Evolution of Mycobacteria under temporally variable environment predominantly selects for efflux mediated resistance Authors: Akanksha1, Sarika Mehra1* Affiliations: 1Indian Institute of Technology, Bombay, Powai, Mumbai-400076 *Corresponding author email address: email@example.com Abstract: Antibiotic resistance has emerged as a global health concern in present scenario leading to increased mortality rates. Moreover, rate of resistance emergence outcompetes rate of antibiotic discovery. Consequently, we are compelled to investigate different factors shaping resistance evolution in bacteria and ultimately identify strategies to restrict the same. In our study we study the effect of temporally varying environment on evolution of resistance. Mycobacterium smegmatis was chosen as the model organism which was subjected to two extreme temporal profiles. In the first case, the concentration of norfloxacin was kept constant (0.5 to 4X WT MIC) through the adaptive evolution process. In the second case, the selection pressure was increased every few generations (from 0.5 to 4X WT MIC). Strains evolved under this condition are referred to as ramp. All the evolved populations had increased resistance against Norfloxacin along with emergence of cross-resistance against some other drugs. Resistance was proportional to the selection strength during evolution with highest in case of ramp population and the constant population that evolved under Norfloxacin concentration of 4X MIC. To gain insight into factors behind increased resistance, Whole Genome Sequencing (WGS) was done for representative colonies from all evolved populations using Illumina platform. To our surprise, no mutation in gyrA (target for norfloxacin) was identified in any of the sequenced strains. Instead, all evolved populations had acquired mutation in a regulatory gene, lfrR, which negatively regulates a common efflux pump, LfrA, in M. smegmatis. This was also reflected in the expression study as lfrA gene was found to be overexpressed in all the evolved strains. Although two different types of mutations were identified in the lfrR gene, surprisingly, the expression level of lfrA was almost similar in all evolved strains suggesting that inactivation of efflux pump regulator is a common and recurrent way of increasing bacterial resistance in natural settings. Further, to understand the extent of resistance imparted by over-expression of LfrA alone, we determined MIC for Norfloxacin in the presence of CCCP, an efflux pump inhibitor. CCCP was able to reduce the resistance of all evolved strains by at least 2-fold. This restored the sensitivity for the low resistance strains similar to that of the WT suggesting that efflux is solely responsible for increased resistance in these strains. However, for attaining higher resistance levels, efflux along with other mutations are required. Consistent with this hypothesis, additional mutations were identified, through WGS, in the high resistance mutants. While the constant 16 population had a mutation in the DNA helicase (involved in DNA replication and repair), mutation in and around global regulatory genes were identified in Ramp population. Overall, this study emphasizes the importance of an alternative but rather conserved efflux mediated pathway for resistance in laboratory evolved strains as opposed to the primary target of fluoroquinolones. However, for high level resistance emergence, additional mutations affecting global regulators and core DNA replication and repair mechanisms are required along with efflux mediated resistance mechanism. Based on the central role of efflux pumps in resistance emergence, efflux pump inhibitors could prove to be excellent adjuvants along with the drugs. This would ultimately reduce the net drug dosage required which would help control and somewhat inhibit resistance emergence as soaring drug concentrations help burgeon high resistance levels in pathogens. However, this work has also highlighted the importance of additional mechanisms which are equally important in imparting resistance and therefore should be given proper attention in order to alleviate resistance emergence in deadly pathogens.