Mab2780c, a TetV-like efflux pump, confers high-level spectinomycin resistance in mycobacterium abscessus.

Mycobacterium abscessus is highly resistant to spectinomycin (SPC) thereby making it unavailable for therapeutic use. Sublethal exposure to SPC strongly induces whiB7 and its regulon, and a ΔMab_whiB7 strain is SPC sensitive suggesting that the determinants of SPC resistance are included within its regulon. In the present study we have determined the transcriptomic changes that occur in M. abscessus upon SPC exposure and have evaluated the involvement of 11 genes, that are both strongly SPC induced and whiB7 dependent, in SPC resistance. Of these we show that MAB_2780c can complement SPC sensitivity of ΔMab_whiB7 and that a ΔMab_2780c strain is ∼150 fold more SPC sensitive than wildtype bacteria, but not to tetracycline (TET) or other aminoglycosides. This is in contrast to its homologues, TetV from M. smegmatis and Tap from M. tuberculosis, that confer low-level resistance to TET, SPC and other aminoglycosides. We also show that the addition of the efflux pump inhibitor (EPI), verapamil results in >100-fold decrease in MIC of SPC in bacteria expressing Mab2780c to the levels observed for ΔMab_2780c; moreover a deletion of MAB_2780c results in a decreased efflux of the drug into the cell supernatant. Together our data suggest that Mab2780c is an SPC antiporter. Finally, molecular docking of SPC and TET on models of TetVMs and Mab2780c confirmed our antibacterial susceptibility findings that the Mab2780c pump preferentially effluxes SPC over TET. To our knowledge, this is the first report of an efflux pump that confers high-level drug resistance in M. abscessus. The identification of Mab2780c in SPC resistance opens up prospects for repurposing this relatively well-tolerated antibiotic as a combination therapy with verapamil or its analogs against M. abscessus infections.

Relationship between methamphetamine-induced behavioral activation and hyperthermia.

Methamphetamine (METH) changes core temperature and induces behavioral activation. Behavioral activation is also known to change core temperature. The purpose of this report was to 1.) evaluate the extent to which the behavioral activation induced by METH showed a temporal relationship to METH-induced hyperthermia; and 2.) describe the temporal pattern of METH-induced hyperthermia over an extended dose range. Rats were treated with saline or METH (0.5-10.0mg/kg) in computer-controlled chambers with ambient temperature maintained at 24°C. Continuous telemetric core temperature measurements were made during a 7h test period. Behavioral observations were made once every 15 min using an 11-point scale ranging from 0 (quiet awake) to 10 (focused licking or biting). The onset of METH-induced behavioral activation occurred at 15-30 min after treatment for all doses and preceded core temperature increases; the onset of METH-induced hyperthermia ranged from 45 min post-treatment to 120 min post-treatment. This behavior-temperature delay was 15-30 min at the lowest (0.5 and 1.0mg/kg) and the highest (7.0, 8.0, and 10.0mg/kg) doses tested; the delay was increased between 1.0 and 4.0mg/kg METH (105 min delay at 4.0mg/kg) and then decreased again from 4.0 to 10.0mg/kg. The strongest relationship between core temperature and behavioral activation occurred at 180 min post-treatment. These data suggest that factors other than behavior are primarily responsible for the observed core temperature effects during the initial post-treatment period (60 min peak); possible effects from movement are masked. For the latter post-treatment period (180 min peak) the stronger relationship between temperature and behavior suggests a role for movement in METH-induced hyperthermia.