Efficacy of Endolysin LysAB1245 Combined with Colistin as Adjunctive Therapy Against Colistin-Resistant Gram-Negative Bacteria

Abstract

Background: Colistin resistance among Gram-negative nosocomial pathogens is an increasing concern. The bacteriophage-encoded lytic enzyme endolysin LysAB1245, which targets bacterial peptidoglycan, was evaluated as a potential antibacterial agent in combination with colistin as a therapeutic approach. Methods: Clinical isolates of Acinetobacter baumannii and Pseudomonas aeruginosa, along with two reference strains, were used to assess the antibacterial activity of LysAB1245 and colistin, individually and in combination. Antibacterial susceptibility was assessed by broth microdilution. Synergistic interactions were determined using checkerboard assays and confirmed by time-kill kinetics. Resistance development was assessed after several rounds of exposure to each agent, either alone or in combination. Results: In this study, the synergistic activity of the LysAB1245/colistin combination therapy was found in some clinical isolates of Acinetobacter baumannii and Pseudomonas aeruginosa, resulting in a reduction in the MICs of both LysAB1245 and colistin. The bactericidal effects, with a significant, more than 3-log reduction in CFU/mL (p < 0.01), were observed in representative synergistic isolates within 4 h of treatment with the combination of LysAB1245 at 1/4 × MIC and colistin at 1/4 × MIC. Scanning electron microscope micrographs confirmed bacterial cell damage upon treatment with the combination. Additionally, treatment with LysAB1245 in combination with colistin had no effect on the development of bacterial resistance after multiple passages. Conclusions: Combining LysAB1245 with a last-resort antibiotic like polymyxins (colistin) could be used as a promising new antibacterial strategy for preventing and controlling antibiotic-resistant Gram-negative bacteria.

Keywords: antibacterial agent; antibiotic-resistant bacteria; colistin; combination; endolysin.

Figure 1

Time-kill curve of Acinetobacter baumannii AB01 isolate after treatment with colistin and endolysin LysAB1245, both individually and in combination, for 18 h. Results obtained from two independent experiments performed in triplicate are expressed as mean ± standard deviation (SD). Statistical analysis was compared with the combination, ** p < 0.01, and NS means non-significant.

Figure 2

Time-kill curve of Pseudomonas aeruginosa PA04 isolate after treatment with colistin and endolysin LysAB1245, both individually and in combination, for 18 h. Results obtained from two independent experiments performed in triplicate are expressed as mean ± standard deviation (SD). Statistical analysis was compared with the combination, ** p < 0.01, and NS means non-significant.

Figure 3

Scanning electron micrographs of Acinetobacter baumannii AB01 after exposure to LysAB1245 at 1/4 × MIC combined with colistin at 1/4 × MIC (A), LysAB1245 at 1/4 × MIC (B), colistin at 1/4 × MIC (C), and the control (D), all shown at a magnification of 5000×.

Figure 4

Scanning electron micrographs of Acinetobacter baumannii AB01 after exposure to LysAB1245 at 1/4 × MIC combined with colistin at 1/4 × MIC (A), LysAB1245 at 1/4 × MIC (B), colistin at 1/4 × MIC (C), and the control (D), all shown at a magnification of 20,000×.

Figure 5

Scanning electron micrographs of Acinetobacter baumannii AB01 after exposure to LysAB1245 at 1/4 × MIC combined with colistin at 1/4 × MIC (A), LysAB1245 at 1/4 × MIC (B), colistin at 1/4 × MIC (C), and the control (D), all shown at a magnification of 40,000×. The presence of cell membrane damage is indicated by a circle symbol.

Figure 6

Scanning electron micrographs of Pseudomonas aeruginosa PA04 after exposure to LysAB1245 at 1/4 × MIC combined with colistin at 1/4 × MIC (A), LysAB1245 at 1/4 × MIC (B), colistin at 1/4 × MIC (C), and the control (D), all shown at a magnification of 5000×.

Figure 7

Scanning electron micrographs of Pseudomonas aeruginosa PA04 after exposure to LysAB1245 at 1/4 × MIC combined with colistin at 1/4 × MIC (A), LysAB1245 at 1/4 × MIC (B), colistin at 1/4 × MIC (C), and the control (D), all shown at a magnification of 20,000×.

Figure 8

Scanning electron micrographs of Pseudomonas aeruginosa PA04 after exposure to LysAB1245 at 1/4 × MIC combined with colistin at 1/4 × MIC (A), LysAB1245 at 1/4 × MIC (B), colistin at 1/4 × MIC (C), and the control (D), all shown at a magnification of 40,000×. The presence of cell membrane damage is indicated by a circle symbol.