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

Prasetyoputri, A.; Pitt, M. E.; Cao, M. D.; Ramu, S.; Kavanagh, A.; Elliott, A. G.; Ganesamoorthy, D.; Monk, I.; Stinear, T. P.; Cooper, M. A.; Coin, L. J. M.; Blaskovich, M. A. T.

doi:10.1101/2024.12.22.630013

bioRxiv · 10.1101/2024.12.22.630013

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

Prasetyoputri, A.·Pitt, M. E.·Cao, M. D.·Ramu, S.·Kavanagh, A.·Elliott, A. G.·Ganesamoorthy, D.·Monk, I.·Stinear, T. P.·Cooper, M. A.·Coin, L. J. M.·Blaskovich, M. A. T.

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Abstract

SYNOPSISO_ST_ABSBackgroundC_ST_ABSThe 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. ObjectivesOur study sought to identify the rate in which MRSA develop resistance to vancomycin, daptomycin and linezolid in vitro and further determine the mechanisms underpinning resistance. MethodsMRSA was exposed to increasing concentrations of vancomycin, daptomycin, and linezolid 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. ResultsExposure to vancomycin or linezolid for 20 days resulted in a subtle two-fold increase in the MIC, whereas daptomycin exposure yielded daptomycin-nonsusceptible 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 vancomycin, mprF and rpoB for daptomycin, rplC for linezolid), 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. ConclusionsIn vitro selection against vancomycin, daptomycin, or linezolid resulted in the acquisition of mutations similar to those correlated with clinical resistance, including the associated phenotypic alterations.

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Prasetyoputri, A., Pitt, M. E., Cao, M. D., Ramu, S., Kavanagh, A., Elliott, A. G., Ganesamoorthy, D., Monk, I., Stinear, T. P., Cooper, M. A., Coin, L. J. M., Blaskovich, M. A. T.. 2024-12-23. Characterisation of in vitro resistance selection against second-/last-line antibiotics in methicillin-resistant Staphylococcus aureus. https://doi.org/10.1101/2024.12.22.630013

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