Recombination Blurs Phylogenetic Groups Routine Assignment in Escherichia coli: Setting the Record Straight

The characterization of population structures plays a main role for understanding outbreaks and the dynamics of bacterial spreading. In Escherichia coli, the widely used combination of multiplex-PCR scheme together with goeBURST has some limitations. The purpose of this study is to show that the combination of different phylogenetic approaches based on concatenated sequences of MLST genes results in a more precise assignment of E. coli phylogenetic groups, complete understanding of population structure and reconstruction of ancestral clones. A collection of 80 Escherichia coli strains of different origins was analyzed following the Clermont and Doumith''s multiplex-PCR schemes. Doumith''s multiplex-PCR showed only 1.7% of misassignment, whereas Clermont''s-2000 protocol reached 14.0%, although the discrepancies reached 30% and 38.7% respectively when recombinant C, F and E phylogroups were considered. Therefore, correct phylogroup attribution is highly variable and depends on the clonal composition of the sample. As far as population structure of these E. coli strains, including 48 E. coli genomes from GenBank, goeBURST provides a quite dispersed population structure; whereas NeighborNet approach reveals a complex population structure. MLST-based eBURST can infer different founder genotypes, for instance ST23/ST88 could be detected as the founder genotypes for STC23; however, phylogenetic reconstructions might suggest ST410 as the ancestor clone and several evolutionary trajectories with different founders. To improve our routine understanding of E. coli molecular epidemiology, we propose a strategy based on three successive steps; first, to discriminate three main groups A/B1/C, D/F/E and B2 following Doumith''s protocol; second, visualization of population structure based on MLST genes according to goeBURST, using NeighborNet to establish more complex relationships among STs; and third, to perform, a cost-free characterization of evolutionary trajectories in variants emerging along the clonal expansion using parsimony methods of phylogenetic analysis.     

Spread and evolution of natural plasmids harboring transposon Tn5

Abstract: The presence of transposon Tn 5 was studied in 730 Enterobacteriaceae strains from clinical and sewage origin. From these strains, twenty-five conjugative plasmids harboring transposon Tn 5 were isolated. These plasmids were compared with pJR67 and pRYC119, the only previously studied plasmids harboring Tn 5 . A phylogenetic tree of the evolution of all different plasmids was proposed. Irrespective of their bacterial host and geographical place of isolation, some of the plasmids were shown to be identical. All of them can be included in only eight different prototypical plasmid species. Twenty-two plasmids (88%) carried an IncI1 incompatibility determinant as judged form DNA hybridization experiments. The presence of some other common resistance genes suggested that these plasmids are descendants of a common ancestor. These IncI1 plasmids could be grouped in six prototypical species. The results presented here suggest that Tn 5 spread in nature may be dependent on the conjugative ability of the IncI plasmids harboring the transposon, rather than on the efficiency of Tn 5 transposition between different replicons.     

Measurement of 'in situ' mitochondrial membrane potential in Ehrlich ascites tumor cells during aerobic glycolysis

(1) A method is presented for continuous and simultaneous monitoring of the 'in situ' mitochondrial membrane potential (delta psi m) and respiration rate of Ehrlich ascites tumor cells. The method involves permeabilization of the plasma membrane, achieved by treatment with low digitonin concentration, and the use of a TPP+ selective electrode attached to an oxygraph vessel. Binding of the probe inside the cells was analyzed assuming a proportional relationship between the amount of bound TPP+ and the free concentration of the lipophilic cation. (2) Evidence is reported that the addition of glucose to digitonin-permeabilized Ehrlich ascites tumor cells causes a decrease of mitochondrial membrane potential that coincided with a transient enhancement of the respiration rate and remained unchanged during the subsequent Crabtree effect. We have characterized the effect of glucose on delta psi m by determining its dependent on the glycolytic pathway and its sensitivity towards oligomycin. The mutual relationships between glucose and ADP effects on the mitochondrial membrane potential were also studied. A plausible mechanism underlying the depolarization of mitochondrial membrane induced by glucose is presented.     

Challenges: Selective compartments for resistant microorganisms in antibiotic gradients

The development of bacterial resistance to antibiotics is one of the best documented examples of contemporary biological evolution. Variability in the mechanisms of resistance depends on the diversity of genotypes in the huge bacterial populations, and also on the diversity of selective pressures that are produced along the antibiotic concentration gradients formed in the highly compartmentalized human body during therapy. These antibiotic gradients can be conceived as comprising selective compartments, each one of them defined as the concentration able to select a particular genetic variant. In vitro experimental models confirm that some antibiotic resistant variants are selected only at certain selective concentrations of antibiotics. The correspondence between selective compartments and selectable variants could offer a way of describing more accurately the antibiotic selective landscapes and for taking measures to prevent the development of a major threat to the future of modern medicine.     

Generational coexistence and ancestor's inhibition in bacterial populations

Generational coexistence in structured environments raises the possibility of a competition between ancestors and descendents. This type of kin competition, and in particular, the possibility that descendents might actively repress the ancestor's dominance, has been rarely considered in microbial evolutionary ecology. The recent discovery of the phenomenon of stationary-phase contact-dependent inhibition of bacterial ancestor cells by late descendents provides a new theoretical perspective to analyze intrapopulational evolutionary changes. The ancestor's inhibition effect might accelerate such changes, particularly when the descendents have acquired small adaptive advantages that are insufficient to rapidly displace the well-settled ancestors in a complex niche. Besides this effect of triggering selection of small genetic differences, the opportunities for intergenerational coexistence in bacteria, where ancestor's inhibition might occur, are reviewed in this work. A theoretical analysis is provided about the explanatory possibilities of the ancestor's inhibition effect in the controversies about intraspecific (in a large sense, including intrapopulational) genetic diversification, and the discontinuities observed in such processes, giving rise to the emergence of individualities and therefore differential units of selection.     

Comparative distribution of human and avian type sialic acid influenza receptors in the pig

Background  

A major determinant of influenza infection is the presence of virus receptors on susceptible host cells to which the viral haemagglutinin is able to bind. Avian viruses preferentially bind to sialic acid α2,3-galactose (SAα2,3-Gal) linked receptors, whereas human strains bind to sialic acid α2,6-galactose (SAα2,6-Gal) linked receptors. To date, there has been no detailed account published on the distribution of SA receptors in the pig, a model host that is susceptible to avian and human influenza subtypes, thus with potential for virus reassortment. We examined the relative expression and spatial distribution of SAα2,3-GalG(1-3)GalNAc and SAα2,6-Gal receptors in the major organs from normal post-weaned pigs by binding with lectins Maackia amurensis agglutinins (MAA II) and Sambucus nigra agglutinin (SNA) respectively.     

Mixed biodiversity benefits of agri-environment schemes in five European countries

Agri-environment schemes are an increasingly important tool for the maintenance and restoration of farmland biodiversity in Europe but their ecological effects are poorly known. Scheme design is partly based on non-ecological considerations and poses important restrictions on evaluation studies. We describe a robust approach to evaluate agri-environment schemes and use it to evaluate the biodiversity effects of agri-environment schemes in five European countries. We compared species density of vascular plants, birds, bees, grasshoppers and crickets, and spiders on 202 paired fields, one with an agri-environment scheme, the other conventionally managed. In all countries, agri-environment schemes had marginal to moderately positive effects on biodiversity. However, uncommon species benefited in only two of five countries and species listed in Red Data Books rarely benefited from agri-environment schemes. Scheme objectives may need to differentiate between biodiversity of common species that can be enhanced with relatively simple modifications in farming practices and diversity or abundance of endangered species which require more elaborate conservation measures.     

Differentiation of an adult neuron cell line increases susceptibility to rabies infection

A wide variety of in vitro models have been used for studying rabies infection, however, currently, no central nervous system (CNS) adult neuron cultures are available. The current study determined the susceptibility to rabies infection in an adult CNS neuron cell line (CAD-R1). Cultures of CAD-R1 cells were held for 5 days in medium containing serum (undifferentiated CAD-R1 cells) or in serum-free medium (differentiated CAD-R1 cells). They were then infected with highly neurotropic rabies virus (RV) strain (CVS), obtained from fibroblastic cells (CVS-BHK) or from adult mouse brain (CVS-MB). Undifferentiated and differentiated cells were infected with the two RV strains, but the percentage of infected cells in differentiated cultures was significantly greater (83% and 79%, respectively) than in undifferentiated cells (51% and 60%) (Student's t test<0.05). Susceptibility to infection apparently depended on cellular differentiation state, possibly due to acquisition of additional morphological and biochemical characteristics during the differentiation process that made them more susceptible to RV infection. Therefore, CAD R1 cells may represent a good model for RV infection, making them a useful tool for studying RV neurotropism, infection pathogeny, isolation of street virus or producing safer and most potent vaccines.