Characterisation of in vitro resistance selection against second-/last-line antibiotics in methicillin-resistant Staphylococcus aureus ATCC 43300 strain

Background and objectives
The increasing occurrence of MRSA clinical isolates harbouring reduced susceptibility to mainstay antibiotics has escalated the use of second and last line antibiotics. Hence, it is critical to evaluate the likelihood of MRSA developing clinical resistance to these antibiotics. Our study sought to characterize the development of resistance to vancomycin (VAN), daptomycin (DAP) and linezolid (LZD) in MRSA ATCC 43300 in vitro and further determine the mechanisms underpinning resistance.

Methods
MRSA was exposed to increasing concentrations of VAN, DAP and LZD for 20 days, with eight replicates for each antibiotic conducted in parallel. The resulting day 20 (D20) isolates were subjected to antimicrobial susceptibility testing, whole genome sequencing, autolysis assays, and growth curves to determine bacterial fitness.

Results
Exposure to VAN or LZD for 20 days resulted in a subtle 2-fold increase in the MIC, whereas DAP exposure yielded DAP-non-susceptible isolates with up to 16-fold MIC increase. The MIC increase was accompanied by variable changes in relative fitness and reduced resistance to autolysis in some isolates. D20 isolates harboured mutations in genes commonly associated with resistance to the respective antibiotics (e.g. walK for VAN, mprF and rpoB for DAP, rplC for LZD), along with several previously unreported variants. Introduction of key mutations to these identified genes in the parental strain via allelic exchange confirmed their role in the development of resistance.

Conclusions
In vitro selection against VAN, DAP or LZD resulted in the acquisition of mutations similar to those correlated with clinical resistance, including the associated phenotypic alterations.