Combination with a FtsZ inhibitor potentiates the in vivo efficacy of oxacillin against methicillin-resistant Staphylococcus aureus

Abstract

Oxacillin is a first-line antibiotic for the treatment of methicillin-sensitive Staphylococcus aureus (MSSA) infections but is ineffective against methicillin-resistant S. aureus (MRSA) due to resistance. Here we present results showing that co-administering oxacillin with the FtsZ-targeting prodrug TXA709 renders oxacillin efficacious against MRSA. The combination of oxacillin and the active product of TXA709 (TXA707) is associated with synergistic bactericidal activity against clinical isolates of MRSA that are resistant to current standard-of-care antibiotics. We show that MRSA cells treated with oxacillin in combination with TXA707 exhibit morphological characteristics and PBP2 mislocalization behavior similar to that exhibited by MSSA cells treated with oxacillin alone. Co-administration with TXA709 renders oxacillin efficacious in mouse models of both systemic and tissue infection with MRSA, with this efficacy being observed at human-equivalent doses of oxacillin well below that recommended for daily adult use. Pharmacokinetic evaluations in mice reveal that co-administration with TXA709 also increases total exposure to oxacillin. Viewed as a whole, our results highlight the clinical potential of repurposing oxacillin to treat MRSA infections through combination with a FtsZ inhibitor.

Graphical Abstract

Oxacillin Kills Vancomycin-Resistant Staphylococcus aureus (VRSA) When Combined with the FtsZ Inhibitor TXA707

Introduction

Oxacillin is a member of the β-lactam family of semi-synthetic penicillins that were the gold standard for treatment of staphylococcal infections in the clinic [1–3]. Oxacillin was developed as a 2nd-generation penicillin capable of targeting Staphylococcus aureus isolates resistant to 1st-generation penicillins [4, 5]. These penicillin-resistant S. aureus isolates derived their resistance phenotype from the acquisition of genes encoding for β-lactamase enzymes capable of hydrolyzing the β-lactam ring of penicillin [4, 6, 7]. The semi-synthetic penicillins, which include oxacillin, nafcillin, and methicillin, were designed to contain bulky substituents that provided steric hindrance to the binding of staphylococcal β-lactamases [4, 8, 9]. Such β-lactamase-resistant β-lactams became first-line agents for the treatment of S. aureus infections in the clinic [4, 10]. Moreover, as oxacillin can be delivered parenterally, it has proven particularly useful for the treatment of invasive staphylococcal infections that mandate intravenous antibiotic administration [3].

In recent years, the emergence and spread of methicillin-resistant S. aureus (MRSA) infections has limited the clinical value of oxacillin [11, 12], with its utility currently restricted to the treatment of methicillin-sensitive S. aureus (MSSA) infections [13, 14]. Combination therapy with a synergistic drug offers a strategy for repurposing antibiotics that have been rendered clinically ineffective due to resistance [15–17]. Synergistic combination therapy also presents additional advantages that include a reduced potential for both toxicity and the emergence of resistance. FtsZ inhibitors have the potential to act synergistically with select β-lactam antibiotics against MRSA [18–20]. β-lactam antibiotics target the penicillin binding proteins (PBPs), a family of enzymes that catalyze key steps in the synthesis of the peptidoglycan component of bacterial cell walls [21–23]. S. aureus expresses four native PBPs (PBP1, PBP2, PBP3, and PBP4) [21, 23, 24]. Our previous studies have shown that β-lactam antibiotics with a high affinity for S. aureus PBP2 exhibit the greatest degree of synergy with the benzamide FtsZ inhibitor TXA707 in vitro [18] and its 1-methylpiperidine-4-carboxamide prodrug (TXA709) in vivo [19, 25].

Oxacillin targets S. aureus PBP2 with a high degree of affinity [18]. Here we demonstrate that TXA707 potentiates the bactericidal activity of oxacillin against a broad range of MRSA clinical isolates that have become resistant to current standard-of-care antibiotics, including vancomycin-intermediate S. aureus (VISA), vancomycin-resistant S. aureus (VRSA), and linezolid-resistant S. aureus (LRSA). We also show that co-administration of the TXA709 prodrug potentiates the in vivo efficacy of oxacillin in mouse models of both systemic and tissue (thigh) infection with MRSA, with this in vivo potentiation being observed at equivalent human doses of oxacillin well below the recommended daily dose of the drug for the treatment of staphylococcal infections [10]. In addition, a pharmacokinetic analysis reveals that co-administration with TXA709 in mice increases exposure to oxacillin. Taken together, our results highlight combination therapy with the FtsZ-targeting prodrug TXA709 as a promising means of repurposing oxacillin for use in the clinical management of MRSA infections.

Results and discussion

TXA707 potentiates the bactericidal activity of oxacillin against a panel of 45 clinical isolates of MRSA, VRSA, VISA, and LRSA

Given that β-lactams targeting S. aureus PBP2 with high affinity exhibit the greatest synergy with TXA707 [18], we sought to evaluate the potential of TXA707 to potentiate the activity of oxacillin against clinical isolates of MRSA, VRSA, VISA, and LRSA. For these studies we selected 13 USA100 MRSA isolates, 11 USA300 MRSA isolates, 11 VRSA isolates, 6 VISA isolates, and 4 LRSA isolates for each subtype of resistant S. aureus. Our studies confirmed that all 45 isolates were intrinsically resistant to oxacillin, with corresponding modal MIC values for each class of isolates ranging from 128 to 512 μg/mL (Table 1). Significantly, the presence of 0.5x MIC TXA707 resulted in significantly lower modal MIC values (ranging from 0.063 to 0.5 μg/mL) for oxacillin against every isolate class tested (Table 1). Thus, TXA707 potentiates the activity of oxacillin on the order of 500- to 8000-fold against MRSA clinical isolates of S. aureus that are normally resistant to oxacillin and even the standard-of-care antibiotics vancomycin and linezolid.

Table 1.

Impact of TXA707 on the activity of oxacillin against clinical isolates of MRSA, VRSA, VISA, and LRSA

Isolates	MIC of Oxacillin Alone (μg/mL)		MIC of Oxacillin in the Presence of TXA707 at 0.5x MICa (μg/mL)
	Range	Modal	Range	Modal
MRSA, USA100 (n = 13)	128–512	256	0.063–2	0.125
MRSA, USA300 (n = 11)	32–256	128	0.063–2	0.25
VRSA (n = 11)	64–512	512	0.063–1	0.063
VISA (n = 6)	128–512	256	0.125–4	0.125/0.25b
LRSA (n = 4)	32–512	512	0.125–0.5	0.5

^a0.5x MIC of TXA707 reflects concentrations ranging from 0.5 to 1 μg/mL

^bBimodal behavior

In addition to MIC assays, we also used a time-kill approach to characterize the impact of TXA707 on the activity of oxacillin against MRSA, VRSA, VISA, and LRSA isolates. For these studies, we selected one USA100 MRSA isolate (NRS705), one USA300 MRSA isolate (NRS643), one VRSA isolate (VRS5), one VISA isolate (NRS27), and one LRSA isolate (NRS127) as representative examples, with the resulting kill curves for each isolate being shown in Fig. 1. For each of the isolates investigated, neither TXA707 alone at 0.5 μg/mL (0.5x MIC) nor oxacillin alone at 2 μg/mL (the clinical breakpoint concentration for oxacillin activity against S. aureus [26]) resulted in any killing, with varying degrees of bacterial growth being observed instead. In striking contrast, the combination of TXA707 and oxacillin is associated with bactericidal activity in a dose-dependent manner. Significantly, for all five isolates, treatment with oxacillin at a concentration equivalent to its clinical breakpoint (2 μg/mL) and TXA707 results in approximately 5-logs of kill in 24 h. These collective observations establish that the enhanced actions of oxacillin against the MRSA, VRSA, VISA, and LRSA isolates afforded by combination with TXA707 are bactericidal.

Fig. 1.

Time-kill curves for MRSA NRS705 (USA100) (A), MRSA NRS643 (USA300) (B), VRSA VRS5 (C), VISA NRS27 (D), and LRSA NRS127 (E) showing the potentiation of oxacillin activity resulting from the synergistic combination with TXA707. Bacteria were treated with DMSO vehicle, oxacillin alone at 2 μg/mL (0.004x–0.03x MIC), TXA707 alone at 0.5 μg/mL (0.5x MIC), or a combination of TXA707 at 0.5 μg/mL and oxacillin at a range of concentrations from 0.125–2 μg/mL

The combination of TXA707 and oxacillin also reduces the frequency of resistance (FOR) in MRSA, VISA, VRSA, and LRSA isolates

Combination therapy can offer an additional advantage of reduced potential for the emergence of resistance. To examine how the combination of TXA707 with oxacillin impacts the frequency of resistance (FOR) in S. aureus, we determined the FOR in each of the five clinical isolates described above following treatment with 4 μg/mL (4x MIC) TXA707 alone or in combination with oxacillin at 2 μg/mL. The FOR to TXA707 alone was on the order of 10⁻⁹ to 10⁻⁸ in all five isolates examined (Table 2). Our previous studies have indicated that this FOR results from specific mutations in the ftsZ gene [19, 25]. In marked contrast to the FOR observed with TXA707 alone, the FOR observed with the combination of TXA707 and oxacillin was below detectable levels in four of the five isolates examined and was reduced by an order of magnitude (to a value on the order of 10⁻¹⁰) in the fifth isolate (Table 2). Thus, combination with TXA707 not only potentiates the bactericidal activity of oxacillin against MRSA, VRSA, VISA, and LRSA isolates, but also reduces the potential for the emergence of resistance in these isolates.

Table 2.

Resistance frequencies of representative MRSA, VISA, VRSA, and LRSA isolates to TXA707 alone or in combination with oxacillin

Isolatea	Frequency of Resistance (FOR) to:
	4 μg/mL TXA707 Aloneb	4 μg/mL TXA707 + 2 μg/mL Oxacillinc
MRSA NRS705 (USA100)	2.59 × 10−8	<9.95 × 10−10
MRSA NRS643 (USA300)	7.49 × 10−9	4.28 × 10−10
VRSA VRS5	3.86 × 10−8	<6.02 × 10−10
VISA NRS27	5.75 × 10−8	<5.52 × 10−10
LRSA NRS127	4.46 × 10−8	<3.66 × 10−10

^aEach isolate is indicated by its NARSA designation

^b4 μg/mL TXA707 reflects 4x MIC

^c2 μg/mL oxacillin reflects the clinical breakpoint concentration of the drug for activity against S. aureus

Changes in cell morphology and PBP2 localization induced in MRSA by oxacillin when combined with TXA707 are similar to those induced in MSSA by oxacillin alone

We used fluorescence microscopy to explore the mechanism underlying the potentiation of oxacillin activity against MRSA by TXA707. Recall that our previous studies revealed β-lactam antibiotics with a high affinity for PBP2 to be associated with the greatest degree of synergy with TXA707 [18]. We therefore generated strains of MRSA LAC (USA300) and MSSA RN4220 that express a sfGFP-tagged PBP2 protein and probed the impact of treatment with oxacillin and TXA707 alone or in combination on the cellular localization of PBP2. MRSA LAC (sfGFP-PBP2) cells were treated for 3 h with either vehicle, 0.25 μg/mL (0.25x MIC) TXA707, 0.06 μg/mL (0.001x MIC) oxacillin, or a combination of 0.25 μg/mL TXA707 and 0.06 μg/mL oxacillin, and then examined by differential interference contrast (DIC) and fluorescence microscopy (Fig. 2). Corresponding studies with MSSA RN4220 (sfGFP-PBP2) cells are included for comparative purposes.

Fig. 2.

DIC and fluorescence micrographs of MSSA RN4220 cells (A–H) and MRSA LAC cells (I–P) expressing sfGFPPBP2. Cells were treated for 3 h with DMSO vehicle (A, E, I, M), 0.06 μg/mL TXA707 alone (B, F, J, N), 0.06 μg/mL oxacillin alone (C, G, K, O), or a combination of 0.06 μg/mL TXA707 and 0.06 μg/mL oxacillin (D, H, L, P). Scale bars reflect 1 μm

Vehicle-treated MSSA and MRSA cells appear as cocci 0.6–0.7 μm in diameter, with a majority having distinct septa at mid-cell (Fig. 2A, E, I, M). This morphology is typical of normally dividing S. aureus. Note that much of the fluorescence signal associated with sfGFP-PBP2 is localized to the septum at midcell, an observation consistent with previous reports identifying the septum of S. aureus as the primary site of peptidoglycan synthesis and PBP localization [23, 27–30]. MSSA and MRSA cells treated with sub-MIC concentrations of TXA707 alone also exhibited normal PBP2-associated septa in a manner similar to that of vehicle-treated cells (Fig. 2B, F, J, N), albeit appearing slightly enlarged (0.8–0.9 μm in diameter). MRSA cells treated with 0.06 μg/mL oxacillin alone also exhibited a similar morphology of normal PBP2-associated septa (Fig. 2J, N), consistent with their oxacillin-resistant phenotype. Notably, the morphology of MSSA cells was markedly impacted by exposure to 0.06 μg/mL oxacillin, with the oxacillin-treated MSSA cells being 2-3-fold larger (1.5–2.0 μm in diameter) than vehicle-treated cells and lacking the characteristic septa at midcell (Fig. 2C, G). In addition, the PBP2 fluorescence signal in these cells is primarily associated with the peripheral cell wall, indicating that oxacillin treatment of MSSA cells induces the mislocalization of PBP2 from the septa to the peripheral cell wall. Consistent with previous studies [31], this behavior suggests that oxacillin treatment of MSSA cells causes PBP2 mislocalization and the concomitant disruption of cell division. MSSA cells treated with the combination of oxacillin and TXA707 appear morphologically similar to the MSSA cells treated with oxacillin alone (Fig. 2D, H).

While treatment of MRSA cells with TXA707 or oxacillin alone had minimal impact on cell morphology and PBP2 localization, treatment of MRSA cells with the combination of oxacillin and TXA707 induced marked changes in both cell morphology and PBP2 localization (Fig. 2L, P) similar to those induced in MSSA cells by treatment with oxacillin alone. The similar morphologies and PBP2 localization patterns of oxacillin-treated MSSA cells and combination-treated MRSA cells suggests that the presence of TXA707 renders MRSA susceptible to the same antibacterial actions of oxacillin as MSSA, even at an oxacillin concentration well below the 2.0 μg/mL clinical breakpoint for antistaphylococcal activity. Thus, TXA707 effectively repurposes oxacillin for use against normally resistant staphylococcal strains.

Administration in combination with TXA709 renders oxacillin efficacious in vivo in both systemic and tissue models of MRSA infection in mice

We used a mouse model of systemic (peritonitis) infection with MRSA to determine whether the TXA707-induced potentiation of oxacillin activity against MRSA observed in vitro is also observable in vivo. For the in vivo studies, TXA707 was administered in the form of its prodrug (TXA709), the latter being more soluble and easier to formulate for in vivo administration relative to TXA707 [25].

In the systemic infection experiments, groups of five mice were infected intraperitoneally with a lethal inoculum of MRSA NRS705 (USA 100), followed by intravenous administration of either vehicle, TXA709 alone, oxacillin alone, or the combination of both oxacillin and TXA709. TXA709 was administered at a fixed dose of 20 mg/kg, while oxacillin was administered at three escalating doses of 60, 90, and 150 mg/kg. Treatment of MRSA-infected mice with vehicle, TXA709 alone, or each dose of oxacillin alone was associated with 0% survival (0 out of 5 mice) (Fig. 3). By contrast, co-administration of TXA709 with the different doses of oxacillin revealed a dose-dependent impact on survival. Combination treatment of 20 mg/kg TXA709 with 60, 90, and 150 mg/kg of oxacillin resulted in 0%, 40%, and 100% survival, respectively. Thus, doses of oxacillin and TXA709 that are ineffective against systemic MRSA infections when administered alone become highly efficacious when administered in combination with TXA709. These findings confirm that the potentiation of oxacillin activity by TXA707 observed in vitro (Fig. 1) is maintained in vivo (Fig. 3).

Fig. 3.

TXA709 potentiates the in vivo efficacy of oxacillin against MRSA NRS705 (USA100) in a mouse peritonitis model of systemic infection. All agents were administered intravenously at the indicated doses

It is of interest to gauge how the oxacillin doses used in our mouse models of systemic MRSA infection relate to oxacillin doses that have been shown to be safe and effective for use in humans. To this end, we used an allometric scaling method by which a human-equivalent dose can be estimated by dividing the corresponding mouse dose by 12.3 [32]. Applying this method to the 60, 90, and 150 mg/kg doses used in our studies yields human-equivalent oxacillin doses of 4.9, 7.3, and 12.2 mg/kg, respectively. Thus, TXA709 rendered oxacillin 100% efficacious against MRSA at a human-equivalent dose of 12.2 mg/kg. Significantly, this dose is >15-fold lower than the daily dose of 200 mg/kg recommended for oxacillin in the treatment of staphylococcal infections in humans [10]. The oxacillin doses that would therefore be required for combination therapy with TXA709 to treat systemic MRSA infections in a clinical setting should be readily achievable.

We also used a mouse model of tissue (thigh) infection with MRSA to probe for TXA709-induced potentiation of oxacillin efficacy in vivo, as skin and soft tissue infections are one of the most common clinical manifestations of S. aureus infections [1, 3]. In these studies, the thighs of neutropenic mice were inoculated with MRSA NRS705 (USA100), and groups of five mice were treated intravenously with either vehicle, TXA709 alone, oxacillin alone, or a combination of both oxacillin and TXA709. An intravenous dose of neither 60 mg/kg TXA709 alone nor 600 mg/kg oxacillin alone induced an efficacious response, with thigh bacterial burdens (CFU counts/g thigh after 24 h) similar to those associated with vehicle treatment (Fig. 4). By contrast, the combination of 60 mg/kg TXA709 and 600 mg/kg oxacillin yielded a significant efficacious response, reducing the thigh bacterial burden by approximately 2.3-logs relative to that associated with vehicle treatment. Thus, synergistic actions with TXA709 render oxacillin efficacious not only for the treatment of systemic MRSA infections, but also for the treatment of tissue MRSA infections. Using the same allometric scaling method described above, the human-equivalent dose of oxacillin used in our thigh infection model was 48.8 mg/kg, a value 4-times higher than the corresponding human-equivalent doses of oxacillin used in our systemic infection model (12.2 mg/kg), but still well below the daily dose of oxacillin (200 mg/kg) recommended for use in a clinical setting.

Fig. 4.

TXA709 potentiates the in vivo efficacy of oxacillin against MRSA NRS705 (USA100) in a mouse thigh model of tissue infection. All agents were administered intravenously at the indicated doses. The indicated values of log(CFU/g thigh) reflect the number of CFUs recovered from infected thighs after 24 h. The double asterisk denotes a statistically significant difference (P ≤ 0.003) in mean log(CFU/g thigh) relative to that obtained with the vehicle

Co-administration with TXA709 also increases total exposure to oxacillin in mice

We also explored the impact of TXA709 co-administration on the pharmacokinetics of oxacillin in mice. Female Swiss mice were intravenously administered a 10 mg/kg dose of oxacillin alone or in combination with a 2 mg/kg intravenous dose of TXA709 and plasma concentrations of oxacillin were monitored over time. The resulting mean plasma concentration versus time data are listed in Table 3 and graphically depicted in Fig. 5. Analysis of these results yielded the pharmacokinetic parameters summarized in Table 4. Note that the area under the curve (AUC_last) for oxacillin when co-administered with TXA709 (7128 ng•h/mL) is greater than the corresponding value of AUC_last for oxacillin when administered alone (5243 ng•h/mL). In addition, the clearance (CL) of oxacillin when co-administered with TXA709 (23.35 mL/min/kg) is decreased relative to that of oxacillin when administered alone (31.73 mL/min/kg). Collectively, these results suggest that co-administration with TXA709 increases exposure to oxacillin in vivo.

Table 3.

Time-dependent plasma concentrations of oxacillin following intravenous administration alone or co-administration with TXA709

Time (h)	Plasma Concentration of Oxacillin (ng/mL)a
	10 mg/kg Oxacillin Administered Alone	2 mg/kg TXA709 Co-Administered with 10 mg/kg Oxacillin
0.08	17506.55 ± 1521.68	17624.52 ± 774.48
0.25	5045.40 ± 179.55	10231.57 ± 1329.16
0.5	1299.89 ± 374.97	3354.34 ± 899.89
1	314.65 ± 141.14	790.00 ± 424.83
2	27.55 ± 8.55	29.70 ± 1.12b

^aEach concentration reflects a mean value of three separate samples from three different mice, with the indicated uncertainties reflecting the standard deviations from the mean. The lower limit of quantitation (LLOQ) is 10.08 ng/mL

^bAverage of two values considered for analysis

Fig. 5.

Time-dependent plasma concentrations of oxacillin following intravenous administration oxacillin alone at 10 mg/kg (filled circles) or co-administration of oxacillin at 10 mg/kg and TXA709 at 2 mg/kg (open circles) to female Swiss mice. The indicated mean plasma concentrations and associated standard deviations (represented by the error bars) are derived from Table 3

Table 4.

Impact of TXA709 co-administration on the pharmacokinetics of oxacillin in female Swiss mice^a

Agent	Dose (mg/kg)	Dosing Regimen	AUClast (ng•h/mL)	t½ (h)	CL (mL/min/kg)	Vss (L/kg)
Oxacillin	10	Administered Alone	5243	0.13	31.79	0.35
		Co-administered with TXA709b	7128	0.19	23.38	0.39

^aAll agents were administered via the intravenous route

^bTXA709 was administered at a dose of 2 mg/kg

The primary route by which oxacillin (a β-lactam carboxylate) is cleared from the body has been well established to be secretion via organic anion transporters (OATs) present in the renal tubules [33–35]. We have previously shown that one of the principal in vivo metabolites of TXA709 is an anionic carboxylate derivative of TXA707 [25] (see structure in Fig. 6). It is possible that this anionic metabolite may competitively inhibit the OATs that are involved in the renal secretion of oxacillin. In this connection, the uricosuric agent probenecid (also an anionic carboxylate compound) is known to inhibit OATs and thereby reduce clearance and increase plasma levels of oxacillin and other penicillins [36–39]. In fact, probenecid has been approved by the FDA as an adjuvant to therapy with penicillins for elevation and prolongation of plasma levels of the antibiotics [40]. The carboxylate metabolite of TXA709 may increase exposure to oxacillin in a manner analogous to probenecid.

Fig. 6.

Chemical structures of the FtsZ inhibitor TXA707, its prodrug TXA709, and their previously identified carboxylate metabolite [25]

Concluding remarks

Oxacillin has been used safely in the clinic for over 45 years, with the pharmacokinetic and pharmacodynamic features of the drug having been well-characterized. Our results presented here offer a promising approach to repurpose oxacillin for use against MRSA infections through combination with a synergistic partner, the FtsZ-targeting prodrug TXA709. Significantly, both agents can be administered intravenously, and together, reduce the potential for the emergence of resistance relative to treatment with a single agent. The combination of oxacillin and TXA709 provides a therapeutic option for the treatment of multidrug-resistant staphylococcal infections, particularly infections caused by MRSA isolates that have become resistant to current standard-of-care antibiotics.

Materials and methods

Bacterial isolates and antibacterial agents

Clinical isolates of MRSA, VISA, VRSA and LRSA were provided by the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) for distribution by BEI Resources, NIAID, NIH. The NARSA website (http://www.narsa.net) describes the location of origin of each isolate. MRSA LAC was a gift from Alexander R. Horswill (University of Colorado School of Medicine, Aurora, CO) and MSSA RN4220 containing the plasmid pLL2787 was a gift from Chia Y. Lee (University of Arkansas for Medical Sciences, Little Rock, AK). TXA707 and TXA709 were synthesized as described previously [25]. Oxacillin (sodium salt) was obtained from Sigma-Aldrich (St. Louis, MO).

In vitro susceptibility assays

In vitro susceptibility assays to determine the minimal inhibitory concentrations (MICs) of TXA707 and oxacillin were conducted using the broth microdilution susceptibility method in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines [41]. Briefly, 96-well microtiter plates containing 0 or 0.5 μg/mL TXA707 and 2-fold serial dilutions of oxacillin in cation-adjusted Mueller-Hinton (CAMH) broth were inoculated with log-phase bacteria (at a final concentration of 5 × 10⁵ CFU/mL). The volume in each well was 0.1 mL, and each compound concentration was present in duplicate. MICs are defined as the lowest compound concentrations with no visual growth after aerobic incubation for 18–24 h at 37 °C.

Time-kill assays

Exponentially growing bacteria were diluted in CAMH broth to a final count of approximately 10⁶ CFU/mL. The colony count of each culture at time zero was verified by plating serial dilutions on tryptic soy agar (TSA) plates (Becton-Dickinson). The cultures were distributed into 4 tubes containing vehicle (dimethyl sulfoxide, DMSO), 0.5 μg/mL TXA707, 2 μg/mL oxacillin, or a combination of 0.5 μg/mL TXA707 and 0.125–2 μg/mL oxacillin. All tubes were placed in an incubator at 37 °C with shaking, and aliquots removed at 3, 6, 9, and 24 h post-exposure. The CFU/mL at each time point was determined by plating serial dilutions of the aliquots on TSA plates and counting colonies after incubation at 37 °C for 24 h.

Frequency of resistance (FOR) assays

A large inoculum approach described previously [42–44] was used to determine the frequency of resistance (FOR) of the MRSA, VISA, VRSA, and LRSA isolates to TXA707 alone or in combination with oxacillin. For these determinations, approximately 10⁹–10¹⁰ CFUs of bacteria were plated on TSA plates containing 4 μg/mL TXA707 (4x MIC) alone or in combination with 2 μg/mL oxacillin. All plates were incubated at 37 °C and examined for the emergence of resistant colonies after 48 h.

Differential interference contrast (DIC) and fluorescence microscopy

All differential interference contrast (DIC) and fluorescence microscopy experiments were conducted using an Olympus BX50 microscope equipped with an X-cite Exacte 200 W mercury lamp, a 100x Olympus UPLSAPO oil immersion objective (1.40 aperture), and a Chroma ET-EGFP (FITC/Cy2) filter. Images were captured using a QImaging Retiga R3 charge-coupled device (CCD) camera and the Ocular-Version 2.0 software package (QImaging).

MSSA RN4220 and MRSA LAC cells expressing sfGFP-tagged PBP2 were generated as previously described [31]. Each strain was grown to log phase in 5 mL of tryptic soy broth (TSB) supplemented with 10 μg/mL erythromycin and then diluted to an optical density at 600 nm (OD₆₀₀) of 0.1 in 5 mL of TSB supplemented with 10 μg/mL erythromycin. The log-phase cultures were then treated for 3 h at 37 °C with 10 nM IPTG and either DMSO vehicle, 0.06 μg/mL TXA707 alone, 0.06 μg/mL oxacillin alone, or a combination of 0.06 μg/mL TXA707 and 0.06 μg/mL oxacillin. Each culture was then centrifuged at 15,000g for 1 min and washed twice with 1 mL of phosphate-buffered saline (PBS). Cells were then resuspended in 200 μL of PBS and 8 μL of this final cell suspension was spread on a 0.25-mm layer of 1.5% high-resolution agarose in PBS, which was mounted on a standard 75 × 25 × 1 mm microscope slide (Azer Scientific) using a 1.7 × 2.8 × 0.025 cm Gene Frame (ThermoFisher). A 24 × 40 mm cover slip (Azer Scientific) was then applied to the agarose pad to prepare the slide for microscopic visualization.

In vivo efficacy assays: murine peritonitis model

All murine studies were conducted in full compliance with the standards established by the US National Research Council’s Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (IACUC) of Rutgers Robert Wood Johnson Medical School. Groups of 5 female Swiss-Webster mice (Taconic Biosciences, Rensselaer, NY) with an average weight of 25 g were infected intraperitoneally with a lethal inoculum (2.5 × 10⁷ CFUs) of NRS705 (USA100) in saline (0.9% NaCl) containing 5% (w/v) porcine mucin (Sigma–Aldrich). Inocula were prepared by combining overnight cultures with sterile 10% mucin to achieve the desired bacterial CFU and mucin percentage. Colony counts for all inocula were verified by plating serial dilutions on TSA plates. All compounds and vehicles were administered intravenously by tail vein injection, with the vehicle for TXA709 being 10 mM citrate (pH 2.6) and the vehicle for oxacillin being saline. The dosing volume for all the intravenous administrations was 10 mL/kg.

The body temperatures of all mice were monitored for a period of five days after infection. Body temperatures were recorded at the Xiphoid process using a noninvasive infrared thermometer (Braintree Scientific, Inc.). Infected mice with body temperatures ≤28.9 °C were viewed as being unable to recover from the infection [45] and were euthanized.

Infected mice were randomly assigned into nine groups of 5 mice, with each group treated as follows: Group (1) – citrate and saline vehicle; Group (2) – 16 mg/kg vancomycin; Group (3) – 20 mg/ kg TXA709; Group (4) – 60 mg/kg oxacillin; Group (5) – 90 mg/kg oxacillin; Group (6) – 150 mg/kg oxacillin; Group (7) – a combination of 60 mg/kg oxacillin and 20 mg/kg TXA709; Group (8) – a combination of 90 mg/kg oxacillin and 20 mg/kg TXA709; and Group (9) – a combination of 150 mg/kg oxacillin and 20 mg/kg TXA709. When used, TXA709 was administered 15 min post-infection, while oxacillin was administered 30 min post-infection.

In vivo efficacy assays: murine thigh model

For the mouse thigh model studies of MRSA infection, TXA709 and oxacillin were formulated as described above and administered intravenously by tail vein injection. Female Swiss-Webster mice (Taconic Biosciences, Rensselaer, NY) with an average weight of 25 g were initially immunosuppressed by two intraperitoneal injections with cyclophosphamide (Sigma–Aldrich). The first cyclophosphamide injection was administered at 150 mg/kg four days before infection, while the second was administered at 100 mg/kg one day before infection. The right thigh of each neutropenic mouse was inoculated intramuscularly with 10⁴ CFUs of MRSA NRS705 (USA100) (0.1 mL bacterial suspension in PBS per thigh). Mice with infected thighs were divided into four groups of five, and treated intravenously as follows: Group (1) – citrate vehicle; Group (2) – 60 mg/kg TXA709 (in 3 divided doses of 20 mg/kg at 1, 2, and 3 h post-infection); Group (3) – 600 mg/kg oxacillin (in 4 divided doses of 150 mg/kg at 1.5, 2.5, 3.5, and 4.5 h post-infection); Group (4) – a combination of both 60 mg/kg TXA709 and 600 mg/kg oxacillin, with the TXA709 and oxacillin administration regimens being identical to those when the compounds were administered by themselves. The dosing volume for both TXA709 and oxacillin was 8 mL/kg. Mice were euthanized 24 h post-infection, and the right thigh muscle aseptically harvested, weighed, and homogenized. Serial dilutions of the homogenates were plated on TSA plates for CFU determination.

Pharmacokinetic studies

Pharmacokinetic experiments in healthy female Swiss albino mice (8–12 weeks old and weighing between 20 and 35 g) were conducted by SAI Life Sciences Ltd. (Pune, India). All procedures were conducted in accordance with the guidelines specified by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and approved by the Institutional Animal Ethics Committee (IAEC). Oxacillin was formulated in normal saline at a concentration of 1 mg/mL and TXA709 was formulated in a solution of normal saline (90%), Solutol (Kolliphor^®) HS 15 (5%), and N-methylpyrrolidone (5%) at a concentration of 0.2 mg/mL. 27 mice were divided into three groups, with nine mice in each group. Animals in Group 1 were administered the TXA709 formulation intravenously at a dose of 2 mg/kg. Animals in Group 2 were administered the oxacillin formulation intravenously at a dose of 10 mg/kg. Animals in Group 3 were intravenously administered the TXA709 formulation at a dose of 2 mg/kg followed by the oxacillin formulation at a dose 10 mg/kg with a dosing interval of 15 min. The dosing volume was 10 mL/kg. Blood samples of approximately 60 μL were collected from the retro-orbital plexus of three different mice at 0.08, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h. Plasma was immediately harvested from the blood by centrifugation at 4000 rpm for 10 min at 4 °C. Plasma samples were extracted by addition of acetonitrile at a 4:1 volumetric ratio. The samples were then vortexed for 5 minutes and centrifuged at 4,000 rpm for 10 min at 4 °C. 100 μL of the resultant supernatants were then transferred to 96-well microtiter plates and analyzed by LC-MS/MS. Pharmacokinetic parameters were determined using the non-compartmental analysis tool of the Phoenix WinNonlin^® software package version 7.0. Area under the plasma concentration versus time curve (AUC_last) was determined according to the linear trapezoidal rule. Clearance (CL) was calculated by Dose/AUC_last.