Identification of Markers of Antibiotic Resistance in Daptomycin Non-Susceptible Strains of Staphylococcus aureus
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Issue Date
2014
Authors
Shah, Riddhi Shantilal '14
Degree
MS in Pharmaceutical Sciences
Advisor
Malik, Meenakshi
Committee Members
Voigt, Jeffrey M.
Nicasio, Anthony
Pai, Amit
Nicasio, Anthony
Pai, Amit
Journal Title
Journal ISSN
Volume Title
Abstract
Methicillin resistant Staphylococcus aureus (MRSA) is a major health care concern worldwide and is responsible for a variety of nosocomial infections. Antibiotic resistance in MRSA has posed a major threat in recent years resulting in treatment failures. Daptomycin is often used to treat MRSA infections, but recent reports indicate an increasing incidence of resistance to this antimicrobial agent. A combination therapy involving the use of an anti-staphylococcal β-lactam antibiotic plus daptomycin has been explored to circumvent this problem.
We investigated the putative genetic determinants corresponding to the daptomycin non-susceptible (DNSA) and susceptible (DSSA) Staphylococcus aureus phenotypes using an isogenic pair of strains in response to daptomycin alone or a combination antibiotic therapy. First, we compared the expression of cell membrane charge, cell wall synthesis, autolysis and penicillin binding protein (PBP) genes in DNSA and DSSA strains treated with daptomycin and ceftaroline individually or in combination. In drug-free conditions, DNSA strain showed an upregulation of genes responsible for cell membrane charge (mprF, dltABCD) and cell-wall synthesis (VraS and femB) as compared to the DSSA strain; whereas (atl and lytM) showed upregulation in DSSA compared to DNSA strain. A combination therapy using daptomycin and ceftaroline synergistically down-regulated these genes in DNSA and DSSA strains respectively. Transcriptional analysis of PBPs revealed an upregulation of pbp2a gene in DNSA strain which was downregulated when combination therapy was used.
Next, we investigated if alterations in gene expression involved in antibiotic resistance are due to genetic mutations in the DNSA strains and if altered gene expression in DNSA following Ceftaroline+Daptomycin combination treatment is due to reversion of the genetic mutations that DNSA strain has acquired over a period of time. We found four mutations in mprF gene of DNSA strain when compared with DSSA or standard Mu50 strain of S. aureus. However, monotherapy or combination therapy did not reverse these mutations in the mprF gene. Collectively, these results demonstrate that gene expression profiles associated with the development of daptomycin non-susceptibility may serve as important markers in differentiation of DNSA and DSSA strains and combination of daptomycin and ceftaroline may have potential for use as therapy against DNSA strains.
We investigated the putative genetic determinants corresponding to the daptomycin non-susceptible (DNSA) and susceptible (DSSA) Staphylococcus aureus phenotypes using an isogenic pair of strains in response to daptomycin alone or a combination antibiotic therapy. First, we compared the expression of cell membrane charge, cell wall synthesis, autolysis and penicillin binding protein (PBP) genes in DNSA and DSSA strains treated with daptomycin and ceftaroline individually or in combination. In drug-free conditions, DNSA strain showed an upregulation of genes responsible for cell membrane charge (mprF, dltABCD) and cell-wall synthesis (VraS and femB) as compared to the DSSA strain; whereas (atl and lytM) showed upregulation in DSSA compared to DNSA strain. A combination therapy using daptomycin and ceftaroline synergistically down-regulated these genes in DNSA and DSSA strains respectively. Transcriptional analysis of PBPs revealed an upregulation of pbp2a gene in DNSA strain which was downregulated when combination therapy was used.
Next, we investigated if alterations in gene expression involved in antibiotic resistance are due to genetic mutations in the DNSA strains and if altered gene expression in DNSA following Ceftaroline+Daptomycin combination treatment is due to reversion of the genetic mutations that DNSA strain has acquired over a period of time. We found four mutations in mprF gene of DNSA strain when compared with DSSA or standard Mu50 strain of S. aureus. However, monotherapy or combination therapy did not reverse these mutations in the mprF gene. Collectively, these results demonstrate that gene expression profiles associated with the development of daptomycin non-susceptibility may serve as important markers in differentiation of DNSA and DSSA strains and combination of daptomycin and ceftaroline may have potential for use as therapy against DNSA strains.
Citation
Shah RS. Identification of markers of antibiotic resistance in Daptomycin mon-susceptible strains of Staphylococcus Aureus [thesis]. Ann Arbor (MI): Proquest LLC; 2014. 86 p.
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