Bacterial tolerance - the ability to survive under prolonged bactericidal treatments - has been shown to be a major precursor for antibiotic resistance in vitro, but is still overlooked in most clinical cases. One important reason for this overlooking is the lack of a fast and cheap method to measure tolerance.
The Disk diffusion assay is a common technique for testing bacterial sensitivity to different antibiotics. In this test, bacteria are plated on nutrient agar, and a small disk impregnated with antibiotic is placed on the plate. Two processes that occur in parallel - the bacterial growth and the diffusion of the antibiotic out from the disk - result in a growth inhibition zone around the disk. The larger the radius of the inhibition zone, the more sensitive the bacteria are to the tested antibiotic.
Tolerant bacteria, if present, may survive the otherwise lethal antibiotic exposure in the inhibition zone. However, they will not grow even when the antibiotic concentration drops below the MIC (Minimum Inhibitory Concentration), and will therefore remain undetected, because by the time they would start growing, essential nutrients are depleted. In order to detect those surviving bacteria, we developed the TDtest, based on a the disk diffusion assay: a second step is carried out, where nutrients are added to the disk to support growth of the surviving bacteria, and the plate is returned for further incubation. Using the TDtest we were able to distinguish clinical tolerant strain from non-tolerant ones, despite their disk diffusion results looking identical. The TDtest can be easily applied and may improve treatment by adding new information layer to the susceptibility testing.