Variations of Plasmid Content in Rickettsia felis

Background: Since its first detection, characterization of R. felis has been a matter of debate, mostly due to the contamination of an initial R. felis culture by R. typhi. However, the first stable culture of R. felis allowed its precise phenotypic and genotypic characterization, and demonstrated that this species belonged to the spotted fever group rickettsiae. Later, its genome sequence revealed the presence of two forms of the same plasmid, physically confirmed by biological data. In a recent article, Gillespie et al. ( PLoS One. 2007; 2( 3): e266.) used a bioinformatic approach to refute the presence of the second plasmid form, and proposed the creation of a specific phylogenetic group for R. felis. Methodology/ Principal Findings: In the present report, we, and five independent international laboratories confirmed unambiguously by PCR the presence of two plasmid forms in R. felis strain URRWXCal(2)(T), but observed that the plasmid content of this species, from none to 2 plasmid forms, may depend on the culture passage history of the studied strain. We also demonstrated that R. felis does not cultivate in Vero cells at 37 degrees C but generates plaques at 30 degrees C. Finally, using a phylogenetic study based on 667 concatenated core genes, we demonstrated the position of R. felis within the spotted fever group. Significance: We demonstrated that R. felis, which unambiguously belongs to the spotted fever group rickettsiae, may contain up to two plasmid forms but this plasmid content is unstable

Association Between Preexisting Versus Newly Identified Atrial Fibrillation and Outcomes of Patients With Acute Pulmonary Embolism

Background Atrial fibrillation (AF) may exist before or occur early in the course of pulmonary embolism (PE). We determined the PE outcomes based on the presence and timing of AF. Methods and Results Using the data from a multicenter PE registry, we identified 3 groups: (1) those with preexisting AF, (2) patients with new AF within 2 days from acute PE (incident AF), and (3) patients without AF. We assessed the 90-day and 1-year risk of mortality and stroke in patients with AF, compared with those without AF (reference group). Among 16 497 patients with PE, 792 had preexisting AF. These patients had increased odds of 90-day all-cause (odds ratio [OR], 2.81; 95% CI, 2.33-3.38) and PE-related mortality (OR, 2.38; 95% CI, 1.37-4.14) and increased 1-year hazard for ischemic stroke (hazard ratio, 5.48; 95% CI, 3.10-9.69) compared with those without AF. After multivariable adjustment, preexisting AF was associated with significantly increased odds of all-cause mortality (OR, 1.91; 95% CI, 1.57-2.32) but not PE-related mortality (OR, 1.50; 95% CI, 0.85-2.66). Among 16 497 patients with PE, 445 developed new incident AF within 2 days of acute PE. Incident AF was associated with increased odds of 90-day all-cause (OR, 2.28; 95% CI, 1.75-2.97) and PE-related (OR, 3.64; 95% CI, 2.01-6.59) mortality but not stroke. Findings were similar in multivariable analyses. Conclusions In patients with acute symptomatic PE, both preexisting AF and incident AF predict adverse clinical outcomes. The type of adverse outcomes may differ depending on the timing of AF onset.info:eu-repo/semantics/publishedVersio

Effect of Dietary Magnesium Content on Intestinal Microbiota of Rats

Background: Magnesium is a mineral that modulates several physiological processes. However, its relationship with intestinal microbiota has been scarcely studied. Therefore, this study aimed to assess the role of dietary magnesium content to modulate the intestinal microbiota of Wistar male rats. Methods: Rats were randomly assigned one of three diets: a control diet (C-Mg; 1000 mg/kg), a low magnesium content diet (L-Mg; 60 mg/kg), and a high magnesium content diet (H-Mg; 6000 mg/kg), for two weeks. After treatment, fecal samples were collected. Microbiota composition was assessed by sequencing the V3–V4 hypervariable region. Results: The C-Mg and L-Mg groups had more diversity than H-Mg group. CF231, SMB53, Dorea, Lactobacillus and Turibacter were enriched in the L-Mg group. In contrast, the phyla Proteobacteria, Parabacteroides, Butyricimonas, and Victivallis were overrepresented in the H-Mg group. PICRUSt analysis indicated that fecal microbiota of the L-Mg group were encoded with an increased abundance of metabolic pathways involving carbohydrate metabolism and butanoate metabolism. Conclusion: Dietary magnesium supplementation can result in intestinal dysbiosis development in a situation where there is no magnesium deficiency. Conversely, low dietary magnesium consumption is associated with microbiota with a higher capacity to harvest energy from the diet

Genetic variability of <i>R. felis</i> strain URRWXCal₂^T compared to other studied strains

a<p>Sequences from the LSU strain were determined by Bouyer <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002289#pone.0002289-Bouyer1" target="_blank">[25]</a>;</p>b<p>Sequences from the LSU strain were determined by Pornwiroon <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002289#pone.0002289-Pornwiroon1" target="_blank">[14]</a>;</p>c<p>D = different genotype;</p>d<p>I = Identical genotype;</p>e<p> = <i>ompA</i> gene;</p>f<p> = <i>gltA</i> gene.</p

Phylogenetic tree inferred from the comparison of 667 concatenated <i>Rickettsia</i> core protein-coding genes using the maximum parsimony method.

<p>Bootstrap values are indicated at branch nodes. A similar topology was obtained using the Neighbor-Joining analysis method.</p

Cloning of <i>R. felis</i> cells using a plaque assay.

<p>White arrows show individual lysis plaques. Right, a lysis plaque was enlarged.</p

The <i>R. felis</i> plasmids.

<p>Specific PCR amplification of the two <i>R. felis</i> plasmid forms. Lane 1: Molecular size (bp); lane 2: pRFa-pRFb amplicon; lane 3: pRFc-pRFd amplicon; lane 4: pRFa-pRFd amplicon; b) Schematic representation of <i>R. felis</i> plasmids indicating the position of PCR primers used to amplify the pRF (pRFa/pRFb and pRFc/pRFd primer pairs) and pRFδ (pRFa/pRFd primer pair) plasmids.</p

Determination of the <i>R. felis</i> plasmid ratio.

<p>The Southern blot obtained by hybridizing <i>R. felis</i> genomic DNA digested with PvuI and resolved by PFGE with probes specific for each plasmid form <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002289#pone.0002289-Ogata1" target="_blank">[18]</a> was digitalized by transmission scanning (ImageScanner, Amersham Biosciences). The quantification of each labelled plasmid band was estimated by analysis with the ImageMaster 2D Platinium Version 6.0 software (Amersham Biosciences). The pRF and pRFδ spots represented 57% and 43%, respectively, of the hybridization intensity.</p

PCR results obtained by all five tester laboratories

<p>PCR results obtained by all five tester laboratories</p