Detection and Isolation of Ultrasmall Microorganisms from a 120,000-Year-Old Greenland Glacier Ice Core

The abundant microbial population in a 3,043-m-deep Greenland glacier ice core was dominated by ultrasmall cells (<0.1 μm(3)) that may represent intrinsically small organisms or starved, minute forms of normal-sized microbes. In order to examine their diversity and obtain isolates, we enriched for ultrasmall psychrophiles by filtering melted ice through filters with different pore sizes, inoculating anaerobic low-nutrient liquid media, and performing successive rounds of filtrations and recultivations at 5°C. Melted ice filtrates, cultures, and isolates were analyzed by scanning electron microscopy, flow cytometry, cultivation, and molecular methods. The results confirmed that numerous cells passed through 0.4-μm, 0.2-μm, and even 0.1-μm filters. Interestingly, filtration increased cell culturability from the melted ice, yielding many isolates related to high-G+C gram-positive bacteria. Comparisons between parallel filtered and nonfiltered cultures showed that (i) the proportion of 0.2-μm-filterable cells was higher in the filtered cultures after short incubations but this difference diminished after several months, (ii) more isolates were obtained from filtered (1,290 isolates) than from nonfiltered (447 isolates) cultures, and (iii) the filtration and liquid medium cultivation increased isolate diversity (Proteobacteria; Cytophaga-Flavobacteria-Bacteroides; high-G+C gram-positive; and spore-forming, low-G+C gram-positive bacteria). Many isolates maintained their small cell sizes after recultivation and were phylogenetically novel or related to other ultramicrobacteria. Our filtration-cultivation procedure, combined with long incubations, enriched for novel ultrasmall-cell isolates, which is useful for studies of their metabolic properties and mechanisms for long-term survival under extreme conditions

Phylogenetic Analysis of Anaerobic Psychrophilic Enrichment Cultures Obtained from a Greenland Glacier Ice Core

The examination of microorganisms in glacial ice cores allows the phylogenetic relationships of organisms frozen for thousands of years to be compared with those of current isolates. We developed a method for aseptically sampling a sediment-containing portion of a Greenland ice core that had remained at −9°C for over 100,000 years. Epifluorescence microscopy and flow cytometry results showed that the ice sample contained over 6 × 10(7) cells/ml. Anaerobic enrichment cultures inoculated with melted ice were grown and maintained at −2°C. Genomic DNA extracted from these enrichments was used for the PCR amplification of 16S rRNA genes with bacterial and archaeal primers and the preparation of clone libraries. Approximately 60 bacterial inserts were screened by restriction endonuclease analysis and grouped into 27 unique restriction fragment length polymorphism types, and 24 representative sequences were compared phylogenetically. Diverse sequences representing major phylogenetic groups including alpha, beta, and gamma Proteobacteria as well as relatives of the Thermus, Bacteroides, Eubacterium, and Clostridium groups were found. Sixteen clone sequences were closely related to those from known organisms, with four possibly representing new species. Seven sequences may reflect new genera and were most closely related to sequences obtained only by PCR amplification. One sequence was over 12% distant from its closest relative and may represent a novel order or family. These results show that phylogenetically diverse microorganisms have remained viable within the Greenland ice core for at least 100,000 years

Role of some biomarkers in determining the risk of mortality of hospitalized patients with community- acquired pneumonia

Introduction: Various biomarkers are used to determine the severity and risk of mortality in community-acquired pneumonia (CAP). The aim of this article is to evaluate the prognostic value for in-hospital mortality of leukocyte count (Leuk), C-reactive protein (CRP), procalcitonin (PCT), and mid-regional proadrenomedullin (MR-proADM) in CAP patients.Materials and Methods: This was a prospective study including a total of 92 CAP patients hospitalized in the Clinic of Pneumology and Phthisiatry at St. Marina University Hospital of Varna. Biomarkers were determined at hospitalization, Leuk - by automated methodology, CRP - by latex-enhanced immuno-turbidimetric method, and both MR-proADM and PCT - by standard ELISA. CAP severity was estimated by Pneumonia Severity Index (PSI) and CURB-65.Results: The patients were at a mean age of 59.2±16.8 years, 68.5% were men. In-hospital mortality was 7.6%. The optimal cut-off value of MR-proADM for in-hospital mortality was 0.88 ng/mL (sensitivity 85.7% and specificity 85.8%). The positive predictive value was 33.3% and the negative predictive value was 98.6%. The optimal cut-off value of PCT was 1.84 ng/mL (sensitivity 71.4% and specificity 81.1%). The positive predictive value was 23.8% and the negative predictive value was 97.1%. Cut-off values for CRP and Leuk could not be established. By performing ROC curves, MR-proADM, PSI, PCT and CURB-65 were good predictors for in-hospital mortality (AUC 0.91, 0.90, 0.89, and 0.86, respectively).Conclusion: MR-proADM and PCT are promising markers in predicting CAP prognosis. Their predictive value for mortality is similar to that of PSI and CURB-65. CRP and Leuk cannot serve as predictors

Abundance, viability and diversity of the indigenous microbial populations at different depths of the NEEM Greenland ice core

The 2537-m-deep North Greenland Eemian Ice Drilling (NEEM) core provided a first-time opportunity to perform extensive microbiological analyses on selected, recently drilled ice core samples representing different depths, ages, ice structures, deposition climates and ionic compositions. Here, we applied cultivation, small subunit (SSU) rRNA gene clone library construction and Illumina next-generation sequencing (NGS) targeting the V4–V5 region, to examine the microbial abundance, viability and diversity in five decontaminated NEEM samples from selected depths (101.2, 633.05, 643.5, 1729.75 and 2051.5 m) deposited 300–80 000 years ago. These comparisons of the indigenous glacial microbial populations in the ice samples detected significant spatial and temporal variations. Major findings include: (a) different phylogenetic diversity of isolates, dominated by Actinobacteria and fungi, compared to the culture-independent diversity, in which Proteobacteria and Firmicutes were more frequent; (b) cultivation of a novel alphaproteobacterium; (c) dominance of Cyanobacteria among the SSU rRNA gene clones from the 1729.75-m ice; (d) identification of Archaea by NGS that are rarely detected in glacial ice; (e) detection of one or two dominant but different genera among the NGS sequences from each sample; (f) finding dominance of Planococcaceae over Bacillaceae among Firmicutes in the brittle and the 2051.5-m ice. The overall beta diversity between the studied ice core samples examined at the phylum/class level for each approach showed that the population structure of the brittle ice was significantly different from the two deep clathrated ice samples and the shallow ice core.Keywords: Greenland; NEEM ice core; indigenous microbial diversity; isolates; Illumina MiSeq.To access the supplementary material for this article, please see supplementary files (in the column to the right) under Article Tools.(Published: 16 February 2015)Citation: Polar Research 2015, 34, 25057, http://dx.doi.org/10.3402/polar.v34.2505

Geochemical and Microbiological Studies of Nitrous Oxide Variations within the New NEEM Greenland Ice Core During the Last Glacial Period

Deep polar ice cores provide atmospheric records of nitrous oxide (N₂O) and other trace gases reflecting climate history along with a parallel archive of microbial cells transported with mineral dust, marine and volcanic aerosols from around the globe. Our interdisciplinary study of 32 samples from different depths of the recently drilled NEEM Greenland ice core addressed the question whether the identified microorganisms were capable of post-depositional biological production of N₂O in situ. We used high-resolution geochemical and microbiological approaches to examine the N₂O concentrations, the quantitative distributions of dust, Ca⁺², NH₄⁺ and NO₃⁻ ¡ons related to N cycle pathways, the microbial abundance and diversity at specific NEEM core depths from 1758 m to 1867.8 m. Results showed varying concentrations of N₂O (220 –271.5 ppb). Microbial abundance fluctuated between 3.3 x 10⁴ and 3.3 x 10⁶ cells mL⁻¹ in direct correlation with dust and Ca²⁺ concentrations with higher cell numbers deposited during colder periods. The average values of NH₄⁺ and NO₃⁻ indicated that substrates were available for the microorganisms capable of utilizing them. PCR amplification of selected functional genes involved in bacterial and archaeal nitrification and denitrification was not successful. Sanger and Illumina MiSeq sequence analyses of SSU rRNA genes showed variable representation of Alpha-, Beta- and Gammaproteobacteria, Firmicutes, Actinobacteria, chloroplasts and fungi. The metabolic potential of the dominant genera of Proteobacteria and Firmicutes as possible N₂O producers suggested that denitrification activity may have led to in-situ production and accumulation of N₂O