Cefazolin and Ertapenem, a Synergistic Combination Used To Clear Persistent Staphylococcus aureus Bacteremia

Abstract

Ertapenem and cefazolin were used in combination to successfully clear refractory methicillin-susceptible Staphylococcus aureus (MSSA) bacteremia. In addition, recent work has demonstrated activity of combination therapy with beta-lactams from different classes against methicillin-resistant S. aureus (MRSA). The ertapenem-plus-cefazolin combination was evaluated for synergy in vitro and in vivo in a murine skin infection model using an index MSSA bloodstream isolate from a patient in whom persistent bacteremia was cleared with this combination and against a cadre of well-described research strains and clinical strains of MSSA and MRSA. Against the index MSSA bloodstream isolate, ertapenem and cefazolin showed synergy using both checkerboard (fractional inhibitory concentration [FIC] index = 0.375) and time-kill assays. Using a disk diffusion ertapenem potentiation assay, the MSSA isolate showed a cefazolin disk zone increased from 34 to 40 mm. In vitro pharmacokinetic/pharmacodynamic modeling at clinically relevant drug concentrations demonstrated bactericidal activity (>3 log₁₀-CFU/ml reduction) of the combination but bacteriostatic activity of ether drug alone at 48 h. A disk diffusion potentiation assay showed that ertapenem increased the cefazolin zone of inhibition by >3 mm for 34/35 (97%) MSSA and 10/15 (67%) MRSA strains. A murine skin infection model of MSSA showed enhanced activity of cefazolin plus ertapenem compared to monotherapy with these agents. After successful use in clearance of MSSA bacteremia, the combination of ertapenem and cefazolin showed synergy against MSSA in vitro and in vivo This combination may warrant consideration for future clinical study in MSSA bacteremia.

FIG 1

(A) Kill curves of the index MSSA isolate, rus276, employing 1/2 MIC of CZ or ETP alone or in combination. (B) Kill curve assays of MRSA MW2 containing ≤1/4 MIC of cefazolin, ertapenem, or both drugs. (C) Results of checkerboard analysis of susceptibilities to cefazolin MICs in media containing various concentrations of ertapenem for MRSA strains MW2, TCH 1516, and Sanger 252. The error bars indicate standard deviations.

FIG 2

(Left) Microscopy comparing the effects of CZ or ETP alone and in combination on MSSA rus276 at sub-MICs. Sytox Green and DAPI stain uptake was used to measure cellular permeabilization. (Right) Quantification of DAPI and Sytox staining, reflective of increases in cellular permeability with combined ETP and CZ compared to either drug alone at concentrations relative to the MIC.

FIG 3

CZ population analyses performed on MSSA rus276 under low (10⁶ CFU/ml) and high (10⁹ CFU/ml) inocula using Mueller-Hinton II (MH) or BHI agar containing 1/2 and 1/4 MIC ETP. ETP consistently reduced CZ heteroresistance, particularly under high-inoculum conditions.

FIG 4

(A) Kill curve assays of MSSA rus276 using human cathelicidin LL-37, CZ, or ETP alone or in combination. (B) Neutrophil killing assays of MSSA rus276 after growth in antibiotic-free medium (control) or media containing CZ, ETP, or both drugs. The results are expressed as percentages of bacterial survival remaining after 90 min compared to the starting bacterial density (*, P < 0.05 versus the control). The error bars indicate standard deviations.

FIG 5

(A) Simulated pharmacokinetic/pharmacodynamic modeling of MSSA rus276 employing CZ and ETP alone or in combination at clinically relevant concentrations. Only the combination regimen of ETP plus CZ achieved bactericidal activity at 24 and 48 h, and CZ plus ETP showed significantly lower CFU at 48 h than the other groups (P < 0.01; one-way ANOVA with Tukey's post hoc test). The error bars indicate standard deviations. (B) Bacterial counts in excised skin tissue of mice treated with CZ, ETP, or both drugs versus no antibiotics (control). The horizontal bars indicate the medians of the groups. Only the combination treatment group (CZ+ETP) showed bacterial counts less than those with the control (*, P < 0.02; Mann-Whitney U test). Control versus CZ (P = 0.17) and control versus ETP (P = 0.87) were not statistically significant.

FIG 6

Changes in cefazolin disk diffusion zone size with cefazolin replaced at 1 h by ertapenem versus cefazolin alone for clinical bloodstream isolates of MSSA (A) or MRSA (B).