Abstract
In order to expedite the discovery of genes coding for either drug targets or antibiotic resistance, we have developed a functional genomic strategy termed Plas-Seq. This technique involves coupling a multicopy suppressor library to next-generation sequencing. We generated an Escherichia coli plasmid genomic library that was transformed into E. coli. These transformants were selected step by step using 0.25× to 2× minimum inhibitory concentrations for ceftriaxone, gentamicin, levofloxacin, tetracycline or trimethoprim. Plasmids were isolated at each selection step and subjected to Illumina sequencing. By searching for genomic loci whose sequencing coverage increased with antibiotic pressure we were able to detect 48 different genomic loci that were enriched by at least one antibiotic. Fifteen of these loci were studied functionally, and we showed that 13 can decrease the susceptibility of E. coli to antibiotics when overexpressed. These genes coded for drug targets, transcription factors, membrane proteins and resistance factors. The technique of Plas-Seq is expediting the discovery of genes associated with the mode of action or resistance to antibiotics and led to the isolation of a novel gene influencing drug susceptibility. It has the potential for being applied to novel molecules and to other microbial species.
Highlights
The major objective of the global PPL is to guide the prioritization of incentives and funding, help align research and development (R&D) priorities with public health needs and support global coordination in the fight against antibioticresistant bacteria
The World Health Organization was requested by Member States to develop a global priority pathogens list of antibiotic-resistant bacteria to help in prioritizing the research and development (R&D) of new and effective antibiotic treatments
The major objective of the global PPL is to guide the prioritization of incentives and funding, help align R&D priorities with public health needs and support global coordination in the fight against antibioticresistant bacteria
Summary
A coordinating group of eight experts in infectious diseases, clinical microbiology, R&D, public health and infection control were selected to define the protocol. The global WHO-PPL was developed applying a multi-criteria decision analysis (MCDA) technique, which allows the evaluation of different alternatives according to multiple criteria. The MCDA method incorporates both expert opinion and evidence-based data in a transparent, explicit, and deliberative fashion. The main strength of this approach is the relatively high weight given to the evidence retrieved and summarised for each criterion in order to reduce the impact of individual perceptions and beliefs. The prioritization exercise has been performed through the following steps: 1. Selection of antibiotic-resistant bacteria to be prioritized; 2. Scoring of alternatives and weighting of criteria by experts; and 5.
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