M341I substitution of penicillin binding protein 2X contributes to the emergence of high-level penicillin resistant Streptococcus suis in China.

Streptococcus suis is a major zoonotic pathogen, with penicillins being the first-line treatment for S. suis infections. However, increasing reports of penicillin-non-susceptible (PNS) S. suis have raised concerns, yet the underlying resistance mechanisms remain poorly understood. In this study, we analyzed 107 PNS S. suis isolates to investigate the genetic and phenotypic basis of penicillin resistance. Antimicrobial susceptibility testing revealed that PNS S. suis isolates exhibited higher resistant rates to a number of antimicrobials, including tetracyclines, macrolides and lincosamides. Indeed, high-level penicillin-resistant (HLPR) isolates exhibited even higher resistant rates to these antimicrobials. Serotyping and multilocus sequence typing (MLST) revealed the presence of diverse genetic backgrounds, indicating a potential for widespread transmission. A detailed amino acid analysis of penicillin-binding proteins (PBPs) identified specific substitutions in PBP2x-M341I, I373V, and M401I-associated with HLPR. Recombinant expression of PBP2x proteins containing these substitutions allowed further investigation of their binding affinity to β-lactam antibiotics. Acylation efficiency assays revealed that the M341I substitution significantly reduced the binding affinity of PBP2x for penicillins but not for cephalosporins. These findings provide new insights into the molecular mechanisms underlying HLPR in S. suis and underscore the importance of PBP2x substitutions in driving penicillin resistance.