Direct continuous method for monitoring biofilm infection in a mouse model

Abstract

We have developed a rapid, continuous method for real-time monitoring of biofilms, both in vitro and in a mouse infection model, through noninvasive imaging of bioluminescent bacteria colonized on Teflon catheters. Two important biofilm-forming bacterial pathogens, Staphylococcus aureus and Pseudomonas aeruginosa, were made bioluminescent by insertion of a complete lux operon. These bacteria produced significant bioluminescent signals for both in vitro studies and the development of an in vivo model, allowing effective real-time assessment of the physiological state of the biofilms. In vitro viable counts and light output were parallel and highly correlated (S. aureus r = 0.98; P. aeruginosa r = 0.99) and could be maintained for 10 days or longer, provided that growth medium was replenished every 12 h. In the murine model, subcutaneous implantation of the catheters (precolonized or postimplant infected) was well tolerated. An infecting dose of 10 (3) to 10 (5) CFU/catheter for S. aureus and P. aeruginosa resulted in a reproducible, localized infection surrounding the catheter that persisted until the termination of the experiment on day 20. Recovery of the bacteria from the catheters of infected animals showed that the bioluminescent signal corresponded to the CFU and that the lux constructs were highly stable even after many days in vivo. Since the metabolic activity of viable cells could be detected directly on the support matrix, nondestructively, and noninvasively, this method is especially appealing for the study of chronic biofilm infections and drug efficacy studies in vivo.

FIG. 1.

Monitoring the level of bioluminescence activity of P. aeruginosa and S. aureus in a 2-day-old biofilm on catheter segments. Images were acquired with the IVIS camera and are displayed as pseudocolor images, with variations in color representing light intensity at a given location. Red represents the most intense light emission, while blue correspond to the weakest signal. Note the lack of light signal from the wild-type and control catheter, indicating the specificity of the detection system. The color bar indicates relative signal intensity.

FIG. 2.

Phase-contrast micrograph of a wet mount, showing 5-day-old biofilms of P. aeruginosa Xen 5 (A) and S. aureus Xen 29 (B). A small lump of catheter biofilm was detached from the catheter surface to show the bacterial aggregates.

FIG. 3.

Growth and bioluminescence curves of P. aeruginosa Xen 5 and S. aureus Xen 29 grown on catheter surfaces. Viable counts are reported as CFU per catheter, and bioluminescence is represented as RLU measured using the IVIS camera. Each data point is the mean and standard error for three or four catheters. Bioluminescence was determined at each time point immediately prior to determination of the viable-cell count.

FIG. 4.

Scatter plots of viable cells and bioluminescence data to demonstrate the relationship between viable counts and bioluminescence for P. aeruginosa Xen 5 (A) and S. aureus Xen 29 (B).

FIG. 5.

Real-time monitoring of P. aeruginosa Xen 5 (A) and S. aureus Xen 29 (B) biofilms in a mouse model. Precolonized catheters were implanted at subcutaneous sites with doses ranging from 10 ³ to 10 ⁵ CFU, and growth of the biofilm was monitored by detecting photon emission over a 20-day time course using an IVIS camera. Similar results were obtained when the catheter bed was inoculated with doses ranging from 10 ³ to 10 ⁵ CFU after subcutaneous implantation of sterile catheters.

FIG. 6.

Growth and bioluminescence curves of P. aeruginosa Xen 5 (A) and S. aureus Xen 29 (B) biofilms in mice infected with precolonized catheters carrying various inocula. Each data point is the mean and standard error for two or three mice. Each mouse was subjected to implantation of two catheters. The viable counts in each catheter were determined immediately after removal of the catheters from the implanted sites and are shown in the upper quadrants of the plot. Identical results were obtained when similar doses of bacteria (10 ³, 10 ⁴, and 10 ⁵ CFU/catheter) were injected into the implant site after implantation, except that the RLU value peaked 2 days after infection.