Project description:In the last decade, Candida krusei has caused multiple outbreaks of candidemia in Neonatal Intensive Care Units (NICUs) in low-and middle-income countries such as Brazil, India, and South Africa. In India, C. krusei ranks as the sixth cause of candidemia in adult ICUs. Additionally, sporadic outbreaks of nosocomial candidemia in the NICUs are widely reported from India. However, the genetic population of C. krusei causing outbreaks remain largely unknown. In the present study, we used whole genome sequencing to examine the genetic structure of C. krusei population causing candidemia spanning a period of five years (2015-20) in a single NICU in Delhi, India. Further, to evaluate the mechanisms of azole antifungal resistance in C. krusei, we compare the transcriptomic profiles of fluconazole susceptible (FLU-S) and resistant (FLU-R) isolates. Transcriptomic assay was performed in logarithmically growing C. krusei clinical isolates 123/P/19 and 1390/P/18 strains. STAR aligner v.2.5.2b was used to sequence the trimmed reads with the specified reference genome of P. kudriavzevii to determine the unique gene hit counts. A total of 178 genes were differentially expressed by at least 1.5-fold in 1390/P/18 as compared to 123/P/19 isolate. Principal component analysis (PCA) of normalized read counts also depicted almost similar transcriptomic profile between the two C. krusei strains with 53 % variance at principal component 1. Out of 178 differentially expressed genes, 72 were up-regulated and 106 were down-regulated in 1390/P/18 strain compared to 123/P/19 strain. Functionally, genes associated with transport (n=10), mitogen activated protein kinase signaling (MAPK; n=8), transcription factors (TF; n=6) and ergosterol biosynthesis (n=3) were expressed differentially.
Project description:Pichia kudriavzevii causes life-threatening infections in immune compromised hosts including hospitalized neonates. This pathogen is resistant to fluconazole while uncommon, strains resistant to multiple antifungal drugs voriconazole, amphotericin B and echinocandins have been reported in healthcare environments. Understanding how P. kudriavzevii spread, persist, and adapt to healthcare settings could help us develop better management strategies. In this study, whole genome sequencing identifies multiple outbreaks of bloodstream infections caused by P. kudriavzevii in a single neonatal intensive care unit (NICU) over five years. Interestingly, two genetically diverse clusters of P. kudriavzevii population showed frequent loss of heterozygosity (LOH) events between two temporal samples. The first outbreak cluster (during 2015-16) showed LOH at chromosomes 1, 4 and 5 and the other outbreak cluster (year 2020) exhibited marked LOH at chromosome 2. The circulation of two separate strain clusters of P. kudriavzevii suggests nosocomial transmission in the NICU in different time periods. Further, to evaluate the gene expression difference between isolates from two clusters, we compared the transcriptomic profiles of three isolates of cluster I and II and exhibiting distinct fluconazole MICs. While no difference was found at the azole target gene ERG11 or the ATP-binding cassette (ABC) transporter genes, differences in transcript abundance were found between the two isolates in genes coding for cell division and filamentation, repressor of ABC gene, FCR1 and ERG5 gene involved in ergosterol biosynthesis pathway. Our study indicates significant diversity, persistence, and rapid evolution of P. kudriavzevii within a single NICU.