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 bla CTX-M, bla SHV and bla OXA 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 bla CTX-M-15 (29.8%), bla CTX-M-truncated (1.3%), bla CTX-M-27 (0.7%), bla SHV-1 (20.5%), bla SHV-11 (2.0%), bla SHV-42 (0.7%) and bla OXA-1 (9.9%), among which bla CTX-M variants and bla SHV-42 were ESBLs. Phylogenetic analysis predicted strong selection pressure on all bla CTX-M variants, bla SHV-11 and bla SHV-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 K m ) 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. [Copyright &y& Elsevier]