Background: Pseudomonas aeruginosa is an opportunistic multi-drug resistance pathogen implicated as the causative agent in a high-percentage of nosocomial and community acquired bacterial infections. The gene encoding leucyl-tRNA synthetase (LeuRS) from P. aeruginosa was overexpressed in Escherichia coli and the resulting protein was characterized. Methods: LeuRS was kinetically evaluated and the KM values for interactions with leucine, ATP and tRNA were 6.5, 330, and 3.0 μM, respectively. LeuRS was developed into a screening platform using scintillation proximity assay (SPA) technology and used to screen over 2000 synthetic and natural chemical compounds. Results: The initial screen resulted in the identification of two inhibitory compounds, BT03C09 and BT03E07. IC50s against LeuRS observed for BT03C09 and BT03E07 were 23 and 15 μM, respectively. The minimum inhibitory concentrations (MIC) were determined against nine clinically relevant bacterial strains. In time-kill kinetic analysis, BT03C09 was observed to inhibit bacterial growth in a bacteriostatic manner, while BT03E07 acted as a bactericidal agent. Neither compound competed with leucine or ATP for binding LeuRS. Limited inhibition was observed in aminoacylation assays with the human mitochondrial form of LeuRS, however when tested in cultures of human cell line, BT03C09 was toxic at all concentration whereas BT03E07 only showed toxic effects at elevated concentrations. Conclusion: Two compounds were identified as inhibitors of LeuRS in a screen of over 2000 natural and synthetic compounds. After characterization one compound (BT03E07) exhibited broad spectrum antibacterial activity while maintaining low toxicity against human mitochondrial LeuRS as well as against human cell cultures.

IntroductionPseudomonas aeruginosa is an opportunistic pathogen and a common cause of nosocomial infections. Aminoacyl‐tRNA synthetases (aaRSs) are a class of enzymes that catalyze the covalent attachment of amino acids to their cognate tRNAs during protein biosynthesis. We describe here the cloning and enzymatic characterization of the class I leucyl tRNA synthetase (LeuRS) from P. aeruginosa.ResultsLeuRS from P. aeruginosa was cloned and expressed in E. coli and purified to greater than 98% homogeneity. Sequence analysis shows that this protein contains the characteristic motifs of class I aminoacyl‐tRNA synthetases. The ability of LeuRS to function in aminoacylation of its cognate tRNA was determined in tRNA aminoacylation assays. The kinetic parameters for the interaction of P. aeruginosa LeuRS with its three substrates (tRNA, ATP, leucine) were determined. Initial velocities were determined for charging of tRNA using tRNA concentrations between 6.5 and 10 μM tRNALeu. The KM and Vmax for the interaction of LeuRS with the substrate tRNALeu was determined to be 7.14 μM and 0.36 μM min−1, respectively. The ATP:PPi exchange reaction was used to monitor interaction with ATP and leucine.ConclusionLeuRS identified in P. aeruginosa was cloned, expressed and purified and shown to be functional in assays suggesting that it is functional in protein synthesis. Research was partially supported by NIH grant 1SC3GM098173–01A1.

With an increasing obese population nationwide, there has been an increase in the number of laparoscopic surgeries performed. However, few studies have evaluated different methods to reduce pain in bariatric patients after laparoscopic surgery. Bupivacaine infusion pumps (On-Q® pumps) have shown efficacy in reducing postoperative pain in other surgical populations, such as orthopedic patients. Therefore, the purpose of this study was to investigate whether infusion pain pumps improve postoperative pain control and decrease length of hospital stay in bariatric patients. Between January 2015 and August 2016, 125 charts were retrospectively reviewed. Patients were divided into two groups: those who received infusion pain pumps and those who did not. The primary endpoints were hospital length of stay and the amount of postoperative narcotic use. Secondary endpoints included postoperative pain score, use of non-narcotics, and complications due to opioids or bupivacaine. There were 82 patients in the infusion pain pump group and 43 patients in the control group. Baseline characteristics were similar between both groups. There were no statistically significant differences in length of hospital stay (p = 0.39) or postoperative opioid use (p = 0.48) between the two groups. Although the median postoperative pain score was significantly reduced in the infusion pain pump group (3.5) compared to the control group (5) for the first 48 h (p = 0.002), this difference was not statistically significant for the first 24 h (p = 0.06). The use of infusion pain pumps did not significantly reduce the length of hospitalization and use of narcotics postoperatively.