Table 1_LysR-type transcriptional regulator CARR represses the expression of blaCAR-2 and reduces P. diazotrophicus resistance to cefalothin, cefuroxime and cefotaxime.docx

BackgroundP. diazotrophicus was isolated from a newborn with D-galactosemia complicated sepsis. A homologous blaCAR-2 gene, encoding CAR-2, a predicted member of the CAR family subclass B3 metallo-β-lactamases (MBLs), was found in the genome of this strain. This study aimed to explore the identification of a novel CAR-2 protein encoded by the chromosome of P. diazotrophicus Pd1 that exhibits the zinc-binding motifs of subclass B3 enzymes and the regulatory pattern of CARR, located directly upstream of the blaCAR-2 gene, on the blaCAR-2 gene and its impact on antibiotic resistance.
MethodsAntibiotic susceptibility testing was conducted by the plate agar dilution method. Site-directed mutagenesis was conducted using Mut Express II Fast Mutagenesis Kit V2. Kinetic assays were used to determine the hydrolysis of β-Lactam. The construction of bacterial knockout strains was carried out according to the principle of homologous recombination. The detection of the mRNA expression level of the gene was performed by Real-time Quantitative PCR (qPCR).
ResultsThe minimum inhibitory concentrations (MICs) for E.coli DH5α(pHSG398::CAR-2), which expressed blaCAR-2, increased significantly for cefalothin, cefuroxime and cefotaxime sodium by 2-, 16- and 32- fold, respectively, which showed that blaCAR-2 had resistance to these three antibiotics. This protein, which was a MBL, contains two classical zinc-binding sites characteristic of subclass B3, with the amino acid motif His136, His138, His211, Asp140, His141, and His276. Among the six residues, His136, Asp140, and His211 exhibited the highest catalytic activity. We determined that CAR-2 can effectively hydrolyze cefalothin, cefuroxime and cefotaxime. The chloramphenicol resistance of the constructed E. coli DH5α strain was significantly reduced in the presence of CARR than in the absence of CARR. Compared with those for the wild-type P. diazotrophicus, MICs of cefalothin, cefuroxime and cefotaxime for the ΔCARR strain increased by 8-, 8- and 16-fold, respectively, and the expression level of blaCAR-2 also increased by approximately 10-fold.
ConclusionOverall, CAR-2 is a novel subclass B3 MBL of the CAR family that exhibits catalytic activity against cefalothin, cefuroxime and cefotaxime. CARR represses the expression of blaCAR-2, thereby reducing the resistance of P. diazotrophicus to these antibiotics. This provided a theoretical basis for revealing new mechanisms of pathogen resistance.