Comparative Efficacies of Cloxacillin-Daptomycin and the Standard Cloxacillin-Rifampin Therapies against an Experimental Foreign-Body Infection by Methicillin-Susceptible Staphylococcus aureus

Abstract

We compared the efficacies of daptomycin (doses equivalent to 8 to 10 mg/kg of body weight/day in humans) and cloxacillin alone with those of cloxacillin-rifampin and cloxacillin-daptomycin combinations, using a tissue cage methicillin-susceptible Staphylococcus aureus (MSSA) infection model. Monotherapies were less effective than combinations (P < 0.05), and daptomycin resistance emerged. Cloxacillin-daptomycin proved as effective as cloxacillin-rifampin and prevented the appearance of resistance; this combination may be an alternative anti-MSSA therapy, which may offer greater benefits in the early treatment of prosthetic joint infections (PJI).

TEXT

Current guidelines for the treatment of prosthetic joint infections (PJI) caused by methicillin-susceptible Staphylococcus aureus (MSSA) recommend intravenous cloxacillin plus rifampin during the first weeks (1), but the suboptimal efficacy of beta-lactams against high bacterial inocula and the in vitro antagonistic effect of the combination are issues of concern (2–5).

The use of daptomycin at high doses (HD; 8 to 10 mg/kg of body weight/day) in combination against difficult-to-treat methicillin-resistant S. aureus (MRSA) infections is encouraged (6). In particular, daptomycin-cloxacillin has proved efficacy in the treatment of persistent bacteremia caused by MRSA (7, 8) but only modest effectiveness against experimental foreign-body infection (FBI) (9). Taking into account the inherent anti-MSSA killing by cloxacillin, we aimed to study the in vivo efficacy of the combination cloxacillin-daptomycin at high doses, using a rat tissue cage MSSA infection model, and to compare its efficacy with that of the standard treatment with cloxacillin-rifampin.

MSSA strain ATCC 29213 was used for in vitro and in vivo studies. The MICs, minimal bactericidal concentrations (MBCs), and time-kill curves were determined in the log phase (inocula of 10⁵ CFU/ml and 10⁸ CFU/ml) and in the stationary phase, using the methodology described elsewhere (10–13).

Cloxacillin showed bactericidal activity only in the log phase with standard inocula (10⁵ CFU/ml); in accordance with previous findings, the addition of rifampin to bactericidal concentrations of cloxacillin produced an antagonistic effect (Table 1) (2, 4, 14). The addition of daptomycin to cloxacillin finally achieved a bactericidal effect under all study conditions, which required daptomycin concentrations of 1× and 8× MIC with the use of standard inocula (log phase) and high inocula (log phase), respectively, and of 64× MIC (the MBC of daptomycin) with the use of bacteria in the stationary phase (Fig. 1). The bactericidal and synergistic antistaphylococcal activity of the daptomycin-cloxacillin combination against MRSA strains has been documented (7, 8, 15), but little is known about the anti-MSSA efficacy of this combination.

TABLE 1.

Log changes in viable cell counts obtained with various concentrations of cloxacillin alone and in combination with rifampin against standard inocula (10⁵ CFU/ml) of MSSA strain ATCC 29213 in the log phase^a

Time point (h)	Log change [log(CFUt) − log(CFU0)] for:
	CXA (1)	CXA (2)	RIF (0.12)	CXA (1) + RIF (0.12)	CXA (1) + RIF (8)	CXA (2) + RIF (0.12)	CXA (2) + RIF (8)
6	−1.2	−2.5	−1.8	−1.2	−1.3	−1.3	−1.2
24	−3.1	−3.6	−3.3	−1.6	−1.4	−1.8	−1.1

^aExperiments were performed with three replicates. CFU_t, number of CFU at the given time point; CFU₀, number of CFU at time zero; CXA, cloxacillin (purity, 98.1 %; Sigma-Aldrich, Madrid, Spain); RIF, rifampin (purity, 98%; Sigma-Aldrich, Madrid, Spain). Numbers in parentheses represent concentrations in mg/liter.

FIG 1.

Results of time-kill curves in the log phase, with standard (A) and high (B) bacterial inocula, and in the stationary phase (C) for different concentrations of cloxacillin alone and in combination with daptomycin. Concentrations are in mg/liter. CXA, cloxacillin (purity, 98.1%; Sigma-Aldrich, Madrid, Spain); DAP, daptomycin (purity, 100%; Novartis, Basel, Switzerland).

The rat tissue cage infection model (approved by the Ethics Committee for Animal Experiments at the University of Barcelona) had previously been standardized by our group (10, 16). Briefly, the methodology used consisted of subcutaneous implantation in Wistar rats of two Teflon tissue cages with two polymethylmethacrylate coverslips (CV). After 3 weeks, the tissue cage fluid (TCF) was percutaneously infected with 0.1 ml of an MSSA preparation (0.2 × 10⁶ to 2 × 10⁶ CFU/ml). One week later (designated day 1), TCF was obtained to quantify bacterial counts; therapy was then started and administered intraperitoneally for 7 days. One and 4 days after the end of treatment (days 8 and 11, respectively), TCF was again recovered to count bacteria. On day 11, animals were sacrificed, and CV were removed and processed as described previously (10) to quantify adherent bacteria and the infection cure rate (the percentage of samples with bacterial counts under the limit of detection with respect to the total number of samples). The methodology for pharmacokinetic (PK) studies and the PK-pharmacodynamic (PD) parameter values for cloxacillin (200 mg/kg/12 h) and rifampin (25 mg/kg/12 h) have been previously described (10). For the particular case of daptomycin, we used a high-performance liquid chromatography (HPLC) assay (17). Samples, calibrators, or quality controls (100 μl) were mixed with acetonitrile (100 μl) and centrifuged at 12,000 × g for 5 min. The method was successfully validated, and the results were linear, over the concentration range studied (5 to 250 μg/ml). The analytical results for the calibration standards fulfilled all of the acceptance criteria. The PK parameter values for a rat dosage of 45 mg/kg/day of daptomycin in sera and TCF were as follows: peak concentrations, 118 and 41 mg/liter, respectively; through concentrations, <5 and 17 mg/liter, respectively; and areas under the concentration-time curve from 0 h to infinity (AUC_0-∞), 800 and 740 μg·h/ml, respectively, which were equivalent to the values obtained with high doses of 8 to 10 mg/kg/day in humans.

A total of 65 animals were used (130 tissue cages); as some tissue cages were lost due to spontaneous shedding, 122 cages remained at the beginning of the experiments (day 1), with no significant differences between therapeutic groups. The decreases in the bacterial counts from the TCF (days 8 and 11) and the adherent bacterial counts from the CV are shown in Fig. 2. The cure rates on day 11 for the TCF and CV groups were 10.5% and 36.8%, respectively, for cloxacillin-daptomycin and 10.5% and 26.3%, respectively, for cloxacillin-rifampin (no significant differences). There was no cure rate in any other group.

FIG 2.

Decreases in counts of bacteria recovered from TCF at day 8 and day 11 (A) and counts of bacteria recovered from CV at day 11 (B). Bacterial counts are expressed in mean log numbers of CFU/ml (± standard deviation [SD]). *, therapeutic groups achieved higher efficacy than the control (P < 0.05); #, therapeutic groups achieved greater efficacy than the control and monotherapies (P < 0.05); n, number of tissue cages; D8, day 8; D11, day 11; CV, coverslips; CXA, cloxacillin; DAP, daptomycin; RIF, rifampin.

Resistant strains from TCF and CV were screened with agar plates containing 1 μg/ml cloxacillin, rifampin, or daptomycin (supplemented with 50 mg/liter calcium chloride dihydrate), as described previously (10, 16); resistant strains were recovered to determine MICs (11). Heteroresistance to daptomycin was studied by the screening of subpopulations able to grow in the presence of ≥0.5, 1, 2, and 4 mg/liter daptomycin in population analysis profiles (PAPs; determined as described previously) (16). There was emergence of daptomycin and rifampin resistance only when these therapies were used alone; combinations with cloxacillin protected against the appearance of resistance (Table 2).

TABLE 2.

Results for screening of bacteria recovered from TCF and CV for the presence of resistant strains at the end of therapy (day 11)

Treatment group (no. of samples)a	No. of samples with resistant strains/total no. of samples (%)c from:		MIC (mg/liter) of resistant strains isolated
	TCF	CV
Control (21)b	0	0
CXA (20)	0	0
DAP (22)	35	35	2–3
RIF (19)	88	88	2–4
CXA + DAP (20)b	0	0
CXA + RIF (20)	0	0

^aCXA, cloxacillin; DAP, daptomycin; RIF, rifampin.

^bWe did not detect subpopulations of daptomycin with MICs of ≥0.5 mg/liter among the wild-type strain (ATCC 29213) or among strains recovered ex vivo from the control or daptomycin-cloxacillin group.

^cScreening of resistant strains recovered from TCF and CV was performed using the methodology previously described (10,16), with agar plates containing 1 μg/ml cloxacillin, rifampin, or daptomycin. Results were interpreted as positive (some macroscopic growth) or negative (no growth).

To our knowledge, there are no previous data regarding the efficacy of daptomycin alone and in combination with cloxacillin against FBI caused by MSSA. We confirmed that cloxacillin or daptomycin monotherapies were less active than combinations. The activity of cloxacillin alone against experimental FBI caused by MSSA has previously been reported as moderate (10, 18). In contrast, the little previous information on the anti-MSSA activity of daptomycin is limited to the field of experimental endocarditis, in which daptomycin was reported as highly efficacious, even more so than nafcillin (19, 20). In the present work, we used a rat dosage of daptomycin equivalent to high doses in humans (8 to 10 mg/kg/day), but its efficacy was slight and did not protect against the emergence of resistance. In vivo development of daptomycin resistance is currently a matter of concern, which has been widely reported in the setting of MRSA infections, even with no exposition to antibiotics (21), but this result was also found in an experimental model of endocarditis caused by MSSA (20, 22).

The benefits of the cloxacillin-daptomycin combination against MRSA strains have been stressed either in terms of efficacy (7, 8) or in terms of prevention of resistance (23); in fact, oxacillin was the most effective antibiotic in preventing daptomycin resistance over time in a recent in vitro study (23). Given the inherent anti-MSSA killing by cloxacillin, this combination could provide even higher efficacy in these infections. Our data showed that cloxacillin-daptomycin therapy was as effective as the cloxacillin-rifampin combination and had a safety profile of protection against resistance; overall, these data supported the idea that this combination may be considered an intravenous alternative anti-MSSA treatment. Our study has the limitation of having used only one MSSA strain, which was not isolated from a difficult-to-treat-infection; thus, the generalization of these findings to routine clinical practice should be made with caution.

In conclusion, cloxacillin and daptomycin alone showed lower efficacy than the combined therapies against FBI caused by MSSA, and daptomycin resistance emerged. The combination cloxacillin-daptomycin at high doses proved as effective as the standard reference of cloxacillin-rifampin treatment and prevented the appearance of resistance. Our results support the idea that the cloxacillin-daptomycin combination can be considered an alternative anti-MSSA therapy for FBI, which may offer greater benefits in the early treatment of these infections.