Designing phage cocktails to combat the emergence of bacteriophage-resistant mutants in multidrug-resistant Klebsiella pneumoniae

Abstract

In this study, we aimed to design a novel and effective bacteriophage cocktail that can target both wild-type bacteria and phage-resistant mutants. To achieve this goal, we isolated four phages (U2874, phi_KPN_H2, phi_KPN_S3, and phi_KPN_HS3) that recognized different bacterial surface molecules using phage-resistant bacteria. We constructed three phage cocktails and tested their phage resistance-suppressing ability against multidrug-resistant Klebsiella pneumoniae. We argue that the phage cocktail that induces resensitization of phage susceptibility exhibited superior phage resistance-suppressing ability. Moreover, we observed trade-off effects that manifested progressively in phage-resistant bacteria. We hypothesize that such trade-off effects can augment therapeutic efficacy. We also recommend collating phage host range data against phage-resistant mutants in addition to wild-type bacteria when establishing phage banks to improve the efficiency of phage therapy. Our study underscores the importance of phage host range data in constructing effective phage cocktails for clinical use.

Keywords: Klebsiella pneumoniae; bacteriophage; phage cocktail; phage resistance.

Fig 1

Workflow of novel phage isolation. We induced phage-resistant mutants and sequentially isolated a novel phage. KPN_270 is a wild-type bacterial strain. KPN_U2874R, KPN_H2R, and KPN_S3R are first-, second-, and third-stage phage-resistant mutants, respectively. U2874 is the first phage that was previously isolated. phi_KPN_H2, phi_KPN_S3, and phi_KPN_HS3 were, respectively, the second, third, and fourth phages that newly isolated.

Fig 2

Results of adsorption tests depicting the phage-resistance mechanism. All the phage-resistant mutants exhibited reduced adsorption rates compared with that of the original host strain. Adsorption tests were performed in triplicate. (A) Bacteria KPN_270 and KPN_U2874R against phage U2874. (B) Bacteria KPN_U2874R and KPN_H2R against phi_KPN_H2. (C) Bacteria KPN_H2R and KPN_S3R against phi_S3

Fig 3

Phage host spectrums against four bacterial strains used in this study. Phage samples were serially diluted 100-fold, and 10 µL was spotted for each diluent.

Fig 4

TEM images of phage U2874 (A), phi_KPN_H2 (B), phi_KPN_S3 (C), and phi_KPN_HS3 (D). Phage plaque morphologies of U2874 (E), phi_KPN_H2 (F), phi_KPN_S3 (G), and phi_KPN_HS3 (H). Black bars indicate the scale.

Fig 5

Circular map of (A) U2874, (B) phi_KPN_H2, (C) phi_KPN_S3, and (D) phi_KPN_HS3 genomes using DNAPlotter. Outer black lines represent the scales of genome sizes. Yellow arrows indicate forward CDSs and dark blue arrows indicate reverse CDSs. Arrows represent the 5′–3′ direction of each CDSs. Inner rings indicate GC content (black) and GC skew (+, olive color; −, purple). The whole genome sizes and names of phages are represented at the center of the circular map.

Fig 6

Lysis test results of single phage and phage cocktails. (A) MOI 10, (B) MOI 1, (C) MOI 0.1, (D) MOI 0.01, (E) MOI 0.001, and (F) MOI 0.0001. All the tests were performed in quintuplicate. (Single Phage; U2874, Cocktail 2; U2874 +phi_KPN_H2, Cocktail 3; U2874 +phi_KPN_H2 + phi_KPN_S3, Cocktail 4; U2874 +phi_KPN_H2 + phi_KPN_S3 + phi_KPN_HS3)

Fig 7

(A) The 24-hour growth curves of wild-type bacteria and phage-resistant mutants were measured at 600 nm wavelength. (B) Sedimentation of bacterial cells after 30 minutes of incubation with agitation was observed. (C-D) Biofilm formation on the bottom surface was quantified using stained crystal violet and measured at 590 nm. (E-F) Biofilm formation on lids was evaluated. Stained crystal violet was used to quantify the biofilm, and measurements were taken at 590 nm. (G) Bacterial growth in static culture was assessed by measuring optical density at the 24-hour time point, using a wavelength of 600 nm. (H) Survival rates of larvae infected with each bacterial strain were determined. One-way ANOVA followed by Tukey’s multiple comparisons test was used to compare the biofilm formation (***, P < 0.001; **, P = 0.007; ##, P = 0.005)

Fig 8

TEM images of bacterial cell morphology of KPN_270 (A), KPN_U2874R (B), KPN_H2R (C), and KPN_S3R (D). Black bars indicate the scale. Nonsense mutation of gene wbaP (E) and fhuA (F). Red boxes indicate codons where the mutation occurred. Green boxes indicated the premature stop codon.