Evaluation of optimal growth conditions for Enterobacterales thymidine-dependent small-colony variants: A pilot study for the development of antimicrobial susceptibility testing.

Thymidine-dependent small-colony variants (TD-SCVs) are slow-growing subpopulations of bacteria that require thymidine for growth. As they do not grow on Mueller-Hinton media, conducting antimicrobial susceptibility tests on these strains is difficult, increasing the risk of overlooking antimicrobial-resistant strains. In this study, we aimed to evaluate the optimal thymidine concentration for their growth and viable cell counts on various media for thymidine-dependent small-colony variants of Enterobacterales as a preliminary investigation toward development of their antimicrobial susceptibility testing. For 11 TD-SCVs of Enterobacterales, the optimum developmental thymidine concentrations and the viable cell counts in 0.5 McFarland turbidity standard prepared from colonies cultured on various media were determined. Subsequently, we assessed a modified antimicrobial susceptibility test method. In this method, broth microdilutions were supplemented with the optimal thymidine concentration and bacterial suspensions were prepared from the colonies grown on the medium that yielded the highest viable cell count. The tested strains showed the best growth at 10 μg/mL of thymidine. The 0.5 McFarland turbidity standard prepared from colonies on Mueller-Hinton agar supplemented with 10 μg/mL thymidine had the highest number of viable bacteria. Using the modified antimicrobial susceptibility testing method with 10 μg/mL thymidine generally produced reasonable results, including ESBL-producing strains. Therefore, it is important to consider the optimal thymidine concentration for growth and the difference of the viable cell counts on each media to develop antimicrobial susceptibility testing methods for TD-SCVs.