title: Genetic and structural insights into plasmid-mediated extended-spectrum β-lactamase activity of CTX-M and SHV variants among pathogenic Enterobacteriaceae infecting Indian patients.
abstract: Resistance to third-generation cephalosporins (3GCs) mediated by extended-spectrum β-lactamases (ESBLs) in pathogenic Enterobacteriaceae is considered a major public health threat in India. This study deals with the detection of plasmid-mediated blaCTX-M, blaSHV and blaOXA genes, understanding their contribution to the ESBL phenotype, and their molecular interaction with 3GCs. More than 87% of isolates showed 3GC resistance, with ESBL production in 60.0% of Escherichia coli and 47.7% of Klebsiella pneumoniae. Molecular characterisation revealed the presence of blaCTX-M-15 (29.8%), blaCTX-M-truncated (1.3%), blaCTX-M-27 (0.7%), blaSHV-1 (20.5%), blaSHV-11 (2.0%), blaSHV-42 (0.7%) and blaOXA-1 (9.9%), among which blaCTX-M variants and blaSHV-42 were ESBLs. Phylogenetic analysis predicted strong selection pressure on all blaCTX-M variants, blaSHV-11 and blaSHV-42. The instability index and Gibbs free folding energy change (ΔΔG) predicted decreased stability of SHV-11 and SHV-42. Mutations of CTX-M-truncated, SHV-11 and SHV-42 located in the core region of the enzymes were found to be functional/pathogenic in nature. The catalytic pockets of CTX-M-15 and SHV-42 had the greatest molecular surface area, which might explain their expanded substrate spectrum towards oxyimino-cephalosporins. Molecular dynamics analysis indicated different structural flexibility of CTX-M-truncated compared with the other enzymes. Amino acid alterations resulted in a change of orientation of catalytic residues of class A β-lactamases that might affect their catalytic processes. Molecular interactions revealed higher catalytic efficiency (ΔG and Km) of CTX-M-15, CTX-M-truncated, CTX-M-27, SHV-11 and SHV-42 compared with their respective wild-types. This study provides useful insights into ESBL production of pathogenic Enterobacteriaceae in India that might help in the development of new antibiotics.
authors.affiliation: Department of Biochemistry and Medical Biotechnology, Calcutta School of Tropical Medicine, 108 C.R. Avenue, Kolkata 700073, India.
authors.forename: Lena
authors.initials: L
authors.lastname: Dhara
authors.affiliation: Department of Biochemistry and Medical Biotechnology, Calcutta School of Tropical Medicine, 108 C.R. Avenue, Kolkata 700073, India. Electronic address: anusri.stm@gmail.com.
authors.forename: Anusri
authors.initials: A
authors.lastname: Tripathi
mesh_terms: D000595:Amino Acid Sequence; D018550:Cephalosporin Resistance; D004755:Enterobacteriaceae / Q000187:drug effects* / Q000302:isolation & purification; D004756:Enterobacteriaceae Infections / Q000382:microbiology*; D005798:Genes, Bacterial; D006801:Humans; D007194:India; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D010802:Phylogeny; D010957:Plasmids; D011487:Protein Conformation; D016415:Sequence Alignment; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics*
chemical_list: D001618:beta-Lactamases
keywords: Docking; ESBL; Hydrolytic activity; Protein stability; bla(CTX-M); bla(SHV)