Formulation of phage cocktails and evaluation of their interaction with antibiotics in inhibiting carbapenemase-producing Klebsiella pneumoniae in vitro in Kenya

Abstract

Background: The development of alternative control measures, such as phage therapy or adjunctive therapy, is urgently needed to manage the dissemination of carbapenemase-producing Klebsiella pneumoniae.

Objective: This study aimed to evaluate the therapeutic potential of formulated phage cocktails and their interaction with select antibiotics in inhibiting the growth of carbapenemase-producing K. pneumoniae clinical isolate in vitro in Kenya.

Methods: The study was conducted from February 2021 to October 2021 at the Institute of Primate Research, Nairobi, Kenya. Phage cocktails were formulated based on the morphology and biological properties of precipitated Klebsiella phages. The efficacy of individual bacteriophages and phage cocktails as well as their combination with antibiotics were determined for their inhibitory activity on carbapenemase-producing K. pneumoniae (KP20).

Results: The precipitated bacteriophages were members of Myoviridae, Siphoviridae and Podoviridae. Regarding the evaluation of the phage cocktails, the absorbances at 600 nm of the bacterial culture treated with the two-phage cocktail (2φ MA) ranged from 0.173 to 0.246 at 16 h and 20 h whereas it peaked from 2.116 to 2.190 for the positive control. Moreover, the results of the adjunctive therapy showed that the optical density at 600 nm of the bacterial culture treated with 2φ MA was 0.186 at 24 h post-incubation time while it was 0.099 with the bacterial culture treated with imipenem in combination with 2φ MA.

Conclusion: This study demonstrated that the two-phage cocktail in combination with imipenem was able to synergistically delay the increase in carbapenemase-producing K. pneumoniae growth in vitro.

Keywords: Klebsiella pneumoniae; absorbances; antibiotics; carbapenemase; phage cocktail.

FIGURE 1

Transmission electron microscope images of the precipitated Klebsiella bacteriophages isolated from Ruai and Rongai in February 2021. (a) and (b) Bacteriophages belonging to the family of Myoviridae with contractile tails consisting of a sheath and a central tube (CPRSA and CPRSB); (c) and family of Podoviridae short tail (ESBLA).

FIGURE 2

Efficacy of phage cocktails in inhibiting the growth of carbapenem-resistant K. pneumoniae (KP20), Nairobi, Kenya, February 2021 – October 2021. Optical density of KP20 cultures (≈ 10⁶ CFU/ml) infected with (a) Individual phages CPRSA and CPRSB at MOI 1.0, 0.1 and 0.001 (b) the 2-phage cocktail (2φ MA at MOI 1.0, 0.1 and 0.001) or 3-phage cocktail (3φ MB at MOI 1.0, 0.1 and 0.001). The digits 1, 2 and 3 coming next after the ID of the phages represents MOI 1.0, 0.1 and 0.001. The most effective phages were the individual phage CPRSA and phage cocktail 2φ MA at MOI 0.1 (CPRSA2 & 2φ MA2) and 0.001 (CPRSA3 & 2φ MA3).

FIGURE 3

Bactericidal activity of Klebsiella phage cocktail alone or in combination with antibiotics, Nairobi, Kenya, February 2021 – October 2021. (a) Carbapenemase-producing K. pneumoniae culture (OD₆₀₀≈0.6) treated with phage cocktail 2φ MA and 3φ MB at MOI 0.1 or 0.001, Imipenem (IMP, MIC = 4 µg/mL), and Tigecycline (TG, MIC = 1µg/mL), separately. IMP2 and TG2 were considered as double the volume of each antibiotic; (b) Carbapenemase-producing K. pneumoniae culture treated with phage cocktail + imipenem or phage cocktail + tigecycline. IMP2 + 2φ MA3 was the treatment of KP20 culture with the double volume of imipenem combined with two-phage cocktail at MOI 0.001, and TG1 + 3φ MB2 was the treatment of KP20 culture with the single volume of tigecycline combined with the three-phage cocktail at MOI 0.1.