Characterisation of resistance selection against second-/last-line antibiotics in methicillin-resistant 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 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. for VAN, and for DAP, 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

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