TDtest: easy detection of bacterial tolerance and persistence in clinical isolates by a modified disk-diffusion assay

Antibiotic tolerance - the ability for prolonged survival under bactericidal treatments - is a potentially clinically significant phenomenon that is commonly overlooked in the clinical microbiology laboratory. Recent in vitro experiments show that high tolerance can evolve under intermittent antibiotic treatments in as little as eight exposures to high doses of antibiotics, suggesting that tolerance may evolve also in patients. However, tests for antibiotic susceptibilities, such as the disk-diffusion assay, evaluate only the concentration at which a bacterial strain stops growing, namely resistance level. High tolerance strains will not be detected using these tests. We present a simple modification of the standard disk-diffusion assay that allows the semi-quantitative evaluation of tolerance levels. This novel method, the "TDtest", enabled the detection of tolerant and persistent bacteria by promoting the growth of the surviving bacteria in the inhibition zone, once the antibiotic has diffused away. Using the TDtest, we were able to detect different levels of antibiotic tolerance in clinical isolates of E. coli. The TDtest also identified antibiotics that effectively eliminate tolerant bacteria. The additional information on drug susceptibility provided by the TDtest should enable tailoring better treatment regimens for pathogenic bacteria.