| Summary: | <i>Streptococcus pasteurianus</i> is a zoonotic pathogen causing meningitis and bacteremia in animals and humans. A lack of accurate and convenient detection methods hinders preventing and controlling diseases caused by <i>S. pasteurianus</i>. Additionally, there is limited knowledge about its pathogenicity and antimicrobial resistance characteristics, as there are only three complete genome sequences available. In this study, we established a multiplex PCR assay for the detection of <i>S. pasteurianus</i>, which was applied to six fecal samples from cattle with diarrhea and 285 samples from healthy pigs. Out of the samples tested, 24 were positive, including 5 from pig tonsils, 18 from pig hilar lymph nodes, and 1 from cattle feces. Two strains were isolated from positive samples, and their complete genomes were sequenced. The two strains were non-virulent in mice and multidrug-resistant by the antimicrobial susceptibility test. We first found the presence of genes <i>tet(O/W/32/O)</i> and <i>lsa(E)</i> in <i>S. pasteurianus</i>, leading to resistance to lincosamides and tetracyclines. The convenient and specific multiplex PCR assay provides essential technical support for epidemiological research, and the complete genome sequence of two non-virulent strains contributes to understanding this zoonotic bacterium’s genomic characteristics and pathogenesis.
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