UNLABELLED: Accurate species-level identification among Streptococcus suis and its close relatives remains challenging due to limited resolution of existing diagnostic tools and incomplete taxonomic frameworks. We investigated 61 isolates from diseased swine and three from cows identified as S. suis by MALDI-TOF MS, but which tested negative by a recN gene-based PCR assay commonly used for species confirmation. Whole-genome sequencing revealed that only four isolates were S. suis sensu stricto, while most others belonged to Streptococcus parasuis, Streptococcus ruminantium, Streptococcus iners, Streptococcus suivaginalis, Streptococcus hepaticus, and several other newly proposed Streptococcus species. Core genome phylogenies consistently resolved these novel taxa as monophyletic and taxonomically coherent. However, phylogenies based on the recN gene did not reliably track species boundaries due to limited resolution and recombination, which also affects diagnostic assays. As a proof of concept, we identified 38 genes conserved in ≥95% of S. suis sensu stricto genomes that may support future marker-based diagnostics. Our results confirm extensive taxonomic diversity among S. suis-like organisms, support the recognition of a broader S. suis complex, demonstrate the need for genome-based methods to distinguish its members, and provide evidence supporting the potential clinical significance of several newly recognized and recently proposed species within this complex. IMPORTANCE: Several new species closely related genetically to Streptococcus suis have recently been formally recognized or proposed, raising the possibility that they form a broader, previously unrecognized S. suis complex. Yet most clinical laboratories still report such isolates simply as S. suis, due to the limited resolution of current diagnostic tools. Here, we show that two widely used methods, MALDI-TOF MS and a recN-based PCR used for molecular confirmation of MALDI-TOF MS results, can misidentify S. suis-like isolates. We analyzed 61 isolates from diseased swine and three from cows: all were classified as S. suis by MALDI-TOF MS but tested negative by the recN PCR. Exposing a major gap in current diagnostic frameworks, whole-genome sequencing revealed that most isolates were not S. suis sensu stricto but instead belonged to other recognized or recently proposed Streptococcus species. Most swine isolates were recovered from normally sterile sites, suggesting potential but unconfirmed pathogenic relevance. We provide genomic evidence supporting the proposal of a structured S. suis complex and identify S. suis sensu stricto-specific markers that may inform improved molecular diagnostics in the future. Our findings emphasize the need to modernize diagnostics to account for the true diversity and potential importance for animal health of this expanding group of taxa.

Ceva Biovac Beaucouzé France

Département des Sciences Animales Faculté des Sciences de l'Agriculture et de l'Alimentation Université Laval Québec Québec Canada

Department of Microbiology Graduate School of Medicine Kyoto University Kyoto Japan

Department of Veterinary Medicine University of Cambridge Cambridge United Kingdom

Division of Infectious Animal Disease Research National Institute of Animal Health National Agriculture and Food Research Organization Tsukuba Ibaraki Japan

Groupe de recherche sur les maladies infectieuses en production animale and Centre de recherche en infectiologie porcine et avicole Faculté de médecine vétérinaire Université de Montréal Saint Hyacinthe Québec Canada

National Institute for Communicable Disease Control and Prevention Chinese Center for Disease Control and Prevention Beijing People's Republic of China

Research Center for Food Safety Graduate School of Agricultural and Life Sciences The University of Tokyo Tokyo Japan

Veterinary Research Institute Brno Czech Republic

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