Biology subjects

Kean, R.

Publications and source records attributed to Kean, R..

4 recordsLinked to original sources

bioRxiv2023

Dry surface biofilm formation by Candida auris facilitates persistence and tolerance to sodium hypochlorite

Candida auris is an enigmatic fungal pathogen, recently elevated to the critical priority group of pathogens by the World Health Organization. Of key concern is its ability to cause outbreaks within intensive and chronic care units, facilitated through its environmental persistence. We investigated the susceptibility of phenotypically distinct C. auris isolates to sodium hypochlorite (NaOCl) disinfection, and the subsequent role of biofilms in surviving disinfection using a dry-surface biofilm (DSB) model and transcriptomic profiling. Planktonic cells were tested for susceptibility to NaOCl in suspension, with biofilm formation using the DSB model consisting of consecutive 48 hr cycles with/without media across a 12-day period, assessed using viable counts, biomass assays, and microscopy. Disinfection efficacy was assessed using clinically relevant protocols of 500-1000ppm for 1-5min. RNA-sequencing was performed on untreated DSBs in comparison to planktonic cells. Isolates were found to be sensitive to NaOCl planktonically at concentrations [≤]62.5 ppm, and grew robust biofilms using the DSB protocol. Biofilms developed tolerance to all NaOCl treatment parameters, with only 2-4 log10-reductions in viable cells observed at highest concentrations. Transcriptomics identified ABC transporters and iron acquisition pathways as strongly upregulated in DSBs relative to planktonic cells. Our novel findings have optimised a DSB protocol in which C. auris biofilms can mediate tolerance to adverse conditions such as NaOCl disinfection, suggesting a lifestyle through which this problematic yeast can environmentally persist and transmit. Mechanistically it has been shown for the first time that upregulation of small-molecule and iron transport pathways are potential facilitators of environmental survival. IMPORTANCECandida auris is a pathogenic yeast that has been responsible for outbreaks in healthcare facilities across the globe, predominantly affecting vulnerable patients. This organism displays a concerning ability to persist within the healthcare environment that is likely facilitated by attaching onto surfaces and developing protective microbial communities knows as biofilms. These communities allow cells to survive and tolerate disinfection with bleach. In this study, we show that C. auris forms robust biofilms on surfaces which promote survival up to 12 days, even with prolonged drying periods. We also demonstrate that development of these biofilms over time significantly reduces the efficacy of hypochlorite disinfection. By investigating the molecular mechanisms of biofilms, we have shown that these biofilms express efflux pumps, which may actively remove hypochlorite molecules from cells, allowing them to tolerate disinfection, and that uptake of iron from the external environment is also important for survival of these communities.

microbiology↗

bioRxiv2021

Recurrent Vulvovaginal Candidiasis; a dynamic interkingdom biofilm disease of Candida and Lactobacillus

Vulvovaginal Candidiasis (VVC) is the most prevalent Candida infection in humans affecting 75% of women at least once throughout their lifetime. In its debilitating recurrent form, RVVC is estimated to affect 140 million women annually. Despite this strikingly high prevalence, treatment options for RVVC remain limited with many women experiencing failed clinical treatment with frontline azoles. Further, the cause of onset and recurrence of disease is largely unknown with few studies identifying potential mechanisms of failed treatment. This study aimed to assess a panel of clinical samples from healthy women and those with RVVC to investigate the influence of Candida, vaginal microbiome and antagonism between Candida and Lactobacillus on disease pathology. 16S rRNA sequencing characterised disease by a reduction in specific health-associated Lactobacillus such as L. crispatus, coupled with an increase in L. iners. In vitro analysis showed Candida albicans clinical isolates are capable of heterogeneous biofilm formation and show the presence of hyphae and C. albicans aggregates in vaginal lavage. Additionally, the ability of Lactobacillus to inhibit C. albicans biofilm formation and biofilm-related gene expression was demonstrated. Using RNA sequencing technology, we were able to exploit a possible mechanism by which L. crispatus may aim to re-establish a healthy vaginal environment through amino-acid acquisition from C. albicans. This study suggests RVVC is not entirely due to an arbitrary switch in C. albicans from commensal to pathogen and understanding interactions between the yeast and vaginal Lactobacillus species may be more crucial to elucidating the cause of RVVC and developing appropriate therapies.

microbiology↗

bioRxiv2020

Candida auris phenotypic heterogeneity determines pathogenicity in vitro

Candida auris is an enigmatic yeast that provides substantial global risk in healthcare facilities and intensive care units. A unique phenotype exhibited by certain isolates of C. auris is their ability to form small clusters of cells known as aggregates, which have been to a limited extent described in the context of pathogenic traits. In this study, we screened several non-aggregative and aggregative C. auris isolates for biofilm formation, where we observed a level of heterogeneity amongst the different phenotypes. Next, we utilised an RNA-sequencing approach to investigate the transcriptional responses during biofilm formation of a non-aggregative and aggregative isolate of the initial pool. Observations from these analyses indicate unique transcriptional profiles in the two isolates, with several genes identified relating to proteins involved in adhesion and invasion of the host in other fungal species. From these findings we investigated for the first time the fungal recognition and inflammatory responses of a three-dimensional skin epithelial model to these isolates. In these models, a wound was induced to mimic a portal of entry for C. auris. We show both phenotypes elicited minimal response in the model minus induction of the wound, yet in the wounded tissue both phenotypes induced a greater response, with the aggregative isolate more pro-inflammatory. This capacity of aggregative C. auris biofilms to generate such responses in the wounded skin highlights how this opportunistic yeast is a high risk within the intensive care environment where susceptible patients have multiple indwelling lines. ImportanceCandida auris has recently emerged as an important cause of concern within healthcare environments due to its ability to persist and tolerate commonly used antiseptics and disinfectants, particularly when surface attached (biofilms). This yeast is able to colonise and subsequently infect patients, particularly those that are critically ill or immunosuppressed, which may result in death. We have undertaken analysis on two different types of this yeast, using molecular and immunological tools to determine whether either of these has a greater ability to cause serious infections. We describe that both isolates exhibit largely different transcriptional profiles during biofilm development. Finally, we show that the inability to form small aggregates (or clusters) of cells has an adverse effect on the organisms immuno-stimulatory properties, suggestive the non-aggregative phenotype may exhibit a certain level of immune evasion.

microbiology↗

bioRxiv2019

Modelling of pH-dependence to develop a strategy for stabilising mAbs at acidic steps in production

AO_SCPLOWBSTRACTC_SCPLOWEngineered proteins are increasingly being required to function or pass through environmental stresses for which the underlying protein has not evolved. A major example in health are antibody therapeutics, where a low pH step is used for purification and viral clearance. In order to develop a computational model for analysis of pH-stability, predictions are compared with experimental data for the relative pH-sensitivities of antibody domains. The model is then applied to proteases that have evolved to be functional in an acid environment, showing a clear signature for low pH-dependence of stability in the neutral to acidic pH region, largely through reduction of saltbridges. Interestingly, an extensively acidic protein surface can maintain contribution to structural stabilisation at acidic pH through replacement of basic sidechains with polar, hydrogen-bonding groups. These observations form a design principle for engineering acid-stable proteins.

bioinformatics↗