Telavancin in Therapy of Experimental Aortic Valve Endocarditis in Rabbits Due to Daptomycin-Nonsusceptible Methicillin-Resistant Staphylococcus aureus

Abstract

A number of cases of both methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) strains that have developed daptomycin resistance (DAP-R) have been reported. Telavancin (TLV) is a lipoglycopeptide agent with a dual mechanism of activity (cell wall synthesis inhibition plus depolarization of the bacterial cell membrane). Five recent daptomycin-susceptible (DAP-S)/DAP-R MRSA isogenic strain pairs were evaluated for in vitro TLV susceptibility. All five DAP-R strains (DAP MICs ranging from 2 to 4 μg/ml) were susceptible to TLV (MICs of ≤0.38 μg/ml). In vitro time-kill analyses also revealed that several TLV concentrations (1-, 2-, and 4-fold MICs) caused rapid killing against the DAP-R strains. Moreover, for 3 of 5 DAP-R strains (REF2145, A215, and B_2.0), supra-MICs of TLV were effective at preventing regrowth at 24 h of incubation. Further, the combination of TLV plus oxacillin (at 0.25× or 0.50× MIC for each agent) increased killing of DAP-R MRSA strains REF2145 and A215 at 24 h (∼2-log and 5-log reductions versus TLV and oxacillin alone, respectively). Finally, using a rabbit model of aortic valve endocarditis caused by DAP-R strain REF2145, TLV therapy produced a mean reduction of >4.5 log₁₀ CFU/g in vegetations, kidneys, and spleen compared to untreated or DAP-treated rabbits. Moreover, TLV-treated rabbits had a significantly higher percentage of sterile tissue cultures (87% in vegetations and 100% in kidney and spleen) than all other treatment groups (P < 0.0001). Together, these results demonstrate that TLV has potent bactericidal activity in vitro and in vivo against DAP-R MRSA isolates.

INTRODUCTION

Daptomycin (DAP) was approved by the FDA in 2003 for the treatment of skin and soft tissue Staphylococcus aureus infections (including methicillin-resistant S. aureus [MRSA]) and in 2006 for bacteremic syndromes, including right-sided infective endocarditis (IE) (28, 34). Although still relatively uncommon, DAP resistance (DAP-R) among clinical strains isolated during failures of clinical treatment with this agent has been increasingly reported (1, 2, 6, 24, 29). (Although the currently accepted term for reduced in vitro susceptibility to daptomycin is “nonsusceptible,” we will use the terms “daptomycin resistant [DAP-R]” and “daptomycin susceptible [DAP-S]” in this paper for a more facile presentation.) Potential alternative regimens for such infections are limited to the bacteriostatic agent linezolid and the clinically cumbersome agent quinupristin-dalfopristin (Synercid) (17).

Telavancin (TLV) is a novel lipoglycopeptide with bactericidal activity against Gram-positive pathogens, including S. aureus (4, 9, 18, 19). The bactericidal action of TLV results from a dual mechanism that includes both cell wall synthesis inhibition and disruption of the target bacterial membrane (7, 18). In the current investigation, we evaluated (i) in vitro efficacy of TLV against several recent clinical DAP-R MRSA strains, (ii) whether combinations of TLV and oxacillin (OXA) would enhance in vitro efficacy of these single agents against DAP-R MRSA strains, taking advantage of the so-called DAP-OXA “see-saw” phenomenon (38), and (iii) the in vivo efficacy of TLV in a prototypic rabbit IE model of invasive systemic infection caused by a DAP-R MRSA strain.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

The S. aureus strains employed in this study are listed in Table 1. We used five recent clinically derived DAP-S/DAP-R MRSA strain pairs isolated from patients who failed DAP therapy. Two of the isolate pairs were from patients at Harbor-UCLA Medical Center, one pair from the University of Nebraska Medical Center, Omaha, NE (courtesy of Paul Fey), one pair from Kaiser-Hospital, Bellflower, CA (courtesy of Lisa Chang), and one pair from San Francisco General Hospital, San Francisco, CA (courtesy of Henry Chambers). Molecular typing by pulsed-field gel electrophoresis using the SmaI restriction enzyme (data not shown), agr typing, spa typing (data not shown), multilocus sequence typing (MLST), and staphylococcal cassette chromosome mec (SCCmec) typing (Table 1) confirmed that each DAP-R strain was of a clonal type identical to and indistinguishable from that of its respective parental strain.

Table 1.

MRSA strains used in this study

Strain	Origin of clinical isolate	SCCmec type	agr type	CC type	MIC (μg/ml)				Reference(s)
					DAP	TLV	OXA	VAN
MRSA11/11	Endocarditis	IV	I	8	0.38	0.25	32	2	24, 38
REF2145a	Endocarditis	IV	I	8	4	0.38	6	2	24, 38
A214	Bloodstream	IV	I	8	0.5	0.25	64	2	This study
A215	Bloodstream	IV	I	8	3	0.38	16	2	This study
A0.5	Bloodstream	II	II	5	0.5	0.25	24	1.5	2
B2.0	Bloodstream	II	II	5	2	0.38	48	2	2
SA675	Endocarditis	NTb	III	30	0.25	0.38	64	0.5	13
SA684	Endocarditis	NT	III	30	2	0.38	64	2	13
L282	Bloodstream	IV	I	8	0.38	0.25	64	3	This study
L283	Bloodstream	IV	I	8	2	0.38	6	3	This study

^aStrain used in the in vivo endocarditis studies.

^bNT, nontypeable; positive for mecA, but no ccr or mec complex has been detected.

All strains were grown in either tryptic soy broth (TSB) (Difco Laboratories, Detroit, MI) or Mueller-Hinton broth (MHB) (Difco Laboratories) for specific studies. Liquid cultures were grown in Erlenmeyer flasks at 37°C with shaking (225 rpm) in a volume that was no greater than 10% of the flask volume.

Antimicrobial agents and susceptibility testing.

TLV (Theravance, Inc., South San Francisco, CA) was provided by the manufacturer. DAP was purchased from Cubist Pharmaceuticals (Lexington, MA) and reconstituted according to the manufacturer's recommendations. All DAP assays were done in the presence of 50 μg/ml Ca²⁺ as recommended by the manufacturer. The MICs of DAP and TLV were determined by the standard Etest method (bioMérieux, La Balme-les-Grottes, France) according to the manufacturer's recommended protocols. A minimum of three independent experimental runs were performed. Although the Clinical and Laboratory Standards Institute (CLSI) has established MIC guidelines for DAP-S and not officially for DAP-R (3), S. aureus strains with DAP MICs of ≥2 μg/ml were termed DAP-R.

In vitro time-kill curves.

Time-kill experiments were performed using MHB with an initial inoculum of 10⁵ or 10⁷ CFU/ml in the presence of 1×, 2×, and 4× MICs of DAP or TLV. The two different inocula (10⁵ and 10⁷ CFU/ml) were chosen to encompass bacterial counts commonly achieved in all target tissues of animals with experimental IE (32, 33, 35, 36, 38, 39). A minimum of three independent experimental runs were performed.

TLV-OXA combination kill curves.

Several previous studies have shown an inverse relationship between DAP and OXA MICs in some strains of DAP-R MRSA (i.e., decreasing OXA MICs accompanying increased DAP MICs) (23, 37, 38). Thus, bactericidal synergy assays for TLV plus OXA were performed using MHB as described before (26, 38). Briefly, we used an initial inoculum of ∼10⁶ CFU/ml in the presence of 0.25× or 0.5× MIC of OXA and TLV in combination and compared timed killing for the combination versus that for each single agent. Synergy was defined as ≥2-log-fold-greater killing at 24 h by the combination than by the more active of the single antibiotics alone (5, 26, 38). A minimum of two independent experiments were performed.

Experimental IE model.

Animals were maintained in accordance with the American Association for Accreditation of Laboratory Animal Care criteria. The Animal Research Committee (IACUC) of the Los Angeles Biomedical Research Institute at Harbor—UCLA Medical Center approved these animal studies. A well-characterized catheter-induced rabbit model of aortic IE was used as described previously (32, 33, 35) to compare in vivo efficacies of TLV and DAP against a DAP-R MRSA strain, REF2145. This strain was prioritized for use in the in vivo studies based on the following parameters: (i) isolation from a clinical IE patient (24, 38), (ii) highest DAP MIC among the DAP-R strains tested, and (iii) previously shown to be virulent in the same IE model (38). Briefly, female New Zealand White rabbits (2.0 to 2.5 kg body weight; Harlan Laboratories, CA) underwent indwelling transcarotid-transaortic valve catheterization with a polyethylene catheter. Twenty-four hours after catheter placement, animals were infected intravenously (i.v.) with 10⁵ CFU/animal, a 95% infective dose (ID₉₅) established in previous studies (38). At 24 h postinfection, animals were randomized to receive (i) no therapy (control), (ii) TLV at 30 mg/kg, i.v., twice a day (b.i.d.), (iii) DAP at 12 mg/kg, i.v., once daily, or (iv) DAP at 18 mg/kg, i.v., once daily. The TLV dose regimen was estimated to achieve humanlike pharmacokinetics (PK) of the recommended human clinical dose (10 mg/kg, i.v.) (4, 19). The DAP dose regimens (12 mg/kg and 18 mg/kg) mimic humanlike PK of the human standard dose (6 mg/kg once daily) and high dose (10 mg/kg, once daily), respectively (2, 27). Treatments were for 3 days. Control and antibiotic-treated animals were sacrificed by a rapid i.v. injection of sodium pentobarbital (200 mg/kg; Abbott Laboratories) at either 24 h postinfection (untreated controls) or 24 h after the last antibiotic dose, respectively. At the time of sacrifice, cardiac vegetations, kidneys, and spleen were sterilely removed and quantitatively cultured as described before (36). The mean log₁₀ CFU/g of tissue (± standard deviation [SD]) was calculated for each tissue in each group for statistical comparisons. The lower limit of microbiologic detection in the target tissues is ≤1 log₁₀ CFU/g of tissue.

No serum antibiotic levels were obtained, as the pharmacokinetics of the above-mentioned dose regimens for these agents in this animal model have been previously published (2, 8, 19, 31).

Statistical analysis.

To compare tissue MRSA counts of antibiotic-treated and untreated controls and between the different antibiotic treatment regimens (TLV versus DAP), the Student t test was employed. To assess the percentage of sterile tissue cultures among different regimens, two proportional test analyses (χ² and Fisher's exact test) were performed where appropriate. P values of <0.05 were considered statistically significant.

RESULTS

DAP and TLV MICs.

As shown in Table 1, all the DAP-R strains (DAP MICs ranging from 2 to 4 μg/ml) were susceptible to TLV (MICs, ≤0.38 μg/ml) by standard Etest. Four of five DAP-S strains exhibited slightly decreased TLV MICs compared to those of the respective DAP-R strain in the pair, the SA675-SA684 pair being the exception. Of interest, in 3 of 5 strain pairs, the so-called “see-saw” phenomenon was observed between DAP and OXA, whereby the OXA MICs decreased ≥4-fold in the DAP-R strain compared to the parental isolate (although no OXA MIC fell into the “susceptible” range).

In vitro DAP time-kill curves.

At 10⁵-CFU/ml initial inocula, supra-MIC DAP concentrations (2× and 4× MICs) yielded an early decrease in CFU (2 and 6 h) in all DAP-R strains (Fig. 1A and B; data not shown for B_2.0, SA684, and L283, which yielded virtually identical results). However, for all the DAP-R strains, DAP could not prevent substantial regrowth (ranging from 1 to 2 log) between 6 and 24 h of incubation, even at 2× or 4× DAP MICs.

Fig 1.

DAP time-kill analyses. REF2145 (A) and A215 (B) at 10⁵ CFU/ml and REF2145 (C) and A215 (D) at 10⁷ CFU/ml. Time-kill experiments were performed using Mueller-Hinton broth supplemented with 50 μg/ml Ca²⁺ in the presence of 0 (squares), 1× (circles), 2× (triangles), and 4× (inverted triangles) MIC of DAP.

Similar to the 10⁵-CFU/ml inocula mentioned above, supra-MICs of DAP at 10⁷-CFU/ml inocula caused an early killing effect against the DAP-R strains (Fig. 1C and D; data not shown for B_2.0, SA684, and L283). However, as with the 10⁵-CFU/ml inoculum, substantial regrowth was noted in cultures between 6 and 24 h of incubation at the 10⁷-CFU/ml initial inoculum, even at 4× MIC of DAP for most DAP-R strains.

In vitro TLV time-kill curves.

As shown in Fig. 2A and B (data not shown for B_2.0, SA684, and L283, which yielded virtually identical results), at 10⁵-CFU/ml initial inocula, all TLV concentrations exhibited an early decrease in CFU (∼1 to 2 log) against the DAP-R strains. For REF2145, A215, and B_2.0 DAP-R strains, supra-MICs of TLV were effective at preventing regrowth at 24 h of incubation. For SA684 and L283, regrowth at 24 h of incubation was observed even at 4× MIC of TLV.

Fig 2.

TLV time-kill analyses. REF2145 (A) and A215 (B) at 10⁵ CFU/ml and REF2145 (C) and A215 (D) at 10⁷ CFU/ml. Time-kill experiments were performed using Mueller-Hinton broth in the presence of 0 (squares), 1× (circles), 2× (triangles), and 4× (inverted triangles) MIC of TLV.

At 10⁷-CFU/ml inocula, data similar to those of the 10⁵-CFU/ml inoculum study described above were observed, although the extent of the killing effect was less (Fig. 2C and D; data not shown for B_2.0, SA684, and L283). Of note, TLV at 4× MIC prevented regrowth in 4 of the 5 DAP-R strains (the one exception being SA684).

In vitro efficacy of the TLV-OXA combination.

Two clinical DAP-R MRSA strains were selected to determine the potential for enhanced in vitro efficacy of TLV-OXA combinations compared to TLV or OXA alone. The combination of TLV and OXA, each at 0.25× MIC, increased the killing activity in the DAP-R MRSA strains REF2145 and A215 and prevented substantial regrowth at 24 h (Fig. 3A and B). At 0.5× MIC of TLV plus OXA, there was a further increase in killing, as well as prevention of regrowth of strain REF2145 relative to that by any single one of the agents (data not shown).

Fig 3.

In vitro efficacy of TLV-OXA combinations for DAP-R REF2145 (A) and A215 (B) strains. The REF2145 strain was used in the experimental IE studies detailed in the text.

Experimental IE model.

TLV significantly reduced MRSA densities in all three target tissues compared to those in the untreated control and DAP-treated animals (at both 12-mg/kg and 18-mg/kg regimens) (Table 2). The magnitude of these reductions in target tissue MRSA counts was substantive, with at least ∼5 log₁₀-CFU/g decreases observed. Moreover, TLV-treated rabbits had a significantly higher percentage of culture-negative target tissues (87% in vegetations and 100% in kidneys and spleen) than all other groups (P < 0.0001) (Table 2). The two dosage regimens of DAP did not significantly reduce MRSA counts in any target tissues compared to untreated controls (Table 2).

Table 2.

DAP-R MRSA strain REF2145 counts in target tissues with DAP or TLV treatment in the rabbit IE model

Treatment (no. of animals in group)	Density of S. aureus (% sterile tissue cultures) ina:
	Vegetations	Kidneys	Spleen
No treatment (16)	8.84 ± 0.55 (0)	6.57 ± 0.75 (0)	6.27 ± 0.64 (0)
TLV at 30 mg/kg, i.v., b.i.d. (15)	1.11 ± 0.69b (86.7c)	0.36 ± 0.11b (100c)	0.40 ± 0.13b (100c)
DAP at 12 mg/kg, i.v., once daily (13)	8.23 ± 0.85 (0)	6.78 ± 0.77 (0)	6.22 ± 0.75 (0)
DAP at 18 mg/kg, i.v., once daily (15)	7.50 ± 1.82 (0)	5.71 ± 1.63 (0)	5.02 ± 1.44 (0)

^aValues are mean log₁₀ CFU/g ± SD; values in parentheses represent the percentage of sterile tissue cultures after 3 days of treatment in the IE model.

^bP < 1.27E−12 relative to the value for DAP at 12-mg/kg and 18-mg/kg treatments.

^cP < 0.0001 versus no treatment control and DAP at 12-mg/kg and 18-mg/kg treatments.

DISCUSSION

Due to its potent activity against a wide range of Gram-positive organisms, including MRSA strains, vancomycin-intermediate S. aureus (VISA), and hetero-VISA strains, DAP has become a clinical mainstay of treatment against S. aureus infections (28, 34). However, there have been an increasing number of reports in which initially DAP-S S. aureus strains have developed DAP-R in vitro and in vivo as a result of exposures to DAP (6, 10, 12, 20, 25). Given this potentially serious resistance problem, evaluation of alternative and potent bactericidal agents such as TLV has been urgently needed.

TLV is a new semisynthetic lipoglycopeptide with activity against the same in vitro spectrum of susceptible and drug-resistant staphylococcal isolates as listed above for DAP. TLV acts through a dual mechanism that includes inhibition of peptidoglycan synthesis (related to its vancomycin-like domain) and disruption of bacterial membrane potential (9, 30). The potent bactericidal action of TLV has been linked to this dual mechanism of action (4, 11, 21, 29, 30). Although the data are rather limited, TLV appears to be active against clinically derived DAP-R S. aureus strains. For example, Kosowska-Shick et al. identified 12 VISA isolates that were DAP-R; all were TLV-S, in the MIC range of 0.25 to 1 μg/ml, with an MIC₉₀ of 1 μg/ml (14). Similarly, among 98 MRSA isolates, Krause et al. found all TLV MICs to be ≤1 μg/ml. This included two highly DAP-R strains with MICs of 4 and 8 μg/ml, in which the TLV MICs were 0.5 and 0.25 μg/ml, respectively (15). The efficacy assessment of TLV for highly invasive, bacteremic S. aureus infections in animal models is also relatively sparse. The studies of Madrigal et al. (19) and Miró et al. (22) have both evaluated TLV in the same rabbit IE model as the one used in the present investigation. Madrigal et al. (19) employed the same dose regimen as the present study (30 mg/kg, i.v., b.i.d.) and found it to be quite active against an MRSA isolate in the IE model, although not statistically better than vancomycin. In contrast, TLV was very effective in clearing VISA isolates from vegetations of animals compared to vancomycin. Miró et al. (22) also observed TLV to be active in the treatment of experimental VISA IE. Despite these interesting outcomes, there have been no parallel studies of TLV efficacies in experimental IE caused by DAP-R S. aureus isolates.

Our in vitro results demonstrated that all five clinically derived DAP-R MRSA strains tested were susceptible to TLV by Etest (MICs of ≤0.38 μg/ml). In addition to these MIC data, in vitro time-kill curves exhibited an early TLV killing effect against all the DAP-R strains at both 10⁵- and 10⁷-CFU/ml initial inocula (Fig. 2). The in vitro bactericidal activities of TLV observed in this study mirror those in previously published investigations of TLV activity against MRSA, VISA, and hetero-VISA strains (16, 30). Moreover, Steed et al. confirmed TLV's bactericidal activity against DAP-R strains of S. aureus using an in vitro pharmacodynamic model that mimics IE (30). Thus, our current studies were designed to translate the above-mentioned in vitro outcomes into in vivo relevance. In the current study, using a clinical DAP-R MRSA strain (REF2145), we confirmed the potent bactericidal activity of TLV in experimental IE in terms of DAP-R MRSA clearance and sterilization in all major target tissues in this model. In contrast, and paralleling data from Chambers et al. (2), neither “standard-dose” nor “high-dose” DAP regimens (6- or 10-mg/kg once-daily equivalents in rabbits) were able to significantly reduce target tissue MRSA counts.

Lastly, we and others have identified a so-called “see-saw” effect in vitro between DAP and OXA for many MRSA strains, in which OXA MICs fell dramatically as DAP MICs rose. This occurred in both in vitro- and in vivo-derived DAP-R isolates (23, 37, 38). Of interest, OXA MICs fell ≥4-fold in 3 of 5 study isolates. Also, several groups have shown consistently enhanced killing in vitro between DAP and OXA by time-kill curve analyses (26, 38). In addition, the combination of DAP plus OXA was found to enhance DAP-R MRSA clearance from all target tissues (vegetations, kidneys, and spleen) in an experimental IE model relative to monotherapies in the context of only 3 days of treatment (38). These observations prompted us to investigate whether a TLV-OXA combination would enhance the in vitro efficacy of each single agent against DAP-R strains. In the current study, the combination of TLV and OXA (each at sublethal concentrations) was found to increase the in vitro efficacy relative to that of TLV and OXA alone in two DAP-R strains, REF2145 and A215, both of which exhibited the in vitro DAP-OXA “see-saw” phenomenon (Table 1; Fig. 3A and B). Whether the latter combination would exhibit enhanced activity compared to monotherapies in relevant animal models remains to be defined.