Genomic analyses reveal high diversity and rapid evolution of Pichia kudriavzevii within a neonatal intensive care unit in Delhi, India
Ontology highlight
ABSTRACT: 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.
Project description:Previous work has demonstrated that non-synonymous mutations in the gene encoding the putative transcription factor CpMrr1 can influence fluconazole susceptibility; however, the direct contribution of these mutations and how they influence fluconazole resistance in clinical isolates is poorly understood. We identified ten non-synonymous CpMRR1 mutations in a collection of 35 fluconazole resistant clinical isolates. The mutations leading to the A854V, R479K, and I283R substitutions were further examined and found to be activating mutations leading to increased fluconazole resistance. In addition to CpMDR1, we identified another major facilitator superfamily (MFS) transporter gene (CpMDR1B, CPAR2_603010) and an ATP-binding Cassette (ABC) transporter gene (CpCDR1B, CPAR2_304370), as being up-regulated in isolates carrying CpMRR1 activating mutations.
Project description:In Candida albicans, Upc2 is a zinc-cluster transcription factor that targets genes including those of the ergosterol biosynthesis pathway. To date there have been three documented UPC2 gain-of-function (GOF) mutations recovered from fluconazole-resistant clinical isolates that contribute to an increase in ERG11 expression and decreased fluconazole susceptibility. In a group of 62 fluconazole-resistant isolates, we found that 47 of these overexpressed ERG11 by at least two-fold over that of an average expression of 3 unrelated fluconazole susceptible strains. Of those 47 isolates, 29 contained a mutation in UPC2, whereas the remaining 18 isolates did not. Of the isolates containing mutations in UPC2, we recovered eight distinct mutations resulting in single putative amino acid substitutions: G648D, G648S, A643T, A643V, Y642F, G304R, A646V and W478C. Seven of these resulted in increased ERG11 expression, increased cellular ergosterol, and decreased susceptibility to fluconazole as compared to the wild-type strain. Genome-wide transcriptional analysis was performed for the four strongest Upc2 amino acid substitutions (A643V, G648D, G648S and Y642F). Genes commonly upregulated in all four mutations included those involved in ergosterol biosynthesis, in oxidoreductase activity, the major facilitator efflux pump encoded by the MDR1 gene, and the uncharacterized ATP binding cassette transporter CDR11. These findings demonstrate that gain-of-function mutations in UPC2 are more prevalent than previously thought among clinical isolates, make a significant contribution to azole antifungal resistance, but do not account for ERG11 overexpression in all such isolates of C. albicans.
Project description:Purpose: We compared the expression profile of three fluconazole resistant clinical isolates to two fluconazole sensitive clinical isolates, from the same patient, all containing mutation in the trancription factor Mrr1, to define the Mrr1 regulon in Candida lusitaniae.
Project description:In Candida albicans, Upc2 is a zinc-cluster transcription factor that targets genes including those of the ergosterol biosynthesis pathway. To date there have been three documented UPC2 gain-of-function (GOF) mutations recovered from fluconazole-resistant clinical isolates that contribute to an increase in ERG11 expression and decreased fluconazole susceptibility. In a group of 62 fluconazole-resistant isolates, we found that 47 of these overexpressed ERG11 by at least two-fold over that of an average expression of 3 unrelated fluconazole susceptible strains. Of those 47 isolates, 29 contained a mutation in UPC2, whereas the remaining 18 isolates did not. Of the isolates containing mutations in UPC2, we recovered eight distinct mutations resulting in single putative amino acid substitutions: G648D, G648S, A643T, A643V, Y642F, G304R, A646V and W478C. Seven of these resulted in increased ERG11 expression, increased cellular ergosterol, and decreased susceptibility to fluconazole as compared to the wild-type strain. Genome-wide transcriptional analysis was performed for the four strongest Upc2 amino acid substitutions (A643V, G648D, G648S and Y642F). Genes commonly upregulated in all four mutations included those involved in ergosterol biosynthesis, in oxidoreductase activity, the major facilitator efflux pump encoded by the MDR1 gene, and the uncharacterized ATP binding cassette transporter CDR11. These findings demonstrate that gain-of-function mutations in UPC2 are more prevalent than previously thought among clinical isolates, make a significant contribution to azole antifungal resistance, but do not account for ERG11 overexpression in all such isolates of C. albicans. We examined the expression of genes in response to the presence of 4 gain-of-function alleles of the zinc-cluster transcription factor Upc2. The global gene expression of each mutant Upc2 strain was compared to that of the wildtype strain SC5314.
Project description:A food-borne outbreak of haemorrhagic colitis (HC) and HUS caused by E. coli O103:H25 occurred in Norway, 2006. The outbreak included 17 registered cases, of which 10 developed HUS. The aim of this study was to characterize two E. coli O103:H25 isolates from this outbreak. Only one of the isolates carry the stx2 gene (by PCR). Since they have the same typing profile by typing method MLVA, we expect the isolates to have identical gene content except from an Stx2-encoding phage. Therefore, we further investigate whether the Stx2-encoding phage has any impact on the gene expression. Keywords: mixed, gene expression, comparative genomic hybridization
Project description:Salmonella Heidelberg is currently the 9th common serovar and has more than twice the average incidence of blood infections in Salmonella. A recent Salmonella Heidelberg outbreak in chicken infected 634 people during 2013-2014, with a hospitalization rate of 38% and an invasive illness rate of 15%. While the company’s history suggested longstanding sanitation issues, the strains’ characteristics which may have contributed to the outbreak are unknown. We hypothesized that the outbreak strains of S. Heidelberg might possess enhanced stress tolerance or virulence capabilities. Consequently, we obtained nine food isolates collected during the outbreak investigation and several reference isolates and tested their tolerance to processing stresses, their ability to form biofilms, and their invasiveness in vitro. We further performed RNA-sequencing on three isolates with varying heat tolerance to determine the mechanism behind our isolates’ enhanced heat tolerance. Ultimately, we determined that (i) many Salmonella Heidelberg isolates associated with a foodborne outbreak have enhanced heat resistance (ii) Salmonella Heidelberg outbreak isolates have enhanced biofilm-forming ability under stressful conditions, compared to the reference strain (iii) exposure to heat stress may also increase Salmonella Heidelberg isolates’ antibiotic resistance and virulence capabilities and (iv) Salmonella Heidelberg outbreak-associated isolates are primed to better survive stress and cause illness. This data helps explain the severity and scope of the outbreak these isolates are associated with and can be used to inform regulatory decisions on Salmonella in poultry and to develop assays to screen isolates for stress tolerance and likelihood of causing severe illness.
Project description:We compared the transcriptional profiles of 12 E. coli O157:H7 isolates grown to stationary phase in LB broth. These isolates possess the same two enzyme PFGE profile and are related temporally or geographically to the above outbreak. These E. coli O157:H7 isolates included three clinical isolates, five isolates from separate bags of spinach, and single isolates from pasture soil, river water, cow feces, and a feral pig.
Project description:A food-borne outbreak of haemorrhagic colitis (HC) and HUS caused by E. coli O103:H25 occurred in Norway, 2006. The outbreak included 17 registered cases, of which 10 developed HUS. The aim of this study was to characterize two E. coli O103:H25 isolates from this outbreak. Only one of the isolates carry the stx2 gene (by PCR). Since they have the same typing profile by typing method MLVA, we expect the isolates to have identical gene content except from an Stx2-encoding phage. Therefore, we further investigate whether the Stx2-encoding phage has any impact on the gene expression. Keywords: mixed, gene expression, comparative genomic hybridization Triplicate samples of mRNA from a test strain O157:H7 EDL933 and two outbreak strains - one Stx positive and one stx negative were co-hybridized with genomic DNA from the same strain. Triplicate samples of the Stx positive strain grown at acidic conditions was also co-hybridized with genomic DNA from the Stx positive strain. Genomic DNA for each strain is technical replicates only.
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
Project description:Azole resistance was induced in vitro by growth of a susceptible C. parapsilosis isolate in the presence of fluconazole. Whole genome microarrays were used to compare the transcriptional response of the fluconazole-resistant and susceptible isolates.