Mapping the genetic evolution of daptomycin non-susceptibility in methicillin-resistant Staphylococcus aureus (MRSA)
Loading...
Issue Date
2019-07
Authors
Mishra, Smruti '19
Degree
MS in Molecular Biosciences
Advisor
Malik, Meenakshi
Committee Members
Parent, Michelle
O'Donnell, Nicholas
Shakerley, Nicole
O'Donnell, Nicholas
Shakerley, Nicole
Journal Title
Journal ISSN
Volume Title
Abstract
Over the past decade, there has been increased use of daptomycin for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections, which has led to the emergence of daptomycin non-susceptible (DAP-NS) MRSA strains. The mechanism of daptomycin resistance is a complex/multifactorial process which is still not fully understood. Past studies have shown a correlation between daptomycin resistance and genetic mutations associated with alteration in the cell membrane and cell wall phenotypes. However, these studies have not been able to identify a hierarchy of evolutionary genetic changes that lead to daptomycin resistance. In this study, a novel continuous culture bioreactor model was used to study the effects of incremental doses of daptomycin on Staphylococcus aureus (S. aureus) strain N315. The bacteria were grown for 24 hours, following which increasing concentrations of daptomycin were added. Samples were collected at various time points to determine the bacterial densities and minimum inhibitory concentrations (MICs). The bioreactor-derived strains were whole-genome sequenced to map the acquisition of daptomycin resistance. Mutations were found in genes encoding for cell membrane charge and composition in daptomycin-non-susceptible strains. Additional mutations in genes for metabolic functions were also seen, but the acquisition of resistance was primarily dependent on mutations at a few key loci. Further, these mutations that resulted in daptomycin resistance were stable and did not revert back even after eight days of growth in the bioreactor without the selective pressure of daptomycin. We also investigated the molecular basis of the seesaw effect by testing the effect of oxacillin on DAP-NS strains. It was found that oxacillin was able to reduce the frequencies of certain key mutations responsible for daptomycin resistance. RNA-Seq analysis was also employed to investigate the alterations in gene expression profiles of daptomycin treated strains after addition of oxacillin. To conclude, our study successfully demonstrates the use of a novel in vitro bioreactor model to track the evolutionary pathways for the development of antibiotic resistance by simulating in vivo conditions. The knowledge gained from these studies will provide critical insights into the mechanism of daptomycin resistance and will enable the design and development of new strategies that can counteract the problem of antibiotic resistance.
Citation
Mishra S. Mapping the genetic evolution of daptomycin non-susceptibility in methicillin-resistant staphylococcus aureus (MRSA) [thesis]. Ann Arbor (MI): Proquest LLC; 2019. 85 p.
ACPHS Research Commons URI
Description
Click on the Resource Link to find this item in the ACPHS Library catalog.