Efficacy of Novel Antistaphylococcal Ectolysin P128 in a Rat Model of Methicillin-Resistant Staphylococcus aureus Bacteremia

Abstract

Staphylococcus aureus causes systemic infections with high morbidity and mortality, and the emergence of drug-resistant strains is a rapidly growing clinical concern. Novel therapeutic agents are required to tackle S. aureus infections. P128 is a bacteriophage-derived chimeric ectolysin with potent and rapid bactericidal activity against S. aureus In the present study, the efficacy of P128 was evaluated in a newly developed rat model of S. aureus bacteremia. Prior to in vivo testing, P128 was shown to be stable in whole blood by incubation in rat blood for up to 6 h and testing its bactericidal activity against the methicillin-resistant S. aureus isolate USA300. Rats succumbed to intravenous challenge with 10⁹ CFU of S. aureus USA300, resulting in 80 to 100% mortality by day 14. Evaluation of the bacterial load in various organs at 96 h postinfection revealed high bacterial counts in the kidney, and this correlated with the presence of renal abscesses. Treatment of infected animals with P128 either by intravenous bolus administration via tail vein or by 1-h infusion via the jugular vein at 2 h postinfection resulted in the dose-dependent survival of rats. P128 treatment also resulted in very few or no abscesses in the kidneys. These data show that P128 is stable in the physiological milieu and that intravenous treatment with P128 is highly effective in rescuing rats from S. aureus bacteremia. P128 can be a novel therapeutic option for treatment of S. aureus systemic infections.

Keywords: P128; Staphylococcus aureus bacteremia; antibiotic resistance; ectolysin; phage lysins; renal abscesses.

FIG 1

Activity of P128 in rat blood. P128 retains activity against MRSA USA300 in saline (A) and in whole blood (B) as measured by a ≥99.9% reduction in CFU/ml in comparison to controls (n = 3). Results are expressed as mean residual CFU/ml ± the SE. *, significantly different from cell control (P < 0.05).

FIG 2

Establishment of minimum lethal dose of S. aureus USA300 strain. (A) Wistar albino rats (n = 6 per group) were challenged with 10⁸ or 10⁹ CFU of MRSA USA300 per animal and monitored for survival for 14 days. (B) Wistar albino rats (n = 5 per group) were challenged with 10⁹ CFU of MRSA USA300 per animal and treated 2 h postinfection with vancomycin at 120 mg/kg administered subcutaneously (two doses at 12-h interval), and survival was monitored for 14 days.

FIG 3

Bacterial load in blood at different time points after infection. Blood samples were drawn at different time points from rats challenged with 10⁹ CFU of MRSA USA300 per animal to determine bacterial counts, expressed as the mean CFU/ml ± the SE in individual animals. Each animal is represented by a different shape. n = 4/group at 72 and 96 h; n = 5/group at all other time points.

FIG 4

Bacterial burden in tissues and renal abscesses. Wistar rats were challenged with 10⁹ CFU per animal i.v. and euthanized at 96 h postinfection. (A) Liver, spleen, kidney, and lung tissues were homogenized and plated to determine the CFU. Data represent individual CFU counts and means ± the SE for each organ. Each animal is represented by a different shape. (B) Renal abscesses (arrows) were observed at 96 h postinfection. (C and D) Kidneys from control animals showed normal renal histology (C), whereas histological findings after i.v. challenge with S. aureus USA300 included massive infiltration of inflammatory cells and accumulation of inflammatory fluid, marked by arrows (D). The original magnification of panels C and D was ×400.

FIG 5

Efficacy of P128 after i.v. bolus administration. Wistar rats were challenged i.v. with 10⁹ CFU per animal and treated 2 h postinfection with either saline or various doses of P128. (A) Survival was monitored for 14 days. P128 treatment resulted in dose-dependent survival with 100% survival observed at 2.5 mg/kg (P < 0.0001). (B and C) Diffuse renal abscesses (indicated by arrows) were found in animals treated with saline (B), whereas P128 treatment (2.5 mg/kg) resulted in few or no abscesses (C).

FIG 6

Body weights of saline- and P128-treated animals. Wistar rats were challenged i.v. with 10⁹ CFU per animal and treated 2 h postinfection with either saline or various doses of P128. Body weights were recorded on days 1 and 7. Data are represented as means ± the SE. n = 10 or 11 on day 1, and n = 4 to 10 on day 7.

FIG 7

Efficacy of P128 after i.v. infusion. Wistar rats (n = ≥5) were challenged i.v. with 10⁹ CFU per animal and treated 2 h postinfection with a 1-h i.v. infusion of either saline or various doses of P128. (A) Survival was monitored for 14 days. P128 resulted in dose-dependent survival with 80% survival at 10 mg/kg (P < 0.04). (B and C) Renal abscesses (arrows) found in animals 96 h after bacterial challenge and administration of a single dose of saline (B) or P128 at 10 mg/kg per animal (C).