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

INTRODUCTION

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 bla LAP-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 bla LAP-3 revealed that the gene was situated within the multidrug resistance unit of TnpA-tet(A)-bla LAP-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 bla LAP-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.