Efficacy of Artilysin Art-175 against Resistant and Persistent Acinetobacter baumannii

Abstract

Bacteriophage-encoded endolysins have shown promise as a novel class of antibacterials with a unique mode of action, i.e., peptidoglycan degradation. However, Gram-negative pathogens are generally not susceptible due to their protective outer membrane. Artilysins overcome this barrier. Artilysins are optimized, engineered fusions of selected endolysins with specific outer membrane-destabilizing peptides. Artilysin Art-175 comprises a modified variant of endolysin KZ144 with an N-terminal fusion to SMAP-29. Previously, we have shown the high susceptibility of Pseudomonas aeruginosa to Art-175. Here, we report that Art-175 is highly bactericidal against stationary-phase cells of multidrug-resistant Acinetobacter baumannii, even resulting in a complete elimination of large inocula (≥10(8) CFU/ml). Besides actively dividing cells, Art-175 also kills persisters. Instantaneous killing of A. baumannii upon contact with Art-175 could be visualized after immobilization of the bacteria in a microfluidic flow cell. Effective killing of a cell takes place through osmotic lysis after peptidoglycan degradation. The killing rate is enhanced by the addition of 0.5 mM EDTA. No development of resistance to Art-175 under selection pressure and no cross-resistance with existing resistance mechanisms could be observed. In conclusion, Art-175 represents a highly active Artilysin against both A. baumannii and P. aeruginosa, two of the most life-threatening pathogens of the order Pseudomonadales.

FIG 1

Antibacterial effects of Art-175 and combinations of conventional antibiotics with Art-175 on different A. baumannii reference strains. (A and B) Stationary-phase cultures of RUH875 (black), LUH5875 (dark gray), NCTC 13423 (light gray), and RUH134 (white) were treated for 1 h (A) or 24 h (B) with ciprofloxacin (15 μg/ml), tobramycin (60 μg/ml), Art-175 (60 μg/ml), or the combination therapy of Art-175 with these antibiotics. All concentrations correspond to 30× MIC_RUH134. Bacterial survival after 1 h and 24 h of treatment is expressed in log₁₀ CFU per milliliter. ND, not detected. (C) Time-kill curves were obtained for RUH134 with stationary-phase cells that were treated for 20 min, 40 min, 1 h, or 2 h with buffer (control; black line), ciprofloxacin (filled circles; 15 μg/ml), ciprofloxacin plus Art-175 (open circles; 15 μg/ml and 120 μg/ml, respectively), tobramycin (filled triangles; 60 μg/ml), tobramycin plus Art-175 (open triangles; 60 μg/ml and 120 μg/ml, respectively), or Art-175 (open diamonds; 120 μg/ml). All concentrations correspond to 30× MIC_RUH134. Data points represent the mean values (±SEM) from at least three independent repeats. (D) Stationary-phase cells of RUH134 were treated with increasing concentrations of Art-175 for 20 min, 40 min, 1 h, and 2 h. The control consists of buffer (black line). Art-175 was administered at 5× MIC (open circles; 20 μg/ml), 10× MIC (open triangles; 40 μg/ml), 20× MIC (open squares; 80 μg/ml), and 30× MIC (open diamonds; 120 μg/ml). Data points represent the mean values (± SEM) from three independent repeats.

FIG 2

Real-time monitoring of osmotic lysis induced by Art-175. Exponential-phase cells of A. baumannii RUH134 with a high internal osmotic pressure (washed with 20 mM HEPES-NaOH [pH 7.4]–0.5 mM EDTA) (A) or a low internal osmotic pressure (washed with 20 mM HEPES-NaOH [pH 7.4]–0.5 M NaCl–0.5 mM EDTA) (B) were immobilized in a flow cell and exposed to Art-175 (0.4 mg/ml with 0.5 mM EDTA). A time-lapse series is presented with intervals of 3 s (A) or 5 min (B). To visualize the rapid lysis under conditions of high osmotic pressure, three frames before contact with Art-175 are shown (from −00:00:06). The flow of Art-175 is started at 00:00:00. Total durations are 36 s (A) and 1 h (B). Scale bars correspond to 2 μm. Movies S1A and S1B in the supplemental material show the full time-lapse series.

FIG 3

Art-175 kills isolated persister cells. Stationary-phase cultures of RUH 134 were treated with high doses of tobramycin to isolate the surviving persister fraction. The remaining persister cells were treated with 30× MICs of ciprofloxacin (15 μg/ml) and Art-175 (120 μg/ml) and an equimolar dose of KZ144 (108 μg/ml), all in the absence or presence of 0.5 mM EDTA. As a control, untreated persister cells and treatment with 0.5 mM EDTA and 30× MIC tobramycin (60 μg/ml) were taken into account. Persister survival after 5 h is expressed in log₁₀ CFU per milliliter. Mean values are shown (±SEM) from at least 3 independent repeats.

FIG 4

Art-175 is highly refractory to resistance development. A. baumannii strains RUH134 (triangles), RUH875 (circles), and LUH5875 (squares) were serially treated with subinhibitory concentrations of ciprofloxacin (A) or Art-175 (B) to select for decreased susceptibility. Values are the ratios of the MIC after cycle i (MIC_i) over the MIC at the start of the experiment (MIC₀). After 20 cycles, the MIC of Art-175 increased maximally 2-fold, whereas the MIC of ciprofloxacin increased 512-fold (RUH134 and RUH875) or 4-fold (LUH5875).