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  1. As transposon sequencing (TnSeq) assays have become prolific in the microbiology field, it is of interest to scrutinize their potential drawbacks. TnSeq data consist of millions of nucleotide sequence reads that are generated by PCR amplification of transposon-genomic junctions. Reads mapping to the junctions are enumerated thus providing information on the number of transposon insertion mutations in each individual gene. Here we explore the possibility that PCR amplification of transposon insertions in a TnSeq library skews the results by introducing bias into the detection and/or enumeration of insertions. We compared the detection and frequency of mapped insertions when altering the number of PCR cycles, and when including a nested PCR, in the enrichment step. Additionally, we present nCATRAs - a novel, amplification-free TnSeq method where the insertions are enriched via CRISPR/Cas9-targeted transposon cleavage and subsequent Oxford Nanopore MinION sequencing. nCATRAs achieved 54 and 23% enrichment of the transposons and transposon-genomic junctions, respectively, over background genomic DNA. These PCR-based and PCR-free experiments demonstrate that, overall, PCR amplification does not significantly bias the results of TnSeq insofar as insertions in the majority of genes represented in our library were similarly detected regardless of PCR cycle number and whether or not PCR amplification was employed. However, the detection of a small subset of genes which had been previously described as essential is sensitive to the number of PCR cycles. We conclude that PCR-based enrichment of transposon insertions in a TnSeq assay is reliable, but researchers interested in profiling putative essential genes should carefully weigh the number of amplification cycles employed in their library preparation protocols. In addition, nCATRAs is comparable to traditional PCR-based methods (Kendall’s correlation=0.896–0.897) although the latter remain superior owing to their accessibility and high sequencing depth. 
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  2. Freitag, Nancy E. (Ed.)
    ABSTRACT Mutation of purR was previously shown to enhance the virulence of Staphylococcus aureus in a murine sepsis model, and this cannot be fully explained by increased expression of genes within the purine biosynthesis pathway. Rather, the increased production of specific S. aureus virulence factors, including alpha toxin and the fibronectin-binding proteins, was shown to play an important role. Mutation of purR was also shown previously to result in increased abundance of SarA. Here, we demonstrate by transposon sequencing that mutation of purR in the USA300 strain LAC increases fitness in a biofilm while mutation of sarA has the opposite effect. Therefore, we assessed the impact of sarA on reported purR -associated phenotypes by characterizing isogenic purR , sarA , and sarA/purR mutants. The results confirmed that mutation of purR results in increased abundance of alpha toxin, protein A, the fibronectin-binding proteins, and SarA, decreased production of extracellular proteases, an increased capacity to form a biofilm, and increased virulence in an osteomyelitis model. Mutation of sarA had the opposite effects on all of these phenotypes and, other than bacterial burdens in the bone, all of the phenotypes of sarA / purR mutants were comparable to those of sarA mutants. Limiting the production of extracellular proteases reversed all of the phenotypes of sarA mutants and most of those of sarA/purR mutants. We conclude that a critical component defining the virulence of a purR mutant is the enhanced production of SarA, which limits protease production to an extent that promotes the accumulation of critical S. aureus virulence factors. 
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  3. Abstract

    Bloodstream infections, especially those that are antibiotic resistant, pose a significant challenge to health care leading to increased hospitalization time and patient mortality. There are different facets to this problem that make these diseases difficult to treat, such as the difficulty to detect bacteria in the blood and the poorly understood mechanism of bacterial invasion into and out of the circulatory system. However, little progress has been made in developing techniques to study bacteria dynamics in the bloodstream. Here, we present a new approach using anin vivoflow cytometry platform for real‐time, noninvasive, label‐free, and quantitative monitoring of the lifespan of green fluorescent protein‐expressingStaphylococcus aureusandPseudomonas aeruginosain a murine model. We report a relatively fast average rate of clearance forS. aureus(k= 0.37 ± 0.09 min−1, half‐life ~1.9 min) and a slower rate forP. aeruginosa(k= 0.07 ± 0.02 min−1, half‐life ~9.6 min). We also observed what appears to be two stages of clearance forS. aureus, whileP. aeruginosaappeared only to have a single stage of clearance. Our results demonstrate that an advanced research tool can be used for studying the dynamics of bacteria cells directly in the bloodstream, providing insight into the progression of infectious diseases in circulation. © 2019 International Society for Advancement of Cytometry

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