Abstract
Background:Candida auris is an enigmatic yeast recently named as one of four Critical Priority Group fungal pathogens by the World Health Organisation, and has been responsible for outbreaks within healthcare facilities across the world. Genomic sequencing has identified a total of six "phylogeographic" clades of C. auris isolates, each with distinctive characteristics which impact clinical disease outcomes. Importantly, outbreaks of C. auris have been associated with clades I (South Asian), III (African) and IV (South American) to date, whilst isolated cases have been documented for clades II (East Asian), V (Iranian) and VI (Indo-Malayan). Another phenotype of C. auris that is associated with clades I and III is the formation of cellular clusters known as ‘aggregation’, which have been observed in vitro as well as during infection. The mechanisms behind aggregation are not well understood but appear to be a response to cellular stressors that may provide a selective advantage to cells.
C. auris demonstrates a remarkable ability to persist on surfaces, textiles and reusable equipment long-term, often despite rigorous cleaning and disinfection protocols. One phenotype that has been implicated in environmental persistence of C. auris is the ability to form biofilms on a number of abiotic and biotic surfaces. Biofilm development is a known virulence factor of C. auris, and confers tolerance to all available classes of antifungal drugs, however the ability to form biofilms can vary widely between isolates. There is a growing body of evidence to show that environmental biofilm development by C. auris and other healthcare-associated pathogens promotes survival of disinfection. Given that current protocols for infection prevention and control of C. auris are based on other healthcare-associate pathogens such as Clostridioides difficile, there is an urgent need to understand how C. auris survives in the healthcare environment in order to optimise such protocols. The overarching aim of this research, therefore, was to investigate mechanisms that contribute to persistence and transmission of C. auris within the healthcare environment.
Methods:
To understand whether genetic and phenotypic background influence heterogeneity of biofilm formation and antifungal drug susceptibility, we screened a panel of 26 clinical isolates of C. auris for these characteristics (chapter 2). A set of six isolates which represent the variability in biofilm-former and aggregative phenotypes, as well as clades I and III, were selected for ongoing experimental work relating to planktonic and biofilm growth characteristics (chapter 2 & 3).
Mechanisms of growth that facilitate the survival of C. auris in the healthcare environment were explored using a semi-dry surface biofilm (SDB) model (chapter 3). Biofilm-mediated environmental persistence was further investigated by quantifying the immediate and long-term survival of planktonic and biofilm cells following disinfection with sodium hypochlorite (NaOCl) using clinically-relevant parameters. We also performed transcriptomics analysis of SDBs relative to planktonic cells to elucidate potential cellular mechanisms which contribute to SDB formation and tolerance to NaOCl (chapter 4).
Given the clinical reports of environmental persistence following disinfection, planktonic cells were also screened for ability to withstand NaOCl disinfection through entry into a dormant viable-but-non-culturable (VBNC) state (chapter 5). The long-term effect of NaOCl treatment on recovered VBNC cells was investigated through growth as SDBs and transcriptomics on pre-treated SDBs relative to untreated SDBs and planktonic cells.
Results:
Phenotypic heterogeneity was observed for biofilm formation and antifungal susceptibility of all 26 C. auris isolates (chapter 2). This was also evident for planktonic growth rates, biofilm-mediated drug susceptibility and expression of biofilm-related genes amongst the subset of six isolates. Importantly, this variability occurred independently of biofilm-former status, aggregative phenotype or geographic clade.
NaOCl disinfection testing revealed total eradication of planktonic cells using clinically relevant parameters (chapter 3). In contrast biofilm formation as little as 90 minutes was found to facilitate immediate survival to NaOCl disinfection, and development over 24 hours also promoted persistence of cells up to 14 days after disinfection. Isolates were capable of growth as SDBs, and greater numbers of cells survived disinfection with NaOCl following each cycle of growth. Transcriptomics and differential gene expression analysis of SDBs identified fewer than 350 genes that were up- or down-regulated relative to planktonic cells (chapter 4). Functional enrichment analysis revealed that SDBs upregulate pathways for iron acquisition and protein synthesis, and downregulated cell wall remodelling and cell division processes, suggestive of metabolically active cells which are not actively growing.
Deeper examination of the response to disinfection amongst planktonic C. auris cells revealed a subpopulation which were found to be viable using DNA methods (chapter 5). That these cells could not be quantified immediately after disinfection by traditional culture methods, was suggestive of a VBNC phenotype, and this was confirmed by later resuscitation of cells when grown using the SDB protocol. Recovered cells formed SDBs at a slower rate, but eventually the number of viable cells and biomass normalised to the levels of untreated SDBs. Furthermore, no significantly differentially-expressed genes between NaOCl pre-treated and untreated SDBs were identified by transcriptomics, consistent with a conserved mechanism for biofilm development amongst C. auris regardless of cellular background.
Conclusions:
Variability in phenotypes between clinical isolates from diverse backgrounds demonstrates that heterogeneity is an inherent feature of C. auris, which may have important implications for clinical outcomes. The findings from this research also demonstrate the necessity of evaluating disinfectant efficacy against clinically-relevant growth models including biofilms and VBNC cells, and suggest that biofilms formed by C. auris are inherently tolerant to NaOCl at currently-recommended concentrations. Mechanistically, persistence and tolerance may be conferred through acquisition and intracellular sequestration of iron and constitutive up-regulated expression of efflux pumps. Collectively, this thesis provides novel insights into phenotypic characteristics of this resilient yeast that make it a troublesome pathogen.
| Date of Award | 2023 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Ryan Kean (Supervisor), John Butcher (Supervisor) & Lesley Price (Supervisor) |