Population structure, genetic diversity and pathotypes of Streptococcus suis isolated during the last 13 years from diseased pigs in Switzerland

Abstract

Streptococcus (S.) suis is a globally important swine pathogen, which comprises certain zoonotic serotypes. In this study, a detailed characterization of 88 porcine S. suis isolates was performed by analyzing capsular (cps) types, multilocus sequence typing (MLST) and investigation of the minimum core genome (MCG). In order to focus on the virulence potential of presumable invasive disease-associated S. suis isolates, virulence-associated gene profiles were assessed followed by screening a chosen subset of S. suis strains with a molecular pathotyping tool. Results showed a high genetic variability within this strain collection. In total, seventeen cps types were identified with a predominance of cps type 9 (15.9%) and 6 (14.8%). MLST revealed 48 sequence types (STs) including 41 novel ones. The population structure of S. suis was heterogenous and isolates belonged to eight different clonal complexes (CCs) including CC28 (9.1%), CC1109 (8%), CC13/149 (6.8%), CC1237 (5.7%), CC1 (3.4%), CC17 (3.4%), CC87 (2.3%), and CC1112 (1.1%), whereas a significant portion of isolates (60.2%) could not be assigned to any described CCs. Virulence-associated markers, namely extracellular protein factor (epf), muramidase-released protein (mrp), and suilysin (sly), showed a link with STs rather than with cps types. With this study an expanded knowledge about the population structure and the genetic diversity of S. suis could be achieved, which helps to contribute to an optimal public health surveillance system by promoting a focus on strains with an increased virulence and zoonotic potential.

Keywords: Capsular type; Clonal complex; Invasive disease-associated; MCG groups; MLST; Streptococcus suis; Virulence potential.

Figure 1

Sequence type (ST) distribution of porcineS. suisin relation to capsular type. Identified STs are shown in form of a stacked histogram and illustrated in the corresponding bar sections. Nontypeable (NT) isolates could not be identified by multiplex PCR. Isolates with one lacking housekeeping gene identification could not be determined (nd).

Figure 2

MLST-based minimal spanning tree of 88 porcineS. suisisolates. The MLST-based minimum spanning tree is representing the temporal distribution of sequence types determined for S. suis isolates collected during the last 13 years from diseased pigs. The tree was calculated using the goeBURST full MST algorithm in Phyloviz 2.0. Sizes of nodes reflect the number of isolates with a specific MLST profile. Numbers within the nodes indicate the corresponding sequence type. Node colors refer to the year of isolation as represented in the legend. Numbers on lines indicate locus variants between nodes. Black lines indicate single locus variants and grey lines represent multi locus variants. x and y represent isolates with no determined ST due to lacking of housekeeping gene identification.

Figure 3

Population snapshot ofS. suisin Switzerland. Groups at triple locus variants (TLV) level were created by goeBURST v1.2 software using the phyloviz software [41] applying a data set from the PubMLST database (https://pubmlst.org/ssuis). Grey lines define a link at double locus variant (DLV) or triple locus variant (TLV) between the CCs following eBURST rules. Numbers in nodes represent sequence types (ST), whereas light green represents founder groups, blue shows common nodes and red indicates STs identified in Switzerland. Clonal complexes (CCs) identified are indicated in bold. Association of Swiss isolates between capsular types (cps), ST and its corresponding CCs is shown. Identified virulence markers namely extracellular protein factor (epf), muramidase-released protein (mrp), and suilysin (sly) are indicated, highlighting its correlation with according STs and CCs. Isolates harboring mrp + epf + sly + , mrp + (epf +)sly + , sly + , and mrp + are represented in pink, green, orange, and brown, respectively. Variants of mrp are indicated with mrp^S (small variant) and mrp*** (large variant); variant of epf is marked as epf* (large variant). Isolates appearing as singletons or with no determined founder are not represented. CC1109 and CC1237 are shown separately with no connections to the main CCs due to the absence of relation at TLV level. Corresponding minimum core genome (MCG) groups are highlighted in yellow.

Figure 4

Capillary electrophoresis plots illustrating invasive disease-associated and non-disease-associated SwissS. suisisolates. On the left hand side the DNA size marker (100 bp–2.5 kb) is shown. The alignment marker (green) is representing the start and end of electrophoresis. Amplicons sizes obtained from the molecular pathotyping tool [31] are indicated on the right hand side. Lane 1 corresponds to the amplification reaction of a highly virulent isolate of cps type 2 (sequence type 1103) revealing both disease-associated markers (red) predicting a putative copper exporting ATPase 1 (SSU0207) and a type I restriction-modification (RM) system S protein (SSU1589). Lane 2 and 3 represent disease-associated isolates harboring the copper ATPase and a variant form with a 21 base pair (bp) deletion (lane 3), respectively. Lane 4, 5, and 6 demonstrate observed amplification patterns of non-disease-associated samples (blue). Lane 4 shows a PCR amplicon for the putative sugar ABC reporter (SSUST30534) also in the presence of the copper ATPase gene. Samples with no amplification of any markers are considered to be non-disease-associated (lane 5). A sporulation regulator (WhiA) serves as identification control for S. suis (SSU0577).