Genomic and Long-Term Transcriptomic Imprints Related to the Daptomycin Mechanism of Action Occurring in Daptomycin- and Methicillin-Resistant Under Daptomycin Exposure.

Daptomycin (DAP) is one of the last-resort treatments for heterogeneous vancomycin-intermediate (hVISA) and vancomycin-intermediate (VISA) infections. DAP resistance (DAP-R) is multifactorial and mainly related to cell-envelope modifications caused by single-nucleotide polymorphisms and/or modulation mechanisms of transcription emerging as result of a self-defense process in response to DAP exposure. Nevertheless, the role of these adaptations remains unclear. We aim to investigate the comparative genomics and late post-exponential growth-phase transcriptomics of two DAP-resistant/DAP-susceptible (DAP) methicillin-resistant (MRSA) clinical strain pairs to focalize the genomic and long-term transcriptomic fingerprinting and adaptations related to the DAP mechanism of action acquired under DAP pressure using Illumina whole-genome sequencing (WGS), RNA-seq, bioinformatics, and real-time qPCR validation. Comparative genomics revealed that membrane protein and transcriptional regulator coding genes emerged as shared functional coding-gene clusters harboring mutational events related to the DAP-R onset in a strain-dependent manner. Pairwise transcriptomic enrichment analysis highlighted common and strain pair-dependent Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, whereas DAP double-pair cross-filtering returned 53 differentially expressed genes (DEGs). A multifactorial long-term transcriptomic-network characterized DAP MRSA includes alterations in (i) peptidoglycan biosynthesis, cell division, and cell-membrane (CM) organization genes, as well as a B/S autolysin genes; (ii) 2 involved in fermentative metabolism; (iii) CM-potential perturbation genes; and (iv) oxidative and heat/cold stress response-related genes. Moreover, a D-alanyl-D-alanine decrease in cell-wall muropeptide characterized DAP/glycopeptide cross-reduced susceptibility mechanisms in DAP MRSA. Our data provide a snapshot of DAP MRSA genomic and long-term transcriptome signatures related to the DAP mechanism of action (MOA) evidencing that a complex network of genomic changes and transcriptomic adaptations is required to acquire DAP-R.