Understanding vancomycin tolerance in hospital-acquired methicillin-resistant Staphylococcus aureus isolates from patients with sustained bacteraemia

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of healthcare-associated infections including bacteraemia. Vancomycin has traditionally been the antibiotic of choice for treatment, but despite using the minimum inhibitory concentration (MIC) to guide treatment management, some patients still fail antibiotic therapy even when infected with strains that test as susceptible. This is further exacerbated with the emergence of vancomycin intermediate S. aureus (VISA) and heterogeneous vancomycin intermediate S. aureus (hVISA) strains, which exhibit reduced susceptibility to vancomycin. When exposed to sub-lethal concentrations of vancomycin, these VISA and hVISA subtypes are believed to undergo cell wall metabolism changes which increases the thickness of their cell walls and this form of resistance has been associated with sustained bacteraemia and increased mortality. Antibiotic tolerance is a phenotypic trait where an organism is resistant to the lethal killing of an antibiotic despite being inhibited by normal concentrations, and may explain the poorer outcome seen in patients with sustained bacteraemia. In vitro this interaction between an antibiotic concentration and bacterial growth or killing can be measured by the MIC (antibiotic concentration required to inhibit bacterial growth) and the minimum bactericidal concentration (MBC; that antibiotic concentration required to kill the bacterium) respectively. Using these two measures, tolerance is defined when the ratio of an isolates measured MBC to MIC is >32 after 24 hrs of incubation. Despite guidelines for MBC testing being issued in 1999, there is ongoing variation in the methodology used among testing laboratories, and the disparity in reported tolerance rates among S. aureus strains has lead clinicians to question the utility of MBC testing. Although there is conflicting data on the clinical significance of vancomycin-tolerant staphylococci, evidence suggests that tolerance may be an independent risk factor for poorer outcome, and the close association with hVISA and VISA strains may provide an understanding of the mechanisms that drive vancomycin resistance. This study investigated whether variations in the methodology used in MBC and time-kill tests impacted the ability to detect antibiotic tolerance. This was achieved determining the effect of using different media and test conditions on tolerance rates seen in a collection of hVISA and VISA isolates from an ST239 dominant MRSA population. The results from this study demonstrate that MBC results vary between tests and that the detection of antibiotic tolerance is highly dependent on testing conditions. Time-kill assays are the recommended method for detecting tolerance, and if MBC testing were to be performed, then it should be performed after isolates have undergone an in vitro vancomycin pre-exposure step. To better understand antibiotic tolerance and the mechanisms that drive vancomycin reduced susceptibility, MBC testing must be better standardised, and furthermore, the findings from this thesis propose an enhancement to current testing methodology which can be used in future large-scale studies to determine the clinical relevance of antibiotic tolerance.

Date of Award	2017
Original language	English