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Phase-variable Type I Restriction Modification (RM) systems are epigenetic regulatory systems that have been identified in numerous human bacterial pathogens. We previously showed that an emerging zoonotic lineage ofStreptococcus suisacquired a phase-variable Type I RM system named SsuCC20p. The SsuCC20p locus was present in the genome of disease-associated isolates from multiple streptococcal species. This indicates that it is not restricted toS. suisand can be acquired through horizontal gene transfer. We demonstrate that SsuCC20p phase-variability relies on a recombinase present within the locus.In vitro, only SsuCC20p is responsible for the genome methylation profiles that were detected in the representative zoonoticS. suisisolate 861160. In addition, we show that, contrary to previous observations,hsdSgenes located downstream of thehsdMgene and the recombinase gene, can contribute to the SsuCC20p genome methylation profile. SsuCC20p locked mutants expressing a singlehsdSeach showed unique genome methylation profiles. The differential genome methylation of the distinct locked mutants caused phase dependent differences in global gene expression in a growth condition dependent manner. We observed significant differences in virulence betweenhsdSlocked mutants in a zebrafish larvae infection model. These data indicate that the streptococcal phase-variable Type I RM system SsuCC20p can impact bacterial virulence via epigenetic regulation of gene expression and potentially contributes to the zoonotic potential ofS. suis.ImportancePhase-variation contributes to the virulence of bacterial pathogens as it allows a single strain to produce phenotypic diverse subpopulations. Phase-variable Restriction Modification (RM) systems are systems that allow for such phase-variation via epigenetic regulation of gene expression levels. The phase-variable RM system SsuCC20p was found in multiple streptococcal species and was acquired by an emerging zoonotic lineage ofStreptococcus suis. We show that the phase-variability of SsuCC20p is dependent on a recombinase encoded within the SsuCC20p locus. We characterized the genome methylation profiles of the different phases of SsuCC20p and showed that the differential genome methylation within the phases causes differences in gene expression levels and virulence. Altogether, we show that the acquisition of a phase-variable RM system impacts virulence and can potentially contribute to the zoonotic potential ofS. suis. Bacterial pathogens can increase their virulence through acquisition of mobile elements containing epigenetic regulatory systems such as RM systems.

Streptococcus suis is an emerging zoonotic swine pathogen which can cause severe infections in humans. In March 2021, an outbreak of S. suis infections with 19 confirmed cases of septicemia and meningitis leading to two deaths, occurred in Nakhon Ratchasima province, Thailand. We characterized the outbreak through an epidemiological investigation combined with Illumina and Nanopore whole genome sequencing (WGS). The source of the outbreak was traced back to a raw pork dish prepared from a single pig during a Buddhist ceremony attended by 241 people. WGS analysis revealed that a single S. suis serotype 2 strain belonging to a novel sequence type (ST) of the emergent Thai zoonotic clade CC233/379, was responsible for the infections. The outbreak clone grouped together with other Thai zoonotic strains from CC233/379 and CC104 in a global S. suis phylogeny and capsule switching events between serotype 2 zoonotic strains and serotype 7 porcine strains were identified. The outbreak strain showed reduced susceptibility to penicillin corresponding with mutations in key residues in the penicillin binding proteins (PBPs). Furthermore, the outbreak strain was resistant to tetracycline, erythromycin, clindamycin, linezolid and chloramphenicol, having acquired an integrative and conjugative element (ICE) carrying resistance genes tetO and ermB, as well as a transposon from the IS1216 family carrying optrA and ermA. This investigation demonstrates that multi-drug resistant zoonotic lineages of S. suis which pose a threat to human health continue to emerge.

Streptococcus suis is an emerging zoonotic pathogen. Over 100 putative virulence factors have been described, but it is unclear to what extent these virulence factors could contribute to zoonotic potential of S. suis. We identified all S. suis virulence factors studied in experimental models of human origin in a systematic review and assessed their contribution to zoonotic potential in a subsequent genomic meta-analysis. PubMed and Scopus were searched for English-language articles that studied S. suis virulence published until 31 March 2021. Articles that analyzed a virulence factor by knockout mutation, purified protein, and/or recombinant protein in a model of human origin, were included. Data on virulence factor, strain characteristics, used human models and experimental outcomes were extracted. All publicly available S. suis genomes with available metadata on host, disease status and country of origin, were included in a genomic meta-analysis. We calculated the ratio of the prevalence of each virulence factor in human and pig isolates. We included 130 articles and 1703 S. suis genomes in the analysis. We identified 53 putative virulence factors that were encoded by genes which are part of the S. suis core genome and 26 factors that were at least twice as prevalent in human isolates as in pig isolates. Hhly3 and NisK/R were particularly enriched in human isolates, after stratification by genetic lineage and country of isolation. This systematic review and genomic meta-analysis have identified virulence factors that are likely to contribute to the zoonotic potential of S. suis.

The zoonotic pathogen Streptococcus suis can cause septicemia and meningitis in humans. We report five complete genomes of Streptococcus suis serotype 2 and serotype 9, covering the complete phylogeny of serotype 9 Dutch porcine isolates and zoonotic isolates. The isolates include the model strain S10 and the Dutch emerging zoonotic lineage.