Fitness Burden for the Stepwise Acquisition of First- and Second-Line Antimicrobial Reduced-Susceptibility in High-Risk ESKAPE MRSA Superbugs

<b>Background:</b> The fitness costs (FCs) of antimicrobial resistance (AMR) are crucial issues in antimicrobial resistance (AMR) onset, spread, and, consequently, public health. In <i>Staphylococcus aureus</i>, AMR can induce significant FCs due to slow growth, low competitiveness, and virulence. Here, we investigated the genomics and FCs emerging for progressively acquiring daptomycin (DAP) and glycopeptide (GLY) reduced susceptibility in MRSA. <b>Methods:</b> Genomics was carried out using Illumina-MiSeq Whole-genome sequencing and bioinformatics. The biological FCs of isogenic MRSA strain pairs progressively acquiring DAP and GLY-reduced susceptibility, under DAP/GLY mono or combined therapy, were performed by in-vitro independent and competitive mixed growth, phenotypic in-vitro virulence analysis, and in-vivo <i>G. mellonella</i> larvae killing. <b>Results:</b> Genomics evidenced four different extremely resistant high-risk clones, i.e., ST-5 N315 HA-MRSA, ST-398 LA-MRSA, ST-22 USA-100 HA-EMRSA-15, and ST-1 MW2 CA-MRSA. In-vitro fitness assays revealed slow growth, lower competitiveness, and reduced virulence, predominantly in <i>Galleria mellonella</i> killing ability, in DAP-S hGISA, DAP-R GSSA, DAP-R hGISA, and DAP-R GISA strains. <b>Conclusions:</b> The occurrence of glycopeptide and daptomycin reduced susceptibility conferred increasing FCs, paid as a gradual reduction in virulence, competitiveness, and slow growth performance.