Antistaphylococcal Activity of DX-619 Alone and in Combination with Vancomycin, Teicoplanin, and Linezolid Assessed by Time-Kill Synergy Testing

Abstract

Time-kill synergy studies testing in vitro activity of DX-619 alone and with added vancomycin, teicoplanin, or linezolid against 101 Staphylococcus aureus strains showed synergy between DX-619 and teicoplanin at 12 to 24 h in 72 strains and between DX-619 and vancomycin in 28 strains. No synergy was found with linezolid, and no antagonism was observed with any combination.

The emergence of methicillin- and quinolone-resistant staphylococci, and recently glycopeptide-intermediate staphylococci, as well as the propensity of these organisms to cause serious systemic infections in immunocompromised hosts, necessitates other therapeutic modalities (10, 12, 16, 17, 21, 23). During 2002, two clinical strains of vancomycin-resistant Staphylococcus aureus (VRSA) carrying vanA, one from Detroit, MI, and one from our hospital, were isolated. There are now a total of six VRSA strains: four from Michigan, one from Pennsylvania and one from New York (2, 3, 15, 22; M. Rybak, unpublished data).

In contrast, vancomycin-intermediate S. aureus (VISA) strains, first described in Japan by Hiramatsu (13), have been much more widely reported, and hospital colonization leading to systemic infections with these strains has recently been reported (9, 18). A recent lowering of the vancomycin susceptibility breakpoint to ≤2 μg/ml (6) will doubtless result in an even higher reported rate for these organisms, whose incidence is almost certainly under-reported. It also seems that some VISA strains have higher daptomycin MICs than is the case with vancomycin-susceptible strains (7).

The situation is further complicated by the emergence, both in the United States and elsewhere, of virulent clonal (usually USA300 or USA400) Panton-Valentine leukocidin-associated community-acquired methicillin-resistant S. aureus strains (MRSA), which primarily cause abscesses but which have on occasion produced life-threatening and lethal diseases (1, 10, 11). Whether this toxin is the major virulence determinant in community-acquired MRSA disease is not clear at the present time (24).

Most methicillin-resistant staphylococci are also resistant to available quinolones such as ciprofloxacin, levofloxacin, moxifloxacin, and gemifloxacin (10, 14, 19-21, 23). Thus, the latter compounds may not be safely used in empirical therapy of patients with methicillin-resistant staphylococcal infections. DX-619 is a new des-F(6)-quinolone with excellent activity against gram-positive organisms. In a recent study from our laboratory, DX-619 was shown to be potent and bactericidal against quinolone-susceptible and -resistant S. aureus strains, with a low propensity to develop resistance (2).

A previous preliminary study showed synergy between clinafloxacin and glycopeptides against S. aureus by time-kill methodology (5). In the present study, we used time-kill methodology to test the possible synergistic activity of DX-619 when combined with vancomycin, teicoplanin, and linezolid against 101 S. aureus strains with various phenotypes.

The bacterial strains used in this study were all nonduplicate recent clinical isolates (2002 to date) isolated at the Hershey Medical Center and Southwestern Medical Center, Dallas, TX, from wounds, blood, and sputum and identified by standard methods. The 101 organisms tested comprised (i) 50 (including 2 VISA strains) methicillin-susceptible S. aureus (MSSA) strains, 46 of which were quinolone susceptible and 4 of which were quinolone resistant and (ii) 51 MRSA strains. A total of 37 MRSA strains were also quinolone resistant and included 4 VISA and 3 VRSA strains (1 VISA strain and 1 VRSA strain were isolated at the Hershey Medical Center). Strains were stored frozen at −70°C in double-strength skim milk (Difco, Inc., Detroit, MI) until use. DX-619 powder was obtained from Daiichi Pharmaceutical Co., Ltd. (Tokyo, Japan), and other compounds were obtained from their respective manufacturers.

MICs were predetermined by macrodilution in cation-adjusted Mueller-Hinton broth (BBL Microbiology Systems, Cockeysville, MD) according to standard methodology (6). All strains were tested by time-kill methodology, with each compound alone as described previously (5). Concentrations one to two dilutions below the MIC were chosen for synergy testing. Viability counts in synergy tests were performed at 0, 3, 6, 12, and 24 h in a shaking water bath at 35°C, with final inocula between 5 × 10⁵ and 5 × 10⁶ CFU/ml. Drug carryover was addressed by dilution as described previously (5). Synergy was defined as a ≥2-log₁₀ decrease in CFU/ml between the combination and its most active component after 3, 6, 12, and 24 h, the number of surviving organisms in the presence of the combination being ≥2 log₁₀ below the starting inoculum at 0 h. At least one of the drugs had to be present at a concentration that did not significantly affect the growth curve of the test organism when used alone (5).

Compiled results are presented in Table 1, and data for individual strains is provided in the supplemental material. The MICs of the drugs alone were 0.004 to 2 μg/ml (DX-619), 0.5 to >128 μg/ml (vancomycin), 0.25 to 16 μg/ml (teicoplanin), and 1 to 4 μg/ml (linezolid). The results of synergy time-kill analyses at 12 and 24 h are presented and only for DX-619 plus vancomycin (DX-619+vancomycin) and DX-619+teicoplanin because all combinations at 3 and 6 h and all combinations with DX-619+linezolid were additive.

TABLE 1.

Combined results of MIC and time-kill synergy tests

Strain	No. of strains	MIC range (μg/ml)a			% of strainsb
		DX-619	VAN	TEI	DX-619+VAN			DX-619+TEI
					Ant	Add	Syn	Ant	Add	Syn
MSSA	50c	0.004-.06	1-4	0.25-16	0	76	24	0	30	70
MRSA Quin S	14	0.008-0.016	1-2	0.25-2	0	93	7	0	14	86
MRSA Quin R	37d	0.06-2	0.5->128	0.25-16	0	67	33	0	41	59
VISA	6e	0.016-.125	4-8	4-16	0	83	17	0	100	0
VRSA	3f	0.125-0.5	32->128	1-16	0	50	50	0	33	67

^aVAN, vancomycin; TEI, teicoplanin; Quin, quinolone.

^bThat is, the percentage of strains at the 24-h time point demonstrating the labeled effect. Ant, antagonistic; Add, additive; Syn, synergistic.

^cIncludes the two VISA strains 505 and 508. Strains 241, 242, 261, and 508 are quinolone resistant.

^dIncludes the four VISA strains 504, 506, 507, and 555 and the three VRSA strains 509, 510, and 512.

^eTwo isolates are included in the MSSA strains (505 and 508), and four strains are included in MRSA quinolone R (levofloxacin MICs of ≥4 μg/ml): strains 504, 506, 507, and 555.

^fThree isolates are included in the MRSA quinolone-resistant strains: 509, 510, and 512. Isolate 512 was not tested with the DX-619+vancomycin combination. (vancomycin MIC of >128 μg/ml).

Eight of the fifty (16%) MSSA strains showed synergy between DX-619 and vancomycin at 12 h, and twelve of fifty strains (24%), including one quinolone-resistant strain, showed synergy between DX-619 and vancomycin at 24 h. Ten of fifty MSSA strains (20%) showed synergy between DX-619 and teicoplanin at 12 h, and thirty-five of fifty MSSA strains (70%), including three quinolone-resistant strains, showed synergy between DX-619 and teicoplanin at 24 h. The two methicillin-susceptible VISA strains showed additive activity with all combinations.

One of thirty-six (3%) quinolone-resistant MRSA strains showed synergy between DX-619 and vancomycin at 12 h, and twelve of thirty-six strains (33%), including one VISA strain and one Hershey VRSA strain, showed synergy at 24 h between DX-619 and vancomycin. One of the thirty-six strains (a VRSA isolate) was not tested with DX-619 and vancomycin (vancomycin MIC of >128 μg/ml). Five of thirty-seven (13%) quinolone-resistant MRSA (including two VRSA strains, one being the Hershey strain) showed synergy between DX-619 and teicoplanin at 12 h, and twenty-two of thirty-seven quinolone-resistant MRSA strains (59%), including the two previously mentioned VRSA strains, showed synergy between DX-619 and teicoplanin at 24 h. The Hershey VISA strain did not show synergy.

One of fourteen quinolone-susceptible MRSA strains (7%) showed synergy between DX-619 and vancomycin after 24 h. One of fourteen (7%) quinolone-susceptible MRSA strains showed synergy between DX-619 and teicoplanin at 12 h, and twelve of fourteen (86%) quinolone-susceptible MRSA strains showed synergy between DX-619 and teicoplanin at 24 h. The synergy between DX-619 with both vancomycin and teicoplanin is depicted graphically in Fig. 1.

FIG. 1.

Time-kill graphs. (A) DX-619/vancomycin quinolone-resistant MRSA strain 019; (B) DX-619/teicoplanin-quinolone-resistant MRSA strain 019; (C) DX-619/vancomycin-VRSA strain 510; (D) DX-619/teicoplanin-VRSA strain 510.

Additive results were obtained with the five VISA strains and one VRSA strain tested with all combinations at all time points. All other combinations (including all between DX-619 and linezolid) were additive, and no antagonism with any combination was observed. The bacteriostatic nature of linezolid is well known (4, 8).

The results of this study confirm the potency of DX-619 against all S. aureus strains tested irrespective of phenotype (2). DX-619 and teicoplanin were synergistic at sub-MICs of DX-619 in 69 of the 101 strains (68%) after 24 h. These strains comprised 35 MSSA strains; of these 3 were quinolone resistant, 12 were MRSA and quinolone susceptible, and 22 were MRSA and quinolone resistant (including 2 VRSA) strains.

In contrast, only 25 of 100 strains (25%) showed synergy between DX-619 and vancomycin after 24 h. We have no explanation for why DX-619 was more synergistic in combination with teicoplanin than in combination with vancomycin. An examination of the data in the supplemental material did not reveal any obvious correlation between strains showing synergy between DX-619 and one or both glycopeptides. Although clear in vitro synergy between DX-619 and teicoplanin was seen in most strains studied, antimicrobial interactions are known to be strain dependent, and generalizations based upon our results should be avoided, at least for the moment.

In summary, combinations between DX-619 and teicoplanin and (in some cases) vancomycin showed in vitro synergy, as determined by time-kill analyses, against S. aureus strains of all resistotypes. The significance of these findings requires clinical confirmation.