Phage lytic enzyme Cpl-1 as a novel antimicrobial for pneumococcal bacteremia

Abstract

Streptococcus pneumoniae is becoming increasingly antibiotic resistant worldwide, and thus new antimicrobials are badly needed. We report the use of Cpl-1, the lytic enzyme of a pneumococcal bacteriophage, as an intravenous therapy for pneumococcal bacteremia in a mouse model. A 2000- microg dose of Cpl-1 reduced pneumococcal titers from a median of log(10) 4.70 CFU/ml to undetectable levels (<log(10) 2.00 CFU/ml) within 15 min. This dose given 1 h after intravenous infection led to 100% survival at 48 h, compared to the 20% survival of buffer-treated controls. In advanced bacteremia, treatment with two doses at 5 and 10 h still resulted in significantly longer survival (P < 0.0001) and a hazard ratio of 0.29 (95% confidence interval, 0.04 to 0.35). The enzyme is immunogenic, but the treatment efficacy was not significantly diminished after previous intravenous exposure of mice and hyperimmune rabbit serum did not neutralize the activity. Cpl-1 is also very effective as a topical nasal treatment against colonization by S. pneumoniae. In vitro, the enzyme is active against many serotypes of S. pneumoniae, independent of their penicillin resistance, and it is very specific for this species. Bacteriophage enzymes are unusual but extremely effective antimicrobials and represent a new weapon against infections with resistant bacteria.

FIG. 1.

Bactericidal activity of Cpl-1 in the bloodstream of mice. In mice receiving a single bolus containing 2,000 μg of Cpl-1 (○), the bacterial blood titers dropped rapidly below the detection limit (log₁₀ 2.0 CFU/ml) and remained undetectable at 120 min, in contrast to the titers in mice that received only buffer (•).

FIG. 2.

Survival of bacteremic mice after Cpl-1 treatment. (A) Treatment of mice with a single bolus containing 2,000 μg of Cpl-1 (○) (n = 10) at 1 h after infection (arrow) led to 100% survival at 48 h, compared to the significantly lower level of survival of buffer-treated mice (•) (20%; n = 10), which had a median survival time of 25.56 h. (B) Treatment with two doses of Cpl-1 (○) (n = 14) at 5 and 10 h after infection (arrows) significantly extended survival compared to the survival after two administrations of only buffer (•) (n = 14). The hazard ratio for the Cpl-1 treatment was 0.29.

FIG. 3.

Activity spectrum of Cpl-1 against pneumococci and various gram-positive organisms. Treatment with 100 μg of Cpl-1 per ml reduced a 10⁸-CFU suspension of 15 clinical strains of S. pneumoniae (capsular serotypes are indicated by boldface type; see Materials and Methods for strain designations) and three mutants and their ancestor (D39, R36A, R6, and Lyt 4-4) by 2.8 to 4.5 log₁₀. Of the other tested species, only Streptococcus oralis, Streptococcus mitis, and Streptococcus gordonii lysed noticeably, but they lysed to a lesser extent than S. pneumoniae.

FIG. 4.

pH profile of Cpl-1 activity. Activity was tested in a universal buffer with a pH range of 2 to 13. The enzyme exhibited the highest activity at pH 4 and 5. At pH 7 it exhibited approximately 10% of the maximal lytic activity, and at pH 8 only 1% of the maximal lytic activity was found.

FIG. 5.

Activity of Cpl-1 in previously exposed mice and in hyperimmune rabbit serum. (A) In vivo, 15 min after a single bolus containing 200 μg of Cpl-1, the pneumococcal blood titer of mice that had received three doses of 1,000, 2,000, or 4,000 μg of Cpl-1 4 weeks earlier (□) (n = 6) dropped by a median of log₁₀ 0.40 CFU/ml and the titer of unexposed mice (▪) (n = 6) decreased by log₁₀ 0.57 CFU/ml (the difference was not significant). (B) In vitro, the viable count of a pneumococcal suspension exposed to 2,000 μg of Cpl-1 in undiluted preimmune rabbit serum (○) exhibited a rapid 3-log₁₀ CFU/ml reduction within 1 min, and the count continued to decrease more slowly up to 10 min. In hyperimmune serum after 10 min of preincubation (•) and 1 h of preincubation (▪) the killing was less efficient by 0.8 log₁₀ but the lytic activity was not neutralized. □, control reaction with buffer in nonimmune serum.