High-density transposon libraries utilising outward-oriented promoters identify mechanisms of action and resistance to antimicrobials.

The use of bacterial transposon mutant libraries in phenotypic screens is a well-established technique for determining which genes are essential or advantageous for growth in conditions of interest. Standard, inactivating, transposon libraries cannot give direct information about genes whose over-expression gives a selective advantage. We report the development of a system wherein outward-oriented promoters are included in mini-transposons, generation of transposon mutant libraries in Escherichia coli and Pseudomonas aeruginosa and their use to probe genes important for growth under selection with the antimicrobial fosfomycin, and a recently-developed leucyl-tRNA synthase inhibitor. In addition to the identification of known mechanisms of action and resistance, we identify the carbon-phosphorous lyase complex as a potential resistance liability for fosfomycin in E. coli and P. aeruginosa. The use of this technology can facilitate the development of novel mechanism-of-action antimicrobials that are urgently required to combat the increasing threat worldwide from antimicrobial-resistant pathogenic bacteria.

Characterization of an umbilical cord blood sourced product suitable for allogeneic applications.

Aim: Umbilical cord blood (UCB) sourced allografts are promising interventions for tissue regeneration. As applications of these allografts and regulations governing them continue to evolve, we were prompted to identify parameters determining their quality, safety and regenerative potential. Materials & methods: Flow-cytometry, mass-spectrometry, protein multiplexing, nanoparticle tracking analysis and standard biological techniques were employed. Results: Quality attributes of a uniquely processed UCB-allograft (UCBr) were enumerated based on identity (cell viability, immunophenotyping, proteomic profiling, and quantification of relevant cytokines); safety (bioburden and microbiological screening), purity (endotoxin levels) and potency (effect of UCBr on chondrocytes and mesenchymal stem cells derived exosomes). These attributes were stable up to 24 months in cryopreserved UCBr. Conclusion: We identified a comprehensive panel of tests to establish the clinical efficacy and quality control attributes of a UCB-sourced allograft.