Quinupristin-dalfopristin combined with beta-lactams for treatment of experimental endocarditis due to Staphylococcus aureus constitutively resistant to macrolide-lincosamide-streptogramin B antibiotics

Abstract

Quinupristin-dalfopristin (Q-D) is an injectable streptogramin active against most gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). In experimental endocarditis, however, Q-D was less efficacious against MRSA isolates constitutively resistant to macrolide-lincosamide-streptogram B (C-MLS(B)) than against MLS(B)-susceptible isolates. To circumvent this problem, we used the checkerboard method to screen drug combinations that would increase the efficacy of Q-D against such bacteria. beta-Lactams consistently exhibited additive or synergistic activity with Q-D. Glycopeptides, quinolones, and aminoglycosides were indifferent. No drugs were antagonistic. The positive Q-D-beta-lactam interaction was independent of MLS(B) or beta-lactam resistance. Moreover, addition of Q-D at one-fourth the MIC to flucloxacillin-containing plates decreased the flucloxacillin MIC for MRSA from 500 to 1,000 mg/liter to 30 to 60 mg/liter. Yet, Q-D-beta-lactam combinations were not synergistic in bactericidal tests. Rats with aortic vegetations were infected with two C-MLS(B)-resistant MRSA isolates (isolates AW7 and P8) and were treated for 3 or 5 days with drug dosages simulating the following treatments in humans: (i) Q-D at 7 mg/kg two times a day (b.i.d.) (a relatively low dosage purposely used to help detect positive drug interactions), (ii) cefamandole at constant levels in serum of 30 mg/liter, (iii) cefepime at 2 g b.i.d., (iv) Q-D combined with either cefamandole or cefepime. Any of the drugs used alone resulted in treatment failure. In contrast, Q-D plus either cefamandole or cefepime significantly decreased valve infection compared to the levels of infection for both untreated controls and those that received monotherapy (P < 0.05). Importantly, Q-D prevented the growth of highly beta-lactam-resistant MRSA in vivo. The mechanism of this beneficial drug interaction is unknown. However, Q-D-beta-lactam combinations might be useful for the treatment of complicated infections caused by multiple organisms, including MRSA.

FIG. 1

FIC indices for Q-D (Synercid) combined with a variety of antibiotics as determined by the checkerboard method. Median (range) values are indicated for each combination tested against both MLS_B-susceptible (four MSSA and five MRSA isolates; open bars) and C-MLS_B-resistant (four MSSA and five MRSA isolates; plain bars) clinical isolates of S. aureus. The vertical line at 0.5 indicates the limit for synergism (FIC index, ≤0.5). The vertical lines between 0.5 and 1 delineate the area of addition (FIC index, >0.5 but ≤1). The area above 1 indicates indifference (FIC index, >1 but ≤4) (7). No antagonism between Q-D and any of the other drug tested was observed. ND, not determined.

FIG. 2

Population analysis profiles of MRSA AW7 (A and B) and MRSA P8 (C and D) grown on agar plates containing increasing concentrations of either flucloxacillin (A and C) or cefepime (B and D). Large numbers of bacteria (ca. 10⁹ CFU) were spread onto plates supplemented with increasing concentrations of either flucloxacillin or cefepime used alone (triangles) or in combination with a constant subinhibitory concentration of Q-D of 0.125 mg/liter (equal to one-fourth the MIC) (squares). The plates were incubated for 48 h at 35°C before the colonies were counted. The number of colonies was plotted as a function of the β-lactam concentrations present in the plates.

FIG. 3

Time-kill experiments with MRSA AW7 (A and B) and MRSA P8 (C and D) exposed to concentrations of Q-D and/or cefepime that mimic either trough antibiotic levels (A and C) or peak antibiotic levels (B and D) obtained during i.v. treatment in humans or rats. Cultures received either no drug (diamonds), Q-D alone (squares), cefepime alone (triangles), or Q-D and cefepime combined (open circles). Trough concentrations (A and C) of Q-D and cefepime were 0.5 mg/liter (1× the MIC) and 5 mg/liter (1/12× the MIC), respectively. Peak concentrations (B and D) of Q-D and cefepime were 5 mg/liter (20× the MIC) and 160 mg/liter (2.5× the MIC), respectively. Antibiotics were added to the cultures at time zero. At various times before and after antibiotic addition, samples were removed from the cultures, diluted, and plated for colony count determinations, with adequate measures taken to avoid antibiotic carryover (see Materials and Methods). Each dot represents the mean of at least three separate experiments.

FIG. 4

Treatment of experimental endocarditis due to MRSA AW7 with Q-D (Synercid) and cefamandole used alone or in combination. Each dot indicates the bacterial density in the vegetations of independent animals. The horizontal bars in the columns indicate the median values for the group. Treatment groups and treatment duration are indicated at the top of the graph. Control animals were killed at treatment onset, i.e., 12 h after inoculation, in order to determine the frequency and severity of valve infection. Treatment duration was either 3 days (3 d) or 5 days (5 d). A P value of <0.05 indicates that the difference between the groups was statistically significant.

FIG. 5

Treatment of experimental endocarditis due to MRSA AW7 or MRSA P8 with Q-D (Synercid) and cefepime used alone or in combination. Each dot indicates the bacterial density in the vegetations of independent animals. The horizontal bars in the columns indicate the median values for the group. Control animals were killed at treatment onset, i.e., 12 h after inoculation, in order to determine the frequency and severity of valve infection. Animals receiving antibiotics were treated for a total of 5 days. A P value of <0.05 indicates that the difference between the groups was statistically significant.