The Impact of Ser165Glu Substitution Within the Ω-Loop on the Hydrolytic Activity of a Novel Ambler Class A β-Lactamase Variant LAP-3

Kaichun Lin,1 Pan Lü,2 Yining Liu,1 Jingqian Zhou,3 Jian Zhou3 1Department of Otorhinolaryngology, The First People’s Hospital of Yongkang, Jinhua, People’s Republic of China; 2Department of Clinical Laboratory, The First People’s Hospital of Yongkang, Jinhua, People’s Republic of China; 3Department of Pediatrics, The First People’s Hospital of Yongkang, Jinhua, People’s Republic of ChinaCorrespondence: Jian Zhou, Department of Pediatrics, The First People’s Hospital of Yongkang, Jinhua, People’s Republic of China, Email [email protected]: Lactamase activity proteins (LAPs) are narrow-spectrum β-lactamases identified within the Enterobacteriaceae family. In this research, we uncovered a novel variant, LAP-3, while investigating quinolone and carbapenem resistance in Klebsiella pneumoniae. Consequently, we aimed to elucidate the hydrolytic profile and to identify and characterize the new β-lactamase, LAP-3.Methods: Antimicrobial susceptibility was assessed using the agar dilution method. The blaLAP-3 gene was analyzed using PCR, and genomic DNA was extracted for whole-genome sequencing and plasmid mapping. The gene was cloned to analyze the hydrolysis spectrum and biochemical characteristics of LAP-3. Protein structure was analyzed using ChimeraX.Results: Genome sequencing and BLAST analysis revealed a substitution of glutamic acid (Glu165) with serine (Ser165) in LAP-3 compared to LAP-2 within the Ω-loop. The plasmid sequence containing blaLAP-3 revealed that the gene was situated within the multidrug resistance unit of TnpA-tet(A)-blaLAP-3-qnrS1-TnpR. Structural analysis revealed that Glu165 in LAP-3 formed hydrogen bonds with Glu163 and Asn167, in contrast to Ser165 in LAP-1 and LAP-2. The cloned blaLAP-3 gene resulted in elevated MIC levels for Amoxicillin (64-fold), Piperacillin (64-fold), cefuroxime (16-fold), and Cephalothin (32-fold) and while conferring resistance to clavulanic acid and tazobactam by increasing their MICs 8- and 4-fold, respectively, thereby indicating an expanded resistance spectrum.Conclusion: The findings reveal that the novel Ambler class A β-lactamase LAP-3 demonstrates an expanded hydrolysis spectrum against Penicillins, certain cephalosporins, and β-lactamase inhibitors. The Ser165Glu substitution within the Ω-loop may influence the resistance characteristics associated with LAP β-lactamases.Keywords: blaLAP-3, resistance, variant, β-lactamase, Ω-loop