Real-time monitoring of bacterial infection in vivo: development of bioluminescent staphylococcal foreign-body and deep-thigh-wound mouse infection models

Abstract

Staphylococcal infections associated with catheter and prosthetic implants are difficult to eradicate and often lead to chronic infections. Development of novel antibacterial therapies requires simple, reliable, and relevant models for infection. Using bioluminescent Staphylococcus aureus, we have adapted the existing foreign-body and deep-wound mouse models of staphylococcal infection to allow real-time monitoring of the bacterial colonization of catheters or tissues. This approach also enables kinetic measurements of bacterial growth and clearance in each infected animal. Persistence of infection was observed throughout the course of the study until termination of the experiment at day 16 in a deep-wound model and day 21 in the foreign-body model, providing sufficient time to test the effects of antibacterial compounds. The usefulness of both animal models was assessed by using linezolid as a test compound and comparing bioluminescent measurements to bacterial counts. In the foreign-body model, a three-dose antibiotic regimen (2, 5, and 24 h after infection) resulted in a decrease in both luminescence and bacterial counts recovered from the implant compared to those of the mock-treated infected mice. In addition, linezolid treatment prevented the formation of subcutaneous abscesses, although it did not completely resolve the infection. In the thigh model, the same treatment regimen resulted in complete resolution of the luminescent signal, which correlated with clearance of the bacteria from the thighs.

FIG. 1.

Correlation between bacterial infection and bioluminescence in the mouse foreign-body model. (A) Mice implanted with catheters were infected with 10⁸, 10⁷, and 10⁶ CFU of S. aureus Xen8.1 and were imaged immediately after infection (day 0) using the IVIS Imaging System. Pictures of 5 of 10 imaged mice are presented. (B) Correlation between infectious dose and bacterial luminescence at day 0. (C) Correlation between bioluminescent signal detected and the amount of bacteria recovered from the catheters of mice at day 3 after infection with S. aureus Xen8.1. Bioluminescence (black circles) (in relative light units [RLU]) of individual mice (n = 4 to 8) is shown in panels B and C. Geometric mean values (black bars) are shown in panel B.

FIG. 2.

In vivo detection of persistent S. aureus Xen8.1 infection in the foreign-body (A) and the deep-thigh-wound (B) mouse models. Bioluminescence (in relative light units [RLU]) of individual mice (n = 10) is shown on the y axes. Geometric mean values (black bars) are shown. Uninf., uninfected.

FIG. 3.

Recovery of bacteria from infected catheters. (A) Mice with implanted catheters were infected with 5 × 10⁸, 1 × 10⁸, or 1 × 10⁷ CFU of S. aureus Xen8.1. At day 7, the mice were sacrificed and bacteria were recovered from 8 of 9, 8 of 8, and 7 of 10 of the catheters infected with 5 × 10⁸, 1 × 10⁸, and 1 × 10⁷ CFU of S. aureus Xen8.1, respectively. (B) Mice with implanted catheters were infected with different doses of S. epidermidis RP62A. At day 7, the mice were sacrificed, and bacteria was recovered from 8 of 10, 7 of 10, and 5 of 10 of the catheters infected with 5 × 10⁸, 1 × 10⁸, and 1 × 10⁷ CFU of S. epidermidis RP62A, respectively. The CFU of the infected catheters (black circles) and the GMT (black dashes) are shown. Only the infected mice were included in the GMT calculations.

FIG. 4.

Correlation between S. aureus Xen8.1 infection dose and the level of bioluminescence in the deep-wound infection model. (A) Mice implanted with catheters were infected with 1 × 10⁸, 5 × 10⁷, or 1 × 10⁷ CFU of S. aureus Xen8.1 and were imaged immediately after infection (day 0) using the IVIS Imaging System. Pictures of 5 of 10 imaged mice are presented. (B) Correlation between infectious dose and bacterial luminescence at day 0. (C) Correlation between bioluminescent signal detected and the amount of bacteria recovered from the thighs of mice at day 3 after infection with S. aureus Xen8.1. Bioluminescence (black circles) (in relative light units [RLU]) of individual mice (n = 4 to 8) is shown in panels B and C. Geometric mean values (black bars) are shown in panel B.

FIG. 5.

Treatment with linezolid reduced the luminescence of mice in the foreign-body model of S. aureus Xen8.1 infection. BALB/c mice were implanted with catheters and infected with 10⁸ CFU of S. aureus Xen8.1 after 24 h. Ten infected mice were given 20 mg of linezolid per kg by oral gavage at 2, 5, and 24 h following infection. Another group of 10 infected mice was given the same regimen of linezolid but by s.c. injection. Bacterial replication was monitored over time by imaging the mice at days 0, 1, 2, 4, and 7. The data are presented as geometric means of the bioluminescence (in relative light units [RLU]) for the group of 10 mice. Statistically significant differences between the values for the untreated and treated mice were observed at day 1, day 2 (P < 0.01), and day 4 (P < 0.05). The P values for untreated and treated mice at different times after infection are as follows: for day 0, P = 0.8795 for oral treatment and P = 0.1295 for s.c. treatment; for day 1, P = 0.0099 for oral treatment and P = 0.00689 for s.c. treatment; for day 2, P = 0.0001 for oral treatment and P = 0.00007 for s.c. treatment; for day 4, P = 0.0446 for oral treatment and P = 0.03785 for s.c. treatment; for day 7, P = 0.5677 for oral treatment and P = 0.4515 for s.c. treatment. The results from one experiment are shown. Two experiments were performed with similar results. Uninf., uninfected.

FIG. 6.

Effects of linezolid on bioluminescence and bacterial counts in the foreign-body model during the first 24 h of infection. (A) Luminescence (in relative light units [RLU]) of linezolid-treated (black circles) or control (white circles) mice. Mice with implanted catheters were infected with S. aureus Xen8.1, given linezolid or PBS p.o., and imaged at 0, 8, 12, and 20 h. Uninf., uninfected. (B) Bacteria recovered from the catheters of linezolid-treated (black circles) and control (white circles) mice. Mice with implanted catheters were infected with S. aureus Xen8.1, given linezolid or PBS p.o., and sacrificed at 0, 8, 12, and 20 h.

FIG.7.

Complete resolution of luminescence in the deep-thigh-wound model following linezolid treatment of mice infected with S. aureus Xen8.1. BALB/c mice were infected in the thigh with 10⁸ CFU of S. aureus Xen8.1 and subsequently were randomly assigned into five groups. The groups were treated with antibiotic or were given PBS and used as controls as described in Materials and Methods. All mice were imaged at day 0 (after the infection before treatment) and at days 1, 3, 6, and 16. The figure shows mice at days 0 (A), 3 (B), and 16 (C).

FIG. 8.

Resolution of bacterial infection from the thighs of mice infected with S. aureus Xen8.1 and treated with linezolid. Four groups of BALB/c mice were infected in the thigh with 10⁸ CFU of S. aureus Xen8.1. Groups were treated once (at 2 h), twice (at 2 and 5 h), or three times (at 2, 5, and 24 h) with linezolid as described in Materials and Methods. At day 16, all mice were sacrificed, the thighs were removed and ground, and the bacterial titers from the suspensions of cells from individual thighs were determined. The bacterial counts (CFU/thigh) (black circles) and the geometric mean values (black bars) are shown. The P values for the untreated and treated mice were as follows: P = 0.04 for mice treated once, P = 0.03 for mice treated twice, and P < 0.00001 for mice treated three times.

FIG. 9.

Effects of linezolid on bioluminescence and bacterial counts in the deep-wound model during the first 24 h of infection. (A) Luminescence (in relative light units [RLU]) of linezolid-treated (black circles) or control (white circles) mice. Mice were infected with S. aureus Xen8.1 in the thighs, given linezolid or PBS p.o., and imaged at 0, 4, 8 and 12 h. Uninf., uninfected. (B) Bacteria recovered from the thighs of linezolid-treated (black circles) and control (white circles) mice. Mice were infected with S. aureus Xen8.1, given linezolid or PBS p.o., and sacrificed at 0, 4, 8, and 12 h.