Growth-rate dependent response of mycobacteria to relief of inhibition by a bacteriostatic antibiotic

Pathogenic bacteria generally resume growth upon removal of bacteriostatic antibiotics. However, it is poorly understood how pre-growth conditions influence (i) the lag time before growth resumption and (ii) induction of antibiotics tolerance upon removal of the antibiotics. To study these traits in Mycobacterium smegmatis, we used a stringent-response-inducing bacteriostatic inhibitor, serine hydroxamate (SHX), which mimics a growth-rate-reducing antibiotic. We find that SHX-induced, growth-arrested cells display characteristics typical of persister cells, i.e. low ATP levels, increased antibiotic tolerance, and delayed growth-recovery. Single-cell microscopy shows morphological changes in SHX-treated cells (elongation and polar swelling) and growth resumption upon SHX removal after a lag time. During this lag time, the cells displayed increased antibiotic survival, which remained evident after complete recovery from SHX stress. Varying the SHX dose, exposure time, and pre-exposure growth rate indicated that the duration of the lag phase depends on the SHX exposure time and the prior growth rate, not on the SHX dose. The lag phase could be shortened by over two-fold with the addition of amino acids, pyruvate, or spent medium, indicating that growth inhibition induces a relievable metabolic bottleneck. Our findings align with studies of other (evolutionarily distant) microorganisms, suggesting that mycobacteria may be limited by similar phenotypic trade-offs associated with adaptive responses to sudden growth rate reductions.