Human Simulated Studies of Aztreonam and Aztreonam-Avibactam To Evaluate Activity against Challenging Gram-Negative Organisms, Including Metallo-β-Lactamase Producers

Abstract

Secondary to the stability of aztreonam against metallo-β-lactamases, coupled with avibatam's neutralizing activity against often coproduced extended-spectrum β-lactamases (ESBLs) or AmpC enzymes, the combination of aztreonam and avibactam has been proposed as a principal candidate for the treatment of infections with metallo-β-lactamase-producing Gram-negative organisms. Using the neutropenic-mouse thigh infection model, we evaluated the efficacy of human simulated doses of aztreonam-avibactam and aztreonam against 14 Enterobacteriaceae and 13 Pseudomonas aeruginosa isolates, of which 25 produced metallo-β-lactamases. Additionally, six P. aeruginosa isolates were also evaluated in immunocompetent animals. A humanized aztreonam dose of 2 g every 6 h (1-h infusion) was evaluated alone and in combination with avibactam at 375 or 600 mg every 6 h (1-h infusion), targeting the percentage of the dosing interval in which free-drug concentrations remained above the MIC (fT>MIC). Efficacy was evaluated as the change in bacterial density after 24 h compared with the bacterial density at the initiation of dosing. Aztreonam monotherapy resulted in reductions of two of the Enterobacteriaceae bacterial isolates (aztreonam MIC, ≤32 μg/ml; fT>MIC, ≥38%) and minimal activity against the remaining isolates (aztreonam MIC, ≥128 μg/ml; fT>MIC, 0%). Alternatively, aztreonam-avibactam therapy resulted in the reduction of all 14 Enterobacteriaceae isolates (aztreonam-avibactam MICs, ≤16 μg/ml; fT>MIC, ≥65%) and no difference between the 375- and 600-mg doses of avibactam was noted. Similar pharmacodynamically predictable activity against P. aeruginosa was noted in studies with neutropenic and immunocompetent mice, with activity occurring when the MICs were ≤16 μg/ml and variable efficacy noted when the MICs were ≥32 μg/ml. Again, no difference in efficacy between the 375- and 600-mg doses of avibactam was observed. Aztreonam-avibactam represents an attractive treatment option for infections with metallo-β-lactamase-producing Gram-negative pathogens that coproduce ESBLs or AmpC.

INTRODUCTION

While the mainstay of therapy for a whole host of infection types is β-lactams, the production of β-lactamases represents one of the most common mechanisms organisms have developed to threaten the viability of these agents (1–3). Gram-negative organisms, in particular, have exhibited a variety of these enzymes and continually emerge with novel forms. An example of such an enzyme is the New Delhi metallo-β-lactamase (NDM), which was first isolated in 2008 (4) and has now been identified in many countries worldwide. NDM is only one of many types of metallo-β-lactamases; other notable examples include both IMP and VIM (5, 6). Collectively, these enzymes are of particular concern because they are potent hydrolyzers of not only the penicillins and cephalosporins but also the carbapenems (6). This, coupled with other enzymatic and nonenzymatic resistance mechanisms, typically leaves the number of treatment options severely limited.

As the only available β-lactam that is inherently impervious to metallo-β-lactamases, aztreonam would theoretically present an attractive option for the treatment of infections with pathogens that produce these enzymes. Unfortunately, in most cases, these organisms come with an onslaught of other β-lactamases (i.e., CTX-M type, CMY type, etc.) that readily hydrolyze aztreonam (4). However, the availability of novel β-lactamase inhibitors such as avibactam, which is active against a wide variety of these hydrolyzing enzymes but not protective against metallo-β-lactamases (7), may offer an excellent opportunity to marry these two compounds and effectively fill the respective “holes” in their coverage. These suspicions were confirmed in a recent in vitro analysis evaluating the potency of aztreonam-avibactam against a number of metallo-β-lactamase-producing members of the family Enterobacteriaceae (8), but no data describing the activity of this combination in vivo are available. Using the murine thigh infection model, we sought to evaluate human simulated doses of this combination against a variety of multidrug-resistant Gram-negative organisms, the majority of which produce metallo-β-lactamases.

MATERIALS AND METHODS

Antimicrobial test agent.

Commercially available aztreonam (Azactam, lot 1G68718; Bristol-Myers Squibb, Princeton, NJ) was obtained from the Hartford Hospital Pharmacy Department and used for all in vivo studies, while analytical-grade aztreonam (lot 031M0100V; Sigma-Aldrich, St. Louis, MO) was used for ex vivo protein binding studies and in vitro MIC determinations. Analytical-grade avibactam was supplied by AstraZeneca Pharmaceuticals (Waltham, MA). Clinical vials of aztreonam were reconstituted as described in the prescribing information and diluted as appropriate to achieve the desired concentrations. Analytical-grade aztreonam and avibactam powders were weighed in a quantity sufficient to achieve the required concentrations and reconstituted immediately prior to use.

Bacterial isolates.

A total of 27 Gram-negative clinical isolates were used for these studies. Included were 13 Pseudomonas aeruginosa, 12 Escherichia coli, and 2 Klebsiella pneumoniae strains provided by International Health Management Associates, Inc., Schaumburg, IL. All isolates were maintained in double-strength skim milk (BD Biosciences, Sparks, MD) at −80°C. Each isolate was subcultured twice on Trypticase soy agar with 5% sheep blood (BD Biosciences) prior to use in experiments.

Susceptibility testing.

The MICs of aztreonam and aztreonam-avibactam were determined for each isolate by the broth microdilution method as outlined by the Clinical and Laboratory Standards Institute (CLSI) (9). For aztreonam-avibactam, doubling dilutions of aztreonam were used in combination with a fixed 4-μg/ml concentration of avibactam. MIC studies were conducted with a minimum of five replicates, and the modal MICs are reported.

Protein binding studies.

Free-avibactam amounts were calculated by using previously described protein binding values of 18 and 10% for avibactam in humans and mice, respectively (10). The human protein binding of aztreonam was assumed to be 42%, as previously reported for patients (11). Given the lack of available data in the literature, aztreonam murine protein binding was determined over a range of concentrations herein.

Namely, aztreonam protein binding studies were conducted as three independent tests with Amicon Centrifree Micropartition devices (Millipore, Bedford, MA) and filters with a molecular weight cutoff of 30,000 according to the manufacturer's package insert. Aztreonam concentrations of 425, 300, 200, 150, 140, 125, and 25 μg/ml were evaluated, as was nonspecific binding to the filter device at a concentration of 100 mg/liter. Briefly, solutions were made in freshly collected mouse serum, heated at 37°C in a shaking water bath for 10 min, and then centrifuged for 45 min at 10°C at 2,000 × g.

Percent protein binding (%PB) at each prepared concentration was calculated with the equation %PB = [(S − SUF)/S] × 100, where S is the aztreonam concentration in the initial serum solutions and SUF is the concentration in the ultrafiltrate.

Neutropenic-mouse thigh infection model.

The study was reviewed and approved by the Hartford Hospital Institutional Animal Care and Use Committee. Pathogen-free female ICR mice weighing approximately 25 g were acquired from Harlan Sprague-Dawley, Inc. (Indianapolis, IN), and used throughout these experiments. Animals were maintained and used in accordance with National Research Council recommendations and provided food and water ad libitum. Mice were rendered neutropenic with 100- and 150-mg/kg intraperitoneal injections of cyclophosphamide (Cytoxan; Bristol-Myers Squibb, Princeton, NJ) given 1 and 4 days prior to inoculation, respectively. Three days prior to inoculation, mice were also given a single 5-mg/kg intraperitoneal injection of uranyl nitrate, which produces a predictable degree of renal impairment to aid in humanizing the drug regimens (12). Two hours prior to the initiation of antimicrobial therapy, each thigh was inoculated intramuscularly with 0.1 ml of a solution containing the test isolate at approximately 10⁷ CFU/ml.

Immunocompetent-mouse thigh infection model.

Mice used in the immunocompetent-mouse studies underwent the same procedures as outlined above, except that cyclophosphamide was not given and an inoculum of 10⁸ CFU/ml was used to produce thigh infection.

Determination of the in vivo dosing regimen.

In these studies, we determined a mouse dosing regimen that simulated the percentage of the dosing interval for which free-drug concentrations were above the MIC (fT>MIC) profile observed in humans given 2,000 mg aztreonam every 6 h as a 1-h infusion as monotherapy or combined with avibactam at a dose of 375 or 600 mg every 6 h as a 1-h infusion. Aztreonam exposures of patients were derived from a previously published pharmacokinetic model (11), while avibactam exposures were determined from a population pharmacokinetic model (13).

First, single-dose studies with aztreonam-avibactam and aztreonam alone were undertaken with thigh-infected neutropenic mice. For these analyses, animals were dosed with a single, weight-based, 0.2-ml, subcutaneous injection of the study drug(s) and groups of six mice were euthanized at eight time points over the following 12 h. Blood samples were taken via cardiac puncture, and serum was stored at −80°C until analysis. Aztreonam concentrations were analyzed at the Center for Anti-Infective Research and Development (Hartford, CT) with a high-performance liquid chromatography assay (14), while avibactam concentrations were determined by Eurofins Medinet, Inc. (Chantilly, VA), with a liquid chromatography-tandem mass spectrometry assay (15). The intraday and interday coefficients of variation for high- and low-check samples in each assay were ≤5.4%.

Pharmacokinetic parameters for single doses of aztreonam-avibactam and aztreonam alone were calculated by using first-order input and elimination and nonlinear least-squares techniques (WinNonlin version 5.0.1; Pharsight, Mountain View, CA). Compartment model selection and weighting schemes were based on visual inspection of the fit and use of the correlation between the observed and calculated concentrations.

Using pharmacokinetic parameters derived in single-dose studies, regimens that simulated the free-drug exposure profile of patients given aztreonam-avibactam and aztreonam alone were constructed for mice. Confirmatory pharmacokinetic studies were undertaken with infected mice prior to the use of these regimens in the pharmacodynamic analyses, and an assessment of fT>MIC was made from the resulting concentration-time profiles. For these studies, infected neutropenic mice were dosed with the regimens calculated as described above and groups of six mice were euthanized at five time points throughout the first dosing interval (i.e., 6 h) to confirm target exposures.

In vivo efficacy.

For each of the 14 Enterobacteriaceae (12 E. coli and 2 K. pneumoniae) and 13 P. aeruginosa isolates, groups of three mice were administered human simulated regimens of aztreonam or aztreonam-avibactam beginning 2 h after inoculation. All doses were administered as 0.2-ml subcutaneous injections and consisted of four 6-h dosing intervals (i.e., 24 h). To serve as control animals, an additional group of mice were administered normal saline at the same volume, route, and frequency as the treatment regimen. The thighs of all of the animals were harvested 24 h after the initiation of therapy. The thighs of mice that failed to survive for 24 h were harvested at the time of expiration. The harvesting procedure for all study mice began with euthanization by CO₂ exposure, followed by cervical dislocation. After sacrifice, thighs were removed and individually homogenized in normal saline. Serial dilutions of the thigh homogenates were plated on Trypticase soy agar with 5% sheep blood by the spiral-plating techniques for CFU determination. Given the narrow window between the final treatment dose and tissue harvesting, plates were observed for the presence of antibiotic carryover. In addition to the above-mentioned treatment and control groups, the thighs of another group of three infected, untreated mice were harvested at the initiation of dosing and served as 0-h controls. Efficacy, designated as a change in bacterial density, was calculated as the difference in bacterial log₁₀ CFU/ml in treated mice after 24 h from the starting densities observed in 0-h control animals. Given that both stasis and 1-log reductions in bacterial density have been used to predict clinical efficacy in humans, this target range was used throughout as a benchmark to evaluate therapies (16, 17).

RESULTS

Bacterial isolates.

The enzyme production and phenotypic profiles of the 27 isolates included in the efficacy studies are shown in Table 1. Of note, all of the Enterobacteriaceae isolates produced NDM β-lactamases, as well as various extended-spectrum β-lactamases (ESBLs). Moreover, nearly all of the P. aeruginosa isolates were metallo-β-lactamase producers.

Table 1.

Enzyme production and phenotypic data for the E. coli, K. pneumoniae, and P. aeruginosa isolates used in our in vivo efficacy studies

Isolate	Enzyme production or genotype	MIC (μg/ml)
		ATM-AVIb	ATMc
E. coli
396	NDM-1, TEM type, OXA-1	0.125	16
395	AmpC, OXA-2,CTX-M-15, CMY-4, NDM-1, OXA-1, TEM-1	4	>256
406	NDM-1, OXA-1	4	32
407	NDM-1, CTX-M-15	4	>256
394	AmpC, NDM-1, CTX-M-15, TEM-1, OXA-2, CMY-6	8	>256
397	NDM-1, TEM-1, OXA-1, CTX-M type, CMY-2	8	>256
408	NDM-1, CTX-M-15, OXA-1	8	>256
410	NDM-1, CTX-M-15, OXA-1	8	>256
411	NDM-4, CTX-M-15	8	>256
412	NDM-6, CTX-M-15	8	>256
413	NDM-1, TEM-1, SHV-12	8	>256
414	NDM type, CTX-M-15, OXA-1	16	>256
K. pneumoniae
427	NDM-1, TEM-1, CTX-M-15, SHV-2a, DHA-1	0.125	128
449	NDM type, TEM-1, OXA-1, CTX-M-15	0.5	256
P. aeruginosa
1449	NDd	4	32
1461	GES type	4	16
1476	VIM type	4	16
1451a	GES type	8	>128
1471	VIM type	8	8
1472	VIM type	16	16
1474	IMP type	16	16
1447a	ND	32	32
1448a	VIM-2	32	32
1465a	AmpC, cPoxB, VIM-1	32	32
1466a	VIM-2	32	32
1477a	VIM type	32	64
1480	VIM type	32	32

^aIsolate evaluated in neutropenic and immunocompetent studies.

^bATM-AVI, aztreonam-avibactam.

^cATM, aztreonam.

^dND, not determined.

Protein binding studies.

The protein binding of aztreonam was concentration dependent over a range of 25 to 200 mg/liter (range, 90.8 to 40.3%), while protein binding remained constant at concentrations of ≥200 μg/ml with a mean protein binding value of 43.5% ± 6.6%. Nonspecific binding studies revealed no binding of aztreonam to the filter device. Given that the peak concentrations observed during human simulated studies fell within this static range, a set protein binding level of 43.5% was used for free-drug calculations.

Determination of dosing regimen for in vivo studies.

The pharmacokinetics of aztreonam and avibactam were best described by using a one-compartment model with first-order input and elimination. Human simulated regimens consisted of 5 doses in each 6-h dosing interval. The in vivo free-drug pharmacokinetic profiles of 2,000 mg aztreonam and 375 mg avibactam, 2,000 mg aztreonam and 600 mg avibactam, and 2,000 mg aztreonam alone are shown in Fig. 1, 2, and 3, respectively. The comparative aztreonam fT>MIC attained with these regimens in mice and that anticipated in humans are shown in Table 2 and highlight the similarities between the exposures.

Fig 1.

Free-drug concentration-time profile of human simulated 2,000 mg aztreonam (ATM)-375 mg avibactam (AVI) in thigh-infected ICR mice. Circles represent means ± standard deviations.

Fig 2.

Free-drug concentration-time profile of human simulated 2,000 mg aztreonam (ATM)-600 mg avibactam (AVI) in thigh-infected ICR mice. Squares represent means ± standard deviations.

Fig 3.

Free-drug concentration-time profile of human simulated 2,000 mg aztreonam (ATM) in thigh-infected ICR mice. Triangles represent means ± standard deviations.

Table 2.

Human simulated fT>MIC profile of aztreonam in humans compared with that observed in mice^a

MIC (μg/ml)	Aztreonam fT>MIC (%)
	Human	Mouse
2	100	100
4	100	98
8	90	87
16	65	63
32	38	40
64	15	10
128	0	0

^aAztreonam was given as a 1-h infusion of 2,000 mg q6h. This profile is similar irrespective of avibactam administration.

In vivo efficacy.

Enterobacteriaceae studies were conducted only with neutropenic animals; during these evaluations, 0-h control mice displayed a mean bacterial density of 5.87 ± 0.19 log₁₀ CFU, which increased to an average of 8.23 ± 0.90 log₁₀ CFU in untreated mice after 24 h. Infection-related deaths were observed in control animals and a small number of aztreonam-treated mice; all aztreonam-avibactam-treated animals were alive at 24 h. Antibiotic carryover was not observed on any of the treatment plates. The results of these studies are shown in Fig. 4. While aztreonam monotherapy reduced the bacterial density of only the two isolates with aztreonam MICs of ≤32 μg/ml (fT>MIC, ≥38%), secondary to the increased in vitro potency, aztreonam-avibactam treatment resulted in maximal activity against all 14 isolates. Also of note, the efficacies of combination therapy with the two avibactam regimens were similar.

Fig 4.

Comparative efficacy of a human simulated dose of 2,000 mg aztreonam q6h (1-h infusion) as monotherapy (ATM) or combined with 375 mg avibactam q6h (ATM-AVI 375 mg) or 600 mg avibactam q6h (ATM-AVI 600 mg) against Enterobacteriaceae in the neutropenic-mouse thigh infection model.

Pseudomonal studies were conducted with both neutropenic (Fig. 5) and immunocompetent (Fig. 6) animals. The respective bacterial densities in control mice at the initiation of dosing were 4.98 ± 0.24 and 6.44 ± 0.27 log₁₀ CFU, increasing to 7.85 ± 0.85 and 7.69 ± 1.14 log₁₀ CFU after 24 h. A large number of untreated control animals succumbed to infection, as did aztreonam-treated mice infected with P. aeruginosa 1451 (aztreonam MIC, >128 μg/ml); all aztreonam-avibactam-treated mice survived to 24 h. Antibiotic carryover was not observed on any of the treatment plates. In general, neutropenic-mouse studies showed the activity of aztreonam monotherapy and aztreonam-avibactam to be predictable on the basis of the pharmacodynamic profile, with maximal activity occurring when the fT>MIC was ≥65% (i.e., the MICs were ≤16 μg/ml), and variable efficacy was noted when the fT>MIC dropped to ≤38% (i.e., the MICs were ≥32 μg/ml). Similar results were obtained with immunocompetent animals, with a slight enhancement of the activities of both aztreonam and aztreonam-avibactam against organisms against which minimal activity was seen in neutropenic-mouse studies. Regardless of immune status, no differences in efficacy between the 375- and 600-mg doses of avibactam were observed.

Fig 5.

Comparative efficacy of a human simulated dose of 2,000 mg aztreonam q6h (1-h infusion) as monotherapy (ATM) or combined with 375 mg avibactam q6h (ATM-AVI 375 mg) or 600 mg avibactam q6h (ATM-AVI 600 mg) against P. aeruginosa in the neutropenic-mouse thigh infection model.

Fig 6.

Comparative efficacy of a human simulated dose of 2,000 mg aztreonam q6h (1-h infusion) as monotherapy (ATM) or combined with 375 mg avibactam q6h (ATM-AVI 375 mg) or 600 mg avibactam q6h (ATM-AVI 600 mg) against P. aeruginosa in the immunocompetent-mouse thigh infection model.

DISCUSSION

Secondary to the high rates of resistance to our current armamentarium, coupled with the lack of novel agents in development, Gram-negative pathogens bring difficulties to present-day clinical practice and fear for the days to come. While a number of resistance mechanisms contribute to the bleak phenotypic profiles displayed by many of these organisms, the production of β-lactamases, both new and old, plays a vital role. It is for this reason that novel β-lactamase inhibitors such as avibactam are of great interest in a number of development programs (18). While avibactam has a broad spectrum of β-lactamase inhibition, it is inactive against metallo-β-lactamases. Owing to its inherent imperviousness to metallo-β-lactamases, aztreonam combined with avibactam represents a possible approach to the treatment of infections with these organisms. Using the murine thigh infection model, we found that while aztreonam monotherapy was ineffective against a large percentage of metallo-β-lactamase producers, aztreonam-avibactam yielded activity against a large proportion of these isolates.

When evaluating the in vitro MIC data for the Enterobacteriaceae isolates included in this analysis, it is clear that while aztreonam may be impervious to hydrolysis by the NDM enzymes produced by these organisms (6), the multitude of other β-lactamases and/or potential non-enzyme-mediated mechanisms confirmed high levels of resistance to aztreonam. The addition of avibactam to aztreonam resulted in a potency shift of 3 to ≥11 doubling dilutions, rendering all of the organisms quite responsive to combination therapy in vivo. Similar in vitro results were noted in a previous study conducted by Livermore et al. (8) in which 17 NDM-1-producing isolates reveled a median aztreonam MIC of ≥256 μg/ml (range, 0.06 to ≥256 μg/ml) and a median aztreonam-avibactam MIC of 0.25 μg/ml (range, ≤0.03 to 4 μg/ml). Of note, while the present analysis did not include Enterobacteriaceae isolates that produced other metallo-β-lactamases of interest (i.e., IMP, VIM), the distribution of MICs for the isolates tested was inclusive of that anticipated for these organisms on the basis of the data of Livermore et al. (≤4 μg/ml) (8). The aztreonam and aztreonam-avibactam MICs for most of the P. aeruginosa isolates tested, the majority of which produce metallo-β-lactamases, were similar, suggesting that β-lactamases within the inactivation profile of avibactam were playing a minor role in aztreonam resistance.

Using human simulated strategies, we found the activities of aztreonam monotherapy and aztreonam-avibactam to be quite predictive on the basis of the aztreonam fT>MIC profile. Namely, with a dose of 2 g every 6 h (q6h) (1-h infusion), maximal activity was noted when the MICs were ≤16 μg/ml (fT>MIC, ≥65%) and became variable when the MICs were ≥32 μg/ml (fT>MIC, ≤38%). While we did not conduct traditional dose-ranging studies in this analysis, the break-in activity noted on the basis of fT>MIC was similar to targets reported in the literature (i.e., 50 to 60%) for aztreonam alone (19). This observation is of particular interest for aztreonam-avibactam, as it provides in vivo support for the use of a set avibactam concentration of 4 μg/ml during MIC testing and perhaps an early look into a potential clinical breakpoint.

Another important observation of this study was the similarity in activity between the 375- and 600-mg doses of avibactam when combined with aztreonam. This similarity was noted regardless of the organism, the aztreonam-avibactam MIC, the genotype, or the reduction in the MIC of aztreonam-avibactam compared with the reduction in the MIC of aztreonam alone. While previous in vitro hollow-fiber studies of avibactam combined with ceftaroline suggested that avibactam free time above a threshold concentration was required for maximal cephalosporin activity, the specific threshold concentration and/or percentage of time has not been fully elucidated; further, no studies have been published for the combination of aztreonam-avibactam. On the basis of the data described herein, the pharmacodynamic profiles of avibactam at doses of 375 and 600 mg q6h were clearly sufficient to restore aztreonam activity against the isolates evaluated.

When comparing the activities of aztreonam and aztreonam-avibactam against isolates in which the addition of avibactam did not alter the potency of aztreonam, similar activities against a majority of the isolates tested were noted. There were, however, isolates such as P. aeruginosa 1447 (aztreonam and aztreonam-avibactam MIC of 32 μg/ml) in which the activity of aztreonam-avibactam was greater than that of aztreonam monotherapy. (Fig. 5). While the reason for this observation is not entirely clear, it is possible that the addition of avibactam reduced the actual MIC but not enough to make it fall below the next doubling dilution (i.e., 16 μg/ml). In this example, the theoretical reduction of the actual MIC could therefore improve the observed fT>MIC to a value somewhere between 38 and 65%.

It should also be noted that the current CLSI and European Committee on Antimicrobial Susceptibility Testing (EUCAST) aztreonam susceptibility breakpoints for Enterobacteriaceae are ≤4 and ≤1 μg/ml, respectively. However, when using the humanized aztreonam monotherapy dose of 2 g q6h, we saw activity against two isolates with respective aztreonam MICs of 16 and 32 μg/ml. While EUCAST does not make specific comments, CLSI states that this breakpoint recommendation is generated from a regimen of 1 g q8h, which would clearly result in a comparatively reduced pharmacodynamic profile. Similar comments could be made for P. aeruginosa, where the respective CLSI and EUCAST susceptibility breakpoints are ≤8 and ≤1 μg/ml. In these cases, CLSI comments that the breakpoint is based on a dose of 1 g q6h or 2 g q8h and EUCAST denotes the use of “high dose therapy.” As noted previously, the efficacy of both aztreonam and aztreonam-avibactam against isolates with MICs ≤16 μg/ml in the present study was predictable on the basis of the fT>MIC profile of a regimen of 2 g q6h.

With the continued evolution and spread of β-lactamases among Gram-negative pathogens, infected patients are in dire need of novel treatment approaches. Therapeutic options specifically for metallo-β-lactamase producers are of particular interest. Using the murine thigh infection model, we found that while a human simulated aztreonam regimen of 2 g q6h was relatively ineffective against metallo-β-lactamase-producing organisms because of the coproduction of ESBL or AmpC enzymes, the addition of avibactam at a dose of 375 or 600 mg restored the in vivo activity of aztreonam and yielded maximal activity against isolates with MICs of ≤16 μg/ml. On the basis of these finding, aztreonam-avibactam represents an attractive treatment option for infections with metallo-β-lactamase-producing Gram-negative pathogens that coproduce ESBL or AmpC enzymes.