Thesis
Date
2018-11-08
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
ORCID
Type
Thesis
Degree Level
Doctoral
Abstract
Novel In Vitro Susceptibility Measurement of Vancomycin and Other Antibiotics Against Blood Isolates of Staphylococcus Aureus
Staphylococcus aureus is a versatile pathogen causing mild to moderate to severe and life threatening infections including blood stream infections. Methicillin resistant Staphylococcus aureus (MRSA) has evolved as an important nosocomial pathogen and multi-drug resistance limits the available antibiotics for therapy. In many healthcare facilities, vancomycin remains the preferred agent for treating serious MRSA infections; however, other drugs like linezolid and tedizolid are also useful agents. Tedizolid is a newly approved drug and currently only available for clinical use in some countries. Some data suggests that as the minimum inhibitory concentration (MIC) of vancomycin for MRSA increases beyond 2ug/ml, therapeutic failures become more common. We studied 60 blood culture of MRSA and determined MIC and mutant prevention concentration (MPC) values to vancomycin, linezolid and tedizolid. In separate experiments, we preformed in vitro kill studies.
The minimum inhibitory concentration (MIC) is defined as the lowest drug concentration required to inhibit or block the growth of 105 colony forming units per milliliter (CFU/ml) of bacteria and is the international standard for determining susceptibility/resistance in clinical laboratories. The mutant prevention concentration (MPC) defines the antimicrobial drug concentration blocking growth of the least susceptible cell present in high density (≥109 CFU) bacterial populations.
For vancomycin, MIC values ranged from 0.5 to 1.0ug/ml as compared to 1 – 4 µg/ml and 0.125 - 0.5 µg/ml for linezolid and tedizolid respectively. By MPC testing, vancomycin MPC values ranged from 2 to ≥32 µg/ml as compared to 2 – 4 µg/ml for linezolid and 0.25 – 0.5 µg/ml for tedizolid. Such high vancomycin MPC values have not been previously reported. To further characterize strains showing high vancomycin MPC values, we compared pulsed field gel electrophoresis (PFGE) profiles on strains with high vancomycin MPC values to rule in or out, the presence of a single clone. A single clone was not detected. We also compared PFGE profiles on select strains with high vancomycin MPC values to the profiles of the wild type (parental) strains and found the profiles were identical. Cell wall thickness of strains with high vancomycin MPC values was investigated as a possible explanation of the higher MPC values. For MRSA cells taken directly from agar plates containing high vancomycin drug concentrations, increased cell wall thickness was seen. Amplification of cell wall accessory genes by polymerase chain reaction (PCR) failed to identify a genetic marker that could explain the high MPC values and the presence or absence of the Panton-Valentine leukocidin (PVL) gene was not more frequent in strains with high vancomycin MPC values. Additionally, serial passage experiments increasing vancomycin drug concentrations did not result in a strain of MRSA with a stably resistant clone. These experiments did not identify a common characteristic that could be associated with the survival of MRSA bacterial cells in high vancomycin drug concentrations. Therefore, further investigations to determine the mechanism of this observation are necessary. MIC and MPC testing with MRSA strains tested against linezolid and tedizolid gave values consistent with expectations based on values previously generated with linezolid. For tedizolid, the MPC100 was 0.5 µg/ml. MPC values for tedizolid have not been previously reported. In vitro kill experiments were conducted using a range of bacterial densities from 106-109 CFU/ml. Previously reported kill experiments used bacterial densities approximating 105-106 CFU/ml and as such the definition of bactericidal and bacteriostatic may not be relevant when higher bacterial densities are used. For our measurements and depending on the bacterial density tested, linezolid, tedizolid and vancomycin showed both bactericidal and bacteriostatic activity against select strains of MRSA.
In summary, high vancomycin MPC values are concerning and may impact the clinical use of the drug and be responsible for clinical failure in some patients. The low MPC values for tedizolid could potentially indicate a low propensity for resistance selection with this drug.
Description
Keywords
MRSA, MPC, vancomycin
Citation
Degree
Doctor of Philosophy (Ph.D.)
Department
School of Public Health
Program
Vaccinology and Immunotherapeutics