Population screening shows risk of inherited cancer and familial hypercholesterolemia in Oregon.

The Healthy Oregon Project (HOP) is a statewide effort that aims to build a large research repository and influence the health of Oregonians through providing no-cost genetic screening to participants for a next-generation sequencing 32-gene panel comprising genes related to inherited cancers and familial hypercholesterolemia. This type of unbiased population screening can detect at-risk individuals who may otherwise be missed by conventional medical approaches. However, challenges exist for this type of high-throughput testing in an academic setting, including developing a low-cost high-efficiency test and scaling up the clinical laboratory for processing large numbers of samples. Modifications to our academic clinical laboratory including efficient test design, robotics, and a streamlined analysis approach increased our ability to test more than 1,000 samples per month for HOP using only one dedicated HOP laboratory technologist. Additionally, enrollment using a HIPAA-compliant smartphone app and sample collection using mouthwash increased efficiency and reduced cost. Here, we present our experience three years into HOP and discuss the lessons learned, including our successes, challenges, opportunities, and future directions, as well as the genetic screening results for the first 13,670 participants tested. Overall, we have identified 730 pathogenic/likely pathogenic variants in 710 participants in 24 of the 32 genes on the panel. The carrier rate for pathogenic/likely pathogenic variants in the inherited cancer genes on the panel for an unselected population was 5.0% and for familial hypercholesterolemia was 0.3%. Our laboratory experience described here may provide a useful model for population screening projects in other states.

Understanding the Phage-Host Interaction Mechanism toward Improving the Efficacy of Current Antibiotics in Mycobacterium abscessus.

Pulmonary infections caused by Mycobacterium abscessus (MAB) have been increasing in incidence in recent years, leading to chronic and many times fatal infections due to MAB's natural resistance to most available antimicrobials. The use of bacteriophages (phages) in clinics is emerging as a novel treatment strategy to save the lives of patients suffering from drug-resistant, chronic, and disseminated infections. The substantial research indicates that phage-antibiotic combination therapy can display synergy and be clinically more effective than phage therapy alone. However, there is limited knowledge in the understanding of the molecular mechanisms in phage-mycobacteria interaction and the synergism of phage-antibiotic combinations. We generated the lytic mycobacteriophage library and studied phage specificity and the host range in MAB clinical isolates and characterized the phage's ability to lyse the pathogen under various environmental and mammalian host stress conditions. Our results indicate that phage lytic efficiency is altered by environmental conditions, especially in conditions of biofilm and intracellular states of MAB. By utilizing the MAB gene knockout mutants of the MAB_0937c/MmpL10 drug efflux pump and MAB_0939/pks polyketide synthase enzyme, we discovered the surface glycolipid diacyltrehalose/polyacyltrehalose (DAT/PAT) as one of the major primary phage receptors in mycobacteria. We also established a set of phages that alter the MmpL10 multidrug efflux pump function in MAB through an evolutionary trade-off mechanism. The combination of these phages with antibiotics significantly decreases the number of viable bacteria when compared to phage or antibiotic-alone treatments. This study deepens our understanding of phage-mycobacteria interaction mechanisms and identifies therapeutic phages that can lower bacterial fitness by impairing an antibiotic efflux function and attenuating the MAB intrinsic resistance mechanism via targeted therapy.

Mycobacterium abscessus Genetic Determinants Associated with the Intrinsic Resistance to Antibiotics.

Mycobacterium abscessus subsp. abscessus (MAB) is a fast-growing nontuberculous mycobacterium causing pulmonary infections in immunocompromised and immunocompetent individuals. The treatment of MAB infections in clinics is extremely challenging, as this organism is naturally resistant to most available antibiotics. There is limited knowledge on the mechanisms of MAB intrinsic resistance and on the genes that are involved in the tolerance to antimicrobials. To identify the MAB genetic factors, including the components of the cell surface transport systems related to the efflux pumps, major known elements contributing to antibiotic resistance, we screened the MAB transposon library of 2000 gene knockout mutants. The library was exposed at either minimal inhibitory (MIC) or bactericidal concentrations (BC) of amikacin, clarithromycin, or cefoxitin, and MAB susceptibility was determined through the optical density. The 98 susceptible and 36 resistant mutants that exhibited sensitivity below the MIC and resistance to BC, respectively, to all three drugs were sequenced, and 16 mutants were found to belong to surface transport systems, such as the efflux pumps, porins, and carrier membrane enzymes associated with different types of molecule transport. To establish the relevance of the identified transport systems to antibiotic tolerance, the gene expression levels of the export related genes were evaluated in nine MAB clinical isolates in the presence or absence of antibiotics. The selected mutants were also evaluated for their ability to form biofilms and for their intracellular survival in human macrophages. In this study, we identified numerous MAB genes that play an important role in the intrinsic mechanisms to antimicrobials and further demonstrated that, by targeting components of the drug efflux system, we can significantly increase the efficacy of the current antibiotics.