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Multifactorial Azole Resistance, Widespread Emergence and Clonal Transmission of Fluconazole-Resistant Candida tropicalis in Indian Hospitals

ABSTRACT: Candida tropicalis is a leading cause of invasive candidiasis in the Asia-Pacific region, with mortality rates exceeding 50%. The rising prevalence of azole resistant clinical strains, particularly in this region, presents a significant clinical challenge. In this study, we analyzed 1,032 C. tropicalis isolates, including 1,016 clinical isolates collected over nine years from 27 hospitals across India, as well as 16 environmental isolates. Fluconazole resistance was detected in 5.1% of clinical isolates, with more than half also exhibiting cross-resistance to voriconazole and itraconazole. Multilocus sequence typing (MLST) and phylogenomic analysis of 1,571 global isolates, confirmed the clonal emergence and persistence of azole- resistant MLST clade 4 strains in Indian hospitals. Genomic analysis revealed that Indian isolates cluster closely with azole resistant strains from China, Singapore, and Taiwan. Azole resistance was found to be multifactorial, involving well-characterized hotspot mutations in the ERG11 gene (Y132F, S154F), ERG11 gene amplification (2–7.5 copies), and significant overexpression of ERG11. Transcriptomic profiling showed significant up regulation of virulence-associated genes in the fluconazole-resistant isolate. Notably, ALS7, a member of the agglutinin-like sequence (ALS) family involved in cell adhesion along with Secreted Aspartyl Proteinases (SAP) genes SAP7 and SAP9, were significantly upregulated in fluconazole-resistant isolates. Additionally, fluconazole-resistant isolates exhibited significantly enhanced biofilm formation compared to fluconazole-susceptible strains, indicating a higher virulence potential. Furthermore, fluconazole-resistant isolates exhibited reduced β-glucan exposure, a trait linked to immune evasion, and showed greater survival in both neutrophil and macrophage killing assays. Together, our findings provide comprehensive genomic and phenotypic evidence supporting the emergence, persistence, and increased pathogenic potential of azole-resistant C. tropicalis clade 4 in Indian hospitals. A deeper understanding of the epidemiological trends and molecular mechanisms driving drug resistance in C. tropicalis is essential for improving diagnostic accuracy, optimising antifungal susceptibility testing, and informing effective clinical management strategies.

ORGANISM(S): Candida tropicalis

PROVIDER: GSE309448 | GEO | 2026/02/23

REPOSITORIES: GEO

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Project description:In the pathogenic yeast Candida albicans, the zinc cluster transcription factor Upc2p has been shown to regulate expression of ERG11 and other genes involved in ergosterol biosynthesis upon exposure to azole antifungals. ERG11 encodes lanosterol demethylase, the target enzyme of this antifungal class. Over-expression of UPC2 reduces azole susceptibility, whereas its disruption results in hypersusceptibility to azoles and reduced accumulation of exogenous sterols. Constitutive up-regulation of ERG11 is a major cause of resistance to fluconazole in clinical isolates of C. albicans, yet the mechanism for this has yet to be determined. Using genome-wide gene expression profiling, we found UPC2 and other genes involved in ergosterol biosynthesis to be coordinately up-regulated with ERG11 in a fluconazole resistant clinical isolate as compared with a matched susceptible isolate from the same patient. Sequence analysis of the UPC2 alleles of these isolates revealed that the resistant isolate contained a single nucleotide substitution in one UPC2 allele that resulted in a G648D exchange in the encoded protein. Introduction of the mutated allele into a drug susceptible strain resulted in constitutive up-regulation of ERG11 and increased resistance to fluconazole. By comparing the gene expression profiles of the fluconazole resistant isolate and of strains carrying wild-type and mutated UPC2 alleles, we identified target genes that are controlled by Upc2p. Here we show for the first time that a gain-of-function mutation in UPC2 leads to increased expression of ERG11 and imparts resistance to fluconazole in clinical isolates of C. albicans. Keywords: genome-wide expression profiling Clinical isolates S1 (susceptible) and S2 (resistant), genome strain SC5314, UPC2 disruption strains (UPC2M4A and UPC2M4B), UPC2 re-integrant strains of non-mutated UPC2 allele (UPC2M2K21A and UPC2M2K21B), and UPC2 re-integrant strains of mutated UPC2 allele (UPC2M2K31A and UPC2M2K31B) were grown for 3 hours until log phase. RNA was extracted for each isolate/strain. Experiments were performed in duplicate.

Project description:Microarray was used to analyze azole resistance of Candida glabrata oropharyngeal isolates from 7 hematopoietic stem cell transplant recipients receiving fluconazole prophylaxis. Transcriptional profiling of the sequential-paired clinical isolates by microarray revealed 19 genes upregulated in the majority of resistant isolates compared to their paired-susceptible isolates. All seven resistant isolates had greater than two fold upregulation of CgPDR1, a master transcriptional regulator of PDR network, and all 7 resistant isolates showed upregulation of known CgPDR1-target genes. The altered transcriptome can be explained in part by the observation that all 7 resistant isolates had acquired a single nonsynonymous mutation in their CgPDR1 ORF. Four mutations occurred in the regulatory domain (L280P, L344S, G348A, S391L) and one in the activation domain (G943S) while two mutations (N764I, R772I) occurred in an undefined region. Association of azole resistance and the CgPDR1 mutations was investigated in the same genetic background by introducing the CgPDR1 sequences from one sensitive and five resistant isolates into a laboratory azole-sensitive strain (cgpdr1) via integrative transformation. The cgpdr1 strain was restored to wild-type fluconazole susceptibility when transformed with CgPDR1 from the susceptible isolate but became resistant when transformed with CgPDR1 from the resistant isolates. However, despite the identical genetic background, upregulation of CgPDR1 and CgPDR1-target genes varied between the 5 transformants, independent of the domain locations in which the mutations occurred. In sum, gain-of-function mutations in CgPDR1 not only contributed to the clinical azole resistance but different mutations had varying degrees of impact on the CgPDR1-target genes. Seven pairs of oropharyngeal sequential isolates were chosen for study because each pair came from an individual hematopoietic stem cell transplant recipient receiving fluconazole. Seven groups consisted of 5 biological replicates in which a sensitive sample was paired with a resistant sample. Each group included 1 to 2 reciprocally labeled samples.

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Multifactorial Azole Resistance, Widespread Emergence and Clonal Transmission of Fluconazole-Resistant Candida tropicalis in Indian Hospitals

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