Antimicrobial Resistance and Virulence Genes of Escherichia coli Isolates from Swine in Ontario

A total of 318 Escherichia coli isolates obtained from diarrheic and healthy pigs in Ontario from 2001 to 2003 were examined for their susceptibility to 19 antimicrobial agents. They were tested by PCR for the presence of resistance genes for tetracycline, streptomycin, sulfonamides, and apramycin and of 12 common virulence genes of porcine E. coli. Antimicrobial resistance frequency among E. coli isolates from swine in Ontario was moderate in comparison with other countries and was higher in isolates from pigs with diarrhea than in isolates from healthy finisher pigs. Resistance profiles suggest that cephamycinases may be produced by ≥8% of enterotoxigenic E. coli (ETEC). Resistance to quinolones was detected only in enterotoxigenic E. coli (≤3%). The presence of sul3 was demonstrated for the first time in Canada in porcine E. coli isolates. Associations were observed among tetA, sul1, aadA, and aac(3)IV and among tetB, sul2, and strA/strB, with a strong negative association between tetA and tetB. The paa and sepA genes were detected in 92% of porcine ETEC, and strong statistical associations due to colocation on a large plasmid were observed between tetA, estA, paa, and sepA. Due at least in part to gene linkages, the distribution of resistance genes was very different between ETEC isolates and other porcine E. coli isolates. This demonstrates that antimicrobial resistance epidemiology differs significantly between pathogenic and commensal E. coli isolates. These results may have important implications with regards to the spread and persistence of resistance and virulence genes in bacterial populations and to the prudent use of antimicrobial agents.     

RGS9 Concentration Matters in Rod Phototransduction

The transduction of light by retinal rods and cones is effected by homologous G-protein cascades whose rates of activation and deactivation determine the sensitivity and temporal resolution of photoreceptor signaling. In mouse rods, the rate-limiting step of deactivation is hydrolysis of GTP by the G-protein-effector complex, catalyzed by the RGS9 complex. Here, we incorporate a “Michaelis module” describing the RGS9 reaction into the conventional scheme for phototransduction and show that this augmented scheme can account precisely for the dominant recovery rate of intact rods in which RGS9 expression varies over a 20-fold range. Furthermore, by screening the parameter space of the scheme with maximum-likelihood methodology, we tested specific hypotheses about the rate constant for rhodopsin deactivation, and about the forward, reverse, and catalytic constants for RGS9-mediated G-protein deactivation. These tests reliably exclude lifetimes >∼50 ms for rhodopsin, and reveal that the dominant time constant of rod photoresponse recovery is 1/(V_max/K_m) for the RGS9 reaction, with k_cat/K_m ≈ 0.04 μm² s⁻¹ and k_cat > 35 s⁻¹ (or K_m > 840 μm⁻²). All together, the new kinetic scheme and analysis explain how and why RGS9 concentration matters in rod phototransduction, and they provide a framework for understanding the molecular mechanisms that rate-limit deactivation in other G-protein systems.     

Invasion of insect cells by Spiroplasma citri involves spiralin relocalization and lectin/glycoconjugate‐type interactions

Spiroplamas are helical, cell wall‐less bacteria belonging to the Class Mollicutes, a group of microorganisms phylogenetically related to low G+C, Gram‐positive bacteria. Spiroplasma species are all found associated with arthropods and a few, including Spiroplasma citri are pathogenic to plant. Thus S. citri has the ability to colonize cells of two very distinct hosts, the plant and the insect vector. While spiroplasmal factors involved in transmission by the leafhopper Circulifer haematoceps have been identified, their specific contribution to invasion of insect cells is poorly understood. In this study we provide evidence that the lipoprotein spiralin plays a major role in the very early step of cell invasion. Confocal laser scanning immunomicroscopy revealed a relocalization of spiralin at the contact zone of adhering spiroplasmas. The implication of a role for spiralin in adhesion to insect cells was further supported by adhesion assays showing that a spiralin‐less mutant was impaired in adhesion and that recombinant spiralin triggered adhesion of latex beads. We also showed that cytochalasin D induced changes in the surface‐exposed glycoconjugates, as inferred from the lectin binding patterns, and specifically improved adhesion of S. citri wild‐type but not of the spiralin‐less mutant. These results indicate that cytochalasin D exposes insect cell receptors of spiralin that are masked in untreated cells. In addition, competitive adhesion assays with lectins strongly suggest spiralin to exhibit glycoconjugate binding properties similar to that of the Vicia villosa agglutinin (VVA) lectin.     

Discordant mtDNA and cpDNA phylogenies indicate geographic speciation and reticulation as driving factors for the diversification of the genus Picea

The phylogeny of the genus Picea was investigated by sequencing three loci from the paternally inherited chloroplast genome (trnK, rbcL and trnTLF) and the intron 2 of the maternally transmitted mitochondrial gene nad1 for 35 species. Significant topological differences were found between the trnK tree and the rbcL and trnTLF phylogenetic trees, and between cpDNA and mtDNA phylogenies. None of the phylogenies matched morphological classifications. The mtDNA phylogeny was geographically more structured than cpDNA phylogenies, reflecting the different inheritance of the two cytoplasmic genomes in the Pinaceae and their differential dispersion by seed only and seed and pollen, respectively. Most North American taxa formed a monophyletic group on the mtDNA tree, with topological patterns suggesting geographic speciation by range fragmentation or by dispersal and isolation. Similar patterns were also found among Asian taxa. Such a trend towards geographic speciation is anticipated in other Pinaceae genera with similar life history, autecology and reproductive system. Incongruences between organelle phylogenies suggested the occurrence of mtDNA capture by invading cpDNA. Incongruences between cpDNA partitions further suggested heterologous recombination presumably also linked to ancient reticulate evolution. Whilst cpDNA appears potentially valuable for molecular taxonomy and systematics purposes, these results emphasize the reduced value of cpDNA to infer vertical descent and the speciation history for plants with paternal transmission and high dispersal of their chloroplast genome.     

Phenotypes of non-attached Pseudomonas aeruginosa aggregates resemble surface attached biofilm

For a chronic infection to be established, bacteria must be able to cope with hostile conditions such as low iron levels, oxidative stress, and clearance by the host defense, as well as antibiotic treatment. It is generally accepted that biofilm formation facilitates tolerance to these adverse conditions. However, microscopic investigations of samples isolated from sites of chronic infections seem to suggest that some bacteria do not need to be attached to surfaces in order to establish chronic infections. In this study we employed scanning electron microscopy, confocal laser scanning microscopy, RT-PCR as well as traditional culturing techniques to study the properties of Pseudomonas aeruginosa aggregates. We found that non-attached aggregates from stationary-phase cultures have comparable growth rates to surface attached biofilms. The growth rate estimations indicated that, independently of age, both aggregates and flow-cell biofilm had the same slow growth rate as a stationary phase shaking cultures. Internal structures of the aggregates matrix components and their capacity to survive otherwise lethal treatments with antibiotics (referred to as tolerance) and resistance to phagocytes were also found to be strikingly similar to flow-cell biofilms. Our data indicate that the tolerance of both biofilms and non-attached aggregates towards antibiotics is reversible by physical disruption. We provide evidence that the antibiotic tolerance is likely to be dependent on both the physiological states of the aggregates and particular matrix components. Bacterial surface-attachment and subsequent biofilm formation are considered hallmarks of the capacity of microbes to cause persistent infections. We have observed non-attached aggregates in the lungs of cystic fibrosis patients; otitis media; soft tissue fillers and non-healing wounds, and we propose that aggregated cells exhibit enhanced survival in the hostile host environment, compared with non-aggregated bacterial populations.     

Influence of nitrate supply on concentrations and translocation of abscisic acid in barley (Hordeum vulgare)

Spring barley ( Hordeum vulgare L. cv. Golf) was grown at different nitrate supply rates, controlled by using the relative addition rate technique, in order to elucidate the relationship between nitrate-N supply and root and shoot levels of abscisic acid (ABA). The plants were maintained as (1) standard cultures where nitrate was supplied at relative addition rates (RAs) of 0.03, 0.09 and 0.18 day⁻¹, and (2) split-root cultures at RA 0.09 day⁻¹ but with the nitrate distributed between the two root parts in ratios of 100:0, 80:20 and 60:40. Time-dependent changes in root and shoot concentrations of ABA (determined by radioimmunoassay using a monoclonal antibody) were observed in both standard and split-root cultures during 12 days of acclimation to the different nitrate regimes. However, the ABA responses were similar at all nitrate supply rates. Further experiments were performed with split-root cultures where the distribution of nitrate between the two root parts was reversed from 80:20 to 20:80 so that short-term effects to local perturbations of nitrate supply could be studied without altering whole-plant N absorption. Transient increases in ABA concentrations (maximum of 25 to 40% after 3 to 4 h) were observed in both subroot parts, as well as in xylem sap and shoot tissue. By pruning the root system it was demonstrated that the change in ABA had its origin in the subroot part receiving the increased nitrate supply (i.e. switched from 20 to 80% of the total nitrate supply). The data indicate that ABA responses are easily transmitted between different organs, including transmission from one set of seminal roots to another via the shoot. The data do not provide any indication that long-term nitrate supplies or general nitrogen status of barley plants affect, or are otherwise related to, the average tissue ABA concentrations of roots and shoots.