Microevolutionary analysis of Clostridium difficile genomes to investigate transmission

Background

The control of Clostridium difficile infection is a major international healthcare priority, hindered by a limited understanding of transmission epidemiology for these bacteria. However, transmission studies of bacterial pathogens are rapidly being transformed by the advent of next generation sequencing.

Results

Here we sequence whole C. difficile genomes from 486 cases arising over four years in Oxfordshire. We show that we can estimate the times back to common ancestors of bacterial lineages with sufficient resolution to distinguish whether direct transmission is plausible or not. Time depths were inferred using a within-host evolutionary rate that we estimated at 1.4 mutations per genome per year based on serially isolated genomes. The subset of plausible transmissions was found to be highly associated with pairs of patients sharing time and space in hospital. Conversely, the large majority of pairs of genomes matched by conventional typing and isolated from patients within a month of each other were too distantly related to be direct transmissions.

Conclusions

Our results confirm that nosocomial transmission between symptomatic C. difficile cases contributes far less to current rates of infection than has been widely assumed, which clarifies the importance of future research into other transmission routes, such as from asymptomatic carriers. With the costs of DNA sequencing rapidly falling and its use becoming more and more widespread, genomics will revolutionize our understanding of the transmission of bacterial pathogens.     

Subsurface chlorophyll maximum (SCM) location and extension in the water column as governed by a density interface in the strongly stratified Kattegat estuary

The aims of the study were to analyse the relations between the physics of a water column and the location of the subsurface chlorophyll maximum (SCM) peaks in a strongly stratified estuary. Could extension and depth location of the SCM be explained by the physical conditions in terms of water column stratification and density interface? Questions were addressed by obtaining data on water column density (CTD), chlorophyll a (Chl a), nutrients, (F _v/F _m), σ_PSII and K _d(PAR) at 15 positions along a 575 km transect in the Kattegat estuary. Results showed that the estuary was strongly stratified with mixed surface and bottom layers intercepted by a layer where density increased with depth. The SCM occurred only in this density interface, and widths of SCM and density interface were highly correlated. The surface waters were nearly depleted of inorganic nitrogen, phosphate and silicate though with significant higher concentrations in the waters below the interface. The Chl a concentration was comparatively higher in the SCM peak as well as maximum quantum efficiency (F _v/F _m) and functional cross sectional area (σ_PSII). The SCM was maintained at very low light levels and by a diapycnal nitrogen flux, with a stratified water column and nutrient depleted surface waters as predecessors. It was concluded that the depth location and vertical extension of the SCM in the estuary were closely linked to the physical structure of the water column in terms of density interface and stratification.     

Removal of snow cover inhibits spring growth of Arctic ice algae through physiological and behavioral effects

The snow cover of Arctic sea ice has recently decreased, and climate models forecast that this will continue and even increase in future. We therefore tested the effect of snow cover on the optical properties of sea ice and the biomass, photobiology, and species composition of sea ice algae at Kangerlussuaq, West Greenland, during March 2011, using a snow-clearance experiment. Sea ice algae in areas cleared of snow was compared with control areas, using imaging variable fluorescence of photosystem II in intact, unthawed ice sections. The study coincided with the onset of spring growth of ice algae, mainly an increase in two pennate diatoms (Achnanthes taeniata and Navicula directa), as temperature increased and ice thickness and brine volume stabilized. The increase in biomass was accompanied by an increase in minimum variable fluorescence (F _o) and the maximum quantum yield of PSII (F _v /F _m) and filling of brine channels with fluorescing cells. In contrast, in the minus snow area, PAR transmittance increased sixfold and there was an exponential decrease in chl-a and no increase in F _o, and the area of fluorescing biomass declined to become undetectable. This study suggests that the onset of the spring bloom is predominantly due to temperature effects on brine channel volume, and that the algal decline after snow removal was primarily due to emigration rather than photodamage.     

Binding of human factors VII and VIIa to a human bladder carcinoma cell line (J82). Implications for the initiation of the extrinsic pathway of blood coagulation

We have studied the binding of radioiodinated human factor VII and its activated form, factor VIIa, to monolayers of a human bladder carcinoma cell line (J82) that expresses functional cell surface tissue factor. The binding of factors VII and VIIa to these cells was found to be time-, temperature-, and calcium-dependent. In addition, the binding of each protein to J82 cells was specific, dose-dependent, and saturable. The binding isotherms for factors VII and VIIa were hyperbolic, and Scatchard plots of the binding data obtained at 37 degrees C indicated a single class of binding sites for each protein with Kd values of 3.20 +/- 0.51 and 3.25 +/- 0.31 nM, respectively. Factors VII and VIIa, respectively, interacted with 256,000 +/- 39,000 and 320,000 +/- 31,000 binding sites/cell. Competition experiments suggested a common receptor for factors VII and VIIa. Binding of factor VIIa to the cells was completely blocked by preincubation of the cells with polyclonal anti-tissue factor IgG, whereas binding of factor VII was inhibited approximately 90%, suggesting the presence of a small number of tissue factor-independent binding sites specific for factor VII on this cell. Functional studies revealed that factor X activation by increasing amounts of cell-bound factor VII or VIIa was hyperbolic in nature. Half-maximal rates of factor Xa formation occurred at factor VII and VIIa concentrations of 3.7 +/- 0.47 and 3.2 +/- 0.31 nM, respectively. No factor VII- or VIIa-mediated activation of factor X was observed when cells were preincubated with anti-tissue factor IgG. Two-chain 125I-factor VIIa recovered from the cells was identical to the offered ligand as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. In contrast, the offered single-chain 125I-factor VII was progressively converted to two-chain 125I-factor VIIa upon binding to the cells. When the J82 cells were pretreated with anti-tissue factor IgG, both factor VII recovered from the cells and factor VII in the supernatant were in the single-chain form, indicating that cell-surface tissue factor was essential for the activation of factor VII on these cells. These data indicate that binding of factor VII to tissue factor appears to be a prerequisite for its conversion to factor VIIa and the initiation of the extrinsic pathway of coagulation on these cells.     

Autoactivation of human recombinant coagulation factor VII

Single-chain human recombinant factor VII produced by transfected baby hamster kidney cells was purified to homogeneity in the presence of benzamidine. The amidolytic activity of single-chain recombinant factor VII with a peptidylnitroanilide substrate, methoxycarbonyl-D-cyclohexanylglycyl-L-arginine-p-nitroanilide, was less than 1% of that obtained with factor VIIa. Purified single-chain recombinant factor VII spontaneously activated in the absence of inhibitor. The activation reaction was enhanced by at least 2 orders of magnitude in the presence of a positively charged surface, provided either as an anion-exchange matrix or as poly(D-lysine). The progress curve for factor VIIa generation was sigmoidal. Benzamidine inhibits recombinant factor VIIa activity and factor VII activation with identical inhibition constants (Ki) of 11 mM. In contrast, benzamidine inhibition of bovine factor Xa and bovine factor IIa was observed at Ki values equal to 0.3 and 0.5 mM, respectively. Bovine factors Xa and IIa are known activators of factor VII and the most likely contaminants of our recombinant factor VII preparations. Single-chain recombinant factor VII purified from cells cultured in the absence of bovine serum activated at the same rate as factor VII from cells cultured in the presence of bovine serum. This also excluded the possibility that the activation reaction was caused by contaminating bovine proteases. On the basis of these observations, we propose that factor VII is autoactivated in vitro in the presence of a positively charged surface.     

Contrasting levels of DNA polymorphism at the autosomal and X-linked visual color pigment loci in humans and squirrel monkeys

The X-linked color pigment (opsin) locus is known to be highly polymorphic in the squirrel monkey and other New World monkeys. To see whether this is also the case for the autosomal (blue) opsin locus, we obtained 32 squirrel monkey and 30 human blue opsin gene sequences. No amino acid polymorphism was found in either the squirrel monkey sample or the human sample, contrary to the situation at the X-linked opsin locus. This sharp contrast in the level of polymorphism might be due to differences in gene expression between the autosomal and the X-linked loci. At the X-linked locus, heterozygote advantage can occur because, owing to X-inactivation, the two alleles in a heterozygote are expressed in different cone cells, producing two types of cone cell, whereas at the autosomal locus, heterozygote advantage cannot occur because the two alleles in a heterozygote are expressed in the same cone cells, producing only one type of cone cell (i.e., phenotypically a homozygote). From the sequence data, the levels of nucleotide diversity (pi, i.e., the number of nucleotide differences per site) are estimated: for the human sample, pi = 0.00% per nondegenerate site, 0.00% per twofold degenerate site, and 0.04% per fourfold degenerate site in the coding regions and 0.01% per site in intron 4; for the squirrel monkey sample, pi = 0.00% per nondegenerate site, 0.00% per twofold degenerate site, and 0.15% per fourfold degenerate site in the coding regions and 0.17% per site in intron 4. The blue opsin genes from the common and pygmy chimpanzees, the gorilla, the capuchin, and the howler monkey were also sequenced. Features critical to the function of the opsin are well conserved in all known mammalian sequences. However, the interhelical loops are, on average, actually more conservative than the transmembrane helical regions. In addition, these sequence data and those from some other genes indicate that the common and pygmy chimpanzees are not closely related, their divergence data being from one third to one half the date of the human-chimpanzee divergence.      

The M.ADP.P(i) state is required for helical order in the thick filaments of skeletal muscle

The thick filaments of mammalian and avian skeletal muscle fibers are disordered at low temperature, but become increasingly ordered into an helical structure as the temperature is raised. Wray and colleagues (Schlichting, I., and J. Wray. 1986. J. Muscle Res. Cell Motil. 7:79; Wray, J., R. S. Goody, and K. Holmes. 1986. Adv. Exp. Med. Biol. 226:49-59) interpreted the transition as reflecting a coupling between nucleotide state and global conformation with M.ATP (disordered) being favored at 0 degrees C and M.ADP.P(i) (ordered) at 20 degrees C. However, hitherto this has been limited to a qualitative correlation and the biochemical state of the myosin heads required to obtain the helical array has not been unequivocally identified. In the present study we have critically tested whether the helical arrangement of the myosin heads requires the M.ADP.P(i) state. X-ray diffraction patterns were recorded from skinned rabbit psoas muscle fiber bundles stretched to non-overlap to avoid complications due to interaction with actin. The effect of temperature on the intensities of the myosin-based layer lines and on the phosphate burst of myosin hydrolyzing ATP in solution were examined under closely matched conditions. The results showed that the fraction of myosin mass in the helix closely followed that of the fraction of myosin in the M.ADP.P(i) state. Similar results were found by using a series of nucleoside triphosphates, including CTP and GTP. In addition, fibers treated by N-phenylmaleimide (Barnett, V. A., A. Ehrlich, and M. Schoenberg. 1992. Biophys. J. 61:358-367) so that the myosin was exclusively in the M.ATP state revealed no helical order. Diffraction patterns from muscle fibers in nucleotide-free and in ADP-containing solutions did not show helical structure. All these confirmed that in the presence of nucleotides, the M.NDP.P(i) state is required for helical order. We also found that the spacing of the third meridional reflection of the thick filament is linked to the helical order. The spacing in the ordered M.NDP.P(i) state is 143.4 A, but in the disordered state, it is 144. 2 A. This may be explained by the different interference functions for the myosin heads and the thick filament backbone.     

Advection-induced oxygen variability in the North Sea-Baltic Sea transition

Instances of strong oxygen variations are described for two shallow water stations in the Kattegat, situated at the fluctuating frontal zone between outflowing surface water from the Baltic and inflowing bottom water from the Skagerrak/North Sea.The events consist of both a rapid emergence and a rapid disappearance of oxygen-depletion. Changes in oxygen concentration amounted to more than 20 g m^–2 d^–1 for the total water columns. Such high rates of change can not be explained by net local bottom oxygen consumption (0.6 g m^–2 d^–1) or net local water oxygen consumption (1.6 g m^–2 d^–1). The oxygen variations were influenced by the local and regional meteorological conditions. The observed instance of shallow water oxygen-depletion was connected to upward movement of the pycnocline and associated advective transport of oxygen-depleted Kattegat bottom waters to a shallow water area. Similarly, rapid disappearance of the bottom water oxygen deficit in a shallow water area was found to depend more on pycnocline lowering in connection with advective transport, than on the effect of local wind driven mixing.     

Female-limited X-chromosome evolution effects on male pre- and post-copulatory success

Intralocus sexual conflict arises when the expression of shared alleles at a single locus generates opposite fitness effects in each sex (i.e. sexually antagonistic alleles), preventing each sex from reaching its sex-specific optimum. Despite its importance to reproductive success, the relative contribution of intralocus sexual conflict to male pre- and post-copulatory success is not well-understood. Here, we used a female-limited X-chromosome (FLX) evolution experiment in Drosophila melanogaster to limit the inheritance of the X-chromosome to the matriline, eliminating possible counter-selection in males and allowing the X-chromosome to accumulate female-benefit alleles. After more than 100 generations of FLX evolution, we studied the effect of the evolved X-chromosome on male attractiveness and sperm competitiveness. We found a non-significant increase in attractiveness and decrease in sperm offence ability in males expressing the evolved X-chromosomes, but a significant increase in their ability to avoid displacement by other males'' sperm. This is consistent with a trade-off between these traits, perhaps mediated by differences in body size, causing a small net reduction in overall male fitness in the FLX lines. These results indicate that the X-chromosome in D. melanogaster is subject to selection via intralocus sexual conflict in males.