title: Translational Inhibition of CTX-M Extended Spectrum β-Lactamase in Clinical Strains of Escherichia coli by Synthetic Antisense Oligonucleotides Partially Restores Sensitivity to Cefotaxime.
abstract: Synthetic antisense oligomers are DNA mimics that can specifically inhibit gene expression at the translational level by ribosomal steric hindrance. They bind to their mRNA targets by Watson-Crick base pairing and are resistant to degradation by both nucleases and proteases. A 25-mer phosphorodiamidate morpholino oligomer (PMO) and a 13-mer polyamide (peptide) nucleic acid (PNA) were designed to target mRNA (positions -4 to +21, and -17 to -5, respectively) close to the translational initiation site of the extended-spectrum β-lactamase resistance genes of CTX-M group 1. These antisense oligonucleotides were found to inhibit β-lactamase activity by up to 96% in a cell-free translation-transcription coupled system using an expression vector carrying a bla CTX-M-15 gene cloned from a clinical isolate. Despite evidence for up-regulation of CTX-M gene expression, they were both found to significantly restore sensitivity to cefotaxime (CTX) in E. coli AS19, an atypical cell wall permeable mutant, in a dose dependant manner (0-40 nM). The PMO and PNA were covalently bound to the cell penetrating peptide (CPP; (KFF)3K) and both significantly (P < 0.05) increased sensitivity to CTX in a dose dependent manner (0-40 nM) in field and clinical isolates harboring CTX-M group 1 β-lactamases. Antisense oligonucleotides targeted to the translational initiation site and Shine-Dalgarno region of bla CTX-M-15 inhibited gene expression, and when conjugated to a cell penetrating delivery vehicle, partially restored antibiotic sensitivity to both field and clinical isolates.
authors.affiliation: Bacteriology Department, Animal and Plant Health AgencySurrey, UK; Centre for Biomedical Sciences, School of Biological Sciences, Royal Holloway, University of LondonSurrey, UK.
authors.forename: John B
authors.initials: JB
authors.lastname: Readman
authors.affiliation: Centre for Biomedical Sciences, School of Biological Sciences, Royal Holloway, University of London Surrey, UK.
authors.forename: George
authors.initials: G
authors.lastname: Dickson
authors.affiliation: Bacteriology Department, Animal and Plant Health Agency Surrey, UK.
authors.forename: Nick G
authors.initials: NG
authors.lastname: Coldham
keywords: CTX-M; E. coli; ESBL; PMO; PNA; antisense oligonucleotides; translational inhibition