BIO PRESENTATION

TITLE

• Today I will be presenting my project on the genetic analysis of an Irish azole-resistant Candida albicans isolate.

AIMS

• The aim of this project was to assemble and analyse the genome of a Candida albicans isolate.

• This included confirming the species and identifying any SNPs linked to antifungal resistance or virulence.

C. ALBICANS

• Candida albicans is a common opportunistic fungus that normally lives harmlessly in the body.

• However, in immunocompromised individuals, it can cause infections ranging from superficial infections, such as oral or vaginal candidosis, to systemic and potentially life-threatening infections.

• Its success as a pathogen is due to its virulence factors, including its ability to switch morphology, adhere to host cells, form biofilms, and evade the immune system.

MUTATIONS AND ANTIFUNGAL

• Genetic variation in Candida albicans includes mutations such as SNPs, insertions, and deletions.

• These mutations can affect how proteins function or how genes are expressed.

• Antifungal resistance can arise through mechanisms like increased drug efflux or mutations in target genes.

• Key genes involved include ERG11, which is the target of azole drugs, and efflux pump genes such as CDR1 and CDR2.

• Overall, genetic variation plays a major role in adaptation and resistance to antifungal treatments.

GENOME SEQUENCING

• Genome sequencing allows us to determine the complete DNA sequence of an organism.

• It helps identify genetic variation, including SNPs and structural changes.

• In this project, next-generation sequencing was used. This allows fast, high-throughput sequencing of millions of short reads in a single run, unlike Sanger sequencing.

• By comparing the genome to a reference, we can identify mutations linked to antifungal resistance.

METHODS

• To carry out this project, a bioinformatics workflow was used.

• First, reads were quality checked and cleaned to remove low-quality data.

• The cleaned reads were then assembled into a genome using SPAdes.

• Species identification was performed using ITS and ribosomal DNA analysis.

• Aneuploidy and SNP analyses were carried out using established tools such as GATK.

ASSEMBLY

• This table gives a comparison between the original and the cleaned genome.

• After cleaning, the genome assembly improved significantly, with fewer and longer contigs.

• The genome size matched known Candida albicans genomes, approximately 14-15 megabases.

• However, the assembly remained fragmented, indicated by a low N50 score, possibly due to repetitive regions and short-read sequencing.

SPECIES IDENTIFICATION

• This table shows the locations of the ITS and rDNA genes, as well as their database matches.

• Species identification confirmed that the isolate was Candida albicans.

• NCBI results were consistent across all regions analysed.

• Some variation was observed in the UNITE database, but overall, the results validated that all further analyses were carried out on the correct species.

ANEUPLOIDY

• This table gives the results of the aneuploidy analysis, and the graph gives a visual representation, highlighting the analysed contigs.

• Aneuploidy is a genetic condition where cells have an abnormal number of chromosomes due to errors in cell division.

• Aneuploidy analysis showed no major chromosomal abnormalities.

• This suggest that the genome is stable and that resistance in this isolate is more likely due to smaller-scale genetic mutations rather than large chromosomal changes.

SNPS

• This table lists the types and amount SNPs found during analysis and their importance.

• Single Nucleotide Polymorphisms are the most common type of genetic variation and involve a variation at a single position in a DNA sequence.

• A total of over 136,000 variants were identified.

• Most were upstream or synonymous variants with low impact.

• However, over 23,000 missense mutations were detected, which may alter protein function.

• This indicates strong adaptive potential within the genome.

ANTIFUNGAL RESISTANCE GENES

• This tables lists known antifungal genes in Candida albicans and shows how many upstream and missense variants were observed.

• Mutations were identified in key antifungal resistance genes, including ERG11, CDR1, CDR2, and TAC1.

• These genes contribute to resistance through reduced drug binding and increased drug efflux

• Importantly, this isolate was confirmed to be highly azole-resistant, with a fluconazole MIC value of 64 micrograms per milliliter.

• For context, an MIC above 4 is considered resistant, meaning this strain shows very strong resistance.

• The presence of both missense and upstream variants supports this, suggesting changes in both protein function and gene regulation.

ERG GENES

• This table shows the number of missense and upstream variants observed in ergosterol biosynthesis genes.

• Azole antifungal drugs target the ergosterol biosynthesis pathway, particularly the ERG11 enzyme.

• Ergosterol is essential for maintaining the fungal cell membrane.

• In this isolate, variants were identified in multiple ERG genes, include ERG2, ERG3, ERG4, ERG11, ERG24, ERG25, ERG26, and ERG27.

• Missense mutations may alter enzyme structure, while upstream variants could affect gene expression.

• Together, these changes suggest the isolate may maintain ergosterol production or adapt under azole stress, contributing to antifungal resistance.

VIRULENCE GENES

• This table lists virulence genes and shows how many missense and upstream variants were detected.

• Significant variation was also found in virulence-associated genes such as ALS3, HWP1, and SAP2.

• These genes are involved in adhesion, invasion and immune evasion.

• Mutations in these regions may influence pathogenicity, suggesting both resistance and virulence potential in this isolate.

• CONCLUSION

• In summary, the genome assembly was accurate in size but fragmented.

• No aneuploidy was detected, indicating genomic stability.

• However, a high number of SNPS were identified, particularly in resistance and virulence genes.

• Combined with the high fluconazole MIC value, this confirms that the isolate is strongly azole-resistant and has significant adaptive potential.

• These findings highlight the clinical importance of monitoring antifungal resistance, especially as highly resistant strains can limit treatment options.

• However, a limitation of this study is the use of short-read sequencing, which can result in fragmented assemblies and may miss larger structural variations.

FURTHER WORK

• For future work, functional validation of these SNPs is needed to confirm their biological impact.

• Gene expression studies could determine how these mutations affect activity.

• Drug susceptibility testing would confirm resistance.

• Finally, long-read sequencing could improve genome assembly quality.