Electron microscopy studies of the bactericidal effects of quinupristin/dalfopristin on Staphylococcus aureus

Sir, Quinupristin/dalfopristin is a combination of two semisynthetic derivatives of pristinamycin which has been shown to have activity in vitro against a wide range of Gram-positive cocci. Preliminary studies in our laboratory of its activity against staphylococci demonstrated that, following exposure to concentrations ,MICs, the bacterial cells appeared larger and stained more intensely than controls. A further study of bacterial ultrastructure revealed that incubation of Staphylococcus aureus in the presence of quinupristin/dalfopristin resulted in two major cell alterations: increases in cell size and the thickness of the cell wall; the greatest increase in cell size was observed after exposure to a concentration equivalent to 0.5 3 MIC (P , 0.005). Electron micrographs taken after 24 h exposure to the combination at this concentration showed breaks in the walls of several cells and ghosts of lysed cells, in addition to larger cells. Our earlier study was repeated with the aim of performing viable counts and quantifying breaks in the cell wall following incubation of a strain of S. aureus in the presence of 0.5 3 MIC of quinupristin/ dalfopristin. Quinupristin/dalfopristin was provided by RhônePoulenc Rorer (Vitry sur Seine, France) and S. aureus ATCC 25923 was obtained from Difco (Detroit, MI, USA). The S. aureus strain was incubated without quinupristin/ dalfopristin (control) and in the presence of the antibiotic at a concentration of 0.2 mg/L (equivalent to 0.5 3 MIC) as described previously. Aliquots were withdrawn after incubation at 36oC for 6, 12 and 24 h and the numbers of viable bacteria were determined. Samples of the 24 h culture were also examined by electron microscopy according to a method described by Lorian et al. The extent of the cell-wall alterations was assessed by examining photographs of fields containing 30–40 cells at 321,000 magnification. Following exposure of S. aureus to quinupristin/ dalfopristin for 6 h and 12 h, the numbers of viable bacteria fell by 99% and 99.9%, respectively, compared with the control; after 24 h, the viable count increased by 0.5 log10. In a previous study, incubation of some strains of Entero coccus faecium in the presence of the combination at a concentration equivalent to 0.3 3 MIC was associated with 99.9% decreases in the numbers of viable organisms. The apparent paradox that exposure to concentrations ,MICs produced marked bactericidal activity can be accounted for by the technique used to determine the MICs, which is based on measurements of turbidity—i.e. in common with live bacteria, dead cells produce turbidity—whereas the method of quantifying bactericidal activity determines the numbers of viable bacteria only. The Figure shows profound structural alterations, breaks in the cell walls, extrusion of cytoplasm, and cellwall ghosts in 10–20% of cells. Quinupristin/dalfopristin inhibits protein synthesis as a result of its effects on bacterial ribosomes. However, unlike other antibiotics that bind reversibly to the ribosomes, the combination binds irreversibly, thereby exerting a bactericidal effect on susceptible strains of S. aureus. The ultrastructural alterations observed following exposure to quinupristin/dalfopristin for 24 h may be lysis following death and may, as such, represent a visual record of the bactericidal effects of the drug.