Streptococcus suis is an important pathogen in the pig industry, causing meningitis, pneumonia, arthritis, and septicemia in pigs [1], resulting in substantial economic losses for the industry. Moreover, S. suis is also an important zoonotic pathogen that poses a significant threat to public health [2]. Based on capsular polysaccharide (CPS) antigens, S. suis can be classified into 29 serotypes (1–19, 21, 23–25, 27–31, and 1/2). Also, 34 novel cps loci (Chz and NCL1–20, 21a, 21b, 22–32) have been identified from non-typeable isolates based on differences in the cps gene cluste [[3], [4], [5]]. To date, 11 serotypes have been documented as capable of inducing human infections, consisting of serotypes 1, 2, 4, 5, 7, 9, 14, 16, 21, 24, and 31 [6].

Antimicrobial drugs are commonly used to treat and prevent S. suis infections. However, the indiscriminate use of these drugs accelerates the emergence of antimicrobial resistance, leading to a formidable multidrug-resistant state in S. suis [7,8]. The rising rate of β-lactam antibiotics resistance in S. suis has sparked widespread concern [7,9,10]. β-lactam antibiotics encompass penicillins, cephalosporins, monobactams, and carbapenems. These antibiotics inhibit the activity of penicillin-binding proteins (PBPs), which are cell wall transpeptidases that catalyze the cross-linking of bacterial cell wall peptidoglycan, via their β-lactam ring. This inhibition disrupts peptidoglycan synthesis, leading to bacterial cell death [11]. Streptococci exhibit resistance to β-lactam antibiotics primarily through mutations in PBPs [12]. Specifically, streptococci possess five PBPs (PBP1a, PBP1b, PBP2a, PBP2b, and PBP2x) associated with β-lactam antibiotic resistance. Among them, PBP1b, PBP2a, and PBP1a exhibit both transpeptidase and transglycosylase activities, while PBP2b and PBP2x show transpeptidase activity [13]. In Streptococcus species, mutations in PBP1a, PBP2b, and PBP2x are key drivers of penicillin resistance [14,15]. The development of resistance typically requires mutations in both PBP2b and PBP2x, with high-level penicillin resistance being most strongly linked to mutations in PBP1a [[14], [15], [16]]. Current research on β-lactam antibiotic resistance in S. suis has revealed numerous amino acid mutation sites in PBPs associated with this resistance [17,18]. However, most studies primarily rely on bioinformatics analyses to predict mutation sites in S. suis associated with β-lactam antibiotic resistance, often using a single susceptible strain P1/7 as a reference. Additionally, these predictions have not been experimentally validated [[17], [18], [19]]. As a result, accurately identifying the key amino acid mutations linked to β-lactam resistance in S. suis remains a significant challenge.

In this study, we comprehensively analyzed the amino acid sequences of five PBPs in S. suis strains isolated from China, which exhibit varying levels of resistance to penicillin and amoxicillin, with a particular focus on PBP1a. Additionally, molecular docking simulations, site-specific mutant strain construction, and acylation efficiency experiments were performed to identify key amino acid mutations strongly associated with resistance to penicillin and amoxicillin. Our findings offer valuable insights into the mechanisms underlying penicillin and amoxicillin resistance in S. suis.