A single amino acid in PBP1a drives high-level penicillin and amoxicillin resistance in streptococcus suis

Streptococcus suis is an important zoonotic pathogen that causes severe diseases in both humans and pigs, with β-lactam antibiotics serving as the primary treatment. However, resistance to penicillin and amoxicillin has been steadily increasing, and the mechanisms underlying their resistance remain poorly understood. In this study, we analyzed 534 S. suis strains collected from diseased and healthy pigs in China between 2005 and 2024. Among them, 123 strains exhibited high-level resistance to penicillin (minimum inhibitory concentration (MIC) ≥8 µg/mL), and 78 also displayed co-resistance to amoxicillin. Amino acid sequence alignment of penicillin-binding proteins (PBPs), molecular docking, acylation efficiency assays, site-directed mutagenesis, and MIC testing revealed that the S695A mutation in PBP1a significantly reduces its binding affinity to penicillin and amoxicillin, contributing to high-level resistance. Substituting alanine (A) with serine (S) at position 695 in the highly resistant S. suis strain YS682 resulted in a 64-fold reduction in penicillin resistance (MIC decreased from 128 µg/mL to 2 µg/mL) and a 16-fold reduction in amoxicillin resistance (MIC decreased from 32 µg/mL to 2 µg/mL). Furthermore, 95.56% (366/383) of penicillin-susceptible strains carried S at position 695 of PBP1a. This study provides new insights into the molecular mechanisms driving penicillin and amoxicillin resistance in S. suis.