BIOLUMINESCENCE AS A REPORTER OF INTRACELLULAR SURVIVAL OF BORDETELLA-BRONCHISEPTICA IN MURINE PHAGOCYTES

The uptake and persistence of Bordetella bronchiseptica was characteri zed in murine phagocytes by using a novel bioluminescence-based report er system. A mini-Tn5 promoter probe carrying the intact lux operon fr om the terrestrial bacterium Photorhabdus luminescens which allowed me asurement of light output without the addition of exogenous substrate was constructed. It was used to create a pool of bioluminescent fusion strains of B. bronchiseptica. The internalization and persistence in murine macrophages of a constitutive bioluminescent strain of B. bronc hiseptica was monitored by luminometry and by fluorescence and electro n microscopy. The number of bacteria internalized, in a microfilament- dependent process, by a mouse macrophage-like cell line after 2 h was approximately 1% of the inoculum for several different multiplicities of infection (MOI). At an MOI of <500:1 (bacteria to macrophages), via ble numbers of intracellular bacteria declined over a 4-day period. Ho wever, at an MOI of greater than or equal to 500:1, long-term survival was enhanced, with viable bacteria recovered up to 4 days postinfecti on with little decline in numbers, indicating that a critical populati on size may have been essential for intracellular persistence. No evid ence of macrophage killing by intracellular bacteria was detected over the 4-day period. Intracellular bioluminescent B. bronchiseptica orga nisms in mouse peritoneal cells were detected at 24 and 48 h after int raperitoneal injection of mice. Bioluminescence is shown to act as a c onvenient real-time technique for monitoring of intracellular survival of B. bronchiseptica in vitro and may provide a suitable means for ex amining the role of long-term intracellular survival of the bacterium in the host.