Combinatorial Pharmacodynamics of Ceftolozane-Tazobactam against Genotypically Defined β-Lactamase-Producing Escherichia coli: Insights into the Pharmacokinetics/Pharmacodynamics of β-Lactam–β-Lactamase Inhibitor Combinations

Abstract

Despite a dearth of new agents currently being developed to combat multidrug-resistant Gram-negative pathogens, the combination of ceftolozane and tazobactam was recently approved by the Food and Drug Administration to treat complicated intra-abdominal and urinary tract infections. To characterize the activity of the combination product, time-kill studies were conducted against 4 strains of Escherichia coli that differed in the type of β-lactamase they expressed. The four investigational strains included 2805 (no β-lactamase), 2890 (AmpC β-lactamase), 2842 (CMY-10 β-lactamase), and 2807 (CTX-M-15 β-lactamase), with MICs to ceftolozane of 0.25, 4, 8, and >128 mg/liter with no tazobactam, and MICs of 0.25, 1, 4, and 8 mg/liter with 4 mg/liter tazobactam, respectively. All four strains were exposed to a 6 by 5 array of ceftolozane (0, 1, 4, 16, 64, and 256 mg/liter) and tazobactam (0, 1, 4, 16, and 64 mg/liter) over 48 h using starting inocula of 10⁶ and 10⁸ CFU/ml. While ceftolozane-tazobactam achieved bactericidal activity against all 4 strains, the concentrations of ceftolozane and tazobactam required for a ≥3-log reduction varied between the two starting inocula and the 4 strains. At both inocula, the Hill plots (R² > 0.882) of ceftolozane revealed significantly higher 50% effective concentrations (EC₅₀s) at tazobactam concentrations of ≤4 mg/liter than those at concentrations of ≥16 mg/liter (P < 0.01). Moreover, the EC₅₀s at 10⁸ CFU/ml were 2.81 to 66.5 times greater than the EC₅₀s at 10⁶ CFU/ml (median, 10.7-fold increase; P = 0.002). These promising results indicate that ceftolozane-tazobactam achieves bactericidal activity against a wide range of β-lactamase-producing E. coli strains.

INTRODUCTION

The rising prevalence of multidrug-resistant (MDR) Gram-negative organisms has forced urgent efforts to expand the therapeutic armamentarium against these problematic pathogens. Recent endeavors to develop new cephalosporin compounds exhibiting promising antipseudomonal activity led to the discovery of ceftolozane (ceftolozane, previously designated CXA-101 or FR264205) (1). This novel agent has been credited with enhanced stability to popular chromosomally mediated cephalosporin resistance mechanisms (including hyperexpression of AmpC β-lactamase enzymes and efflux pumps) (2), with a low propensity for cross-resistance to other cephalosporins to arise (3).

Antimicrobial inactivation arising from transferable plasmid-mediated β-lactamase hydrolysis presents the principal basis of β-lactam resistance among Enterobacteriaceae. More than 850 structurally diverse β-lactamase enzymes have been identified to date and classified as groups A to D based on their amino acid sequence homology (4). Worryingly, the rapid emergence of extended-spectrum-β-lactamase-producing Escherichia coli strains in recent times has been implicated in community-onset infections as a significant cause of morbidity, mortality, and health care-related costs. Despite ceftolozane's intrinsic benefits over other cephalosporins, the inability of the compound to overcome extended-spectrum β-lactamases (ESBLs) has been documented (5–7). To ameliorate this deficiency, the addition of the β-lactamase inhibitor tazobactam to ceftolozane extends the spectrum of activity of ceftolozane (8). The resulting ceftolozane-tazobactam combination was found to display remarkable activity against a range of MDR Gram-negative species (7) and was subsequently approved by the Food and Drug Administration to treat complicated intra-abdominal and urinary tract infections in adults (9).

Although the activity of ceftolozane-tazobactam against E. coli has been characterized in prior in vitro studies (10–12), a systematic analysis of the combination's performance against strains producing different β-lactamases has yet to be investigated at multiple levels of bacterial burden. Therefore, integrating antimicrobial pharmacokinetics (PK) and pharmacodynamics (PD) to effectively evaluate the bacteriologic response to ceftolozane-tazobactam is warranted (13). Our objective was to utilize time-kill studies to characterize the bacterial killing effects of ceftolozane and tazobactam alone and in combination against different β-lactamase-producing E. coli strains.

MATERIALS AND METHODS

Bacterial strains.

The four isogenic strains of E. coli employed for this study were engineered by Merck & Co. to differentially express a single β-lactamase; these included (i) 2805 (wild type, no β-lactamase), (ii) 2890 (AmpC β-lactamase), (iii) 2842 (CMY-10 β-lactamase), and (iv) 2807 (CTX-M-15 β-lactamase) (Table 1). β-Lactamase expression was modulated by assembly of the enzyme open reading frame per published GenBank sequences (strain 2890, Pseudomonas aeruginosa AmpC and ampR [5′ region], GenBank accession no. X54719.1; strain 2842, E. coli K998298 ESBL precursor [bla_CMY-10], GenBank no. AF381617.1; strain 2807, E. coli strain 405/06 plasmid pKC405 β-lactamase CTX-M-15 [bla_CTX-M-15] and insertion sequence IS26 TnpA, Genbank no. GQ274933.1), insertion into a pBR322 cloning vector (GenBank no. J0749), and replacement of the bla_TEM-1 open reading frame in the β-lactamase-deficient E. coli DH10B parent strain. The native bla_TEM-1 promoter was included in the modified pBR322 plasmid to regulate the expression of the desired β-lactamase. Prior to experiments, subculture of 2805 was performed on tryptic soy agar (TSA) with 5% sheep blood (BD Biosciences, Franklin Lakes, NJ), while 2890, 2842, and 2807 were subcultured in the presence of 25 μg/ml tetracycline to maintain the selection of plasmids.

TABLE 1.

β-Lactamase production and MICs of each strain to ceftolozane in the absence and presence of tazobactam

Strain	β-Lactamase		Ceftolozane MIC with tazobactam concn of (mg/liter):
	Type	Ambler class (4)	0	1	4	16	64
2805	None		0.25
2890	AmpC	C	4	4	1	0.5	0.5
2842	CMY-10	C	8	4	4	1	0.5
2807	CTX-M-15	A	>128	>128	8	2	1

Antibiotics, susceptibility testing, and medium.

Analytical-grade ceftolozane and tazobactam powders were obtained from Merck & Co. (Kenilworth, NJ). Fresh stock solutions were prepared immediately prior to experiments in Milli-Q water. Standard broth microdilution methods were adopted from the Clinical and Laboratory Standards Institute (CLSI) (14) for the determination of MICs. The MIC experiments were performed in quadruplicate, while time-kill experiments were performed in duplicate; all studies were conducted using cation-adjusted Mueller-Hinton broth (CaMHB, at 25.0 mg/liter Ca²⁺ and 12.5 mg/liter Mg²⁺; Difco, Detroit, MI).

Time-kill experiments.

Time-kill experiments were performed for ceftolozane and tazobactam alone and in combination, using previously described methods (15). Briefly, fresh bacterial colonies from overnight growth were added to CaMHB to provide a bacterial suspension, which was diluted with CaMHB to achieve the desired starting inoculum of ∼10⁶ or ∼10⁸ CFU/ml in a 50-ml Falcon tube. A 6 by 5 array of ceftolozane (0, 1, 4, 16, 64, and 256 mg/liter) and tazobactam (0, 1, 4, 16, and 64 mg/liter) concentrations was tested as monotherapy and in combination over a period of 48 h. Thus, 30 treatment regimens were examined in total against each strain at both starting inocula. The choice of studied concentrations was based on clinically achievable targets discerned from free-drug PK profiles in healthy volunteers (16).

Samples were withdrawn at 1, 2, 4, 8, 24, 26, 28, 32, and 48 h after dosing. Colony counts were performed by plating 50-μl aliquots of each diluted sample onto tryptic soy agar (TSA) plates containing 5% sheep blood (Becton Dickinson, Franklin Lakes, NJ) using an automated spiral dispenser (Whitley automatic spiral plater; Don Whitley Scientific Limited, West Yorkshire, England). Plates were incubated at 37°C for 24 h, and viable bacterial counts were determined (log₁₀ CFU/ml) using a laser bacteria colony counter (ProtoCOL version 2.05.02; Synbiosis, Cambridge, England). Bactericidal activity (99.9% kill) was associated with a ≥3.0-log₁₀ CFU/ml decrease in bacterial density compared to the initial inoculum at any time. Experiments were conducted over 48 h (as opposed to the traditional 24-h standard) to provide insight into the pharmacodynamics of each combination beyond 24 h, and also to better discriminate between the killing activities of the different ceftolozane and tazobactam concentrations. As the degradation of ceftolozane and tazobactam at 35°C was of concern, concentrations of ceftolozane and tazobactam were measured in the absence of bacteria over 48 h to calculate the rate of degradation for each agent.

Pharmacodynamic analyses.

The bacterial killing effect (E) of mono- and combination therapies was quantified as the log ratio change in bacterial density (CFU/ml) at 48 h versus preantibiotic exposure at 0 h (see equation 1) (17). Point-based analyses were performed on plots of E versus ceftolozane concentrations. Taking into consideration the general mechanism of action of β-lactam–β-lactam inhibitor combinations (whereby inhibitors are understood to bind to inactivating β-lactamase enzymes, thus allowing β-lactam agents to exert their action), we attributed the majority of the killing activity exerted by the ceftolozane-tazobactam combination to the effect of ceftolozane. Consequently, using nonlinear regression, concentration-effect relationships were fit to Hill-type models for each strain at each fixed tazobactam concentration, according to equation 2, where E₀ is the measured effect in the absence of ceftolozane, E_max is the maximal effect, C is the concentration of ceftolozane, EC₅₀ is the concentration at which there is a 50% maximal effect, and H is the Hill constant. Statistical analyses of E_max and EC₅₀ parameters were conducted to determine the effect of increasing tazobactam concentrations, using a nonparametric Friedman two-way analysis of variance (ANOVA) with pairwise comparisons (P < 0.05). Differences in parameter estimates at 10⁶ versus 10⁸ CFU/ml were determined using hypothesis testing (P < 0.05, F-test). All PD analyses and statistical evaluations were performed using Systat (version 13.00.05; Systat Software, IL).

$$\hspace{0.17em}\text{Log ratio change}={\text{Log}}_{10}\frac{{\text{CFU}}_{49\hspace{0.17em}\text{\hspace{0.17em}h}}}{{\text{CFU}}_{0\hspace{0.17em}\text{\hspace{0.17em}h}}}$$ (1)

$$E=E_0-\frac{E_{\max \hspace{0.17em}\hspace{0.17em}}\times \hspace{0.17em}{C}^H}{\text{EC}{{}_{50}}^H+C^H}$$ (2)

RESULTS

MICs and degradation rates.

The ceftolozane MICs of each strain without tazobactam and in combination with 4 mg/liter tazobactam are presented in Table 1. In the absence of tazobactam, the wild-type non-β-lactamase-producing strain 2805 exhibited the lowest MIC, at 0.25 mg/liter. Strains 2890 and 2842 producing class C β-lactamases exhibited MICs of 4 and 8 mg/liter, respectively, while high resistance to ceftolozane was displayed by the CTX-M-15-producing strain, 2807, with an MIC of >128 mg/liter. In the presence of 4 mg/liter tazobactam (per CLSI guidelines [14]), the MICs of all β-lactamase-producing strains were reduced to 2- to 16-fold.

After 48 h of incubation at 35°C, the degradation rate constants of ceftolozane ranged from 0.00466 to 0.00679 h⁻¹ (half-life range, 102 to 148 h; R² ≥ 0.938). Tazobactam concentrations were constant for the duration of the 48-h experiments.

Time-kill experiments.

Time-kill profiles illustrating the change in bacterial density of the four E. coli strains following exposure to ceftolozane alone and in combination with tazobactam are presented according to fixed tazobactam concentrations at both 10⁶ and 10⁸ CFU/ml in Fig. 1 and 2, respectively).

FIG 1.

Change in bacterial burdens of strains 2805 (A to E), 2890 (F to J), 2842 (K to O), and 2807 (P to T) at a low inoculum (∼10⁶ CFU/ml) over 48 h, following treatment with ceftolozane (0 to 256 mg/liter) at fixed tazobactam concentrations (0 to 64 mg/liter). The colored curves on each graph represent different ceftolozane concentrations, including the untreated growth control (black), tazobactam alone (gray), 1 mg/liter (red), 4 mg/liter (blue), 16 mg/liter (pink), 64 mg/liter (green), and 256 mg/liter (purple).

FIG 2.

Change in bacterial burdens of strains 2805 (A to E), 2890 (F to J), 2842 (K to O), and 2807 (P to T) at a high inoculum (∼10⁸ CFU/ml) over 48 h, following treatment with ceftolozane (0 to 256 mg/liter) at fixed tazobactam concentrations (0 to 64 mg/liter). The colored curves on each graph represent different ceftolozane concentrations, which includes the untreated growth control (black), tazobactam alone (gray), 1 mg/liter (red), 4 mg/liter (blue), 16 mg/liter (pink), 64 mg/liter (green), and 256 mg/liter (purple).

Strain 2805 (no β-lactamase).

For the susceptible strain 2805 at 10⁶ CFU/ml, complete bactericidal activity reaching undetectable limits of eradication was achieved within 24 h in response to monotherapy with 4 mg/liter ceftolozane (−6.48 log₁₀ CFU/ml, Fig. 1A). No improvement in activity was gained at higher concentrations or in combination with tazobactam (Fig. 1B to E). At 10⁸ CFU/ml, bactericidal activity was attained at low tazobactam concentrations (0 to 4 mg/liter) in combination with ceftolozane at ≥64 mg/liter (−6.43 to −8.33 log₁₀ CFU/ml; Fig. 2A to E), or at higher tazobactam concentrations of ≥16 mg/liter in combination with ≥16 mg/liter ceftolozane (−5.32 to −8.23 log₁₀ CFU/ml; Fig. 2D and E).

Strain 2890 (AmpC).

Rapid activity was demonstrated with ceftolozane monotherapy at concentrations of 4 to 256 mg/liter against 2890 at 10⁶ CFU/ml, with a decrease of −2.39 to −3.53 log₁₀ CFU/ml after 4 h, followed by regrowth, whereby bacterial counts mimicked those of the untreated control within 24 h (Fig. 1F). A concentration-dependent trend toward a greater level of ceftolozane killing was observed in combination with increasing tazobactam concentrations. After 48 h, sustained bactericidal activity was attained with combinations of ≥4 mg/liter ceftolozane and ≥16 mg/liter tazobactam (−3.93 to −6.33 log₁₀ CFU/ml, Fig. 1I and J). At 10⁸ CFU/ml, both monotherapy and combination regimens containing ≤4 mg/liter ceftolozane were mostly inactive (below −0.5 log₁₀ CFU/ml), while bacterial counts were reduced to undetectable limits with all regimens containing ceftolozane concentrations of 256 mg/liter (Fig. 2F to J). Bactericidal activity at 48 h was noted for 64 mg/liter ceftolozane in combination with ≥4 mg/liter tazobactam (−4.01, −5.65, and −5.18 log₁₀ CFU/ml, Fig. 2H to J, respectively). The arm with 16 mg/liter ceftolozane was capable of achieving bactericidal activity at 48 h when in the presence of 16 and 64 mg/liter tazobactam (−3.12 and −4.09 log₁₀ CFU/ml, respectively).

Strain 2842 (CMY-10).

Concentration-dependent enhancement of ceftolozane activity in combination with tazobactam was similarly observed for strain 2842 at 10⁶ CFU/ml (Fig. 1K to O). Accordingly, at ≤4 mg/liter tazobactam, activity was negligible in combination with 1 mg/liter ceftolozane, while bactericidal activity was noted within 8 h of exposure to 4 mg/liter ceftolozane (−2.94 to 3.23 log₁₀ CFU/ml) and was followed by rapid regrowth by 24 h (Fig. 1K to M). However, bacteria were driven below detectable limits by all monotherapy and combination regimens containing ≥16 mg/liter ceftolozane (−6.18 log₁₀ CFU/ml; Fig. 1K to O). At 10⁸ CFU/ml, substantial activity at ≤16 mg/liter tazobactam was seen in combination with ≥64 mg/liter ceftolozane (−2.28 to −5.18 log₁₀ CFU/ml; Fig. 2K to N). At the highest tazobactam concentration of 64 mg/liter, bactericidal activity was achieved with ≥16 mg/liter ceftolozane (−4.74 to −5.39 log₁₀ CFU/ml; Fig. 2O).

Strain 2807 (CTX-M-15).

Strain 2807 was resistant to all ceftolozane monotherapy regimens at both inocula, with bacterial counts mirroring the control strain after 48 h. Interestingly, tazobactam maintained the ability to enhance activity in a concentration-dependent manner (Fig. 1 and 2P to T, 10⁶ and 10⁸ CFU/ml, respectively). Against both inocula, ceftolozane concentrations of ≥64 mg/liter achieved bactericidal activity at 48 h at a tazobactam concentration of 16 mg/liter (Fig. 1S and 2S, at or above −4.24 and −3.07 log₁₀ CFU/ml, respectively). In the presence of 64 mg/liter tazobactam, ceftolozane concentrations of ≥4 mg/liter achieved bactericidal activity at 48 h against the 10⁶ CFU/ml inoculum (Fig. 1T, at or above −3.97 log₁₀ CFU/ml), whereas ceftolozane concentrations of ≥16 mg/liter were required for bactericidal activity against the 10⁸ CFU/ml inoculum (Fig. 2T, at or above −3.66 log₁₀ CFU/ml).

PK/PD analyses.

PK/PD analyses of time-kill data were performed to determine the target concentrations of ceftolozane and tazobactam associated with optimal activity. PD relationships were fit to a sigmoidal Hill-type function (equation 2), from which PD parameters were estimated (R² > 0.882, Table 2). The ceftolozane-tazobactam PD relationship was primarily defined by E_max and EC₅₀ parameters, which provide a measure of the maximal capacity of efficacy and half-maximal potency, respectively. Across all strains at both inocula, no significant trends were noted in E_max values with increasing tazobactam concentrations (P > 0.2, ANOVA). However, in the presence of tazobactam, a clear tendency toward lower EC₅₀ values was demonstrated for all strains at both inocula (Table 2). Overall, pairwise comparisons revealed that all EC₅₀ differences at tazobactam concentrations of 0 to 4 mg/liter versus 16 and 64 mg/liter were statistically significant (P < 0.01, ANOVA). The changes in EC₅₀ as tazobactam concentrations increased from 0 to 4 mg/liter were not statistically significant, nor was the difference in EC₅₀s at tazobactam concentrations of 16 versus 64 mg/liter (P > 0.1, ANOVA). Hypothesis testing revealed that the EC₅₀s at 10⁸ CFU/ml were 2.81 to 66.5 times greater than those at 10⁶ CFU/ml (median, 10.7-fold increase; P = 0.002, F-test); however, no significant differences in E_max were observed between the two inocula (P = 0.531, F-test).

TABLE 2.

Hill plot PD parameter estimates describing the effect of ceftolozane in the presence of increasing tazobactam concentrations (0 to 64 mg/liter) at low and high inocula

Strain	Tazobactam concn (mg/liter)	Low inoculum of ceftolozane (SE)					High inoculum of ceftolozane (SE)
		E0	Emax	Ha	EC50	R2	E0	Emax	Ha	EC50	R2
2805	0	2.68	9.16	10.0	1.14	1.00	0.194 (0.343)	8.64 (0.765)	2.46 (0.737)	39.3 (7.58)	0.991
	1	2.62	9.10	10.0	0.948	1.00	0.291 (0.275)	8.84 (0.643)	1.91 (0.388)	33.7 (5.36)	0.995
	4	2.35	8.83	10.0	0.903	1.00	0.104 (0.451)	8.94 (1.23)	1.49 (0.509)	32.8 (9.27)	0.987
	16	2.48	8.96	9.53	0.156	1.00	0.552 (0.645)	9.03 (1.26)	1.00 (0.325)	10.2 (3.47)	0.984
	64	1.83	8.31	10.0	0.136	1.00	0.0450 (0.668)	8.25 (1.25)	1.00 (0.357)	9.04 (3.37)	0.980
2890	0	—b	—	—	—	—	0.629 (0.456)	10 (4.78)	1.00 (0.735)	52.1 (74.3)	0.993
	1	2.61 (0.734)	2.20 (1.12)	1.00 (1.25)	4.44 (5.65)	0.882	0.780 (0.292)	7.32 (0.505)	1.32 (0.271)	68.4 (1.709)	0.938
	4	2.40 (1.56)	9.47 (5.25)	1.00 (1.36)	23.5 (34.7)	0.923	0.497 (0.772)	10.0 (5.56)	1.00 (0.922)	66.0 (7.37)	0.970
	16	2.51 (0.751)	6.69 (0.87)	3.23 (2.72)	1.45 (0.53)	0.981	0.622 (0.554)	7.11 (1.13)	1.43 (0.689)	16.1 (5.53)	0.981
	64	1.96 (1.60)	6.98 (2.07)	5.10 (5.98)	1.27 (1.41)	0.952	0.137 (0.581)	5.81 (0.861)	3.38 (5.64)	12.2 (6.04)	0.970
2842	0	2.93 (0.109)	9.12 (0.140)	10.0 (27.1)	8.52 (1.48)	1.00	0.727 (0.297)	7.37 (2.87)	1.02 (0.478)	88.3 (75.6)	0.989
	1	2.95 (0.100)	9.15 (0.143)	4.57 (3.153)	4.81 (0.620)	1.00	0.179 (0.301)	5.28 (0.603)	4.64 (4.41)	28.8 (17.3)	0.985
	4	2.70 (0.296)	8.95 (0.435)	2.96 (1.45)	4.67 (0.466)	0.987	0.039 (0.308)	5.33 (0.637)	2.85 (1.28)	28.7 (8.93)	0.983
	16	3.10 (0.119)	9.29 (0.100)	10.0 (0.361)	1.04 (0.001)	1.00	0.0451 (0.382)	5.16 (0.806)	2.54 (1.93)	23.1 (8.56)	0.974
	64	2.26 (0.003)	8.44 (0.108)	10.0 (0.112)	0.84 (0.013)	1.00	−0.929 (0.458)	4.52 (0.678)	1.76 (0.867)	6.82 (2.51)	0.973
2807	0	—	—	—	—	—	—	—	—	—	—
	1	2.57 (0.424)	2.00 (4.17)	1.46 (1.95)	34.9 (11.0)	0.986	—	—	—	—	—
	4	3.17 (0.511)	4.16 (0.748)	0.633 (0.196)	2.34 (0.956)	0.995	—	—	—	—	—
	16	2.49 (0.551)	7.27 (0.675)	3.25 (1.36)	1.28 0.421)	0.912	0.0530 (0.473)	5.02 (1.71)	1.00 (0.627)	31.8 (25.0)	0.952
	64	1.82 (0.738)	7.46 (0.876)	2.24 (2.57)	1.65 (0.642)	0.982	1.61 (2.05)	5.08 (2.67)	1.00 (1.19)	1.10 (1.14)	0.913

^aHill's constants were constrained between the limits of 1 and 10.

^b—, no parameter estimates calculated because there was negligible activity that was not modeled well by a Hill-type function.

DISCUSSION

The introduction of ceftolozane-tazobactam has attracted much interest, with in vitro data providing evidence that this combination may present a promising addition to the therapeutic armamentarium against a range of Gram-negative pathogens, including β-lactamase-expressing strains (1–3, 5–8, 18–20). It is well recognized that appropriate antimicrobial dosing strategies necessitate a solid understanding of PK/PD principles to understand the relationship between the concentration-time profile and antimicrobial activity, allowing the design of regimens that maximize bacterial eradication and minimize the development of resistance. In the present study, the in vitro activities of an array of ceftolozane and tazobactam concentrations were tested alone and in combination in a dynamic fashion over 48 h against four strains of E. coli expressing different β-lactamases. Our study design incorporated a wide range of concentrations to ensure that all relevant PD parameters were captured for mathematical modeling purposes in the future.

The ability of inhibitors to inactivate specific β-lactamase enzymes varies markedly within and between classes; for tazobactam, irreversible β-lactamase inhibition (“suicide inhibition”) has mainly been demonstrated against pathogens expressing class A β-lactamases, while reduced activity has been reported for those expressing classes B, C, and D (21). In the present study, however, the time-kill data provide evidence that ceftolozane activity was enhanced by the addition of tazobactam in a concentration-dependent manner, which was observed against all β-lactamase-producing strains at both inocula, irrespective of β-lactamase expression (Fig. 1 and 2). This trend was substantiated by analyses of PD parameters, which revealed significantly reduced EC₅₀ values for all strains, indicating that the potency of ceftolozane increased at both inocula in the presence of increasing tazobactam concentrations. Characteristic differences noted in the time-kill profiles obtained for each strain serve to reinforce the varied extent of inactivation that tazobactam may pose on different β-lactamases (21). Notably, a 10.7-fold median increase in EC₅₀ was observed at the inoculum of 10⁸ CFU/ml compared to EC₅₀ values at 10⁶ CFU/ml; similar trends have been observed for other β-lactam agents against β-lactamase-producing strains in the presence of a high bacterial load (22–24). Mechanistic explanations for the differential PD observed between the two inocula have been hypothesized, including the possible release of larger quantities of β-lactamase enzymes from lysed bacteria (25), cell-to-cell communication, or the decreased presence of active penicillin-binding proteins due to the reduction in bacterial cell wall synthesis at higher bacterial densities (26, 27). Although the clinical implications of these observations have yet to be understood, consideration may be given to the administration of elevated ceftolozane doses when employed for the treatment of infections that entail a higher bacterial burden.

Interestingly, despite previous suggestions of improved stability of ceftolozane against AmpC β-lactamase from Pseudomonas aeruginosa (2, 7, 28), the exposure of 2890 to ceftolozane monotherapy resulted in extensive regrowth, even at extremely high concentrations (256 mg/liter) far in excess of the MIC (Fig. 1F). Owing to the relatively stable ceftolozane concentrations used during the time-killing experiments, regrowth was likely characterized by the amplification of ceftolozane-resistant subpopulations. Indeed, the phenomenon of heteroresistance to colistin (29), vancomycin (30), and carbapenems (31) has been reported and defined as the presence of a resistant subpopulation of bacteria within a susceptible strain based on MICs. Although heteroresistance has yet to be detected in E. coli, it certainly provides a plausible explanation for the regrowth observed here (Fig. 1F). Importantly, for strain 2890, the addition of 4 mg/liter tazobactam to regimens with ≥4 mg/liter ceftolozane resulted in bactericidal activity by 24 h at 10⁶ CFU/ml (Fig. 1H), although regrowth at a concentration of 4 mg/liter ceftolozane was observed by 48 h. Increasing the dose of tazobactam further to 16 mg/liter resulted in sustained killing at the ceftolozane concentration of 4 mg/liter up to 48 h (Fig. 1I). Similarly, the addition of ≥16 mg/liter tazobactam to regimens with ≥64 mg/liter ceftolozane achieved bactericidal activity by 48 h in the ceftolozane-resistant strain 2807 at both inocula. Ceftolozane and tazobactam concentrations of 64 mg/liter and 16 mg/liter reflect achievable maximum concentration of drug (C_max) values obtained in healthy volunteers receiving the equivalent of 1.0-g and 0.5-g doses of ceftolozane and tazobactam, respectively, which were the doses investigated in phase III clinical trials (16, 32, 33). Taken together, these results highlight the utility of tazobactam to increase the susceptibility of β-lactamase-producing E. coli strains to ceftolozane therapy at both inocula.

Although the %T_>threshold has been identified as the PK/PD index most predictive of the activity of ceftolozane-tazobactam, our results show a clear concentration dependence, with enhanced killing at elevated ceftolozane concentrations. The %T_>threshold was first proposed by VanScoy et al. (12) as the PK/PD index that best described ceftolozane-tazobactam killing in E. coli strains expressing the CTX-M-15 β-lactamase (12). Further work with E. coli and Klebsiella pneumoniae strains expressing various β-lactamases led to the unifying conclusion that the percentage of time above the ceftolozane-tazobactam MIC × 0.5 is predictive of the combination's efficacy, regardless of the β-lactamase expression profile of the pathogen (11). An in vivo study utilizing a neutropenic mouse model corroborated %T_>threshold as the ideal PK/PD index, and the authors were able to identify the % time above the MIC (%T_>MIC) targets required to achieve specific magnitudes of bacterial killing (34). In the present study, the concentration dependence observed in the time-kill experiments may partially be ascribed to the elevated bacterial burden (10⁸ CFU/ml) that exceeded the bacterial load utilized in previous in vitro (10⁶ CFU/ml) and in vivo (10^6.2 to 10^7.1 CFU/ml) studies (11, 12, 34). The static concentrations used in the current investigation may also account for some of the discordance with the results from prior studies. However, previous investigations evaluating the PK/PD of β-lactam–β-lactamase inhibitor combinations have asserted that stoichiometric inhibition of β-lactamase enzymes is determined by exposure of the inhibitor over time, which is best predicted by the area under the concentration-time curve (AUC) (35). A prior study utilizing piperacillin and tazobactam also found that dose fractionating the administration of both agents while maintaining the same drug exposure did not alter the killing of a TEM-producing strain of E. coli (36). The activity of ceftolozane-tazobactam may therefore not be completely described by time-dependent killing, and a hybrid index that accounts for the AUC or C_max may improve the predictive capability of the %T_>threshold index.

In summary, we systematically described the concentration-effect relationship of ceftolozane and tazobactam alone and in combination, revealing the ability of ceftolozane-tazobactam to achieve potent bactericidal activity against E. coli strains expressing different types of β-lactamase enzymes. While these results are promising, our time-kill studies utilized static concentrations of ceftolozane-tazobactam, making the extrapolation of our results into the clinical setting difficult. To the best of our knowledge, only two studies, by VanScoy et al. (10, 37), have looked at ceftolozane-tazobactam in a hollow-fiber infection model. Further studies evaluating the performance of the combination regimen against other pathogens containing β-lactamase enzymes are needed to completely understand the niche of ceftolozane-tazobactam among other β-lactams.

Funding Statement

Merck (Merck & Co., Inc.) provided funding to the University at Buffalo.