Large-scale genomic analyses reveal the population structure and evolutionary trends of Streptococcus agalactiae strains in Brazilian fish farms

Abstract

Streptococcus agalactiae is a major pathogen and a hindrance on tilapia farming worldwide. The aims of this work were to analyze the genomic evolution of Brazilian strains of S. agalactiae and to establish spatial and temporal relations between strains isolated from different outbreaks of streptococcosis. A total of 39 strains were obtained from outbreaks and their whole genomes were sequenced and annotated for comparative analysis of multilocus sequence typing, genomic similarity and whole genome multilocus sequence typing (wgMLST). The Brazilian strains presented two sequence types, including a newly described ST, and a non-typeable lineage. The use of wgMLST could differentiate each strain in a single clone and was used to establish temporal and geographical correlations among strains. Bayesian phylogenomic analysis suggests that the studied Brazilian population was co-introduced in the country with their host, approximately 60 years ago. Brazilian strains of S. agalactiae were shown to be heterogeneous in their genome sequences and were distributed in different regions of the country according to their genotype, which allowed the use of wgMLST analysis to track each outbreak event individually.

Figure 1

Map of the distribution of the S. agalactiae sequencing types throughout Brazil, generated in the OpenJump software version 1.6.3.

Figure 2

Reconstruction of evolutionary relationships between piscine S. agalactiae through eBURST analysis from MLST. Points represent STs, circles represent clonal complexes, black lines represent single-locus variants, blue lines represent double-locus variants and isolated points represent singletons. eBurst groups arising exclusively from piscine strains are CC260, CC261 and the group formed by ST-257 and non-typeable strains. CC10 and CC7 arise from piscine and human strains. The dashed area delineates the previously known CC552.

Figure 3

All against all average nucleotide-blast identity from similarity comparison between the studied genomes. Increasing in similarity from red to green. Areas delimited by dashes harbour intra-ST similarities.

Figure 4

Phylogenomic NeighborNet network of wgMLST data. Scale bar measures 100 different alleles. (A) The four major phylogenomic groups, with a focus on Brazilian strains. (B) Zoom of the Brazilian phylogenomic splits. The left side of the network harbours all ST-927 and ST-260 strains, all from grow-out farms with the exception of the SA73 (marked with a star), which is from a hatchery. All strains from those two STs are from the Northeast region with the exception of the marked isolates SA218 and SA245, which both emerged from the Southeast region. The right side of the network harbours all non-typeable strains, which all arise from the Central-South macroregion. The strain SA81 (marked with a circle) was obtained from a diseased catfish, while the remaining strains were obtained from Nile tilapia.

Figure 5

Bayesian evolutionary analysis of S. agalactiae. The upper branch of the tree shows the origin of taxa IV, which contains Brazilian piscine isolates of S. agalactiae and taxa V, which is taxa IV in addition to ST-261. The marked strain SA81 is an exclusive Brazilian catfish isolate, while all the other Brazilian isolates are from tilapia. The time to tMRCA of each taxa of interest is provided and marked with a grey dashed line. The years between brackets are the 95% highest probability density. On the lower region of the tree, within ST-7, the marked strain A909 was isolated from human, while the other ST-7 isolates were from fish.