Ceftolozane/tazobactam disrupts Pseudomonas aeruginosa biofilms under static and dynamic conditions

Abstract

Background: Pseudomonas aeruginosa biofilms limit the efficacy of currently available antibacterial therapies and pose significant clinical challenges. Pseudomonal biofilms are complicated further when other markers of persistence such as mucoid and hypermutable phenotypes are present. There is currently a paucity of data regarding the activity of the newer β-lactam/β-lactamase inhibitor combination ceftolozane/tazobactam against P. aeruginosa biofilms.

Methods: We evaluated the efficacy of ceftolozane/tazobactam against clinical P. aeruginosa isolates, the laboratory isolate PAO1 and its isogenic mutS-deficient hypermutator derivative (PAOMS) grown under static and dynamic biofilm conditions. The clinical isolate collection included strains with mucoid and hypermutable phenotypes.

Results: Ceftolozane/tazobactam exposure led to a bactericidal (≥3 log cfu/cm2) biofilm reduction in 15/18 (83%) clinical isolates grown under static conditions, irrespective of carbapenem susceptibility or mucoid phenotype, with greater activity compared with colistin (P < 0.05). Dynamically grown biofilms were less susceptible to ceftolozane/tazobactam with active biofilm reduction (≥1 log cfu/cm2) observed in 2/3 isolates. Hypermutability did not affect the antibiofilm efficacy of ceftolozane/tazobactam in either static or dynamic conditions when comparing PAO1 and PAOMS. Consistent with the activity of ceftolozane/tazobactam as a potent inhibitor of PBP3, dramatic impacts on P. aeruginosa morphology were observed.

Conclusions: Our data demonstrate that ceftolozane/tazobactam has encouraging properties in the treatment of P. aeruginosa biofilm infections, and its activity is not diminished against mucoid or hypermutable variants at the timepoints examined.

Figure 1.

Ceftolozane/tazobactam activity against static P. aeruginosa biofilms. Combined biofilm cfu reductions of 18 P. aeruginosa clinical isolates and PAO1 following treatment with varying concentrations of ceftolozane/tazobactam, with each isolate tested four times. Results are expressed as log₁₀ reduction of cfu/cm² compared with untreated controls. ***P ≤ 0.001, ****P ≤ 0.0001. C/T, ceftolozane/tazobactam.

Figure 2.

Ceftolozane/tazobactam activity against P. aeruginosa biofilms in a PD model. (a) PK validation of the PD biofilm assay for ceftolozane/tazobactam. The 24 h sample is representative of 8 h post infusion and is represented together with the 8 h result in this position. (b) Bacterial killing by ceftolozane/tazobactam and colistin over 24 h in a PD biofilm assay. Ceftolozane/tazobactam was infused into the reactor over 1 h via a syringe pump at 0, 8 and 16 h. Colistin was supplied as a continuous infusion. Biofilm-embedded coupons were sampled at 0, 8, 16 and 24 h. Results are expressed as log₁₀ reduction of cfu/cm² compared with time 0 (mean ± standard error of the mean). The numbers in brackets indicate the antibiotic MICs for each isolate in mg/L. Each point is representative of three (colistin) or four (ceftolozane/tazobactam) biological replicates. Ceftolozane/tazobactam demonstrated significantly greater efficacy than colistin against isolate MR-2 (P < 0.05, one-way ANOVA). C/T, ceftolozane/tazobactam.

Figure 3.

Effect of ceftolozane/tazobactam treatment on biofilm integrity. (a) Representative CLSM images of LIVE/DEAD stained biofilm embedded cells at 0 and 24 h following ceftolozane/tazobactam treatment in the CBR. Live cells appear green and dead cells appear red. At 0 h there is a dense biofilm present consisting of live cells. By 24 h the biofilm is sparser with many dead cells and altered cell morphology apparent. Images shown are of strain PAO1. (b) Representative SEM images showing the range of cellular morphologies observed following ceftolozane/tazobactam treatment. All isolates have dense biofilms prior to treatment. From 8 h onwards, MR-2 displays a loss of cellular integrity and presence of cellular debris (phenotype 1). From 8 h onwards, MS-6, MI-2, PAO1 and PAOMS display filamentation (phenotype 2) consistent with potent inhibition of PBP3.