Evaluation of Ceftolozane-Tazobactam in Combination with Meropenem against Pseudomonas aeruginosa Sequence Type 175 in a Hollow-Fiber Infection Model

ABSTRACT

The aim of this study was to investigate the efficacy of ceftolozane-tazobactam in combination with meropenem against an extensively drug-resistant (XDR) Pseudomonas aeruginosa high-risk clone, sequence type 175, isolated in a Spanish university hospital. A 14-day hollow-fiber infection model was used to simulate clinical exposure of the two drug regimens alone and in combination, and serial samples were collected to determine drug concentrations and CFU counts. The untreated control failed, as did each study regimen when administered alone. However, when ceftolozane-tazobactam was administered in combination with meropenem, there was a >4-log₁₀ CFU/ml bacterial density reduction and suppression of resistance for the duration of the study. These data suggest that ceftolozane-tazobactam plus meropenem may be a useful combination for treating XDR P. aeruginosa.

TEXT

Pseudomonas aeruginosa continues to be one of the main causes of morbidity and mortality in hospitalized patients. In the current era of multidrug resistance, clinicians often lack effective options with which to treat infections due to antibiotic-resistant P. aeruginosa isolates (1, 2). Without new therapeutic options, the outcomes of patients with many types of diseases will be compromised.

The lack of antipseudomonal agents in the pipeline worsens this situation (3), but over the last few years, there have been some advances in the development of new molecules and new associations with β-lactamase inhibitors (4, 5). The new cephalosporin ceftolozane (formerly, CXA-101) (6), combined with tazobactam, has exhibited promising characteristics for the treatment of P. aeruginosa infection (7).

An in vitro study reported that ceftolozane appears to be stable against the most common mutation-driven resistance mechanisms in this species (8). However, information on the activity of this agent against extensively drug-resistant (XDR) P. aeruginosa clones with high epidemic risk is limited (9). With this in mind, it was our intent to investigate the combination of ceftolozane-tazobactam and meropenem as a new effective regimen against XDR P. aeruginosa high-risk clones.

A single high-risk P. aeruginosa clone showing XDR characteristics (sequence type 175 [ST175]) was recovered from the clinical isolate of a patient with severe infection in a Spanish university hospital and evaluated. Susceptibility studies were performed following Clinical and Laboratory Standards Institute (CLSI) guidelines. MICs were determined by broth microdilution using Sensititre custom plates (Thermo Fisher Scientific), and consensus recommendations were used to classify the isolate as XDR (10).

Clonal relatedness was evaluated by pulsed-field gel electrophoresis (PFGE). Phenotypic detection of AmpC hyperproduction and OprD deficiency was performed using the cloxacillin inhibition test, as previously described (11). The presence of horizontally acquired β-lactamases and chromosomal resistance mechanisms was ruled out through previously established phenotypic and molecular methods, including whole-genome sequencing (12, 13).

A 14-day hollow-fiber experimental infection model was carried out in duplicate to investigate the efficacy of ceftolozane-tazobactam/meropenem combination therapy and the development of antimicrobial resistance (14, 15). In these experiments, we used FiberCell culture cartridges (FiberCell Systems, Inc., Frederick, MD) with a volume of 12 ml. With the use of separate infusion pumps for each compound, ceftolozane, tazobactam, and meropenem were pumped directly into the central reservoir to reach clinically achievable concentrations simulating the human free-drug pharmacokinetic profiles. A 2-g dose of meropenem was administered every 8 h (q8h) over an infusion period of 1 h. A 2-g dose of ceftolozane was administered in combination with 1 g of tazobactam q8h, also for 1 h. All exposures to simulate the steady-state human pharmacokinetics of unbound drugs were based on half-lives of 3, 1, and 1 h for ceftolozane, tazobactam, and meropenem, respectively (15, 16). Protein-binding estimates were 20%, 30%, and 2% for ceftolozane, tazobactam, and meropenem, respectively. All treatment regimens were compared with a no-treatment control.

On days 0, 1, 2, 3, 4, 6, 8, 10, 13, and 14 of the experiment, bacterial samples were obtained from the cartridges, washed, and resuspended in saline solution to minimize the drug carryover effect. Serially diluted samples were quantitatively cultured onto drug-free Mueller-Hinton II agar plates to enumerate the total bacterial population. A portion of the bacterial suspension was also quantitatively cultured onto agar supplemented with either ceftolozane-tazobactam at 3-fold the baseline MIC or meropenem at 3-fold the MIC to assess the effect of each regimen on the less-susceptible bacterial population. Over the first 48 h of the study, all pharmacokinetic samples were assayed by liquid chromatography-tandem mass spectrometry (LC/MS-MS) on a Waters Quattro Ultima instrument.

The MICs of the P. aeruginosa ST175 strain used in the hollow-fiber infection model were 8 mg/liter (intermediate) for meropenem and 2/4 mg/liter (susceptible) for ceftolozane-tazobactam (Table 1). The main antibiotic resistance mechanisms found in the isolate were hyperproduction of the chromosomal β-lactamase AmpC (through an AmpR mutation) and inactivation the carbapenem porin OprD.

TABLE 1.

Antibiotic susceptibility MICs of XDR P. aeruginosa ST175

Antibiotic	MIC (mg/liter)
Ticarcillin	256
Piperacillin-tazobactam	128
Ceftazidime	32
Cefepime	32
Ceftolozane-tazobactam	2/4
Aztreonam	16
Imipenem	32
Meropenem	8
Ciprofloxacin	>16
Tobramycin	32
Amikacin	4
Colistin	2

The simulated drug exposures in this model were considered satisfactory, based on observed r² values of 0.96, 0.97, and 0.97 for ceftolozane, tazobactam, and meropenem, respectively, and slopes with deviations from 1 equaling 4.2%, 9.7%, and 2.8% (Fig. 1). The standard curve for ceftolozane was linear and ranged from 10.0 to 100 mg/liter, and the r² value ranged from 0.968639 to 0.995902. The lower limit of quantification was 10.0 μg/ml. The interassay coefficients of variation (CVs) for the quality control samples at concentrations of 20.0, 50.0, and 75.0 μg/ml were 15.7%, 15.3%, and 12.6%, respectively (Fig. 1A). The standard curve for tazobactam was quadratic and ranged from 0.200 to 32.0 mg/liter, and the r² value ranged from 0.974575 to 0.991352. The lower limit of quantification was 0.200 μg/ml. The interassay CVs for the quality control samples at concentrations of 0.600, 8.00, and 24.0 μg/ml were 26.6%, 17.4%, and 9.73%, respectively (Fig. 1B). The standard curve for meropenem was quadratic and ranged from 0.500 to 80.0 mg/liter, and the r² value ranged from 0.990606 to 0.996273. The lower limit of quantification was 0.500 μg/ml. The interassay CVs for the quality control samples at concentrations of 1.50, 20.0, and 60.0 μg/ml were 13.8%, 6.48%, and 11.3%, respectively (Fig. 1C).

FIG 1.

Relationships between observed and targeted ceftolozane (A), tazobactam (B), and meropenem (C) concentrations.

The effects of monotherapy with meropenem or ceftolozane-tazobactam on the total bacterial population burden are shown in Fig. 2. Meropenem alone failed, as evidenced by a final CFU/ml count similar to that seen in the control samples. The ceftolozane-tazobactam monotherapy regimen resulted in an initial decrease, with a value of 2.95 log₁₀ CFU/ml by day 4, but the microorganism regrew and reached an average of 5.24 × 10⁶ CFU/ml. In contrast, administration of ceftolozane-tazobactam in combination with meropenem led to sustained suppression of the bacterial population. The sustained activity of this combination produced a 4.32-log₁₀ CFU/ml reduction and prevented amplification of the resistant subpopulation up to day 14 (Fig. 3). The density of the resistant subpopulation at doses of 3-fold the MIC was 1 CFU/ml in 8.41× 10⁹ CFU/ml for ceftolozane-tazobactam and 9.8 × 10⁸ CFU/ml for meropenem.

FIG 2.

Effects of the meropenem and ceftolozane-tazobactam regimens alone and in combination in a P. aeruginosa ST175 hollow-fiber infection model. Data express the mean values from duplicate experiments.

FIG 3.

Emergence of resistance during drug administration in P. aeruginosa ST175. (A) Negative control (no treatment) and the corresponding 3-fold MIC meropenem (MER) and ceftolozane-tazobactam (TOL/TAZO) drug plates. (B) Meropenem treatment regimens, comparing the total population versus the resistant population in 3-fold MIC drug plates. (C) Ceftolozane-tazobactam treatment, comparing the total population versus the resistant population in 3-fold MIC drug plates. (D) Ceftolozane-tazobactam plus meropenem, comparing the total population versus the resistant population in 3-fold MIC drug plates.

P. aeruginosa epidemic high-risk clones are an undeniable concern in hospitals around the world. The ST175 clone is of particular relevance in several European countries, such as Spain and France (17). ST175 has been associated with multidrug-resistant isolates and is recognized as a contaminant of the hospital environment and a respiratory tract colonizer in patients with cystic fibrosis, whose treatment is complex (13).

Ceftolozane-tazobactam shows encouraging characteristics for treating P. aeruginosa infection (4). In an in vitro study, development of high-level resistance to ceftolozane-tazobactam occurred only in P. aeruginosa mutant strains (6). The mechanisms involved are the emergence of at least two types of mutations, one leading to overexpression of the chromosomal β-lactamase AmpC and another to structural modification of the microorganism to increase cephalosporin hydrolytic efficiency. In our study, monotherapy with ceftolozane-tazobactam resulted in the development of resistance in the ST175 clone. Although the mechanisms of this effect remain to be defined, note that ST175 already exhibited one of the required mutations, AmpC overexpression, and this likely facilitated resistance development. In a recent study, emergence of ceftolozane-tazobactam resistance due to AmpC mutations was found in several patients infected by the ST175 clone (18).

The combination of β-lactams used here may provide stability to AmpC overexpression. This combination has been less extensively investigated in P. aeruginosa infection than in carbapenem-resistant Enterobacteriaceae isolates, in which a possible indication for the use of double carbapenem regimens has been reported (19). Although a potential enhancing effect of tazobactam on the activity of meropenem and ceftolozane combinations is unlikely according to current knowledge regarding β-lactam resistance mechanisms in P. aeruginosa, this possibility should be ruled out in future studies.

The excellent bacterial cell kill seen in our study, achieved by combining two β-lactams, may be related to an effect on several essential penicillin-binding proteins (PBP2 and PBP3 in P. aeruginosa), which can increase the bactericidal properties of the drugs and induce morphological changes in the bacteria. These effects may be useful in eradicating the microorganism in clinical practice. This concept is the basis for new emerging therapeutic approaches, such as the combination of cefepime with the non-β-lactam PBP2 inhibitor zidebactam (20). Beyond increased effectiveness due to boosted PBP inhibition, our results suggest an additional reason for the enhanced pharmacodynamic effect observed with the ceftolozane-tazobactam plus meropenem combination; i.e., the effectiveness for subpopulation killing would involve one drug killing the subpopulation(s) resistant to the other drug and vice versa.

With regard to the methods used, the dose, dosing interval, and experimental duration were selected to approximate those expected to be used clinically (15). Nonetheless, this study has some limitations. In vitro studies cannot examine toxicity or the different pharmacokinetic/pharmacodynamic effects occurring at the specific site of an infection. Furthermore, only one isolate was evaluated, and only one dosing regimen was used.

In summary, to our knowledge, this is the first study to investigate the novel cephalosporin ceftolozane together with tazobactam used in combination with meropenem against XDR P. aeruginosa. Our results demonstrate excellent bacterial cell kill and suppression of resistance emergence. These in vitro observations provide promising data that are of value as a basis for expanding research in this direction and ultimate evaluation in clinical use. High clinical effectiveness with low rates of resistance selection can be expected with this combination.