Genome-wide saturation mutagenesis of Burkholderia pseudomallei K96243 predicts essential genes and novel targets for antimicrobial development

This is the final version of the article. Available from the publisher via the DOI in this record.Burkholderia pseudomallei is the causative agent of melioidosis, an often fatal infectious disease for which there is no vaccine. B. pseudomallei is listed as a tier 1 select agent, and as current therapeutic options are limited due to its natural resistance to most antibiotics, the development of new antimicrobial therapies is imperative. To identify drug targets and better understand the complex B. pseudomallei genome, we sought a genome-wide approach to identify lethal gene targets. As B. pseudomallei has an unusually large genome spread over two chromosomes, an extensive screen was required to achieve a comprehensive analysis. Here we describe transposon-directed insertion site sequencing (TraDIS) of a library of over 10(6) transposon insertion mutants, which provides the level of genome saturation required to identify essential genes. Using this technique, we have identified a set of 505 genes that are predicted to be essential in B. pseudomallei K96243. To validate our screen, three genes predicted to be essential, pyrH, accA, and sodB, and a gene predicted to be nonessential, bpss0370, were independently investigated through the generation of conditional mutants. The conditional mutants confirmed the TraDIS predictions, showing that we have generated a list of genes predicted to be essential and demonstrating that this technique can be used to analyze complex genomes and thus be more widely applied. IMPORTANCE: Burkholderia pseudomallei is a lethal human pathogen that is considered a potential bioterrorism threat and has limited treatment options due to an unusually high natural resistance to most antibiotics. We have identified a set of genes that are required for bacterial growth and thus are excellent candidates against which to develop potential novel antibiotics. To validate our approach, we constructed four mutants in which gene expression can be turned on and off conditionally to confirm that these genes are required for the bacteria to survive.This work was partially funded by the Defence Science and Technology Laboratories (DSTL)

Genetic diversity and risk factors for the transmission of antimicrobial resistance across human, animals and environmental compartments in East Africa: a review.

BACKGROUND The emergence and spread of antimicrobial resistance (AMR) present a challenge to disease control in East Africa. Resistance to beta-lactams, which are by far the most used antibiotics worldwide and include the penicillins, cephalosporins, monobactams and carbapenems, is reducing options for effective control of both Gram-positive and Gram-negative bacteria. The World Health Organization, Food and Agricultural Organization and the World Organization for Animal Health have all advocated surveillance of AMR using an integrated One Health approach. Regional consortia also have strengthened collaboration to address the AMR problem through surveillance, training and research in a holistic and multisectoral approach. This review paper contains collective information on risk factors for transmission, clinical relevance and diversity of resistance genes relating to extended-spectrum beta-lactamase-producing (ESBL) and carbapenemase-producing Enterobacteriaceae, and Methicillin-resistant Staphylococcus aureus (MRSA) across the human, animal and environmental compartments in East Africa. MAIN BODY The review of the AMR literature (years 2001 to 2019) was performed using search engines such as PubMed, Scopus, Science Direct, Google and Web of Science. The search terms included 'antimicrobial resistance and human-animal-environment', 'antimicrobial resistance, risk factors, genetic diversity, and human-animal-environment' combined with respective countries of East Africa. In general, the risk factors identified were associated with the transmission of AMR. The marked genetic diversity due to multiple sequence types among drug-resistant bacteria and their replicon plasmid types sourced from the animal, human and environment were reported. The main ESBL, MRSA and carbapenem related genes/plasmids were the CTX-Ms (45.7%), SCCmec type III (27.3%) and IMP types (23.8%), respectively. CONCLUSION The high diversity of the AMR genes suggests there may be multiple sources of resistance bacteria, or the possible exchange of strains or a flow of genes amongst different strains due to transfer by mobile genetic elements. Therefore, there should be harmonized One Health guidelines for the use of antibiotics, as well as regulations governing their importation and sale. Moreover, the trend of ESBLs, MRSA and carbapenem resistant (CAR) carriage rates is dynamic and are on rise over time period, posing a public health concern in East Africa. Collaborative surveillance of AMR in partnership with regional and external institutions using an integrated One Health approach is required for expert knowledge and technology transfer to facilitate information sharing for informed decision-making