Evaluation of Bacteriophage Cocktail on Septicemia Caused by Colistin-Resistant Klebsiella pneumoniae in Mice Model

Abstract

Objective: The emergence of resistance against last-resort antibiotics, carbapenem and colistin, in Klebsiella pneumoniae has been reported across the globe. Bacteriophage therapy seems to be one of the most promising alternatives. This study aimed to optimize the quantity and frequency of bacteriophage cocktail dosage/s required to eradicate the Klebsiella pneumoniae bacteria in immunocompetent septicemic mice. Methods: The three most active phages ɸKpBHU4, ɸKpBHU7, and ɸKpBHU14 characterized by molecular and TEM analyses were in the form of cocktail and was given intraperitoneally to mice after inducing the septicemia mice model with a constant dose of 8 × 10⁷ colony-forming unit/mouse (CFU/mouse) Klebsiella pneumoniae. After that, the efficacy of the phage cocktail was analyzed at different dosages, that is, in increasing, variable, constant, and repeated dosages. Furthermore, interleukin-6 and endotoxin levels were estimated with variable doses of phage cocktail. Results: We have elucidated that phage therapy is effective against the Klebsiella pneumoniae septicemia mice model and is a promising alternative to antibiotic treatments. Our work delineates that a single dose of phage cocktail with 1 × 10⁵ plaque-forming unit/mouse (PFU/mouse) protects the mice from fatal outcomes at any stage of septicemia. However, a higher phage dosage of 1 × 10¹² PFU/mice is fatal when given at the early hours of septicemia, while this high dose is not fatal at the later stages of septicemia. Moreover, multiple repeated dosages are required to eradicate the bacteria from peripheral blood. In addition, the IL-6 levels in the 1 × 10⁵ PFU/mouse group remain lower, but in the 1 × 10¹² PFU/mouse group remains high at all points, which were associated with fatal outcomes. Conclusion: Our study showed that the optimized relatively lower and multiple dosages of phage cocktails with the strict monitoring of vitals in clinical settings might cure septicemia caused by MDR bacteria with different severity of infection.

Keywords: IL-6; Klebsiella pneumoniae; endotoxin; phage cocktail; septicemia.

FIGURE 1

Transmission electron micrographs of phages. ɸKpBHU4 (A) and ɸKpBHU14 (C) belong to Siphoviridae family, and ɸKpBHU7 (B) belongs to Tectiviridae family. The bar indicates 200 nm.

FIGURE 2

One-step growth curve of bacteriophages; ɸKpBHU4 (A), ɸKpBHU7 (B), ɸKpBHU14 (C) for estimation of burst size.

FIGURE 3

(A) Fingerprinting of ɸKpBHU4, ɸKpBHU7, and ɸKpBHU14 bacteriophages genomic DNA with RAPD PCR. Lane-1 (L1): molecular marker (1 kb); lane-2 (L2): ɸKpBHU4; lane-3 (L3): ɸKpBHU7; lane-4 (L4): ɸKpBHU14; and lane-5 (L5): molecular marker (100 bp). (B) Restriction endonuclease digestion of bacteriophages genomic DNA. The phage DNA was digested with restriction enzyme EcoRI and run on 1% agarose gel. Lane-1: molecular marker (1 kb) (L1); lane-2 ɸKpBHU4 (L2): lane-3 ɸKpBHU7 (L3): lane-4: ɸKpBHU14 (L4); and lane-5: molecular marker (100 bp) (L5).

FIGURE 4

Mouse experiment flowchart (A) Depicts the induction of septicemia through intraperitoneal route with K. pneumoniae at the dose of 8 × 10⁷ CFU/mouse of KpnBHU101. After 24 h, mice were killed, and organs were homogenized and cultured on MacConkey plates to see the presence of bacteria. (B) Outline of the efficacy of the bacteriophage cocktail dosage with varying concentrations at a different time point after the initial bacterial challenge. Each group has 5 mice; (M) depicts the number of dead mice and (S) depicts the number of mice surviving.

FIGURE 5

Outcome of the phage therapy on Klebsiella pneumoniae septicemia at different time points and phage dosage. The X-axis represents the time point of assessment after administration of phage cocktail and Y-axis shows the grading of severity of disease ranging from 1- normal; 2- slight illness, lethargy, ruffled fur; 3- moderate illness, severe lethargy, ruffled fur, and hunched back; 4- a severe illness with aforementioned signs, exudative accumulation around eyes; 5- death. (A) Phage cocktail (1 × 10², 1 × 10³, 1 × 10⁴, 1 × 10⁵, 1 × 10¹² PFU/mouse) given 6 h after bacterial challenge, 1 × 10² PFU/mouse denoted by an open circle (○), 1 × 10³ PFU/mouse denoted by an open triangle (∆), 1 × 10⁴ PFU/mouse denoted by an open square (□), 1 × 10⁵ PFU/mouse denoted by an closed circle (•), 1 × 10¹² PFU/mouse denoted by an closed triangle (▲). (B) Phage cocktail (1 × 10³, 1 × 10⁵ PFU/mouse) given simultaneously, 1 × 10³ PFU/mouse denoted by the closed circle (•) and 1 × 10⁵ PFU/mouse denoted by an open circle (○). (C) Phage cocktail (1 × 10⁵ PFU/mouse) given simultaneously denoted by a closed square (■), 6 h before bacterial challenge denoted by open square (□), 6 h after bacterial challenge denoted by an open triangle (∆), 12 h after bacterial challenge denoted by a closed circle (•), and 24 h after bacterial challenge denoted by an open circle (○). The bacterial challenge dose is constant for the entire group with 8 × 10⁷ CFU/mouse concentration. (D) Phage cocktail (1 × 10¹² PFU/mouse) given 24 h after bacterial challenge denoted by an open circle (○).

FIGURE 6

(A) Assessment of bacterial and bacteriophage count in the mice blood after simultaneous injection. The bacterial challenge given 8 × 10⁷ CFU/mouse and bacteriophage 1 × 10⁵ PFU/mouse, Open circle (○) denotes PFU/mL, and the closed circle (•) denotes CFU/mL. (B) Assessment of bacterial and bacteriophage count in the mice blood up to 144 h bacteriophage cocktail was given 6 h after bacterial challenge. The bacteriophage cocktail was given daily for 5 days. The bacterial challenge given 8 × 10⁷ CFU/mouse and bacteriophage 1 × 10⁵ PFU/mouse. Open circle (○) denotes PFU/mL, and the closed circle (•) denotes CFU/mL.

FIGURE 7

Assessment of interleukin-6 at the different dosages of phage cocktail given 6 h after bacterial challenge. The open circle (○) denotes (1 × 10¹² PFU/mouse), and the closed circle (•) denotes (1 × 10⁵ PFU/mouse).

FIGURE 8

Assessment of endotoxin at the different dosages of phage cocktail given 6 h after bacterial challenge. The open circle (○) denotes (1 × 10¹² PFU/mouse), and the closed circle (•) denotes (1 × 10⁵ PFU/mouse).