Transcriptome-based design of antisense inhibitors re-sensitizes CREE. colito carbapenems

Abstract

12Carbapenems are a powerful class of antibiotics, often used as a last-resort treatment to eradicate 13 multidrug-resistant infections. In recent years, however, the incidence of carbapenem-resistant 14Enterobacteriaceae (CRE) has risen substantially, and the study of bacterial resistance 15 mechanisms has become increasingly important for antibiotic development. Although much 16 research has focused on genomic contributors to carbapenem resistance, relatively few studies 17 have examined CRE pathogens through changes in gene expression. In this research, we used 18 transcriptomics to study a CRE Escherichia coli clinical isolate that is sensitive to meropenem but 19 resistant to ertapenem, to both explore carbapenem resistance and identify novel gene 20 knockdown targets for carbapenem re-sensitization. We sequenced total and small RNA to 21 analyze gene expression changes in response to treatment with ertapenem or meropenem, as 22Keywords: Carbapenem-resistant E. coli, transcriptome, small RNA sequencing, genome 36 sequence, antibiotic resistance. 37 through the expression of low-affinity or mutated penicillin-binding proteins, [24][25][26][27] or through the 62 overproduction of efflux pumps. 28,29 63 Other research has sought to understand resistance by tracking transcriptomic changes 64 in carbapenem-resistant pathogens. Two studies on the gene expression profile of carbapenem-65 resistant Acinetobacter baumanii found significant upregulation of transposable elements, 66 recombinase, and other mutation-encouraging factors, in addition to the expected upregulation of 67 efflux pump and β-lactamase genes. 30,31 Four recent studies that examined transcriptomic profiles 68 of CRE-Enterobacter cloacae, K. pneumoniae (2), and Escherichia coli-identified less 69 consistent responses, although downregulation of porin genes and upregulation of cell survival 70 and β-lactamase genes were observed. 32-35 Of this prior research, only one study 32 attempts to 71 evaluate the transcriptomic response of a resistant Enterobacteriaceae strain under antibiotic 72 challenge, the majority examining only the constitutive gene expression levels of an untreated 73 resistant pathogen. 74In this study, we both explore and counter carbapenem resistance by examining the short-75 term (<1 hr) transient transcriptomic response of a clinical isolate of multidrug resistant (MDR) E. 76 coli (referred to from here as E. coli CUS2B) to carbapenem treatment. The strain demonstrates 77 resistance to ertapenem, but sensitivity to meropenem and doripenem, and we sought to use this 78 partial carbapenem resistance phenotype to help understand the development of resistance in 79Enterobacteriaceae. We used a combination of total and small RNA sequencing to assess the 80 response of E. coli CUS2B to either ertapenem or meropenem treatment and identify genes that 81 were potentially important to the pathogen's resistance profile. We then used these genes, 82together with genome sequencing of the clinical isolate, as inputs for the FAST platform. 36 ...