Combining Genome-Scale Experimental and Computational Methods To Identify Essential Genes in Rhodobacter sphaeroides.

Rhodobacter sphaeroides is one of the best-studied alphaproteobacteria from biochemical, genetic, and genomic perspectives. To gain a better systems-level understanding of this organism, we generated a large transposon mutant library and used transposon sequencing (Tn-seq) to identify genes that are essential under several growth conditions. Using newly developed Tn-seq analysis software (TSAS), we identified 493 genes as essential for aerobic growth on a rich medium. We then used the mutant library to identify conditionally essential genes under two laboratory growth conditions, identifying 85 additional genes required for aerobic growth in a minimal medium and 31 additional genes required for photosynthetic growth. In all instances, our analyses confirmed essentiality for many known genes and identified genes not previously considered to be essential. We used the resulting Tn-seq data to refine and improve a genome-scale metabolic network model (GEM) for R. sphaeroides. Together, we demonstrate how genetic, genomic, and computational approaches can be combined to obtain a systems-level understanding of the genetic framework underlying metabolic diversity in bacterial species. IMPORTANCE Knowledge about the role of genes under a particular growth condition is required for a holistic understanding of a bacterial cell and has implications for health, agriculture, and biotechnology. We developed the Tn-seq analysis software (TSAS) package to provide a flexible and statistically rigorous workflow for the high-throughput analysis of insertion mutant libraries, advanced the knowledge of gene essentiality in R. sphaeroides, and illustrated how Tn-seq data can be used to more accurately identify genes that play important roles in metabolism and other processes that are essential for cellular survival.

Pilot plant demonstration of stable and efficient high rate biological nutrient removal with low dissolved oxygen conditions.

Aeration in biological nutrient removal (BNR) processes accounts for nearly half of the total electricity costs at many wastewater treatment plants. Even though conventional BNR processes are usually operated to have aerated zones with high dissolved oxygen (DO) concentrations, recent research has shown that nitrification can be maintained using very low-DO concentrations (e.g., below 0.2 mg O2/L), and therefore, it may be possible to reduce energy use and costs in BNR facilities by decreasing aeration. However, the effect of reduced aeration on enhanced biological phosphorus removal (EBPR) is not understood. In this study, we investigated, at the pilot-scale level, the effect of using minimal aeration on the performance of an EBPR process. Over a 16-month operational period, we performed stepwise decreases in aeration, reaching an average DO concentration of 0.33 mg O2/L with stable operation and nearly 90% phosphorus removal. Under these low-DO conditions, nitrification efficiency was maintained, and nearly 70% of the nitrogen was denitrified, without the need for internal recycling of high nitrate aeration basin effluent to the anoxic zone. At the lowest DO conditions used, we estimate a 25% reduction in energy use for aeration compared to conventional BNR operation. Our improved understanding of the efficiency of low-DO BNR contributes to the global goal of reducing energy consumption during wastewater treatment operations.

Oral fosfomycin for treatment of urinary tract infection: a retrospective cohort study.

BACKGROUND: Fosfomycin is increasingly called upon for the treatment of multi drug-resistant (MDR) organisms causing urinary tract infection (UTI). We reviewed oral fosfomycin use for UTI treatment in a large UK hospital. The primary goal was to audit our clinical practice against current national guidelines. Secondary aims were to identify factors associated with treatment failure, and to investigate the potential for using fosfomycin in patients with co-morbidities. METHODS: We retrospectively studied 75 adult patients with UTI who received 151 episodes of treatment with fosfomycin from March 2013 to June 2015. We collected clinical data from our electronic patient record, and microbiology data pre- and post- fosfomycin treatment. We recorded additional data for patients receiving prolonged courses in order to make a preliminary assessment of safety and efficacy. We also reviewed >18,000 urinary tract isolates of Escherichia coli and Klebsiella spp. processed by our laboratory over the final year of our study period to determine the prevalence of fosfomycin resistance. RESULTS: There was a significant increase in fosfomycin treatment episodes over the course of the study period. Co-morbidities were present in 71 % of patients. The majority had E. coli infection (69 %), of which 59 % were extended spectrum beta-lactamase (ESBL)-producers. Klebsiella infections were more likely than E. coli to fail treatment, and more likely to be reported as fosfomycin resistant in cases of relapse following treatment. There were no adverse events in five patients treated with prolonged fosfomycin. Among all urinary isolates collected over a year, fosfomycin resistance was documented in 1 % of E. coli vs. 19 % of Klebsiella spp. (p < 0.0001). CONCLUSIONS: We report an important role for oral fosfomycin for MDR UTI treatment in a UK hospital population, and based on the findings from this study, we present our own local guidelines for its use. We present preliminary data suggesting that fosfomycin is safe and effective for use in patients with complex comorbidities and over prolonged time periods, but may be less effective against Klebsiella than E. coli.