Expanding the Diversity of Bacterioplankton Isolates and Modeling Isolation Efficacy with Large-Scale Dilution-to-Extinction Cultivation.

Cultivated bacterioplankton representatives from diverse lineages and locations are essential for microbiology, but the large majority of taxa either remain uncultivated or lack isolates from diverse geographic locales. We paired large-scale dilution-to-extinction (DTE) cultivation with microbial community analysis and modeling to expand the phylogenetic and geographic diversity of cultivated bacterioplankton and to evaluate DTE cultivation success. Here, we report results from 17 DTE experiments totaling 7,820 individual incubations over 3 years, yielding 328 repeatably transferable isolates. Comparison of isolates to microbial community data for source waters indicated that we successfully isolated 5% of the observed bacterioplankton community throughout the study; 43% and 26% of our isolates matched operational taxonomic units and amplicon single-nucleotide variants, respectively, within the top 50 most abundant taxa. Isolates included those from previously uncultivated clades such as SAR11 LD12 and Actinobacteria acIV, as well as geographically novel members from other ecologically important groups like SAR11 subclade IIIa, SAR116, and others, providing isolates in eight putatively new genera and seven putatively new species. Using a newly developed DTE cultivation model, we evaluated taxon viability by comparing relative abundance with cultivation success. The model (i) revealed the minimum attempts required for successful isolation of taxa amenable to growth on our media and (ii) identified possible subpopulation viability variation in abundant taxa such as SAR11 that likely impacts cultivation success. By incorporating viability in experimental design, we can now statistically constrain the effort necessary for successful cultivation of specific taxa on a defined medium.IMPORTANCE Even before the coining of the term "great plate count anomaly" in the 1980s, scientists had noted the discrepancy between the number of microorganisms observed under the microscope and the number of colonies that grew on traditional agar media. New cultivation approaches have reduced this disparity, resulting in the isolation of some of the "most wanted" bacterial lineages. Nevertheless, the vast majority of microorganisms remain uncultured, hampering progress toward answering fundamental biological questions about many important microorganisms. Furthermore, few studies have evaluated the underlying factors influencing cultivation success, limiting our ability to improve cultivation efficacy. Our work details the use of dilution-to-extinction (DTE) cultivation to expand the phylogenetic and geographic diversity of available axenic cultures. We also provide a new model of the DTE approach that uses cultivation results and natural abundance information to predict taxon-specific viability and iteratively constrain DTE experimental design to improve cultivation success.

Follow-up Recommendation Rates Associated With Spectral Detector Dual-Energy CT of the Abdomen and Pelvis: A Retrospective Comparison to Single-Energy CT.

BACKGROUND: Dual-energy CT image sets have many applications in abdominopelvic imaging but no demonstrated clinical effect. PURPOSE: To determine the effect of dual-energy CT iodine maps on abdominopelvic imaging follow-up recommendation rates. MATERIALS AND METHODS: Retrospective study of abdominopelvic CTs acquired from April 2017 through June 2018. CT reports were analyzed for radiologic follow-up recommendation and follow-up recommendation reason. Follow-up MRI reports were analyzed for benign or nonbenign diagnosis. CT scans with iodine maps (CTIMs) and conventional CT scans (CCTs) subgroups were compared using χ2 testing. RESULTS: In all, 3,221 abdominopelvic CT scans of 2,401 patients (1,326 men, 1,075 women, mean age 54.1 years) were analyzed; 1,423 were CTIMs and 1,798 were CCTs. Follow-up recommendation rates were not significantly different for CTIMs and CCTs (19.5% and 21.4%, respectively, P = .19). Follow-up recommendations because of incomplete diagnosis were significantly lower in CTIMs (9.1%) than in CCTs (11.9%, P = .01). Follow-up recommendations for MRI and PET/CT were significantly lower in CTIMs (9.6%) than CCTs (13.0%, P = .003). Follow-up MRI outcomes (n = 111) were not different between CTIMs (61.2% benign) and CCTs (59.6%, P = .87). CONCLUSION: Dual-energy CT iodine maps are associated with decreased follow-up examinations because of incomplete diagnosis and decreased recommendations for follow-up MRI, suggesting that abdominopelvic iodine maps may benefit patient care and decrease institutional cost.

Draft Genome Sequence of Strain LSUCC0135, an Early Diverging Member of the Order Methylophilales in the Phylum Betaproteobacteria.

We present the draft genome of Methylophilales sp. strain LSUCC0135, isolated using high-throughput dilution-to-extinction culturing methods from the coast of Freshwater City, Louisiana, USA. The genome indicates metabolic flexibility for differing oxygen concentrations and electron donors.

Artificial Seawater Media Facilitate Cultivating Members of the Microbial Majority from the Gulf of Mexico.

High-throughput cultivation studies have been successful at bringing numerous important marine bacterioplankton lineages into culture, yet these frequently utilize natural seawater media that can hamper portability, reproducibility, and downstream characterization efforts. Here we report the results of seven experiments with a set of newly developed artificial seawater media and evaluation of cultivation success via comparison with community sequencing data from the inocula. Eighty-two new isolates represent highly important marine clades, including SAR116, OM60/NOR5, SAR92, Roseobacter, and SAR11. For many, isolation with an artificial seawater medium is unprecedented, and several organisms are also the first of their type from the Gulf of Mexico. Community analysis revealed that many isolates were among the 20 most abundant organisms in their source inoculum. This method will expand the accessibility of bacterioplankton cultivation experiments and improve repeatability by avoiding normal compositional changes in natural seawater. IMPORTANCE The difficulty in cultivating many microbial taxa vexes researchers intent on understanding the contributions of these organisms to natural systems, particularly when these organisms are numerically abundant, and many cultivation attempts recover only rare taxa. Efforts to improve this conundrum with marine bacterioplankton have been successful with natural seawater media, but that approach suffers from a number of drawbacks and there have been no comparable artificial alternatives created in the laboratory. This work demonstrates that a newly developed suite of artificial-seawater media can successfully cultivate many of the most abundant taxa from seawater samples and many taxa previously only cultivated with natural-seawater media. This methodology therefore significantly simplifies efforts to cultivate bacterioplankton and greatly improves our ability to perform physiological characterization of cultures postisolation.

Correction for Henson et al., Artificial Seawater Media Facilitate Cultivating Members of the Microbial Majority from the Gulf of Mexico.

[This corrects the article DOI: 10.1128/mSphere.00028-16.].