Bacteriophage therapy rescues mice bacteremic from a clinical isolate of vancomycin-resistant Enterococcus faecium

Abstract

Colonization of the gastrointestinal tract with vancomycin-resistant Enterococcus faecium (VRE) has become endemic in many hospitals and nursing homes in the United States. Such colonization predisposes the individual to VRE bacteremia and/or endocarditis, and immunocompromised patients are at particular risk for these conditions. The emergence of antibiotic-resistant bacterial strains requires the exploration of alternative antibacterial therapies, which led our group to study the ability of bacterial viruses (bacteriophages, or phages) to rescue mice with VRE bacteremia. The phage strain used in this study has lytic activity against a wide range of clinical isolates of VRE. One of these VRE strains was used to induce bacteremia in mice by intraperitoneal (i.p.) injection of 10(9) CFU. The resulting bacteremia was fatal within 48 h. A single i.p. injection of 3 x 10(8) PFU of the phage strain, administered 45 min after the bacterial challenge, was sufficient to rescue 100% of the animals. Even when treatment was delayed to the point where all animals were moribund, approximately 50% of them were rescued by a single injection of this phage preparation. The ability of this phage to rescue bacteremic mice was demonstrated to be due to the functional capabilities of the phage and not to a nonspecific immune effect. The rescue of bacteremic mice could be effected only by phage strains able to grow in vitro on the bacterial host used to infect the animals, and when such strains are heat inactivated they lose their ability to rescue the infected mice.

FIG. 1.

Lethality of the MLD of VRE in this model. Mice were inoculated i.p. with 10⁹ CFU of VRE strain CRMEN 44. A score of 5 indicates normal health, while 0 indicates death (see the text for a full description of the scale). Each bar indicates the state of health of a single BALB/c mouse. All animals were dead within 48 h.

FIG. 2.

Dose effect of phage ENB6 in rescuing mice from lethal VRE bacteremia. The mice were scored for their state of health as described in the text. Each bar indicates the state of health of mice after i.p. administration of the MLD of VRE followed by a single dose of phage at the indicated concentration 45 min after the bacterial challenge.The group on the far right (four mice) was an untreated control, given SM buffer i.p. instead of phage. The group on the far left (two animals) was a phage control, injected only with phage (at the high dose) and not infected with bacteria, in order to determine if the phage preparation itself produces adverse effects in mice.

FIG. 3.

Delayed phage treatment of bacteremic mice. A single i.p. injection of the highest dose of the phage preparation (3 × 10⁹ PFU) was administered to bacteremic mice at the indicated times after bacterial challenge. The mice were scored for their state of health as described in the text. Black and gray bars show their condition at the time of phage treatment and following phage treatment, respectively. When treatment was delayed for 24 h, all the animals were moribund, but approximately half of them not only survived but also went on to recover completely as a result of the single dose of phage.

FIG. 4.

Comparison of phage strains ENB6 and C33, which cannot and can form plaques in vitro on VRE strain CRMEN 19, respectively, with their ability to rescue mice that are bacteremic with CRMEN 19. Groups A and B were healthy mice treated with a single i.p. injection of 9 × 10⁹ PFU of ENB6 and C33, respectively. Groups C and D were treated with 9 × 10⁹ PFU of ENB6 and C33, respectively, 90 min after bacterial infection. Group E was an untreated control, injected i.p. with PBS buffer 90 min after the bacterial challenge instead of phage. Treatment with phage strain C33 resulted in 100% recovery from bacteremia. This recovery rate is significantly better than the survival rates of 20 and 50% for the infected mice treated with ENB6 and with PBS, respectively.

FIG. 5.

Comparison of the ability of phage and nonfunctional heat-inactivated phage to rescue bacteremic mice. One group of 10 mice was treated with functional (plaque-forming) phage, while the other two groups were treated either with no phage or heat-inactivated phage. Eighty percent of the bacteremic mice treated with functional phage survived, while only 10% of the mice in each of the other two groups survived. The animals were monitored for 20 more days, and no changes in their state of health were noted.

FIG. 6.

Antibody responses in mice to repeated injections of phage ENB6. Phage were injected i.p. at the indicated time points. The resulting titers of anti-ENB6 IgG (•) and IgM (○) antibodies are indicated.

FIG. 7.

Electron micrograph of phage ENB6 negatively stained with uranyl acetate. The phage head is elongated, with a length of 99.7 nm and width of 84.4 nm. The contractile tail is 199.1 nm in length.