chemother. 2014, 58, 4690–4696. [Google Scholar] [CrossRef] [Green Version]
- Rivero-Menendez, O.; Navarro-Rodriguez, P.; Bernal-Martinez, L.; Martin-Cano, G.; Lopez-Perez, L.; Sanchez-Romero, I.; Perez-Ayala, A.; Capilla, J.; Zaragoza, O.; Alastruey-Izquierdo, A. Clinical and Laboratory Development of Echinocandin Resistance in Candida glabrata: Molecular Characterization. Front. Microbiol. 2019, 10, 1585. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Perlin, D.S. Current Perspectives on Echinocandin Class Drugs. Future Microbiol. 2011, 6, 441–457. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Healey, K.R.; Paderu, P.; Hou, X.; Jimenez Ortigosa, C.; Bagley, N.; Patel, B.; Zhao, Y.; Perlin, D.S. Differential Regulation of Echinocandin Targets Fks1 and Fks2 in Candida glabrata by the Post-Transcriptional Regulator Ssd1. J. Fungi 2020, 6, 143. [Google Scholar] [CrossRef] [PubMed]
- Hou, X.; Healey, K.R.; Shor, E.; Kordalewska, M.; Ortigosa, C.J.; Paderu, P.; Xiao, M.; Wang, H.; Zhao, Y.; Lin, L.Y.; et al. Novel FKS1 and FKS2 modifications in a high-level echinocandin resistant clinical isolate of Candida glabrata. Emerg. Microbes Infect. 2019, 8, 1619–1625. [Google Scholar] [CrossRef] [PubMed] [Green Version]
Primers | Sequences (5′-3′) | Purposes |
---|---|---|
ERG11-F | ATGTCCACTGAAAACACTTC | ERG11 amplification and sequencing |
ERG11-R | CTAGTACTTTTGTTCTGGATGTC | |
PDR1-F | GGTAAAGTCATTCTTTAGCTACG | PDR1 amplification and sequencing |
PDR1-R | TACAGGCTATGCACACTGTCT | |
FKS1-F | ATGTCTTACAATAATAACGGAC | FKS1 amplification and sequencing |
FKS1-W1 | TTCTCCGATTTCAGCAGTTAC | FKS1 sequencing |
FKS1-W2 | ACTCCAATCGAAAGAGTTCGT | |
FKS1-W3 | AGTTTCATCCAACTTCTAGCT | |
FKS1-W4 | TCAACACTGTCTTTTCCGTTG | |
FKS1-W5 | GATCAAGATCCTGAGAAGGAA | |
FKS1-W6 | TCGATGCTAACCAAGACAACT | |
FKS1-W7 | TGCTTTGATTTTCTACAGAGG | |
FKS1-W8 | CCTGGTTTCCATTTGAATAAC | |
FKS1-W9 | CTTCTTGGATTACAGAGACTA | |
FKS1-R | TTATTTGATTGTAGACCAGGTC | FKS1 amplification and sequencing |
FKS2-F | ATGTCTTACGATCAAGGTGG | FKS2 amplification and sequencing |
FKS2-W1 | CAAGGTCAAATGCCACAACAA | FKS2 sequencing |
FKS2-W2 | ACAAAAAAGCAATGGAAGAGG | |
FKS2-W3 | TCTCCTACTTTCTACACTCAC | |
FKS2-W4 | GATTGCTACAGATTTCATTTTG | |
FKS2-W5 | TGTTAAGGATACCAAGATTCTG | |
FKS2-W6 | TTGATGCTAACCAAGACAACTA | |
FKS2-W7 | CTGGTTTCCATTTGAATAACTT | |
FKS2-W8 | AGATGGTTATCAAGAGGTAACA | |
FKS2-W9 | TTGGACTCAACCAATGAGAG | |
FKS2-R | TTATTTTATAGTGGACCAGGTCTT | FKS2 amplification and sequencing |
RND5.8a | CTTGGTTCTCGCATCGATGA | real-time PCR for RND5.8 |
RND5.8b | GGCGCAATGTGCGTTCA | |
RND5.8pr | 6FAM-ACGCAGCGAAATGCGATACGTAATGTG-TAMRA | |
CDR1a | TAGCACATCAACTACACGAACGT | real-time PCR for CDR1 |
CDR1b | AGAGTGAACATTAAGGATGCCATG | |
CDR1pr | 6FAM-TGCTGCTGCTTCTGCCACCTGGTT-TAMRA | |
CDR2a | GTGCTTTATGAAGGCTACCAGATT | real-time PCR for CDR2 |
CDR2b | TCTTAGGACAGAAGTAACCCATCT | |
CDR2pr | 6FAM-TACCTTTGCGTGCTGGGCGTCACC-TAMRA | |
SNQ2a | ACCATGTGTTCTGAATCAATCAAT | real-time PCR for SNQ2 |
SNQ2b | TCGACATCATTACAATACCAGAAA | |
SNQ2pr | 6FAM-AACTAATCGCCGCAGGTTGTGACA-TAMRA | |
ERG11a | ATTGGTGTCTTGATGGGTGGTC | real-time PCR for ERG11 |
ERG11b | TCTTCTTGGACATCTGGTCTTTCA | |
ERG11pr | 6FAM-ACTTCCGCTGCTACCTCCGCTTGG-TAMRA | |
FKS1a | TACCAACCAGAAGACCAACAGAATGG | real-time PCR for FKS1 |
FKS1b | TCACCACCGCTGATGTTTGGGT | |
FKS1pr | 6FAM-TGGTCAAGCCATGTACGGTGACG-TAMRA | |
FKS2a | CAATTGGCAGAACACCGATCCCAA | real-time PCR for FKS2 |
FKS2b | AGTTGGGTTGTCCGTACTCATCGT | |
FKS2pr | 6FAM-CCAGAACAACAACAAGGTGGTGAAGGT-TAMRA |
Isolates | POS 1 | FLC | ITC | VRC | CAS | MCF | ANF | AMB | |||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
− | + | − | + | − | + | − | + | − | + | − | + | − | + | − | |
ATCC2001 | 0.25 | 0.03 | 2 | 1 | 0.25 | 0.03 | 0.06 | 0.03 | 0.25 | 0.03 | 0.06 | 0.03 | 0.06 | 0.03 | 1 |
BMU10720 | 4 | 0.12 | 4 | 2 | 2 | 0.06 | 0.12 | 0.06 | 32 | 8 | 8 | 4 | 4 | 2 | 1 |
BMU10722 | 4 | 0.12 | 4 | 2 | 2 | 0.06 | 0.12 | 0.06 | 32 | 8 | 8 | 4 | 4 | 2 | 1 |
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Wang, Q.; Li, Y.; Cai, X.; Li, R.; Zheng, B.; Yang, E.; Liang, T.; Yang, X.; Wan, Z.; Liu, W. Two Sequential Clinical Isolates of Candida glabrata with Multidrug-Resistance to Posaconazole and Echinocandins. Antibiotics 2021, 10, 1217. https://doi.org/10.3390/antibiotics10101217
Wang Q, Li Y, Cai X, Li R, Zheng B, Yang E, Liang T, Yang X, Wan Z, Liu W. Two Sequential Clinical Isolates of Candida glabrata with Multidrug-Resistance to Posaconazole and Echinocandins. Antibiotics. 2021; 10(10):1217. https://doi.org/10.3390/antibiotics10101217
Chicago/Turabian StyleWang, Qiqi, Yun Li, Xuan Cai, Ruoyu Li, Bo Zheng, Ence Yang, Tianyu Liang, Xinyu Yang, Zhe Wan, and Wei Liu. 2021. "Two Sequential Clinical Isolates of Candida glabrata with Multidrug-Resistance to Posaconazole and Echinocandins" Antibiotics 10, no. 10: 1217. https://doi.org/10.3390/antibiotics10101217