In vitro visualizing of S. aureus, using a light emitting probe

Introduction and background Surgical Site Infections (SSI) and Biomaterial Associated Infections (BAI) are a major concern to any surgeon because they lead to an increase in mortality and morbidity of the patient, and usually result in removal of the biomaterial. In the USA alone over a 100.000 implants per year become infected, adding 1.5 billion to the annual healthcare bill. These infections require prompt treatment in order to prevent the formation of a biofilm, which renders antibiotics 1000 times less effective. The detection of surgical site infections, including biomaterial associated infections (BAI) is difficult due to limitations of current imaging modalities, such as radiologic and nuclear imaging techniques. The use of bioluminescent bacteria - bacteria that emit light due to an enzymatic reaction - in combination with a special imaging system creates an opportunity for in vivo imaging of infections. The limitation in studies with bioluminescent bacteria is that these bacteria are genetically modified and do not cause infections in the human environment. To bypass this limitation wild type bacteria can be targeted with a fluorescent near-infrared probe, PSVue794tm. The objectives of this study are: 1. Target S. aureus in vitro specifically by a Zn(II)-probe (PSVue794), both in a bacterial suspension and when attached to biomaterial. 2. Estimate the amount of bacteria present through the amount of light emitted by the probe. Methods PSVue794 as such was imaged to find a correlation between the amount of molecules and the quantity of light captured by the in vivo imaging system. This was followed by adding various concentrations PSVue794 to three concentrations of a bacterial suspension. These probe-bacterial suspensions were centrifuged and resuspended in order to determine the affection of PSVue794 for bacteria. Finally, several types of biomaterial were incubated in PSVue794 with and without pre-incubation with bacteria. These biomaterials were covered with consecutively chicken breast, human skin and phantom tissue to evaluate the proof-of-concept: Visualizing infected biomaterials easy, non-invasively and spatiotemporal through tissue. Results 1. At fixed concentrations above 0,5 μM/L PSVue794 there is a linear correlation between volume and flux (photons/second) (p , 0,001) 2. Between 100 and 250 μM/L PSVue794 a turning point is found. Below 100 μM/L there is a positive correlation between the bacterial load and the flux, whereas above 250 μM/L there is a negative correlation between the bacterial load and flux. 3. It has been made possible with PSVue794 to prove infected biomaterial object through various tissues with fluorescent imaging. Conclusion PSVue794 is a promising probe for targeting bacteria in a bacterial suspension. In the presence of biomaterial, PSVue794 binds equally to biomaterials with and without a biofilm. The data prove the concept that infected biomaterial can be visualized through tissue through fluorescence. However, the next step before bringing this application to the bedside would be to quantify the affinity of PSVue794 for biomaterials versus bacteria.