Characterization of 29 newly isolated bacteriophages as a potential therapeutic agent against IMP-6-producing Klebsiella pneumoniae from clinical specimens

Abstract

Carbapenemase-producing Enterobacteriaceae (CPE) are one of the most detrimental species of antibiotic-resistant bacteria globally. Phage therapy has emerged as an effective strategy for the treatment of CPE infections. In western Japan, the rise of Klebsiella pneumoniae strains harboring the pKPI-6 plasmid encoding bla _IMP-6 is of increasing concern. To address this challenge, we isolated 29 phages from Japanese sewage, specifically targeting 31 K. pneumoniae strains and one Escherichia coli strain harboring the pKPI-6 plasmid. Electron microscopy analysis revealed that among the 29 isolated phages, 21 (72.4%), 5 (17.2%), and 3 (10.3%) phages belonged to myovirus, siphovirus, and podovirus morphotypes, respectively. Host range analysis showed that 18 Slopekvirus strains within the isolated phages infected 25-26 K. pneumoniae strains, indicating that most of the isolated phages have a broad host range. Notably, K. pneumoniae strain Kp21 was exclusively susceptible to phage øKp_21, whereas Kp22 exhibited susceptibility to over 20 phages. Upon administering a phage cocktail composed of 10 phages, we observed delayed emergence of phage-resistant bacteria in Kp21 but not in Kp22. Intriguingly, phage-resistant Kp21 exhibited heightened sensitivity to other bacteriophages, indicating a "trade-off" for resistance to phage øKp_21. Our proposed phage set has an adequate number of phages to combat the K. pneumoniae strain prevalent in Japan, underscoring the potential of a well-designed phage cocktail in mitigating the occurrence of phage-resistant bacteria. IMPORTANCE The emergence of Klebsiella pneumoniae harboring the bla _IMP-6 plasmid poses an escalating threat in Japan. In this study, we found 29 newly isolated bacteriophages that infect K. pneumoniae strains carrying the pKPI-6 plasmid from clinical settings in western Japan. Our phages exhibited a broad host range. We applied a phage cocktail treatment composed of 10 phages against two host strains, Kp21 and Kp22, which displayed varying phage susceptibility patterns. Although the phage cocktail delayed the emergence of phage-resistant Kp21, it was unable to hinder the emergence of phage-resistant Kp22. Moreover, the phage-resistant Kp21 became sensitive to other phages that were originally non-infective to the wild-type Kp21 strains. Our study highlights the potential of a well-tailored phage cocktail in reducing the occurrence of phage-resistant bacteria.

Keywords: Klebsiella pneumoniae; bacteriophages; bla IMP-6; phage library; phage-resistant bacteria; trade-off.

Fig 1

Transmission electron microscopy images of 29 isolated phages. Each sample was negatively stained and magnified at ×50,000. Representative myovirus morphotype phages, øKp_1, øKp_21, øKp_22, and øKp_32, are displayed. All podovirus morphotype phages and siphovirus morphotype phages are shown. The scale bar in each image represents 200 nm.

Fig 2

Network images of the newly isolated phages based on shared protein ortholog families. vConTACT2 v0.11.3 was used for clustering each phage. vConTACT2 assigned viral cluster (VC) to each phage. Our phages were divided into nine VCs. Each VC, including our phages, is highlighted in different colors.

Fig 3

A heatmap illustrating the host range of each phage. The X- and Y-axes represent phages and host strains, respectively. Klebsiella pneumoniae ATCC BAA 1705 and ATCC BAA 1706 served as standard strains, whereas Escherichia coli SK191 and BL21 served as control strains. The color in the heatmap represents the EOP. Bar charts on the X- and Y-axes represent the number of infections in each phage and host, respectively.

Fig 4

OD₆₀₀ kinetics of indicator bacteria incubated with phages. Bacterial strains were incubated until reaching an OD₆₀₀ of 0.1, after which each phage was added at a concentration of 10⁹ pfu/mL. OD₆₀₀ measurements were taken at appropriate time intervals for up to 24 h. All experiments in this section were performed in triplicate. Gray and magenta represent the OD₆₀₀ without (negative control) and with phages, respectively.

Fig 5

Cocktail experiment of Kp21r with Kp22r. Cocktail experiment of Kp21r with Kp22r. OD₆₀₀ kinetics of Kp21 and Kp21r combined with phage cocktail are shown in panels A and B, respectively. OD₆₀₀ kinetics of Kp22 and Kp22r combined with phage cocktail are shown in panels C and D, respectively. Phage-resistant Kp21 (Kp21r) and Kp22 (Kp22r) strains were obtained from the culture medium after 24 h of incubation with øKp_21 or øKp_22. The cocktail comprised 10 phages, each at a concentration of 10⁷ pfu/mL. OD₆₀₀ measurements were taken at appropriate time intervals for up to 24 h. All experiments in this section were performed in triplicate.

Fig 6

Analysis of the shift in susceptibility to the phage cocktail in Kp21 and Kp21r. (A) The host range of Kp21 and Kp21r against a phage cocktail comprising 10 phages. “S” denotes a sparse bacterial lawn. (B) Colony-forming units are mentioned under each phage. Kp21 or Kp21r were mixed with individual phages, and 2 h after phage addition, samples were diluted to 10⁻² and 10⁻⁴ and lawned onto LB plates. “n.d.” indicates that no colonies were detected at the 10⁻² dilution.

Fig 7

Characterization of the phage-resistant Kp21r strain. (A) Adsorption assay of øKp_21 against Kp21 and Kp21r strains. Kp21 and Kp21r were incubated until reaching an OD₆₀₀ of 0.5. Subsequently, øKp_21 was added at an MOI of 0.01, and the mixture was incubated at 37°C with shaking at 200 rpm. After 5 min, 200 µL of the mixture was withdrawn and centrifuged at 9,100 × g for 1 min. The number of phages in the supernatant was measured. (B) 1.0 × 10⁹ pfu of øKp_21 were mixed with 100 µL of overnight culture of Kp21 or Kp21r. Then, 5 mL of 0.6% YT (yeast extract and tryptone)-soft agar was added to the host and phage mixture and incubated at 37°C overnight. “n.d.” indicates that no plaques were detected. (C) Nucleotide sequences of cpsA in Kp21 and Kp21r were aligned using ClustalW (https://www.genome.jp/tools-bin/clustalw). The insertion mutation (A) is indicated by an arrow, and the stop codons of cspA in Kp21 and Kp21r are shown by a square.