Introduction

The emergence of Gram-negative multi-drug resistance is an ongoing threat. Pediatric patients are not spared from these resistant organisms. Of particular concern is emerging Gram-negative resistance among pediatric patients with cystic fibrosis (CF), who are frequently exposed to broad-spectrum antibiotic therapy for the treatment of acute pulmonary exacerbations. Multi-drug-resistant Pseudomonas aeruginosa often leaves CF patients with few remaining treatment options and has a significant detrimental impact on patient care, including increased mortality [1]. New antimicrobial agents have been developed for the treatment of multi-drug-resistant organisms (MDRO), but data in pediatric patients are extremely limited.

Ceftolozane/tazobactam (Zerbaxa™) was approved by the FDA in December 2014 for the treatment of complicated intra-abdominal and urinary tract infections in adults [2]. Ceftolozane/tazobactam is an extended-spectrum cephalosporin/beta-lactamase inhibitor combination that has been shown to maintain activity against MDR P. aeruginosa strains, including respiratory strains from CF patients [3–6]. Further evaluation of ceftolozane/tazobactam drug activity against MDR Pseudomonas isolates from pediatric patients is warranted, given the potential utility of this antibiotic in the treatment of MDR Pseudomonas infection with limited remaining treatment options. To better understand the antimicrobial susceptibility and potential role of ceftolozane/tazobactam in children—used alone, and in combination with tobramycin or amikacin—we performed Minimum inhibitory concentrations (MICs) testing and time-kill analyses against P. aeruginosa isolates from pediatric CF patients.

Methods

Microbiology

Five unique, non-duplicate clinical respiratory isolates of MDR P. aeruginosa (resistant to ≥3 classes as defined by MIC testing in the hospital microbiology laboratory) from four pediatric CF patients age 6–17 years were included. These isolates were collected at the discretion of the physician providing clinical care at Connecticut Children’s Medical Center from 2014 to 2016. Isolates were identified through retrospective review of the monthly institution MDRO surveillance report and were obtained from the hospital microbiology laboratory. Once attained by the Center for Anti-Infective Research and Development (Hartford Hospital, Hartford, CT, USA), isolates were transferred onto Trypticase Soy Agar with 5% sheep blood (Becton, Dickinson, Sparks, MD, USA). The minimum inhibitory concentrations (MICs) were determined in triplicate using broth microdilution methodology, and are listed in Table 1. As recommended by Clinical Laboratory Standards Institute (CLSI), P. aeruginosa 27853 was utilized as a quality control strain and colony counts were performed on each isolate to verify the correct inoculum. All isolates were phenotypically assessed against ceftolozane/tazobactam, tobramycin, amikacin and other antibiotics according to CLSI guidelines [7, 8]. The modal MIC was used to characterize the isolate MICs. Merck (Kenilworth, NJ, USA) provided ceftolozane powder; tazobactam and all other antibiotics were purchased from Sigma Chemical (St. Louis, MO, USA).

Table 1 Minimum inhibitory concentrations of five P. aeruginosa isolates against cefepime (FEP), ceftazidime (CAZ), ciprofloxacin (CIP), colistin (CST), aztreonam (ATM), piperacillin/tazobactam (TZP), meropenem (MEM), amikacin (AMK), fosfomycin (FOF), tobramycin (TOB) and ceftolozane/tazobactam (C/T)
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Time-Kill Analyses

Time-kill analyses were performed on each of the 5 P. aeruginosa isolates. Each isolate was subcultured twice on Trypticase Soy Agar with 5% sheep blood, (Becton, Dickinson) and a bacterial suspension of 108 CFU/mL was prepared. Mueller–Hinton Broth (MHB) was inoculated with the bacterial suspension to a final suspension approximately 5 × 105 CFU/mL. Ceftolozane/tazobactam was added to achieve concentrations of 0.5×, 1×, 2×, 4×, 8×, 16× and 32× ceftolozane/tazobactam MICs alone, and combinations of ceftolozane/tazobactam 2× and 8× the MIC with 30 mg/L tobramycin and ceftolozane/tazobactam 2× and 8× the MIC with 80 mg/L amikacin for all isolates. Aminoglycoside concentrations were determined to mimic peak concentrations achieved with once-daily dosing in children. [9] Control experiments without active compound were conducted simultaneously with the time-kill studies. Final volumes for each bacterium-drug concentration were 10 mL and incubated at 37 °C. Samples were taken from each sample at 0, 3, 6, and 24 h from the time of adding the drug. Multiple 1:10 dilutions were made in saline and sub-cultured onto blood agar plates and incubated for 18–24 h, and mean bacterial densities were determined for each isolate. The minimal, accurately countable number of CFU/mL was determined to be 5 × 10−1 CFU/mL. [10] All studies were conducted in duplicate on different days, and the combined data are presented as mean bacterial density (CFU/mL) for all isolates. Bactericidal activity was defined as a decrease of ≥3-log10 from baseline bacterial density. Synergy was defined as a ≥2-log10 decrease in CFU/mL between the antibiotic combination and its most active constituent after 24 h when the number of surviving organisms in the presence of the combination was ≥2-log10 CFU/mL below the starting inoculum [10].

Compliance with Ethics Guidelines

This study was approved by the Connecticut Children’s Medical Center Institutional Review Board, and does not contain any new studies with human or animal subjects performed by any of the authors.

Results

Isolates had variable susceptibilities to ceftolozane/tazobactam (range 0.5–8 mg/L), tobramycin (range 2 to >64 mg/L) and amikacin (range 8 to >256 mg/L). One isolate (1555) was identified as a mucoid strain. Mean bacterial densities over 24 h for all P. aeruginosa isolates tested against multiples of their ceftolozane/tazobactam MICs alone and in combination with either tobramycin or amikacin are shown in Fig. 1. Time-kill analyses revealed an average of 0.71 (range −0.6 to 4.4), 1.50 (range 0.8–2.0) and 2.1 (range 1.2–3.4) log-kill at 4×, 8× and 16× the ceftolozane/tazobactam MIC, respectively. Bactericidal killing was generally not seen until 16× ceftolozane/tazobactam MICs. At a tobramycin MIC of 2 mg/L, combination therapy with tobramycin and ceftolozane/tazobactam 2× and 8× the MIC showed a 4.3 and 3.7 log-kill, respectively. At a tobramycin MIC of 32 mg/L, combination therapy showed synergistic benefit when the isolate was ceftolozane/tazobactam susceptible (3.8 and 4.0 log-kill with 2× and 8× a ceftolozane/tazobactam MIC of 2 mg/L, respectively). No benefit of tobramycin combination therapy was seen among tobramycin non-susceptible isolates at a tobramycin MIC >64 mg/L.

Fig. 1
figure 1figure 1

Mean bacterial densities of five P. aeruginosa isolates over 24 h when exposed to multiple concentrations of C/T alone and in combination with TOB and AMK. a C/T MIC = 4 mg/L; TOB MIC >64 mg/L; AMK MIC = 32 mg/L, b C/T MIC = 4 mg/L; TOB MIC >64 mg/L; AMK = 16 mg/L; mucoid strain, c C/T MIC = 0.5 mg/L; TOB MIC = 2 mg/L; AMK MIC = 16 mg/L, d C/T MIC = 2 mg/L; TOB MIC = 32 mg/L; AMK MIC >256 mg/L, e C/T MIC = 8 mg/L; TOB MIC = 32 mg/L; AMK MIC = 8 mg/L. MIC minimum inhibitory concentration; for other abbreviatioms, see Table 1

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When compared to the extent of killing with amikacin therapy alone, there was no additional benefit of amikacin and ceftolozane/tazobactam combination therapy at an amikacin MIC of 16 mg/L. Combination therapy with amikacin and ceftolozane/tazobactam 2× and 8× the MIC at an amikacin MIC of 8 mg/L resulted in a 2.2 and 4.6 log-kill, respectively, despite the isolate being ceftolozane/tazobactam non-susceptible (MIC 8 mg/L). Despite amikacin non-susceptibility, synergistic activity was seen with amikacin and ceftolozane/tazobactam combination therapy at an amikacin MIC >256 mg/L and ceftolozane/tazobactam MIC of 2 mg/L (4.7 log-kill at 2× C/T MIC and 4.0 log-kill at 8× ceftolozane/tazobactam MIC).

Discussion

To the best of our knowledge, this is the first study to perform time-kill analyses of ceftolozane/tazobactam alone and in combination with other agents against P. aeruginosa isolates from pediatric CF patients. These data suggest that killing of P. aeruginosa isolates appears to require concentrations of at least 8× the ceftolozane/tazobactam MIC, with the extent of ceftolozane/tazobactam activity decreasing as MICs increase. Given the multiples of ceftolozane/tazobactam MICs tested, these data are likely in agreement with the concentration/MIC ratio of 6.6 suggested to optimize the in vivo antibiotic effect of ceftazidime against P. aeruginosa isolates from CF patients [11]. Further, the retained activity of ceftolozane/tazobactam against the tested mucoid P. aeruginosa strain supports previously published data, and suggests a potential role of this antimicrobial against mucoid strains from CF respiratory isolates [12]. The pharmacokinetics of ceftolozane/tazobactam in pediatric CF patients are yet to be explained, but a phase-one, open-label study evaluating the pharmacokinetics and safety of a single IV dose of ceftolozane/tazobactam in pediatric patients is currently enrolling (NCT02266706) [13]. Yet, the preserved ceftolozane/tazobactam MICs and susceptibility of P. aeruginosa isolates from pediatric CF patients reported here and in other studies is promising when considering the likelihood of attaining adequate ceftolozane/tazobactam pharmacodynamic targets [5].

The effect of tobramycin at concentrations equal to the MIC appears greater in isolates with a lower ceftolozane/tazobactam MIC. Overall, a synergistic effect of ceftolozane/tazobactam and tobramycin was evident among P. aeruginosa isolates that were largely ceftolozane/tazobactam and tobramycin susceptible and among isolates with low-level tobramycin resistance that remained ceftolozane/tazobactam susceptible. While the addition of tobramycin does not appear to enhance ceftolozane/tazobactam activity in isolates that are highly tobramycin resistant, the addition of amikacin to ceftolozane/tazobactam appears to provide synergistic activity against highly-amikacin resistant, ceftolozane/tazobactam-susceptible strains. Additionally, synergistic activity of amikacin and ceftolozane/tazobactam at 8× the MIC was seen when the isolate was ceftolozane/tazobactam resistant but amikacin susceptible.

The size of this study is a limitation, as only a small number of isolates (n = 5) from a small number of patients (n = 4) were evaluated.

Conclusion

Ceftolozane/tazobactam appears to be a promising treatment option for treatment of MDR P. aeruginosa in pediatric CF patients. Interestingly, the benefit of ceftolozane/tazobactam combination therapy with amikacin may be more pronounced than with the addition of tobramycin. Further evaluation of such combination regimens is warranted in pediatric CF patients, specifically when a better understanding of ceftolozane/tazobactam pharmacokinetics in this population has been elucidated and dosing optimization strategies can be determined.