Antibacterial effect of phage cocktails and phage-antibiotic synergy against pathogenic Klebsiella pneumoniae

ABSTRACT The global rise of antibiotic resistance has renewed interest in phage therapy, as an alternative to antibiotics to eliminate multidrug-resistant (MDR) bacterial pathogens. However, optimizing the broad-spectrum efficacy of phage therapy remains a challenge. In this study, we addressed this issue by employing strategies to improve antimicrobial efficacy of phage therapy against MDR Klebsiella pneumoniae strains, which are notorious for their resistance to conventional antibiotics. This includes the selection of broad host range phages, optimization of phage formulation, and combinations with last-resort antibiotics. Our findings unveil that having a broad host range was a dominant trait of isolated phages, and increasing phage numbers in combination with antibiotics significantly enhanced the suppression of bacterial growth. The decreased incidence of bacterial infection was explained by a reduction in pathogen density and emergence of bacterial resistance. Furthermore, phage-antibiotic synergy (PAS) demonstrated considerable broad-spectrum antibacterial potential against different clades of clinical MDR K. pneumoniae pathogens. The improved treatment outcomes of optimized PAS were also evident in a murine model, where mice receiving optimized PAS therapy demonstrated a reduced bacterial burden in mouse tissues. Taken together, these findings offer an important development in optimizing PAS therapy and its efficacy in the elimination of MDR K. pneumoniae pathogens. IMPORTANCE The worldwide spread of antimicrobial resistance (AMR) has posed a great challenge to global public health. Phage therapy has become a promising alternative against difficult-to-treat pathogens. One important goal of this study was to optimize the therapeutic efficiency of phage-antibiotic combinations, known as phage-antibiotic synergy (PAS). Through comprehensive analysis of the phenotypic and genotypic characteristics of a large number of CRKp-specific phages, we developed a systematic model for phage cocktail combinations. Crucially, our finding demonstrated that PAS treatments not only enhance the bactericidal effects of colistin and tigecycline against multidrug-resistant (MDR) K. pneumoniae strains in in vitro and in vivo context but also provide a robust response when antibiotics fail. Overall, the optimized PAS therapy demonstrates considerable potential in combating diverse K. pneumoniae pathogens, highlighting its relevance as a strategy to mitigate antibiotic resistance threats effectively.

(0.2<index≤0.5) and high bactericidal activity (index≤0.2),were determined as the ratios of bacterial density between treated and non-treated group (OD600-treated/OD600-non-treated), respectively.Three independent replicates were performed for each treatment and the abbreviations for phage cocktail combinations were indicated in main Fig. 3a."P" denotes phage cocktails with the subsequent number indicating the number of phages used in the phage cocktails, for example, P3 indicates a cocktail composed of three different phages.

Fig.S10
In vitro antimicrobial activity of four phage cocktails (P3-P6) with or without colistin (4 mg/L) against 88 MDR Kp strains.The infection index was indicated as the ratio of bacterial density between treated and non-treated groups (OD600-treated/OD600-non-treated).The threshold value for high bactericidal activity was 0.2, which indicate that the phage-cocktail combination effectively suppressed a higher number of MDR Kp strains."P" denotes phage cocktails with the subsequent number indicating the number of phages used in the phage cocktails, for example, P3 indicates a cocktail composed of different phages.Three independent replicates were performed for each treatment and the abbreviations for phage cocktail combinations were indicated in main Fig. 3a.The threshold value for high bactericidal activity was 0.2, indicating the similar bactericidal effectiveness was observed in tigecycline and phage-tigecycline combinations."P" denotes phage cocktails with the subsequent number indicating the number of phages used in the phage cocktails, for example, P3 indicates a cocktail composed of three different phages.Three independent replicates were performed for each treatment and the abbreviations for phage cocktail combinations were indicated in main Fig. 3a.

Fig. S1
Fig.S1 Plaque morphologies and representative microscopic images of seven phages selected for phage cocktail combination.Clear plaque morphologies depict the distinct shapes and sizes formed by each phage on bacterial lawns, indicative of their lytic activity and propagation characteristics.Their representative microscopic images provide a detailed visualization of the morphology of each phage, showing their typical capsid structure and tail morphology.The scale bar for TEM images was 50 nm.

Fig
Fig.S2 The circular genomic map of phage P85 with annotated features.The color-coded functional categories are indicated as follow: virion morphogenesis (purple), nucleic acid metabolism (blue), host lysis (red), DNA packing (yellow) and other functions (wathet).The first two inner circles show the GC skew (+ green, -crimson) and GC content (black), respectively.Proteins with unknown functions are marked with gray.The genome size of the phage is indicated in the inner most circle.

Fig
Fig.S3The circular genomic map of phage P61 with annotated features.The color-coded functional categories are indicated as follow: virion morphogenesis (purple), nucleic acid metabolism (blue), host lysis (red), DNA packing (yellow) and other functions (wathet).The first two inner circles show the GC skew (+ green, -crimson) and GC content (black), respectively.Proteins with unknown functions are marked with gray.The genome size of the phage is indicated in the inner most circle.

Fig
Fig.S4 The circular genomic map of phage P52 with annotated features.The color-coded functional categories are indicated as follow: virion morphogenesis (purple), nucleic acid metabolism (blue), host lysis (red), DNA packing (yellow) and other functions (wathet).The first two inner circles show the GC skew (+ green, -crimson) and GC content (black), respectively.Proteins with unknown functions are marked with gray.The genome size of the phage is indicated in the inner most circle.

Fig
Fig.S5 The circular genomic map of phage P60 with annotated features.The color-coded functional categories are indicated as follow: virion morphogenesis (purple), nucleic acid metabolism (blue), host lysis (red), DNA packing (yellow) and other functions (wathet).The first two inner circles show the GC skew (+ green, -crimson) and GC content (black), respectively.Proteins with unknown functions are marked with gray.The genome size of the phage is indicated in the inner most circle.

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Fig.S6 The circular genomic map of phage P28 with annotated features.The color-coded functional categories are indicated as follow: virion morphogenesis (purple), nucleic acid metabolism (blue), host lysis (red), DNA packing (yellow) and other functions (wathet).The first two inner circles show the GC skew (+ green, -crimson) and GC content (black), respectively.Proteins with unknown functions are marked with gray.The genome size of the phage is indicated in the inner most circle.

Fig
Fig.S7The circular genomic map of phage P67 with annotated features.The color-coded functional categories are indicated as follow: virion morphogenesis (purple), nucleic acid metabolism (blue), host lysis (red), DNA packing (yellow) and other functions (wathet).The first two inner circles show the GC skew (+ green, -crimson) and GC content (black), respectively.Proteins with unknown functions are marked with gray.The genome size of the phage is indicated in the inner most circle.

Fig. S8
Fig.S8 The circular genomic map of phage P79 with annotated features.The color-coded functional categories are indicated as follow: virion morphogenesis (purple), nucleic acid metabolism (blue), host lysis (red), DNA packing (yellow) and other functions (wathet).The first two inner circles show the GC skew (+ green, -crimson) and GC content (black), respectively.Proteins with unknown functions are marked with gray.The genome size of the phage is indicated in the inner most circle.

Fig. S11
Fig.S11 In vitro antimicrobial activity of four different phage cocktails (P3-P6) with or without tigecycline (4 mg/L) against 88 MDR Kp strains.The infection index was indicated as the ratio of bacterial density between treated and non-treated groups (OD600-treated/OD600-non-treated).The threshold value for high bactericidal activity was 0.2, indicating the similar bactericidal effectiveness was observed in tigecycline and phage-tigecycline combinations."P" denotes phage cocktails with the subsequent number indicating the number of phages used in the phage cocktails, for example, P3 indicates a cocktail composed of three different phages.Three independent replicates were performed for each treatment and the abbreviations for phage cocktail combinations were indicated in main Fig.3a.