Person:

Sykes, Sean

The degree to which molecular epidemiology reveals information about the sources and transmission patterns of an outbreak depends on the resolution of the technology used and the samples studied. Isolates of Escherichia coli O104:H4 from the outbreak centered in Germany in May–July 2011, and the much smaller outbreak in southwest France in June 2011, were indistinguishable by standard tests. We report a molecular epidemiological analysis using multiplatform whole-genome sequencing and analysis of multiple isolates from the German and French outbreaks. Isolates from the German outbreak showed remarkably little diversity, with only two single nucleotide polymorphisms (SNPs) found in isolates from four individuals. Surprisingly, we found much greater diversity (19 SNPs) in isolates from seven individuals infected in the French outbreak. The German isolates form a clade within the more diverse French outbreak strains. Moreover, five isolates derived from a single infected individual from the French outbreak had extremely limited diversity. The striking difference in diversity between the German and French outbreak samples is consistent with several hypotheses, including a bottleneck that purged diversity in the German isolates, variation in mutation rates in the two E. coli outbreak populations, or uneven distribution of diversity in the seed populations that led to each outbreak.

This study examined a large cohort of clinical and environmental strains of Cryptococcus neoformans var. grubii to better understand its population structure and identify potential markers significantly associated with clinical isolates or increased virulence capacity. C. neoformans var. grubii is an opportunistic fungal pathogen infecting nearly 1 million patients a year, mostly immunocompromised individuals. Previous work using multi-locus sequence typing analysis identified three sublineages: VNI, VNII, and VNB. Unlike the other two sublineages, the VNB sublineage is constrained to sub-Saharan Africa, exhibits high genetic variability, and encompasses a mix of environmental and clinical isolates. Nearly 400 diverse isolates were selected for sequencing and SNP genotype data were generated and provided as input to the STRUCTURE and smartPCA software to further clarify the population structure. This population analysis revealed a split in the VNB sublineage along with the presence of hybrid and introgressed strains. These genotype data and population information were used with the GEMMA software to identify potential markers significantly associated with clinical strains. GWAS identified twelve markers significantly associated with clinical strains, several within known virulence genes. Additional GWAS analysis utilized phenotypic characterization data for oxidative stress, fluconazole drug resistance, and melanization along with the genotype data. This revealed that three clinical strains harbored significantly associated mutations in BZP4 and exhibited decreased melanin production similar to the melanin-deficient Lac1 deletion strain. These data highlight the utility of GWAS in the analysis of complex traits of pathogenic fungi and provide new markers for further virulence studies.