Pharmacodynamics of ClpP-Activating Antibiotic Combinations against Gram-Positive Pathogens

It is often difficult to cure endocarditis, osteomyelitis, and device-associated infections caused by Gram-positive pathogens, despite therapy with clinically appropriate antibiotics. This may be due to antibiotic tolerance or resistance development. Acyldepsipeptides (ADEPs) are a class of bactericidal compounds active against a variety of clinically important Gram-positive bacteria, including staphylococci, streptococci, and enterococci.

ABSTRACT

It is often difficult to cure endocarditis, osteomyelitis, and device-associated infections caused by Gram-positive pathogens, despite therapy with clinically appropriate antibiotics. This may be due to antibiotic tolerance or resistance development. Acyldepsipeptides (ADEPs) are a class of bactericidal compounds active against a variety of clinically important Gram-positive bacteria, including staphylococci, streptococci, and enterococci. ADEPs activate caseinolytic protease P (ClpP), killing high-density, nondividing cultures of bacteria that are tolerant to approved classes of antibiotics. Acyldepsipeptide analog 4 (ADEP4) was active against a panel of drug-resistant Gram-positive pathogens in MIC assays, with no preexisting resistance detected. Killing of stationary-phase cultures was observed when ADEP4 was combined with multiple classes of approved antibiotics. Additionally, a hollow-fiber infection model was used to assess the effects of ADEP4 antibiotic combinations on bacterial killing and resistance development. These studies were performed on high-density cultures of methicillin-resistant S. aureus (MRSA), methicillin-susceptible S. aureus (MSSA), and vancomycin-resistant Enterococcus faecalis (VRE). None of the approved antibiotics linezolid, ampicillin, and oxacillin tested alone had bactericidal activity under these conditions. ADEP4 initially caused killing, but regrowth of the culture was apparent within 96 h due to resistance. Combinations of ADEP4 with linezolid or oxacillin caused substantially improved killing of MRSA or MSSA cultures, respectively, and no regrowth due to resistance was observed. The combination of ADEP4 and ampicillin eradicated cultures of VRE to the limit of detection within 52 h. These data suggest that combining ClpP activators with traditional antibiotics may be a good strategy to treat complicated Gram-positive infections.

INTRODUCTION

Staphylococcus aureus and Enterococcus faecalis are important Gram-positive pathogens predominantly causing bacteremia, urinary tract infections, and infective endocarditis (IE). There is a high unmet need for novel antibiotics to combat drug-resistant Gram-positive infections (1). A systemic review of hospital-based studies, including data from IE epidemiology analyses performed globally over the last half-century, revealed that in the last decade, S. aureus and enterococcal IE frequencies have increased significantly (2). Even with current medical advances, the in-hospital mortality rate for patients with IE is 15% to 20% (3), with 1-year mortality approaching 40% (4). In addition, there is a high incidence of and a high mortality rate associated with other complicated infections such as bacteremia, osteomyelitis, diabetic ulcers, pneumonia, and prosthetic joint-related and catheter-related infections (5, 6).

There are several factors that may contribute to antibiotic failures beyond antibiotic resistance, including drug tolerance, protection within biofilms, and inoculum effects. For example, bacteria can remain viable by escaping the effects of traditional antibiotics, even at high concentrations for prolonged periods, by growing slowly or becoming dormant (7). These dormant persister cells represent a form of antibiotic tolerance and may be responsible for the recalcitrance of some infections. Current antibiotics are also suboptimal against complicated Gram-positive infections due to the recalcitrance of biofilms and inactivity against high-density and nongrowing cells (7). In vitro and in vivo inoculum effects where antibiotics are less effective against high cell densities have been described previously for β-lactams, glycopeptides, and lipopeptide antibiotics (8–10). In neutropenic murine thigh infection models with S. aureus and Streptococcus pneumoniae, the antibiotics vancomycin (VAN), ceftobiprole, daptomycin (DAP), and linezolid (LZD) were all found to be less effective against high bacterial burdens (11). Furthermore, the long-term treatment of complicated infections with current antibiotics undoubtedly contributes to the development and spread of resistance (12). Clinically, salvage therapy with a combination of antibiotics is recommended for patients with refractory or complicated infections caused by S. aureus and vancomycin-resistant enterococci (VRE) (13–15). However, the choice of antibiotics is largely empirical as support for specific combination regimens and optimal treatment strategies has not been conclusively demonstrated by appropriately powered randomized clinical trials.

Given the high mortality and nonresponse rates for antibiotic-resistant and complicated Gram-positive infections, new approaches and new drugs are needed. Acyldpepsipeptide (ADEP) antibiotics cause bacterial cells to self-digest through activation of caseinolytic protease P (ClpP) in a growth-phase and energy-independent manner (16, 17). This target-activating mechanism of action is unique compared to other classes of antibiotics. ADEPs are active against drug-resistant Gram-positive pathogens, including methicillin-resistant S. aureus (MRSA), VRE, and S. pneumoniae. As ClpP is not essential, resistance development occurs at a high rate in vitro, but this can be mitigated with antibiotic combinations. Indeed, the combination of acyldepsipeptide analog 4 (ADEP4) and rifampin (RIF) was effective in treating a mouse model with established MRSA biofilms where standard antibiotics failed (17). These data raise the possibility that a form of ClpP-activating combination therapy may yield clinical benefits compared to the current standard of care, especially in the context of treating complicated, antibiotic-resistant, and recalcitrant infections.

In this study, the antimicrobial activity and potential for resistance development were evaluated for ADEP4 antibiotic combinations against stationary-phase cultures of MRSA, methicillin-susceptible S. aureus (MSSA), vancomycin-intermediate S. aureus (VISA), borderline oxacillin (OXA)-resistant S. aureus (BORSA), and VRE isolates using 72 h time-dependent killing assays. Representative bactericidal and bacteriostatic antibiotics were partnered with ADEP4 and evaluated using a pharmacokinetic/pharmacodynamic (PK/PD) hollow-fiber infection model (HFIM) against high inocula of MRSA, MSSA, and VRE strains. The high inocula which were tested in these studies mimic severe infections with high bacterial burdens (e.g., 10⁹ to 10¹⁰ CFU/ml bacteria) and maximize the potential for resistance development.

RESULTS

MICs against recent clinical isolates of S. aureus, Enterococcus, and multidrug-resistant isolates of S. aureus.

A panel of recently isolated antibiotic-resistant Enterococcus and S. aureus isolates, collected in 2017 and 2018 from bloodstream infections (38%), urinary tract infections (23%), skin and skin structure infections (20.4%), pneumonia in hospitalized patients (12%), intra-abdominal infections (3.7%), and other infections (2.8%), was tested for sensitivity to ADEP4 in MIC assays. All tested isolates were sensitive to ADEP4 (Table 1). The MICs for ADEP4 was also determined for a panel of BORSA (n = 27) and VISA (n = 14) strains obtained from the CDC through the FDA Antibiotic Resistance Isolate Bank. In addition to the VAN and OXA resistance phenotypes, many of these isolates were nonsusceptible to DAP, RIF, and levofloxacin (LVX), among other drugs. Despite the high degree of multidrug resistance phenotypes, the BORSA and VISA isolates were all sensitive to ADEP4 (Table 1).

TABLE 1.

ADEP4 MICs against drug-resistant clinical isolates

Strain category	No. of isolates	ADEP4 MIC (μg/ml)
		Range	MIC50	MIC90
E. faecalis clinical isolates (VRE)	28	0.008–0.06	0.015	0.03
E. faecium clinical isolates (VRE)	27	0.03–0.12	0.06	0.12
MRSA clinical isolates	53	0.25–1	0.5	0.5
BORSA strainsa	27	0.06–0.5	0.25	0.5
VISA strainsb	14	0.25–1	0.5	1

^aStaphylococcus with borderline oxacillin susceptibility panel (29).

^bVancomycin-intermediate Staphylococcus aureus panel (29).

Stationary-phase time-killing assays.

ADEP4 was tested alone and in combination with approved antibiotics in time-kill assays against stationary-phase cultures of MRSA, MSSA, VISA, and BORSA. The antibiotics tested and the concentrations used are shown in Tables 2 and 3 (see also Table S1 in the supplemental material). Each of the approved antibiotics tested, including members of bactericidal classes, killed less than 2 log₁₀ CFU/ml of a stationary-phase culture of MRSA ATCC 33591 over 72 h (Fig. 1; see also Fig. S1 in the supplemental material). ADEP4 alone killed 3 log₁₀ CFU/ml over 24 h; however, there was subsequent regrowth due to ADEP4 resistance as samples from the 72 h time point grew on agar plates containing 10-fold the MIC of ADEP4. The combination of ADEP4 with each antibiotic except for VAN resulted in a greater than 3 log₁₀ CFU/ml reduction in viable counts of MRSA over 72 h (Fig. 1; see also Fig. S1). The addition of a second antibiotic prevented resistance outgrowth for every antibiotic except VAN. A slight increase in CFU counts per milliliter was observed between 48 and 72 h for the DAP combination.

TABLE 2.

MICs against MRSA strain ATCC 33591 and antibiotic concentrations used for time-kill studies

Antibiotic	MIC (μg/ml)	Exposure (μg/ml)	Multiple of MIC
ADEP4	0.125–0.25	4	16×
Tigecycline	1	7.81	∼8×
Linezolid	1–2	15.63	∼8×
Daptomycina	1	15.63	∼16×
Vancomycin	2	20	10×

^aCa²⁺ at 50 μg/ml was included with daptomycin in MIC and time-kill assays.

TABLE 3.

MICs against MSSA ATCC 29213 and antibiotic concentrations used for time-kill studies

Antibiotic	MIC (μg/ml)	Exposure (μg/ml)	Multiple of MIC
ADEP4	0.25	2.5	10×
Oxacillin	0.25	2.5	10×
Linezolid	2	20	10×

FIG 1.

Time-kill studies for stationary-phase MRSA ATCC 33591. The antibiotics tested were acyldepsipeptide analog 4 (ADEP4), daptomycin (DAP), linezolid (LZD), tigecycline (TGC), and vancomycin (VAN) and were used singly (A) or in combinations (B). Antibiotic concentrations are given in Table 2. Ca2⁺ at 50 μg/ml was included in all DAP-exposed cultures. Data represent averages of results from at least three independent experiments. The error bars represent standard deviations, and the x axis represents the limit of detection.

Stationary-phase cultures of MSSA were exposed to ADEP4 and OXA, alone and in combination (Fig. 2). The combination of ADEP4 and OXA reduced viable counts by over 5 log₁₀ CFU/ml in the first 48 h. A slight but reproducible increase in the CFU level was detected between 48 and 72 h.

FIG 2.

Time-kill studies for stationary-phase MSSA ATCC 29213. The antibiotics tested were acyldepsipeptide analog 4 (ADEP4), linezolid (LZD), and oxacillin (OXA). The antibiotic concentrations are given in Table 3. Data represent averages of results from at least three independent experiments. Error bars represent standard deviations, and the x axis is the limit of detection.

Next, the killing kinetics of ADEP4 antibiotic combinations were determined for BORSA and VISA strains. Two extensively multidrug-resistant strains from the MIC panel were chosen. According to the CDC, BORSA strain 0485 is resistant to clindamycin, erythromycin (ERY), LVX, penicillin (PEN), and RIF. VISA strain 0226 has intermediate resistance to VAN; is DAP nonsusceptible; and is resistant to cefoxitin, ERY, OXA, PEN, and RIF. In combination with LZD, ADEP4 reduced viable counts of stationary-phase cultures of these strains by greater than 4 log₁₀ CFU/ml within 24 h and by 5 to 6 log₁₀ CFU/ml over 72 h (Fig. 3). Resistant outgrowth was not detected for the VISA 0226 strain in the time-dependent killing studies conducted with ADEP4 alone.

FIG 3.

Time-kill studies for stationary-phase BORSA AR bank 0485 (A) and VISA AR bank 0226 (B) (29). The antibiotics tested were acyldepsipeptide analog 4 (ADEP4) and linezolid (LZD). Drug concentrations are based on the peak concentrations for hollow-fiber studies—ADEP4 at 16.4 μg/ml and LZD at 10.42 μg/ml. Data represent averages of results from at least three independent experiments. The error bars represent standard deviations, and the x axis represents the limit of detection.

In vitro PK/PD model.

The time-kill data suggested that multiple classes of antibiotics may be suitable partners for ClpP activators. However, resistance development is more likely to emerge in a dynamic system in which drug concentrations fluctuate above and below the MIC. Thus, the efficacy of ADEP4 antibiotic combinations was tested in the dynamic HFIM using PK parameters based on human dosing regimens and their resulting antibiotic exposures. The β-lactams ampicillin (AMP) and OXA were chosen as bactericidal agents and LZD was selected as a bacteriostatic drug for S. aureus and E. faecalis based on the time-kill data and their clinical use against relevant Gram-positive pathogens.

The desired exposure for each antibiotic was established in the HFIM prior to adding bacteria to the hollow-fiber cartridge. PK parameters, including peak concentrations, area under the concentration-time curve (AUC), and half-lives, were optimized for each regimen, and on average, drug concentrations were within 20% of their desired targets (Fig. S2). Survival of MRSA, MSSA, and VRE after exposure to simulated human doses of various antibiotics over 96 h was plotted (Fig. 4), and log decreases in viability were summarized (Table 4). The approved antibiotics LZD, AMP, and OXA were ineffective when tested alone, and the observed killing was less than 1 log₁₀ CFU/ml over 5 days. In contrast, ADEP4 caused an initial killing of 1 to 4 log₁₀ CFU/ml within 48 h, but outgrowth of resistance was apparent within 96 h. ADEP4 initially exhibited more killing against VRE than against S. aureus. Plating on ADEP4-containing plates confirmed that resistant mutants took over the culture within the first 2 days of the experiment (Table S2). Surviving colonies recovered from plates containing ADEP4 had MICs of ≥8 μg/ml.

FIG 4.

Hollow-fiber infection model against high-density cultures of MRSA ATCC 33591 (A), MSSA ATCC 29213 (B), and VRE ATCC 700802 (C). Antibiotics tested were acyldepsipeptide analog 4 (ADEP4), ampicillin (AMP), linezolid (LZD), and oxacillin (OXA). Data represent averages of results from two independent experiments. The error bars represent standard deviations, and the x axis represents the limit of detection.

TABLE 4.

Activity of antibiotics against MRSA, MSSA, and VRE over 96 h in a hollow-fiber infection model

Study	Mean log10 CFU/ml ± SD
	MRSA ATCC 33591		MSSA ATCC 29213		VRE ATCC 700802
	96 h	Change from 0 h	96 h	Change from 0 h	96 h	Change from 0 h
Growth control	10.79 ± 0.03	+0.85 ± 0.02	11.05 ± 0.03	+1.96 ± 0.11	10.70 ± 0.02	+0.95 ± 0.01
ADEP4	10.85 ± 0.07	+0.42 ± 0.09	10.98 ± 0.05	+1.22 ± 0.21	10.62 ± 0.04	+0.67 ± 0.04
LZD	10.27 ± 0.23	−0.49 ± 0.16	NDb	ND	9.30 ± 0.05	−0.75 ± 0.02
ADEP4 + LZD	5.52 ± 0.17	−4.56 ± 0.23	ND	ND	3.65 ± 0.01	−6.13 ± 0.07
OXA	ND	ND	10.05 ± 0.09	−0.04 ± 0.13	ND	ND
ADEP4 + OXA	ND	ND	2.32 ± 0.62a	−8.06 ± 0.70a	ND	ND
AMP	ND	ND	ND	ND	9.20 ± 0.16	−0.85 ± 0.23
ADEP4 + AMP	ND	ND	ND	ND	1.00 ± 0.00a	−8.80 ± 0.05a
LZD + AMP	ND	ND	ND	ND	9.03 ± 0.03	−1.07 ± 0.03

^aThe limit of detection was 10 CFU/ml.

^bND, not determined.

The combination of ADEP4 and LZD caused a 4 log₁₀ CFU/ml reduction and a 6 log₁₀ CFU/ml reduction over 96 h against MRSA (Fig. 4A) and VRE (Fig. 4C), respectively. The combination of ADEP4 and OXA caused an 8 log₁₀ CFU/ml reduction over 96 h against MSSA (Fig. 4B). The bactericidal activity appeared to be initially mediated by ADEP4, with OXA killing the remaining cells as the culture’s density declined. The combined effect was killing at a level near the limit of detection, with no resistance development detected for either drug. The combination of ADEP4 and AMP against VRE caused an 8 to 9 log₁₀ CFU/ml decrease to the limit of detection within 52 h (Fig. 4C). In sharp contrast, the combination of LZD and AMP did not cause a reduction in log₁₀ CFU per milliliter for VRE over 96 h.

DISCUSSION

ClpP-activating antibiotics may have unique clinical benefits because the target-activating mechanism of action is distinct compared to currently approved agents. Combining ClpP-activating agents with traditional antibiotics may prevent resistance development and enhance the effectiveness of current therapies. Using the representative ClpP activator ADEP4, the antimicrobial effects of activation of ClpP alone and in combination with traditional antibiotics were determined against a panel of Gram-positive pathogens, including those that are drug resistant. MIC testing of recent clinical isolates and of a panel of drug-resistant isolates from the CDC indicated no preexisting resistance to ADEP4, even among highly problematic clinical strains of MRSA, VISA, BORSA, and VRE. The novel mechanism of action and the associated virulence defects described for ClpP mutations support these findings (18, 19). In time-dependent killing experiments, ADEP4 was bactericidal against high-density cultures of S. aureus, including multidrug-resistant VISA and BORSA strains. Approved drugs, including VAN, OXA, tigecycline (TGC), and DAP, were largely ineffective against high-density cultures in stationary-phase time-dependent killing studies, even though the same strains were susceptible to each drug according to MIC assays. The limited effectiveness of most antibiotics against high-density cultures is not widely recognized. In contrast, ADEP4 was bactericidal, an indication that ClpP dysregulation is detrimental even under nongrowing, stationary-phase conditions. The use of high-density cultures also provides an improvement in the ability to detect resistance development. Indeed, outgrowth of ADEP4-resistant mutants was detected within 72 h for MRSA strain ATCC 33591, MSSA strain ATCC 29213, and BORSA strain 0485 but not for VISA strain 0226 in the time-dependent killing assays. This raises the possibility that there are increased fitness costs for ClpP mutations in this strain or for VISA backgrounds in general. A slight increase in the CFU count per milliliter was observed between 48 and 72 h for ADEP4 combinations with DAP and OXA against MRSA and MSSA, respectively. The rebound with DAP was possibly due to the inoculum effect lowering drug activity or to drug instability (10, 20), whereas the slight CFU increase in OXA combination experiments may be due to the instability of β-lactams (21). ADEP4 resistance outgrowth was not detected for any of the antibiotic combinations tested, with the exception of ADEP4 with VAN, indicating there are multiple classes of antibiotics which may be suitable for partnering with ClpP activators to prevent resistance development. It is unclear why VAN fails to provide protection from ClpP mutant outgrowth. Antagonism is one possibility, as ClpP mutations were reported to have reduced susceptibility to VAN (22). A previous study found that oritavancin, another member of the glycopeptide class, was unable to prevent ADEP4-resistant outgrowth in an in vitro VRE biofilm killing experiment (23), which may indicate that members of this class represent poor partners for ClpP-activating antibiotics in general.

Based on the time-kill studies, representative bactericidal and bacteriostatic antibiotics were selected for evaluation using a HFIM. High inocula were achieved by infusing the cultures with fresh media overnight prior to antibiotic challenge. In the HFIM, even repeated exposures to traditional antibiotics were insufficient for bactericidal activity under these conditions. In the literature, typical hollow-fiber studies begin antibiotic challenges with densities between 10⁶ and 10⁸ CFU/ml, with studies conducted at the higher end of that range being considered to represent high density (24–26). We are unaware of any publications testing the effects of antibiotics on higher cell densities and speculate that the ineffectiveness of traditional antibiotics under these conditions may be the reason.

The pharmacodynamic effects of ADEP4 containing antibiotic combinations in the HFIM depended on both the pathogen and the partnering antibiotic tested; however, some general trends were noted. Initially, the killing kinetics resulting from the drug combinations appeared to be mediated by ADEP4 as they closely matched the effects caused by ADEP4 alone. The effect of the partnering antibiotics was apparent only at subsequent time points. ADEP4 in combination with the bacteriostatic antibiotic LZD caused 4 and 6 log₁₀ CFU/ml of killing against MRSA and VRE, respectively; however, additional killing was not detected, as the survivors consisted entirely of ADEP4-resistant mutants. The presence of LZD prevented the outgrowth of ADEP4-resistant mutants but did not facilitate additional killing. Dual-drug ADEP4 and LZD resistance did not occur within the 96 h time frame of these experiments. Combining ADEP4 with the bactericidal agents OXA or AMP resulted in more than 8 log₁₀ CFU/ml of killing of MSSA and VRE cultures, respectively. The pharmacodynamic effects were more rapid and prominent for VRE, as the bacterial CFU dropped below the limit of detection by 52 h. For MSSA, while more than 8 log₁₀ CFU/ml of killing was achieved for the combination of ADEP4 and OXA by 96 h, colony counts remained above the limit of detection. The different pharmacodynamic effects observed for MSSA and VRE may be explained by differences in ADEP4 sensitivity. Indeed, the ADEP4 MIC for MSSA strain ATCC 29213 is 0.25 μg/ml, which is more than 10-fold higher than the MIC for VRE ATCC 700802 at 0.016 μg/ml.

There were some limitations to these studies, like others with similar designs. Due to practical considerations, the HFIM studies were run for a duration of only 96 h. The pharmacodynamic effects observed during this time period were encouraging; however, extending the studies would enable us to confirm bacterial eradication and also increase the ability to detect resistance development (27). It is also likely that optimal ADEP4 pharmacodynamic exposures were not achieved in these studies. The exposures were based on previously published animal dosing regimens, as potential human exposure levels and the pharmacodynamic driver of efficacy are currently unknown. In our studies, ADEP4 concentrations were predicted to drop below the MIC after 13.25 h for MRSA and 19.2 h for VRE. Other ClpP-activating antibiotics may also have pharmacokinetics superior to ADEP4 pharmacokinetics (28).

In summary, ADEP4 was active against panels of multidrug-resistant S. aureus as well as MSSA and VRE. Testing ADEP4 in combination with approved antibiotics enhanced bactericidal activity without an outgrowth of resistant mutants both in time-dependent kill studies and in the HFIM. These results suggest that there is potential to treat complicated, drug-resistant Gram-positive infections with ClpP-activating antibiotic combinations.

MATERIALS AND METHODS

Bacterial strains.

S. aureus strains were grown in Mueller-Hinton broth (MHB) and on Mueller-Hinton agar (MHA) plates. E. faecalis was cultured in brain heart infusion (BHI) broth and agar. Liquid cultures were grown at 37°C with shaking at 180 rpm. Cultures grown on agar plates were incubated at 37°C. Bacterial stocks were maintained at –80°C in 30% glycerol.

MRSA strain ATCC 33591, MSSA strain ATCC 29213, and VRE faecalis strain ATCC 700802 (V583) were obtained from the American Type Culture Collection. VISA and BORSA isolates were obtained from the CDC & FDA Antibiotic Resistance Isolate Bank (29).

Antimicrobials.

Stock solutions of LZD, duloxetine (DLX), fusidic acid (FA), rifabutin (RFB), trimethoprim-sulfamethoxazole (SXT), and TGC, as well as of ADEP4, which was custom synthesized, were prepared in dimethyl sulfoxide (DMSO) at 10 mg/ml and stored at –20°C. AMP, DAP, VAN, OXA, and fosfomycin (FOF) were dissolved in sterile deionized water (0.9% saline solution for DAP), and single-use aliquots were stored at –20°C. For hollow-fiber experiments, AMP and OXA were prepared fresh daily in deionized water at 10 mg/ml.

MIC assays.

MIC assays were performed using the broth microdilution method (30). Briefly, 2-fold serial dilutions of antibiotics were prepared in DMSO or water as appropriate at 100× the final concentration. The diluted drugs were subsequently added into cation-adjusted MHB (CAMHB) in a 96-well plate. Colonies from an agar plate were resuspended in CAMHB, and the optical density (OD) at 600 nm was measured. The cultures were diluted to achieve a final concentration of approximately 5 × 10⁵ CFU/ml in the MIC plate. Plates were incubated at 37°C for 18 to 20 h (24 h for VAN). The MIC was the lowest concentration of drug that inhibited growth based on the OD at 600 nm. The medium was supplemented with 50 μg/ml Ca²⁺ for DAP assays and 25 μg/ml d-glucose-6-phosphate for FOF assays. Additional MIC testing was performed at JMI Laboratories (North Liberty, IA) using CLSI methodology.

Stationary-phase time-dependent killing assays.

S. aureus colonies grown on MHA were inoculated into 25 ml of BHI broth and grown for 18 to 20 h in a baffled flask at 37°C in a shaking incubator. Cultures were subsequently divided into 3-ml aliquots in 14-ml polystyrene culture tubes. Prior to the addition of antibiotics, an aliquot was removed, diluted, and plated for the initial colony counts. Antibiotics were added at various concentrations (Table 2) (see also Table 3 and Table S1 in the supplemental material). At each time point, an aliquot was removed, washed with phosphate-buffered saline (PBS), serially diluted, and plated for colony counts. Samples from the 72 h time point were also plated on agar containing 10-fold the MIC of ADEP4 to determine resistance development. Plates were incubated 24 to 48 h at 37°C prior to enumeration. Data were log transformed, and the means and standard deviations were plotted using Prism software (version 8; GraphPad, San Diego, CA).

In vitro PK/PD model.

Dynamic time-kill experiments were performed in a HFIM with a starting density of approximately 10¹⁰ CFU/ml. Hollow-fiber C3008 cellulosic cartridges were obtained from FiberCell Systems (New Market, MD). MHB medium was used for experiments with S. aureus, and BHI medium was used for experiments with E. faecalis. Fresh medium was supplied and removed from the central compartment using a peristaltic pump, and a duet pump continuously circulated media between the central compartment and the inoculated cartridge. A supplemental compartment was used in experiments involving two antibiotics with different half-lives to keep both drugs at the target concentrations throughout the experiments. The flow rates, drug concentrations, and PK parameters were determined from initial PK experiments. Cultures of VRE, MRSA, or MSSA were inoculated into the extracapillary space of the cartridge 1 day prior to the start of the experiment to allow time for the culture to reach high density. Antibiotics were added through injection ports connected to the central and supplement compartments. Hollow-fiber experiments were performed at 37°C. The data presented represent averages of results from the two independent experiments.

Pharmacodynamics.

Bacterial cultures were sampled directly from the cartridge at various time points from 0 to 96 h. Each sample was washed with PBS to minimize antibiotic carryover during plating. Dilution plating was performed in triplicate, and colony counts were enumerated after plates were incubated at 37°C for 24 h. Data were log transformed, and means and standard deviations were plotted using Prism software.

Pharmacokinetics.

Hollow-fiber PK optimizations were initially performed in the absence of bacteria to determine if the concentration of each antibiotic was on target. Antibiotic parameters were based on the following: AMP (2 g every 6 h [q6h]: maximum concentration of free, unbound fraction in serum [fC_max]: 100 μg/ml, half-life: 1.5 h, protein binding: 18%), LZD (600 mg q12h: fC_max: 10.42 μg/ml, half-life: 4.8 h, protein binding: 31%), OXA (1 g q4h: fC_max: 5.16 μg/ml, half-life: 0.75 h, protein binding: 94%), and ADEP4 (2 doses daily, 4 h apart: 25 mg/kg followed by a second dose at 35 mg/kg; first dose C_max: 11.7 μg/ml, second dose C_max: 16.4 μg/ml, half-life: 1.5 h). The exposures of conventional antibiotics were modeled to simulate human PK parameters on the basis of package inserts and published data (31–35). ADEP4 concentrations were derived from the doses which were effective in animal models based on previously published methods (17).

Pharmacokinetic analysis.

Antibiotic concentrations were quantified at Drumetix Laboratories (Greensboro, NC) using validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) protocols. Samples were purified by protein precipitation with acetonitrile after propranolol was added as an internal standard. After vortex mixing and centrifugation, supernatant was injected onto an AB Sciex API 4000 (API 3200 for OXA) LC-MS/MS system. LC separation was carried out on a Phenomenes Polar-RP column with a gradient elution (mobile phase A: 0.1% acetic acid–1 mM ammonium acetate–water; mobile phase B: acetonitrile). Except for OXA, analytes and IS were detected by the use of a triple-quadrupole mass spectrometer operating in electrospray positive-ion mode (negative-ion mode for OXA) with detection by selected reaction monitoring (multiple reaction monitoring [MRM]) using the transition m/z 771.2 to 478.3 for ADEP4; 350.1 to 106 for AMP; 400.2 to 159 for OXA; 338.2 to 296 for LZD; and 226.1 to 116.1 for propranolol. The peak concentrations, half-lives, and AUC of drugs were determined by the trapezoidal method using Phoenix software (version 8.1; Certara, Princeton, NJ).

Resistance development.

Samples from the 0, 24, 48, 72, and 96 h time points in the HFIM were plated on agar containing 5-fold to 10-fold the MIC of ADEP4, AMP, LZD, or OXA alone or in combination as appropriate for each experiment. The antibiotic-containing plates were incubated at 37°C for 24 to 48 h. Colonies were counted and compared to antibiotic-free agar plate counts to determine the resistance frequency. MIC testing was performed on resistant colonies to confirm and determine the extent of resistance.