MOLECULAR AND GENOMIC BASED INSIGHTS INTO THE EVOLUTION OF ENTEROCOCCUS FAECIUM FROM COMMENSAL TO HOSPITAL-ADAPTED PATHOGEN

The basis for the recent transition of Enterococcus faecium from a primarily commensal organism to one of the leading causes of hospital-acquired infections in the United States is not yet understood. To address this, the first part of my project assessed isolates from early outbreaks in the USA and South America using sequence analysis, colony hybridizations, and minimal inhibitory concentrations (MICs) which showed clinical isolates possess virulence and antibiotic resistance determinants that are less abundant or lacking in community isolates. I also revealed that the level of ampicillin resistance increased over time in clinical strains. By sequencing the pbp5 gene, I demonstrated an ~5% difference in the pbp5 gene between strains with MICs <4ug/ml and those with MICs >4µg/ml, but no specific sequence changes correlated with increases in MICs within the latter group. A 3-10% nucleotide difference was also seen in three other genes analyzed, which suggested the existence of two distinct subpopulations of E. faecium. This led to the second part of my project analyzing concatenated core gene sequences, SNPs, the 16S rRNA, and phylogenetics of 21 E. faecium genomes confirming two distinct clades; a community-associated (CA) clade and hospital-associated (HA) clade. Molecular clock calculations indicate that these two clades likely diverged ~ 300,000 to > 1 million years ago, long before the modern antibiotic era. Genomic analysis also showed that, in addition to core genomic differences, HA E. faecium harbor specific accessory genetic elements that may confer selection advantages over CA E. faecium. The third part of my project discovered 6 E. faecium genes with the newly identified “WxL” domain. My analyses, using RT-PCR, western blots, patient sera, whole-cell ELISA, and immunogold electron microscopy, indicated that E. faecium WxL genes exist in operons, encode bacterial cell surface localized proteins, that WxL proteins are antigenic in humans, and are more exposed on the surface of clinical isolates versus community isolates (even though they are ubiquitous in both clades). ELISAs and BIAcore analyses also showed that proteins encoded by these operons bind several different host extracellular matrix proteins, as well as to each other, suggesting a novel cell-surface complex. In summary, my studies provide new insights into the evolution of E. faecium by showing that there are two distantly related clades; one being more successful in the hospital setting. My studies also identified operons encoding WxL proteins whose characteristics could also contribute to colonization and virulence within this species.

Geochemistry and sea-surface temperature reconstruction for sediment cores of the Iberian Margin

Biogenic records of the marine palaeoproductivity (carbonates, organic carbon, and C37 alkenones) and the molecular stratigraphy of past sea surface temperatures (SSTs; UK'37) were studied at high resolution in two cores of the Iberian Margin. The comparison of these records indicates that the oceanographic conditions switched abruptly during the past 160 kyr between three kinds of regimes. A first regime with high (17-22°C) SST and low productivity typifies the interglacial periods, marine isotopic stages (MIS) 5 and 1. Several periods during MIS 6, 2, and the terminations II and I are characterised by about 4-5°C colder SST and a higher organic matter accumulation, both of which define the second regime. This anticorrelation between SST and marine productivity suggests that these variations are related to the intensity of the coastal upwelling. By contrast with this upwelling behaviour, extremely low biological productivity and very cold SST (6-12°C) occurred during short phases of glacial MIS 6, 4, and 2, and as abrupt events (~1 kyr or less) during MIS 3. The three oceanographic regimes are consistent with micropalaeontological changes in the same cores based on foraminifera and diatoms. The general trend of these hydrologic changes follows the long-term glacial/interglacial cycle, but the millennium scale variability is clearly related to Heinrich events and Dansgaard-Oeschger cycles. Strengthening of the upwelling corresponds probably to an intensification of the subtropical atmospheric circulation over the North Atlantic which was influenced by the presence of continental ice sheets. However, extreme glacial conditions due to massive discharges of icebergs interrupted the upwelling. Interestingly, both terminations II and I coincided with strong but transient intensification of the upwelling.

Hydrocarbons in surface layer bottom sediments from the North Dvina Riverand the southern part of the Dvina Bay, White Sea

Analysis of molecular composition of alkanes in bottom sediments of the southern part of Dvina Bay (White Sea) in October 2001 revealed the following main peculiarities of hydrocarbon behavior in the estuary: dominating of high molecular C23-C45 compounds and irregular distribution of hydrocarbons in bottom sediments as a result of high sedimentation rate and active hydrodynamics in the studied area.

Organic-walled dinoflagellate cysts analysis and statistical analysis of 116 surface sediment samples from the eastern Indian Ocean

Assemblages of organic-walled dinoflagellate cysts (dinocysts) from 116 marine surface samples have been analysed to assess the relationship between the spatial distribution of dinocysts and modern local environmental conditions [e.g. sea surface temperature (SST), sea surface salinity (SSS), productivity] in the eastern Indian Ocean. Results from the percentage analysis and statistical methods such as multivariate ordination analysis and end-member modelling, indicate the existence of three distinct environmental and oceanographic regions in the study area. Region 1 is located in western and eastern Indonesia and controlled by high SSTs and a low nutrient content of the surface waters. The Indonesian Throughflow (ITF) region (Region 2) is dominated by heterotrophic dinocyst species reflecting the region's high productivity. Region 3 is encompassing the area offshore north-west and west Australia which is characterised by the water masses of the Leeuwin Current, a saline and nutrient depleted southward current featuring energetic eddies.

Organic compounds and bitumen in surface sediments from the Pechora Bay

Detailed geochemical investigations of bottom sediments in the Pechora Bay were carried out under a monitoring regime. The regional geochemical background is characterized. Organic matter of sediments is found to be clastic, formed under the influence of Pechora River run-off, and determined by genetic and lithofacial factors. An application of geochemical methodology is suggested for estimating hydrocarbon contamination of bottom sediments. As an example consequences of the accidental Usinsky oil spill are studied. They are based on analysis of composition of molecular markers. It is shown that increasing technogenic impact on Pechora Bay sediments is fixed only at molecular level and is not disastrous.

Sea surface temperature variations in the western Mediterranean Sea

A high-resolution sea surface temperature (SST) reconstruction of the western Mediterranean was accomplished using two independent, algae-based molecular organic proxies, i.e. the UK'37 index based on long-chain unsaturated ketones and the novel long-chain diol index (LDI) based on the relative abundances of C28 and C30 1,13- and 1,15-diols. Two marine records, from the western and eastern Alboran Sea basin, spanning the last 14 and 20 kyr, respectively, were studied. Results from the surface sediments suggest that the two proxies presently reflect seasons with similar SST, or simply annual mean SST. Both proxy records reveal the transition from the Last Glacial Maximum to the Holocene in the eastern Alboran Sea with an SST increase of ca. 7 °C for UK'37 and 9 °C for LDI. Minimum SSTs (10-12 °C) are reached at the end of the Last Glacial Maximum and during the last Heinrich event with a subsequent rapid SST increase in LDI-SST towards the beginning of the Bölling period (20 °C), while UK'37-SST remains constantly low (~12 °C). The Bölling-Alleröd is characterized by a rapid increase and subsequent decrease in UK'37-SST, while the LDI-SST decrease continuously. Short-term fluctuations in UK'37-SST are probably related to availability of nutrients and seasonal changes. The Younger Dryas is recorded as a short cold interval followed by progressively warmer temperatures. During the Holocene, the general lower UK'37-derived temperature values in the eastern Alboran (by ca. 1.5-2 °C) suggest a southeastward cold water migration by the western Alboran gyre and divergence in the haptophyte blooming season between both basins.

Lignin and chemical elements in the surface layer of bottom sediments from the Kandalaksha Bay of the White Sea

Chemical composition of the upper layer of sediments (0-1 cm) in the Kolvits and Knazhaya inlets, and also in the deep-water part of the Kandalaksha Bay is considered. It is shown that silts are richer in Fe, TOC, and heavy metals, than sands. The highest concentration of these elements is found in sediments under mixing zones of riverine and sea waters. Correlations of P, Zn, Cd, and Cu with iron are high, and correlations of Pb and Cu with organic carbon are also high. Very high concentration of Pb in the Kandalaksha Bay indicate technogenic pollution of sediments. Lignin makes significant contribution to formation of organic matter in the sediments. Composition of lignin in bottom sediments of the Kandalaksha Bay is defined by composition of lignin in soils and aerosols. Vanillin and syringyl structures prevail in molecular composition of lignin in bottom sediments. Their sources are coniferous vegetations, soils, and mosses. Ratios of certain types of phenol compounds indicate pollution of the upper layer of sediments by technogenic lignin. Lead and copper correlate well with this technogenic lignin.

Response of the calcifying coccolithophore Emiliania huxleyi to low pH/high pCO2: from physiology to molecular level, 2011

The emergence of ocean acidification as a significant threat to calcifying organisms in marine ecosystems creates a pressing need to understand the physiological and molecular mechanisms by which calcification is affected by environmental parameters. We report here, for the first time, changes in gene expression induced by variations in pH/pCO2 in the widespread and abundant coccolithophore Emiliania huxleyi. Batch cultures were subjected to increased partial pressure of CO2 (pCO2; i.e. decreased pH), and the changes in expression of four functional gene classes directly or indirectly related to calcification were investigated. Increased pCO2 did not affect the calcification rate and only carbonic anhydrase transcripts exhibited a significant down-regulation. Our observation that elevated pCO2 induces only limited changes in the transcription of several transporters of calcium and bicarbonate gives new significant elements to understand cellular mechanisms underlying the early response of E. huxleyi to CO2-driven ocean acidification.

Quantification and molecular characterization of dissolved organic sulfur in water samples obtained during POLARSTERN cruise ANT-XXV (PS73) to the East Atlantic and the Southern Ocean

Although sulfur is an essential element for marine primary production and critical for climate processes, little is known about the oceanic pool of non-volatile dissolved organic sulfur (DOS). We present a basin-scale distribution of solid phase extractable DOS in the East Atlantic Ocean and the Atlantic sector of the Southern Ocean. While molar DOS versus dissolved organic nitrogen (DON) ratios of 0.11 ± 0.024 in Atlantic surface water resembled phytoplankton stoichiometry (S/N ~ 0.08), increasing dissolved organic carbon (DOC) versus DOS ratios and decreasing methionine-S yield demonstrated selective DOS removal and active involvement in marine biogeochemical cycles. Based on stoichiometric estimates, the minimum global inventory of marine DOS is 6.7 Pg S, exceeding all other marine organic sulfur reservoirs by an order of magnitude.