Fig 1.
Population structure analysis inferred using sequencing data of the multilocus sequence genotypes of 102 Cryptococcus neoformans isolates using K = 4.
Legends: ST–sequence type and MT–molecular type.
Fig 2.
Phylogenetic analysis of Brazilian Cryptococcus neoformans isolates inferred by neighbour-joining (NJ), maximum likelihood (ML) and unweighted pair group method with arithmetic mean (UPGMA) methods, using the concatenated data set of the seven MLST loci (CAP59, GPD1, LAC1, PLB1, SOD1, URA5, and the IGS1 region).
The analysis involved a total of 43 C. neoformans isolates, 17 from this study and 26 controls isolates (*) previously published and obtained from MLST database (mlst.mycologylab.org). The phylogenetic tree is drawn to scale, with branch lengths measuring the number of substitutions per site. Codon positions 1st+2nd+3rd+Noncoding were included. There were a total of 4044 positions in the final dataset. Numbers at each branch indicate bootstrap values >50% based on 1,000 replicates by each of the three (ML/NJ/UPGMA) algorithms which presented similar topologies. The isolates identification is described as follows: sequence type number (ST), molecular type, isolate name, followed by isolation source.
Table 1.
Sources of isolates within the different genotypic groups identified based on the ISHAM-MLST concatenated sequences obtained amongst the 102 Cryptococcus neoformans isolates.
Table 2.
DNA polymorphisms in different groups and populations analysed based on the ISHAM-MLST concatenated sequences of 102 Cryptococcus neoformans isolates studied.
Fig 3.
Split decomposition analysis of the concatenated dataset of the 7 MLST loci of the studied Cryptococcus neoformans isolates applying the neighbour-net algorithm using the uncorrect-P parameter model and evidencing the diversity and branching ambiguities attributable to recombination events.
The observation that isolates are linked to each other by multiple pathways and are forming an interconnected network rather than a single bifurcating tree is suggestive of recombination. The phi test for recombination implemented in the software SplitsTree showed significant evidence (p<0.0001) for recombination. The STs belonging to the main clusters identified in the previous phylogenetic analysis were also separated using the split decomposition and are highlighted as follow: VNII: purple; VNB: black; Pop1: red; Pop2: green; Pop3: blue; and Pop4: yellow.
Fig 4.
Median-joining haplotype network (A) of 102 clinical and environmental C. neoformans isolates based on concatenated nucleotide sequences of the seven MLST loci. The major molecular types found in this study (VNI, VNII, VNIV, and VNB) are indicated by circles of different colours. Each circle represents a unique Sequence Type (ST), and the circumference is proportional to sequence type frequency (ST93: 77 isolates; ST77: 19; ST504: 5; ST40: 4; ST53: 2; ST527: 2; ST311: 2; ST212: 2; ST528: 1; ST526: 1; ST525: 1; ST160: 1; ST121: 1; ST39: 1; ST15: 1; ST5: 5; ST3: 1). Brown dots (median vectors) are hypothetical missing intermediates. Minimum spanning trees (B) using the goeBURST algorithm among C. neoformans isolates determined by median-joining network analysis. The size of the circle corresponds to the number of isolates within that haplotype, and the numbers between haplotypes represent the genetic distance of each haplotype, excluding the gaps. The figure shows the distribution of sequence types according to the clinical site and environmental origin.
Fig 5.
Comparison of the antifungal susceptibility patterns (μg/mL) among VNI populations of Cryptococcus neoformans found in this study for the antifungals tested.
Statistically significant differences (p <0.05) are marked with *, Kruskal-Wallis test followed by Dunn's test. The internal horizontal lines represent the median, the bars 25–75% percentiles and the horizontal lines percentiles 10–90%. Larger statistical data available in S3 Table.
Table 3.
Comparison of phenotypic characteristics found among populations.