With NC3Rs funding, Professor Shiranee Sriskandan, from Imperial College London, has used bioluminescence to enable longitudinal imaging of bacterial infections in mice over a four day period, reducing the use of animals by avoiding the need to cull cohorts at specific time points.
Principal Investigator: Shiranee Sriskandan, Professor of Infectious Diseases
Organisation: Imperial College London
Award: £276,722, in 2008, over 24 months
Title: Exploiting bioluminescence in mouse studies of bacterial infection
Read more about Professor Sriskandan's research.
There are 616 million cases of pharyngitis caused by the bacteria Streptococcus pyogenes worldwide each year
The bacterium, group A streptococcus (or Streptococcus pyogenes) causes a wide spectrum of diseases from mild skin infections and pharyngitis to life threatening invasive conditions such as sepsis and necrotising fasciitis. 20% of patients with invasive group A streptococcus infection die. Aberrant immune responses lead to some of the most feared complications, such as toxic shock, and also the heart valve-damaging disease, rheumatic fever, that can follow untreated streptococcal pharyngitis.
The main treatment for Streptococcus pyogenes infection is penicillin, yet despite this the infection remains hyperendemic in many communities throughout the developing world, and rheumatic valvular heart disease kills almost half a million people per year worldwide. Invasive infections often progress extremely rapidly, such that antibiotic treatment is never started, or starts too late to be of any use. Research is therefore focused on the development of better therapeutics or a vaccine.
Bioluminescence imaging can be used to refine and reduce animal studies of bacterial infection
Studies of bacterial pathogenesis typically involve infecting the mice and subsequently euthanizing them at specific time points to determine the number of viable bacteria present in specific tissues and organs.
In recent years, bioluminescence imaging has enabled bacteria, genetically modified to express luciferin or green fluorescent protein, to be tracked in mice using specialist equipment. This has provided real-time information on bacterial infection and colonisation, leading to novel scientific insights. Bioluminescence imaging also has the advantage that it is non-invasive.
The ability to monitor bioluminescent bacteria in real-time and non-invasively has provided opportunities to reduce and refine mouse studies. Mice can be serially monitored and it is therefore possible to conduct longitudinal studies without the need to euthanize groups of animals at specific time points for data collection. As a result, the number of mice used per study can be reduced typically from 18–36 to 6–8 animals. Each mouse acts as its own control, avoiding the potentially confounding effects of using different groups of animals for each time point.
The reproducibility of studies is further enhanced by the use of bioluminescence imaging to quantify the dose of bacteria given to each mouse. In conventional studies, the dose is determined by optical density and this can lead to log-fold differences in the amount of bacteria injected into one mouse compared to another. The ability to quantify the bacterial burden means that it is also possible to identify mice in which the infection is progressing rapidly, long before any adverse effects are apparent, allowing the use of humane endpoints and prevention of unnecessary suffering.
Applying the benefits of bioluminescence imaging to streptococcal research
With NC3Rs funding, Professor Shiranee Sriskandan and Dr Siouxsie Wiles*, Imperial College London, have developed a molecular ‘tool kit’ for the application of bioluminescence imaging to study the pathogenesis of Streptococcus pyogenes, focusing on the four main clinically important serotypes. Bioluminescence imaging has not previously been widely used in streptococcal research and the only commercially available bioluminescent strain is a non-clinical serotype.
Most mouse models for Streptococcus pyogenes result in the death of the animals within a few days. The Imperial team has recently pioneered a new mouse asymptomatic nasopharyngeal carriage model to enable better understanding of streptococcal infection. Dosing and sampling in this model have been refined using bioluminescent bacteria.
Bioluminescent Streptococcus pyogenes are introduced into the nostrils of the mouse resulting in nasopharyngeal infection. By quantifying the bioluminescence it has been possible to refine the volumetric dose required for nasal infection, avoiding the spread of the bacteria into the lungs. After approximately four days, however, the bioluminescent signal is too low to adequately detect in the mouse. In order to continue monitoring the mice longitudinally and non-invasively a new simple sampling method, where the mouse’s nostril is gently placed on the surface of a blood agar plate, has been developed. The streptococcal bacteria can then be identified and quantified on the plate.
This proof-of-concept study has shown for the first time the feasibility of bioluminescent imaging in refining the study of streptococcal infections in mice. It should now be possible to extend this to also reduce the number of mice used by four-fold. Moreover, the bioluminescent imaging has already revealed evidence of bacterial transmission from the nasal site of infection, which may be of clinical relevance.
*The grant was originally awarded to Dr Siouxsie Wiles and Professor Sriskandan. It transferred to Professor Sriskandan when Dr Wiles moved to New Zealand.
This case study was published in a review of our research portfolio in September 2011.