This study aimed to investigate novel β-lactam resistance genes in soil and elucidate the underlying mechanisms of resistance, thus providing valuable clinical alerts, and insights that contribute to the development of new β-lactam drugs.The positive clone was screened from soil metagenomic library, and resistance gene sequence was obtained by subclone construction and sequencing.Sequence alignment, phylogenetic tree and three-dimensional structure modeling were used to analyze the drug resistance gene.Microbroth dilution method was used to evaluate drug resistance.The gene was expressed heterologously, and the hydrolysis ability of the purified protein was tested.Sequence analysis of the subclone revealed that pZF700 contained one novel β-lactamase gene, gob-54, exhibiting 52.3% amino acid sequence identity with the known β-lactamase genes.This gene encodes the broad-spectrum β-lactam enzyme GOB-54.The three-dimensional modeling of GOB-54 generated using SWISS-MODEL, revealed striking structural similarities with GOB-18 from clinical sources.The kinetic parameters indicated that GOB-54 had the highest affinity and catalytic activity for piperacillin among the β-lactam antibiotics tested.The Km and Kcat/Km values were found to be 121.82 μmol/L and 49.15 L/(mmol·s), respectively.In this study, the novel β-lactam resistance gene gob-54 was discovered from soil by functional metagenomics serves as a valuable reference for clinical monitoring and prevention of the dissemination of β-lactam antibiotic resistance genes.
[1] TOOKE C L, HINCHLIFFE P, BRAGGINTON E C, et al.β-lactamases and β-lactamase inhibitors in the 21 st century[J].Journal of Molecular Biology, 2019, 431(18):3472-3500.
[2] THOMAS C A, CHENG Z S, BETHEL C R, et al.The directed evolution of NDM-1[J].Antimicrobial Agents and Chemotherapy, 2023, 67(11):e0071423.
[3] DONG X T, LIU W L, DONG Y X, et al.Metallo-β-lactamase SMB-1 evolves into a more efficient hydrolase under the selective pressure of meropenem[J].Journal of Inorganic Biochemistry, 2023, 247:112323.
[4] BOYD S E, LIVERMORE D M, HOOPER D C, et al.Metallo-β-lactamases:Structure, function, epidemiology, treatment options, and the development pipeline[J].Antimicrobial Agents and Chemotherapy, 2020, 64(10):e00397-e00320.
[5] DO NASCIMENTO A P B, MEDEIROS FILHO F, PAUER H, et al.Characterization of a SPM-1 metallo-beta-lactamase-producing Pseudomonas aeruginosa by comparative genomics and phenotypic analysis[J].Scientific Reports, 2020, 10(1):13192.
[6] KADERABKOVA N, BHARATHWAJ M, FURNISS R C D, et al.The biogenesis of β-lactamase enzymes[J].Microbiology (Reading), 2022, 168(8):001217.
[7] LAU C H F, VAN ENGELEN K, GORDON S, et al.Novel antibiotic resistance determinants from agricultural soil exposed to antibiotics widely used in human medicine and animal farming[J].Applied and Environmental Microbiology, 2017, 83(16):e00989-e00917.
[8] STREIT W R, DANIEL R.Metagenomics Methods and Protocols[M].New York:Springer Nature, 2023.
[9] WANG S C, GAO X, GAO Y J, et al.Tetracycline resistance genes identified from distinct soil environments in China by functional metagenomics[J].Frontiers in Microbiology, 2017, 8:1406.
[10] JEAN B, BRANDI L, ROBIN P, et al.Performance Standards for Antimicrobial Susceptibility Testing[M].USA:Clinical and Laboratory Standards Institute, 2018.
[11] LUO H Y, LI M R, YANG Z S, et al.Characterization of RASA-1, a novel class A extended-spectrum beta-lactamase from Riemerella anatipestifer[J].Veterinary Microbiology, 2022, 270:109456.
[12] HORSFALL L E, IZOUGARHANE Y, LASSAUX P, et al.Broad antibiotic resistance profile of the subclass B3 metallo-β-lactamase GOB-1, a di-zinc enzyme[J].FEBS Journal, 2011, 278(8):1252-1263.
[13] BURNARD D, GORE L, HENDERSON A, et al.Comparative genomics and antimicrobial resistance profiling of Elizabethkingia isolates reveal nosocomial transmission and in vitro susceptibility to fluoroquinolones, tetracyclines, and trimethoprim-sulfamethoxazole[J].Journal of Clinical Microbiology, 2020, 58(9):e00730-e00720.
[14] DELMAS J, ROBIN F, BITTAR F, et al.Unexpected enzyme TEM-126:role of mutation Asp179Glu[J].Antimicrobial Agents and Chemotherapy, 2005, 49(10):4280-4287.
[15] WANG S C, WEI L, GAO Y J, et al.Novel amikacin resistance genes identified from human gut microbiota by functional metagenomics[J].Journal of Applied Microbiology,2022, 133(2):898-907.
[16] HU R X, ZHANG Q, GU Z M.Whole-genome analysis of the potentially zoonotic Elizabethkingia miricola FL160902 with two new chromosomal MBL gene variants[J].Journal of Antimicrobial Chemotherapy, 2020, 75(3):526-530.
[17] MORÁN-BARRIO J, LISA M N, LARRIEUX N, et al.Crystal structure of the metallo-β-lactamase GOB in the periplasmic dizinc form reveals an unusual metal site[J].Antimicrobial Agents and Chemotherapy, 2016, 60(10):6013-6022.
[18] LEIROS H K S, BORRA P S, BRANDSDAL B O, et al.Crystal structure of the mobile metallo-β-lactamase AIM-1 from Pseudomonas aeruginosa:Insights into antibiotic binding and the role of Gln157[J].Antimicrobial Agents and Chemotherapy, 2012, 56(8):4341-4353.
[19] SHROPSHIRE W C, ENDRES B T, BORJAN J, et al.High-level ceftazidime/avibactam resistance in Escherichia coli conferred by the novel plasmid-mediated β-lactamase CMY-185 variant[J].The Journal of Antimicrobial Chemotherapy.2023, 78(10):2442-2450.
[20] FRÖHLICH C, SØRUM V, TOKURIKI N, et al.Evolution of β-lactamase-mediated cefiderocol resistance[J].Journal of Antimicrobial Chemotherapy, 2022, 77(9):2429-2436.
[21] HOOBAN B, JOYCE A, FITZHENRY K, et al.The role of the natural aquatic environment in the dissemination of extended spectrum beta-lactamase and carbapenemase encoding genes:A scoping review[J].Water Research, 2020, 180:115880.
[22] KIEFFER N, GUZMÁN-PUCHE J, POIREL L, et al.ZHO-1, an intrinsic MBL from the environmental Gram-negative species Zhongshania aliphaticivorans[J].Journal of Antimicrobial Chemotherapy, 2019, 74(6):1568-1571.
[23] ZHANG P, SHI Q, HU H, et al.Emergence of ceftazidime/avibactam resistance in carbapenem-resistant Klebsiella pneumoniae in China[J].Clinical Microbiology and Infection, 2020, 26(1):124.e1-124.e4.
