Combinations of Daptomycin plus Ceftriaxone, but Not Ascending Daptomycin Dose-Regimens, Are Effective in Experimental Endocarditis Caused by Streptococcus mitis-oralis Strains: Target Tissue Clearances and Prevention of Emergence of Daptomycin-Resistance

ABSTRACT

The Streptococcus mitis-oralis subgroup of the viridans group streptococci (VGS) are the most common cause of infective endocarditis (IE) in many parts of the world. These organisms are frequently resistant in vitro to standard β-lactams (e.g., penicillin; ceftriaxone [CRO]), and have the notable capacity for rapidly developing high-level and durable daptomycin resistance (DAP-R) during exposures in vitro, ex vivo, and in vivo. In this study, we used 2 prototypic DAP-susceptible (DAP-S) S. mitis-oralis strains (351; and SF100), which both evolved stable, high-level DAP-R in vitro within 1 to 3 days of DAP passage (5 to 20 μg/mL DAP). Of note, the combination of DAP + CRO prevented this rapid emergence of DAP-R in both strains during in vitro passage. The experimental rabbit IE model was then employed to quantify both the clearance of these strains from multiple target tissues, as well as the emergence of DAP-R in vivo under the following treatment conditions: (i) ascending DAP-alone dose-strategies encompassing human standard-dose and high-dose-regimens; and (ii) combinations of DAP + CRO on these same metrics. Ascending DAP-alone dose-regimens (4 to 18 mg/kg/d) were relatively ineffective at either reducing target organ bioburdens or preventing emergence of DAP-R in vivo. In contrast, the combination of DAP (4 or 8 mg/kg/d) + CRO was effective at clearing both strains from multiple target tissues (often with sterilization of bio-burdens in such organs), as well as preventing the emergence of DAP-R. In patients with serious S. mitis-oralis infections such as IE, especially caused by strains exhibiting intrinsic β-lactam resistance, initial therapy with combinations of DAP + CRO may be warranted.

INTRODUCTION

The Streptococcus mitis-oralis subgroup of the viridans streptococci includes a cadre of oropharyngeal organisms, including S. mitis, S. oralis, S. gordonii, and S. parasanguinis (1–4). This subgroup represents an important human pathogen group, being the most common overall cause of infective endocarditis (IE) in the developing world (4–6), and the predominant cause of the “streptococcal toxic shock syndrome” in immunocompromised patients (5–9). The in vitro antimicrobial susceptibility profiles of S. mitis-oralis strains often present therapeutic conundrums, as a high per cent of such isolates are resistant to penicillins and/or cephalosporins (including third generation agents such as ceftriaxone [CRO]) (10–14). Interestingly, ‘vancomycin tolerance’ in S. mitis has also been described (9). These scenarios usually raise the notion of using daptomycin (DAP) as alternative therapy for such infections. However, several recent studies have confirmed the unique capacity of S. mitis-oralis strains to rapidly develop high-level and durable DAP resistance (DAP-R) when exposed to this agent in vitro, ex vivo in simulated IE models, and in vivo in experimental IE (4, 15–17). Thus, in 2013, Garcia-de-la-Maria et al. (4) first documented that a substantial proportion of S. mitis-oralis clinical bloodstream isolates (>25%) had the notable property to rapidly (within 48 h in vitro exposures) evolve high-level, stable DAP-R. In addition, this research group demonstrated that such high-level DAP-R could also emerge in vivo during DAP-alone therapy of experimental IE (4). Further, they showed that combination therapy with DAP + gentamicin was able to synergistically clear DAP-susceptible (DAP-S) parental strains from cardiac vegetations in this model, while forestalling evolution of DAP-R subpopulations during therapy (4). Furthermore, in vitro and ex vivo studies featuring DAP combination regimens have shown the ability of several regimens to either synergistically kill S. mitis-oralis strains and/or prevent the emergence of DAP-R during DAP exposures. Such regimens have included DAP + either gentamicin, CRO, ceftaroline, or trimethoprim-sulfamethoxazole (4, 15–17). Of note, the combination therapy outcomes in vitro and ex vivo were most impressive with DAP + either ceftaroline or CRO.

This study was designed to utilize the experimental IE model caused by 2 distinct and well-characterized DAP-S S. mitis-oralis strains, in order to answer 2 key questions: (i) could ascending DAP-alone dose-regimens that encompassed “high-dose” human dose-regimens increase target organ clearances (vegetations, kidneys, and spleen), and also prevent emergence of DAP-R; and (ii) could DAP combination regimens with CRO achieve these same goals? The results of these investigations clearly showed the inability of increasing DAP-alone dosing strategies, on the one hand, to exert such salutary impacts on in vivo efficacy and DAP-R emergence, while DAP + CRO regimens were quite effective in achieving both such outcomes.

The term “DAP resistance (DAP-R)” (instead of “DAP-nonsusceptibility”) is used throughout the manuscript for ease of presentation. The term “ascending” DAP doseregimens indicates increasing doses used within separate treatment groups of animals (not within the same animal treatment groups).

RESULTS

MICs.

The previously published DAP and CRO MICs of the DAP-S 351 and SF100 strains were reconfirmed. The 351 and SF100 strains were susceptible to DAP, each exhibiting DAP MICs of 0.5 μg/mL, however, the 351 strain was resistant to CRO (MIC = 8 μg/mL), while the SF100 strain was susceptible to CRO (MIC = 0.125 μg/mL) (16).

Prevention of emergence of DAP-R during in vitro passage studies.

We determined the MICs of bacterial samples daily at 24 h intervals during a 10 day passage period in DAP-alone or DAP + CRO to assess potential prevention of evolution of DAP-R (Table 1 and 2). Daily passage of each DAP-S strain in distinct concentrations (5, 10, and 20 μg/mL) of DAP-alone resulted in rapid emergence of high-level DAP-R populations by the first 1 to 2 days of passage (Table 1 and 2). In contrast, when the DAP-S 351 and SF100 parental strains were passaged in DAP + CRO, DAP MICs were maintained at relatively low levels (1 to 4 μg/mL), even at higher DAP dose-regimens (Table 1 and 2). None of the postpassaged strains were resistant to CRO. In addition, 351-derived and SF100-derived day 10 (D10) DAP-R passage strains exhibited stable DAP MICs when passaged for 5 additional days in antibiotic-free media (data not shown) (18–20). Any postpassaged strains re-passaged in antibiotic-free media exhibited DAP MIC values ≥ 2 μg/mL were considered DAP-R.

TABLE 1.

In vitro serial passaging of 351 S. mitis-oralis strain in combination of distinct concentrations of DAP with CRO versus DAP alone

Distinct concentrations (μg/mL) of DAP alone and DAP + CRO	DAP MICs (μg/mL) of strain 351 passaged in ascending concentrations of DAP alone or DAP + CRO on distinct days
	D1	D2	D3	D4	D5	D6	D7	D8	D9	D10
DAP (5)	4	256	256	256	256	256	256	256	256	256
DAP (10)	>256	>256	>256	>256	>256	>256	>256	>256	>256	>256
DAP (20)	>256	>256	>256	>256	>256	>256	>256	>256	>256	>256
DAP (5) + CRO (1)	2	4	4	4	4	4	4	4	4	4
DAP (10) + CRO (1)	2	2	2	2	2	2	2	2	2	2
DAP (20) + CRO (1)	2	2	2	2	2	2	2	2	2	2

TABLE 2.

In vitro serial passaging of SF100 S. mitis-oralis strain in combination of distinct concentrations of DAP with CRO versus DAP alone

Distinct concentrations (μg/mL) of DAP alone and DAP + CRO	DAP MICs (μg/mL) of strain 351 passaged in ascending concentrations of DAP alone or DAP + CRO on distinct days
	D1	D2	D3	D4	D5	D6	D7	D8	D9	D10
DAP (5)	2	2	>256	>256	>256	>256	>256	>256	>256	>256
DAP (10)	2	>256	>256	>256	>256	>256	>256	>256	>256	>256
DAP (20)	>256	>256	>256	>256	>256	>256	>256	>256	>256	>256
DAP (5) + CRO (0.0156)	1	1	1	1	4	4	4	4	4	4
DAP (10) + CRO (0.0156)	1	1	1	1	2	2	2	2	2	2
DAP (20) + CRO (0.0156)	0.5	4	4	4	4	4	4	4	4	4

Experimental IE.

For the DAP-S parental strain 351, although treatments with ascending doses of DAP-alone (4 to 18 mg/kg/d) statistically reduced counts in vegetations and other target tissues compared to untreated controls, the bioburden reductions were modest, at best (Table 3). Moreover, the majority of vegetations now contained high proportions of DAP-R subpopulations after 4 days of therapy, even at the highest DAP-alone dose-regimens.

TABLE 3.

Treatment of 351 S. mitis-oralis strain with ascending dosages of DAP in combination of CRO versus DAP and CRO alone in in vivo IE model^a

Treatment	Vegetation [sterile] (/= Growth on DAP 2 mg/L plates)b	Kidney	Spleen
Untreated controls (7)	8.49 ± 0.65	5.27 ± 0.71	5.26 ± 0.36
DAP 4 mg/kg i.v. once daily × 4 d (7)	7.66 ± 0.87/6.14 ± 1.47c	4.16 ± 0.78c	4.20 ± 0.99c
DAP 6 mg/kg (7)	7.43 ± 1.06d/6.09 ± 0.93d	3.90 ± 0.67d	5.06 ± 1.22
DAP 8 mg/kg (6)	8.24 ± 0.82/6.50 ± 1.53e	4.71 ± 0.91	4.94 ± 0.80
DAP 10 mg/kg (6)	7.50 ± 1.08/5.53 ± 0.51f	4.18 ± 0.49f	4.13 ± 0.52f
DAP 12 mg/kg (7)	7.14 ± 1.04g/6.63 ± 1.04g	3.96 ± 0.59g	3.79 ± 0.53g
DAP 18 mg/kg (6)	8.12 ± 0.79/7.79 ± 0.83	3.94 ± 0.51	4.99 ± 0.30
CRO 40 mg/kg i.v. twice daily × 4 d (7)	7.81 ± 0.65	3.94 ± 0.51h	4.53 ± 1.04
DAP (4 mg/kg) + CRO (6)	5.51 ± 1.18i,j,k /0.62 ± 0.07i,j,k	1.93 ± 0.72i,j,k	2.49 ± 0.48i,j,k
DAP (8 mg/kg) + CRO (6)	0.62 ± 0.07l,m,n /0.62 ± 0.07 [6/6]l,m,n	0.69 ± 0.08 [6/6]l,m,n	0.76 ± 0.13 [6/6]l,m,n

^aStatistical differences for Untreated controls versus DAP 4, 6, 8, 10, 12 mg/kg; Untreated controls versus CRO alone; Untreated controls versus DAP + CRO; combination of DAP + CRO versus DAP alone; combination of DAP + CRO versus CRO alone were determined by Student's t test.

^bThese designations define the number of sterile target tissues/number sampled (e.g., 6/6 = all 6 target tissues were sterilized), as well as the proportion of vegetations having DAP-R subpopulations as defined by growth on DAP plates (2 mg/L).

^cP < 0.05 untreated controls versus DAP 4 mg/kg.

^dP < 0.05 untreated controls versus DAP 6 mg/kg.

^eP < 0.05 untreated controls versus DAP 8 mg/kg.

^fP < 0.01 untreated controls versus DAP 10 mg/kg.

^gP < 0.05 untreated controls versus DAP 12 mg/kg.

^hP < 0.005 untreated controls versus CRO alone.

ⁱP < 0.005 DAP 4 mg/kg + CRO versus DAP 4 mg/kg.

^jP < 0.005 untreated controls versus DAP 4 mg/kg + CRO.

^kP < 0.01 DAP 4 mg/kg + CRO.

^lP < 0.0001 DAP 8 mg/kg + CRO. Parenthesis () = number of rabbits.

^mP < 0.0001 untreated controls versus DAP 8 mg/kg + CRO.

ⁿP < 0.0005 DAP 8 mg/kg + CRO versus DAP 8 mg/kg.

CRO alone was also relatively ineffective at bacterial clearances in any target tissues (Table 3); no CRO-R subpopulations were detected. In contrast, combination therapy with DAP + CRO was highly effective at clearing strain 351 from multiple target tissues in a DAP dose-dependent manner (DAP 4 mg/kg/d versus 8 mg/kg/d) (Table 3). At the DAP 4 mg/kg dose + CRO, there were essentially no intravegetation DAP-R subpopulations, despite moderate residual CFU/g bioburdens persisting in this target tissue (Table 3). Of note, the DAP 8 mg/kg dose-regimen + CRO sterilized all target tissues (Table 3).

Treatment outcomes for DAP-S parental strain, SF100, were somewhat similar to strain 351 (Table 4), with several important differences. As with strain 351, ascending DAP-alone dosing exerted very modest impacts on significantly reducing target tissue counts; in addition, substantial proportions of the vegetation bioburdens emerged as DAP-R in these DAP-alone treatment groups. As opposed to strain 351, however, CRO alone was effective at significantly (albeit modestly) reducing vegetation bioburdens of strain SF100 (Table 4); moreover, CRO alone sterilized most kidney and spleen infection (Table 4). As seen with strain 351, the combination of DAP + CRO further cleared vegetations of strain SF100 in a DAP dose-dependent manner (Table 4). Both DAP + CRO regimens were highly effective at significantly reducing tissue bioburdens within kidneys and spleens, sterilizing all lesions in both target organs (Table 4).

TABLE 4.

Treatment of SF100 S. mitis-oralis strain with ascending dosages of DAP in combination of CRO versus DAP and CRO alone in in vivo IE model^a

Treatment (no. of animals)	Vegetation [sterile] (/ = Growth on DAP 2 mg/L plates)b	Kidney	Spleen
Untreated Controls (8)	7.93 ± 0.57	4.58 ± 0.52	4.70 ± 0.85
DAP 4 mg/kg i.v. once daily × 4 days (6)	7.80 ± 0.81/1.00 ± 0.06c	3.50 ± 0.49c	3.54 ± 0.28c
DAP 6 mg/kg (6)	7.42 ± 1.08/3.53 ± 2.63d	4.51 ± 0.63	4.45 ± 0.49
DAP 8 mg/kg (6)	7.69 ± 0.75/4.30 ± 2.78e	3.24 ± 0.41e	3.40 ± 0.90e
DAP 10 mg/kg (6)	7.51 ± 0.80/5.72 ± 1.98f	3.41 ± 0.63f	3.64 ± 0.84f
DAP 12 mg/kg (6)	7.01 ± 1.07/4.28 ± 2.57g	3.86 ± 0.76	3.64 ± 0.79g
DAP 18 mg/kg (6)	7.25 ± 0.84/5.74 ± 1.28	4.42 ± 0.78	4.77 ± 0.45
CRO 40 mg/kg i.v. once daily × 4 days (6)	5.68 ± 0.93 [0/6]h	0.95 ± 0.41 [5/6]h	1.13 ± 0.48 [5/6]h
DAP (4 mg/kg) + CRO (6)	3.75 ± 1.59 [1/6]i,j,k	0.76 ± 0.07 [6/6]i,j	0.89 ± 0.12 [6/6]i,j
DAP (8 mg/kg) + CRO (7)	0.89 ± 0.19 [7/7]l,m,n	0.92 ± 0.11 [7/7]m,n	0.92 ± 0.08 [7/7]m,n

^aStatistical differences for Untreated controls versus DAP 4, 6, 8, 10, 12 mg/kg; Untreated controls versus CRO alone; Untreated controls versus DAP + CRO; combination of DAP + CRO versus DAP alone; combination of DAP + CRO versus CRO alone were determined by Student's t test.

^bThese designations define the number of sterile target tissues/number sampled (e.g., 6/6 = all 6 target tissues were sterilized), as well as the proportion of vegetations having DAP-R subpopulations as defined by growth on DAP plates (2 mg/L).

^cP < 0.01 untreated controls versus DAP 4 mg/kg.

^dP < 0.01 untreated controls versus DAP 6 mg/kg.

^eP < 0.05 untreated controls versus DAP 8 mg/kg.

^fP < 0.05 untreated controls versus DAP 10 mg/kg.

^gP < 0.05 untreated controls versus DAP 12 mg/kg.

^hP < 0.001 untreated controls versus CRO alone.

ⁱP < 0.001 DAP 4 mg/kg + CRO versus DAP 4 mg/kg.

^jP < 0.001 untreated controls versus DAP 4 mg/kg + CRO.

^kP < 0.05 DAP 4 mg/kg + CRO.

^lP < 0.0001 DAP 8 mg/kg + CRO. Parenthesis () = number of rabbits.

^mP < 0.00001 untreated controls versus DAP 8 mg/kg + CRO.

ⁿP < 0.01 DAP 8 mg/kg + CRO versus DAP 8 mg/kg.

DISCUSSION

These investigations assessed if either increasing DAP-alone dosing strategies or DAP + CRO combinations could improve in vivo efficacy and avoid DAP-R emergence in experimental S. mitis-oralis IE. The notion of utilizing DAP-based antibiotic combination regimens for S. mitis-oralis therapy has been previously pursued both in vitro and ex vivo (in simulated IE models) (15, 16). Zapata et al. (15) recently evaluated a broad range of DAP combination regimens for their abilities to both synergistically kill and/or prevent emergence of DAP-R during passage, utilizing the same DAP-S parental strain (351) employed in this study. The combination antibiotics included: DAP + either gentamicin, rifampin, trimethoprim-sulfamethoxazole, imipenem, ceftaroline, tedizolid, and linezolid. Only the combination of DAP + either gentamicin or ceftaroline were both bactericidal and synergistic in early time-kill curve interactions. Of note, the combinations of DAP + gentamicin, ceftaroline, or trimethoprim-sulfamethoxazole prevented DAP-R emergence during serial passage experiments. Yim et al. (17) 20 used the same 2 DAP-S strains employed in this study (351 and SF100) in their simulated IE model ex vivo. As opposed to the in vitro results noted above, the addition of gentamicin to DAP (given in human-simulating PK-PD dose-regimens) in this ex vivo model yielded neither synergistic nor bactericidal interactions against either S. mitis-oralis strain; moreover, this regimen did not forestall the evolution of DAP-R over the 96 h exposure period in this model. Of interest, combinations of DAP + either ceftaroline or CRO accomplished both these latter desired metric outcomes. This ex vivo data led us to examine the combined efficacy of DAP plus CRO in the experimental IE model in vivo in this investigation.

Several notable observations emerged from this study. First, we hypothesized that DAP-alone dose-escalation, in the presence of an intact host defense system microenvironment (e.g., antimicrobial peptides, macrophages-neutrophils, etc.), might: (i) synergize well to clear S. mitis-oralis from infected target tissues; and (ii) override the propensity for S. mitis-oralis strains to develop DAP-R. However, this notion proved to be false. This speaks to the rapid adaptive capacity of these organisms to circumvent DAP exposures. We have previously defined the mechanism(s) driving DAP-R evolution in S. mitis-oralis; these are strain-specific and multifactorial, involving rapid and major phenotypic, metabolic, and genotypic adaptations, including: (i) reduction in the major membrane phospholipids, phosphatidylglycerol and cardiolipin (both of which participate in initial interactions of DAP with target bacterial membranes, and which also “guide” DAP to the divisome site-of-action for DAP) (21, 22). These perturbations are accompanied by loss-of-function mutations in key genes within the cardiolipin biosynthetic pathway, including pgsA and cdsA (21, 22); (ii) preferential accumulation of DAP in selected bacterial cells within streptococcal chains, presumably sequestering the drug in a minority of the subpopulation, while “protecting” the rest of the bacterial population from its bactericidal activity (18); (iii) exaggeration of the fermentative glycolytic pathway in these organisms, which lack a traditional TCA cycle, reducing generation of biometabolic intermediates and ATP (18, 19); and (iv) modifications of the organism’s membrane and cell surface phenotypes, including enhanced fluidity and surface charge, both of which can negatively influence DAP’s bactericidal mechanisms (17, 21, 22). Clearly, these adaptations occur very rapidly during DAP-alone exposures in selected DAP-S S. mitis-oralis strains that appear to be “destined” or “predisposed” to evolve DAP-R, and are not preventable by merely increasing the initial DAP-alone exposure concentrations.

Second, as was seen previously in vitro and ex vivo (15, 16, 22), combining selected agents with DAP can provide an outcome advantage viz-a-viz both streptococcal clearances and prevention of DAP-R. These combinatorial impacts were recapitulated with the DAP + CRO combinations used in the experimental IE model. Notably, this combination was highly effective at clearing all target tissues of both S. mitis-oralis parental strains, and also rendered most of these organs (e.g., vegetations, spleen, kidney) culture-negative. This combination also prevented the rapid emergence of DAP-R or CRO-R subpopulations within target organs in this model. It should be noted that the DAP + CRO efficacy in this model was most prominent in clearing strain SF100 (which was CRO-susceptible in vitro) from multiple target tissues.

Lastly, as alluded to above, both of the study strains (351 and SF100) are organisms that appear “destined” to rapidly evolve high-level and stable DAP-R in vitro, ex vivo, and in vivo during DAP-alone exposures. This latter phenotype occurs in at least 25% of S. mitis-oralis strains (4). We are currently examining those phenotypic, metabolic, and genotypic characteristics that distinguish S. mitis-oralis strains that appear predisposed to evolve DAP-R (or not). If a specific and predictive set of phenotypic or genotypic biomarkers can be identified up-front, that correlate with risk of eventual evolution of DAP-R, this could be a major advance forward in precision selection of agents to treat such infections.

Our study had several limitations. It was limited to only 2 prototype S. mitis-oralis strains; additional S. mitis-oralis strains need to be tested in this same model, especially those that are apparently “not predisposed” to evolve DAP-R in vitro (which represent the majority of S. mitis-oralis strains [4]). In addition, follow-up studies in this same model should also examine higher CRO alone dose-regimens to see if there is an optimal strategy to eradicate multiple S. mitis-oralis strains from all target tissues with such therapy.

In conclusion, the combination of DAP + CRO was effective at clearing both S. mitis-oralis strains in vivo from multiple target tissues, as well as in preventing the emergence of DAP-R. In patients with S. mitis-oralis IE, especially caused by strains exhibiting intrinsic β-lactam resistance, initial therapy with combinations of DAP + CRO may be warranted.

MATERIALS AND METHODS

Bacterial strains.

The well-characterized DAP-S S. mitis-oralis strains, 351 and SF100, were used in this study (4, 15–17, 21). Both strains were initially isolated from the bloodstream of patients with IE without prior DAP exposures. Strain SF100 was kindly provided by Dr. Paul Sullam (San Francisco, CA). The penicillin MIC for the 351 strain has been previously reported by both Etest and broth microdilution assays as 8 μg/mL (penicillin-resistant) 4); the penicillin MIC for strain SF100 was 0.25 μg/mL (penicillin-susceptible). In addition, both 351 and SF100 strains are ceftaroline-susceptible, as tested previously (15, 16).

MICs.

DAP was purchased from Merck & Co., Inc. DAP MIC testing was performed by the CLSI-recommended broth microdilution techniques, with 50 μg/mL of CaCl₂ added to BHI broth (Difco). Also, BHI agar supplemented with 5% lysed horse blood (Difco) was used for quantitative agar plate colony counts. DAP MICs were also determined in parallel by standard Etest (bioMérieux) on Mueller-Hinton agar (MHA) plates supplemented with 50 μg/mL of CaCl₂ (Difco Laboratories). There are no formal CLSI DAP breakpoints against VGS strains; however, streptococcal strains with DAP MICs ≥ 2 μg/mL are considered as DAP-R (4, 15–17, 21–23). The MICs of CRO against S. mitis-oralis strains 351 and SF100 were determined by broth microdilution methods; VGS strains with CRO MICs ≥ 4 μg/mL are defined as CRO-resistant. A minimum of 3 independent broth microdilution-based MICs were carried out on different days for all agents, with the average MIC reported.

Prevention of emergence of DAP a resistance in vitro.

We evaluated the impacts of longer-term DAP +/− CRO exposures of the 2 DAP-S S. mitis-oralis strains on the potential emergence and stability of DAP-R (15–19, 21–23). For these latter studies, the DAP-S parental strains 351 and SF100 were cultured overnight in BHI broth. The initial inoculum of strain SF100 and 351 (OD₆₀₀ = 1.00 = ~108 CFU/mL) were exposed to 5, 10, or 20 μg/mL of DAP in BHI + 50 μg/mL CaCl₂ alone or with the combination of CRO (1/4 × MICs of each strain); surviving colonies were serially passaged for a 10-day period under the same conditions (15). Surviving colonies after each day’s passage were stored at −80°C for subsequent MIC testing. The stability of DAP-R was determined by growing such strains for 5 additional days in antibiotic-free media and repeating the DAP MICs as above.

The DAP and CRO MICs of strains 351 and SF100 following serial passage in DAP alone versus combination of DAP + CRO regimens were determined by broth dilution supplemented with 5% lysed horse blood (as recommended by CLSI for S. mitis-oralis) (15). We also assessed the stability of DAP-R isolates that appeared during the 10-day passage period. Individual postpassage isolates were again serially passaged for 5 days in antibiotic-free BHI media (15). The DAP and CRO MICs of these antibiotic-free postpassage isolates were evaluated by broth dilution assay (15); A minimum of 3 independent experiments were performed on separate days to analyze the MIC data.

Experimental IE model.

To verify the in vivo translatability of the relationship between emergence of DAP-R during either ascending DAP-alone or combined DAP-CRO treatment of S. mitis-oralis strains in vitro, the well-characterized rabbit model of indwelling catheter-induced aortic valve IE was used (4). Rabbits were i.v. infected at 48 h after catheter placement with: 1 × 10⁷ CFU/animal for strain 351; and 8 × 10⁶ CFU/animal for strain SF100. These inocula represent the ID₉₅ for inducing IE and establishing infection in key extracardiac target tissues (kidneys and spleen) as determined by extensive pilot experiments for each strain. At 24 h postinfection, animals were randomized into either an untreated control group (sacrificed at this time point as a therapeutic baseline) or the following antibiotic therapy groups: (i) DAP-alone, given in separate groups at ascending doses of 4 to 18 mg/kg/d i.v. for 4 days. These dose-regimens encompass subtherapeutic to human-mimicking therapeutic doses in humans; for example, 12 to 18 mg/kg DAP dosing regimens equate to 6 to 10 mg/kg human dose PK-PD, respectively; this human dose-range encompasses the current FDA-approved dose-regimen for serious infections (6 mg/kg/d), as well as the human “high-dose” DAP regimens (10 mg/kg/d) (17, 20, 21); (ii) CRO alone (40 mg/kg; bid, iv) has been successfully used in prior experimental streptococcal IE studies (16, 17), and mimics human PK-PD (16, 17); and (iii) the combination of DAP (4 or 8 mg/kg/d) plus CRO (40 mg/kg/d iv, bid). Serum levels of DAP and CRO were not assessed, as these have been previously published in this model using similar dose-regimens (20, 24).

In all studies, at 24 h after the last antibiotic treatment (to circumvent any antibiotic carryover effects), animals were sacrificed, and their cardiac vegetations, kidneys, and spleen were aseptically removed and quantitatively cultured on TSA plates, as previously described (22). Counts were expressed as mean log₁₀ CFU per gram of tissue (±SD). The limit of detection in target organ cultures in this model, based on average target tissue weights, is ≤2 log₁₀ CFU/g. All culture-negative vegetations, although designated as “sterile,” were still assigned a limit-of-detection log₁₀ CFU/g designation based on their weights, for statistical purposes, to enable comparisons between groups. This is a standard procedure used in this model. To assess for in vivo development of DAP or CRO resistances in cardiac vegetations during treatments, serial dilutions of these homogenates were parallel-plated on either DAP-containing agar plates (2 μg/mL) or CRO-containing agar plates (8 μg/mL), depending on the specific treatment groups. Only cardiac vegetations were parallel-plated for emergence of antibiotic resistances, given the high organ bioburdens usually achieved in this infection site. Thus, any potential emergence of DAP-R or CRO-R would be most likely to occur in this anatomic site.

Statistics.

Data were presented, unless otherwise indicated, as the sample mean log₁₀ CFU/g tissue ±SD. All head-to-head statistical comparisons were made using the unpaired Student's t test. P values < 0.05 were considered statistically significant.

Study approval.

Female, New Zealand White rabbits weighing 2.2 to 2.5 kg were used in all animal studies (Irish Farms; Riverside, CA). Rabbits were maintained in accordance with the American Association for Accreditation of Laboratory Animal Care criteria. The Institutional Animal Care and Use Committee of the Lundquist Institute for Biomedical Innovation at Harbor-UCLA Medical Center approved all animal study protocols.