Efficacy of bacteriophage therapy in experimental sepsis and meningitis caused by a clone O25b:H4-ST131 Escherichia coli strain producing CTX-M-15

Abstract

We evaluated phage therapy in experimental infections due to S242, a fatal neonatal meningitis Escherichia coli strain belonging to the worldwide-distributed O25b:H4-ST131 clone that produces extended-spectrum beta-lactamase CTX-M-15. A lytic phage, EC200(PP), active against S242, was isolated from environmental water. After determining in vitro and ex vivo stabilities and pharmacokinetic properties of EC200(PP) in rat pups, we assessed the therapeutic efficacy of a single dose of 10(8) PFU using models of sepsis and meningitis in which fatality was 100%. EC200(PP) was partially neutralized by human serum. In contrast to the high concentration of phage in the spleen and the kidney, low titers in urine and the central nervous system were observed. Nevertheless, in the sepsis model, EC200(PP) administered 7 h or 24 h postinfection resulted in 100% and 50% pup survival, respectively. In the meningitis model, EC200(PP) administered 1 h or 7 h postinfection rescued 100% of the animals. The most delayed treatments were associated with the selection of phage-resistant S242 mutants. However, a representative mutant was highly sensitive to killing serum activity and avirulent in an animal model. EC200(PP) is a potential therapeutic agent for sepsis and meningitis caused by the widespread E. coli O25:H4-ST131 multidrug-resistant clone.

Fig 1

Transmission electron micrograph of a particle (size, 200 nm) of bacteriophage EC200^PP, a member of the Podoviridae family. Magnification, ×48,000.

Fig 2

In vitro characterization of phage EC200^PP comprising an adsorption rate test (A), single-step growth experiment where latent time and burst size of phage EC200^PP were inferred from the curve with a triphasic pattern (L, latent phase; R, rise phase; P, plateau phase) (B), thermal stability test (C), and pH stability (D).

Fig 3

Stability of EC200^PP inoculated at 10⁷ PFU (black arrow) in different media (A) and in sera of different donors (B) during 24 h (*, P < 0.001 versus initial inoculum).

Fig 4

Pharmacokinetic study of EC200^PP after intraperitoneal injection (A) or subcutaneous injection (B) of 10⁸ PFU in rat pups. Representation of mean (n = 3 to 5) PFU/ml or PFU/g and standard error of the mean (SEM) in different organs or biologic fluids at 2, 6 and 24 h: ▲, spleen; ♦, brain; ■, kidney; △, blood; ♢, CSF; and □, urine. Detection thresholds: blood and urine (gray arrow) 20 PFU/ml; spleen, kidney, brain, and CSF (black arrow) (200 PFU/ml or/g).

Fig 5

(A) Survival curves after intraperitoneal injection of 2 × 10⁴ CFU of E. coli S242 in rat pups without treatment (●; n = 30) or treated at 7 h (■; n = 10) or 24 h (▲; n = 10) after infection by 10⁸ PFU of EC200^PP subcutaneously administered (*, P < 0.001 versus no treatment). Ratios on the figure indicate at different time points the rate of positive blood culture. (B) Survival curves after i.p. injection of 2 × 10⁴ CFU of E. coli S242 (●; n = 30) or 2 × 10⁴ CFU of O4-EC200^PPR (▼; n = 10) in rat pups (*, P < 0.001 versus S242). Ratios on the figure indicate at different time points the rate of positive blood culture.

Fig 6

(A) Survival curves after intrathecal injection of 200 CFU of S242 in rat pups without treatment (●; n = 10) or treated at 1 h (■; n = 5) or 7 h (□; n = 10) after infection by 10⁸ PFU of EC200^PP by i.p. injection. Ratios on the figure indicate at different time points the rate of positive CSF cultures (*, P < 0.001 versus no treatment). (B) Survival curves after intrathecal injection of 2 × 10⁶ CFU of S242 in rat pups without treatment (●; n = 10) or treated at 1 h (■; n = 5), 2 h (▲; n = 3), or 4 h (▼; n = 3) postinfection by 10⁸ PFU of EC200^PP by i.p. injection. Ratios on the figure indicate at different time points the rate of positive CSF cultures (*, P < 0.005 versus no treatment).