Protocols of manuscript “Identification and Functional Analysis of the Novel β-Lactamase CumAII: An In-depth Exploration of Resistance Mechanisms” v1

The rising incidence of antibiotic resistance among pathogenic bacteria presents a significant public health challenge, particularly with regard to Proteus vulgaris (_P. vulgaris_). This study aimed to investigate the molecular mechanisms underlying the intrinsic resistance of P. vulgaris to β-lactam antibiotics, focusing specifically on the novel β-lactamase variant CumAII. Employing antimicrobial susceptibility testing, whole-genome sequencing, gene cloning, and kinetic analyses, we characterized the hydrolytic spectrum and biochemical properties of CumAII, revealing an 84.87% homology with its predecessor CumA. The isolate EC6-1 demonstrated high resistance levels, with minimum inhibitory concentrations (MICs) for Ampicillin, Cefuroxime, and Cephalothin exceeding 512 μg/mL. The introduction of CumAII into transformants decreased MICs compared to the parent strain, while co-expression with regulatory proteins CumRII and AmpD enhanced susceptibility to certain antibiotics. Molecular docking studies indicated that CumAII exhibits a varied affinity for β-lactams, reflecting changes in interacting residues. The findings elucidate the role of CumAII as a novel β-lactamase with an expanded hydrolytic spectrum against β-lactams, emphasizing the need for further research into the regulation and potential inhibition of such variants to combat the growing threat of multidrug-resistant P. vulgaris_.