Relative fitness of wild-type and phage-resistant pyomelanogenic P. aeruginosa and effects of combinatorial therapy on resistant formation

Abstract

Bacteriophages, the natural predators of bacteria, are incredibly potent candidates to counteract antimicrobial resistance (AMR). However, the rapid development of phage-resistant mutants challenges the potential of phage therapy. Understanding the mechanisms of bacterial adaptations to phage predation is crucial for phage-based prognostic applications. Phage cocktails and combinatorial therapy, using optimized dosage patterns of antibiotics, can negate the development of phage-resistant mutations and prolong therapeutic efficacy. In this study, we describe the characterization of a novel bacteriophage and the physiology of phage-resistant mutant developed during infection. M12PA is a P. aeruginosa-infecting bacteriophage with Myoviridae morphology. We observed that prolonged exposure of P. aeruginosa to M12PA resulted in the selection of phage-resistant mutants. Among the resistant mutants, pyomelanin-producing mutants, named PA-M, were developed at a frequency of 1 in 16. Compared to the wild-type, we show that PA-M mutant is severely defective in virulence properties, with altered motility, biofilm formation, growth rate, and antibiotic resistance profile. The PA-M mutant exhibited reduced pathogenesis in an allantoic-infected chick embryo model system compared to the wild-type. Finally, we provide evidence that combinatory therapy, combining M12PA with antibiotics or other phages, significantly delayed the emergence of resistant mutants. In conclusion, our study highlights the potential of combinatory phage therapy to delay the development of phage-resistant mutants and enhance the efficacy of phage-based treatments against P. aeruginosa.

Keywords: Anti-phage mutation; Antibiotics; Antimicrobial resistance; Phage cocktails; Pigments; Pyomelanin.

Graphical abstract

Fig. 1

A. Lytic plaques produced by M12PA B. TEM image of M12PA phage belonging to the Myoviridae family.

Fig. 2

MOI optimization for M12PA against P. aeruginosa (PA-WT). MOI ranging from 100 to 0.000001 was used to infect the host. The box and whiskers graph represent the least to the highest obtained values for each MOI and the statistical significance is represented (ns-non significant, ∗ p value < 0.05, ∗∗ <0.01, ∗∗∗ <0.005, ∗∗∗∗ <0.0001).

Fig. 3

Phage priming – A second dose of phage (MOI 1) was provided at hour 6 h, 7 h, 8 h, 9 h, 10 h, 11 h and 12 h from the initial dosage time. The growth curve depicts the least cellular abundance by 18th hour with early dosage. Priming with cocktail was effective for an extended period of 6 h after which resistant mutants emerged.

Fig. 4

A) Structure of pyomelanin molecule, C₃-C₆ (β-bindings) B) IR spectrum of the sample recorded in Spectrum Two FT-IR Spectrometer (PerkinElmer) C) ¹H NMR Spectra of the sample recorded in Bruker 400 MHz instrument -Avance NEO {Solvent d₆-DMSO, number of scans −32}D) ¹³C NMR Spectra of the sample recorded in Bruker 400 MHz instrument -Avance NEO {Solvent d₆-DMSO, number of scans −15360 E) DEPT-135 ¹³C NMR Spectra of the sample recorded in Bruker 400 MHz instrument -Avance NEO {Solvent d₆-DMSO, number of scans −4625}.

Fig. 5

Phage adsorption percentage in 5 min against PA-WT in presence and absence of pyomelanin.

Fig. 6

Resistant profiles of PA-WT and PAM.

Fig. 7A

Combination therapy using gentamicin and phage Fig. 7B: Combination therapy using streptomycin and phage Fig. 7C: Combination therapy using ampicillin and phage.

Fig. 7A

Combination therapy using gentamicin and phage Fig. 7B: Combination therapy using streptomycin and phage Fig. 7C: Combination therapy using ampicillin and phage.

Fig. 7A

Combination therapy using gentamicin and phage Fig. 7B: Combination therapy using streptomycin and phage Fig. 7C: Combination therapy using ampicillin and phage.

Fig. 8

(A) and (B) Exponential and logistic growth curve of PA-WT and PAM with growth rate and carrying capacity derived using growthcurver package in R software and plotted using Geogebra graphing tool. The growth rate of PA-WT and PA-M was 0.359 and 0.324 with carrying capacity at 20 h being 99 % and 66 % respectively.

Fig. 9

Positive and negative correlations among the bacterial populations, PA-WT in LB broth and PA-M grown in presence of phage show a higher degree of positive correlation (represented by thicker lines) whereas no other conditions produced positive correlation of growth model with PA-WT in LB and PAM in phage stress denoting the extent of variation of growth pattern for both strains in other niche.

Fig. 10

PA-WT and PA-M competition in solid media indicates no production of antagonistic metabolites.

Fig. 11

A, B, C, D, E, F) MTT assay on biofilm formed by PA-WT and PA-M treated using gentamicin, streptomycin and ampicillin G) The MTT wells for PA-WT and PA-M, where PA-M did not develop any purple formazan product denoting the absence of viable cells. H) Difference in the dry weight of biomass obtained in a catheter represented using violin plots. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 12

(A) Degree of pathogenesis and structural deformity in eggs infected with PBS, PA-WT and PA-M. PA-WT shows extensive damage of egg yolk, allantoic cavity and the embryo. (B) Kaplan-Meier survival curve depicts the percentage of eggs survived with respect to time of infection. The p-value indicated the significant difference between the survival percentage of infected eggs. Log-Rank (Mantel-Cox) test was used to determine the significance.