Virulence Potential and Genomic Mapping of the Worldwide Clone Escherichia coli ST131

Recently, the worldwide propagation of clonal CTX-M-15-producing Escherichia coli isolates, namely ST131 and O25b:H4, has been reported. Like the majority of extra-intestinal pathogenic E. coli isolates, the pandemic clone ST131 belongs to phylogenetic group B2, and has recently been shown to be highly virulent in a mouse model, even though it lacks several genes encoding key virulence factors (Pap, Cnf1 and HlyA). Using two animal models, Caenorhabditis elegans and zebrafish embryos, we assessed the virulence of three E. coli ST131 strains (2 CTX-M-15- producing urine and 1 non-ESBL-producing faecal isolate), comparing them with five non-ST131 B2 and a group A uropathogenic E. coli (UPEC). In C. elegans, the three ST131 strains showed intermediate virulence between the non virulent group A isolate and the virulent non-ST131 B2 strains. In zebrafish, the CTX-M-15-producing ST131 UPEC isolates were also less virulent than the non-ST131 B2 strains, suggesting that the production of CTX-M-15 is not correlated with enhanced virulence. Amongst the non-ST131 B2 group isolates, variation in pathogenic potential in zebrafish embryos was observed ranging from intermediate to highly virulent. Interestingly, the ST131 strains were equally persistent in surviving embryos as the non-ST131-group B2 strains, suggesting similar mechanisms may account for development of persistent infection. Optical maps of the genome of the ST131 strains were compared with those of 24 reference E. coli strains. Although small differences were seen within the ST131 strains, the tree built on the optical maps showed that these strains belonged to a specific cluster (86% similarity) with only 45% similarity with the other group B2 strains and 25% with strains of group A and D. Thus, the ST131 clone has a genetic composition that differs from other group B2 strains, and appears to be less virulent than previously suspected

Limitation de l'hypertension artérielle pulmonaire par l'exercice physique chronique chez le rat (implication du métabolisme de la L-arginine)

L'hypertension artérielle pulmonaire (HTAP) est une pathologie des artères pulmonaires où l'élévation progressive de la pression artérielle et de la résistance circulatoire aboutit à un remodelage de la paroi vasculaire et une défaillance cardiaque droite. L'HTAP est caractérisée par une altération de la relaxation artérielle pulmonaire induite par une moindre production de monoxyde d'azote (NO), impliquant à un des niveaux de régulation, le métabolisme de la L-arginine. alors que l'exercice physique chronique, qui potentialise la vasorelaxation médiée par le NO, est largement utilisé dans l'approche thérapeutique de nombreuses maladies cardio-vasculaires, il ne fait pas partie de l'approche thérapeutique classique de l'HTAP. Nous avons émis l'hypothèse que l'exercice physique en endurance, associé à une supplémentation en L-arginine, pourrait limiter l'HTAP induite par l'hypoxie, les effets de l'exercice physique chronique aérobie par l'étude in-vivo de la pression artérielle pulmonaire et ex-vivo des propriétés vasomotrices d'anneaux isolés d'artère pulmonaire. La pression artérielle pulmonaire des rats HTAP entraînés était 18% inférieure à celle des rats non entraînés. Les anneaux d'artère pulmonaire des rats HTAP montraient une altération de la vasorelaxation et une potentialisation des propriétés de vasoconstriction, notamment par altération de la libération endothéliale de NO et diminution de la sensibilité du muscle lisse au NO. L'addition de L-arginine améliorait la vasorelaxation des artères de rats HTAP entraînés mais non des rats HTAP sédentaires, suggérant une altération de la biodisponiblité et/ou de l'utilisation de la L-arginine.Ces résultats pourraient avoir une implication importante pour le traitement de l'HTAP en suggérant l'intérêt de combiner exercice et supplémentation en L-argininePulmonary hypertension (PHT) involves pulmonary arteries with progressively increasing blood pressure and circulatory resistance leading to remodelling of pulmonary vessel wall and right heart failure. Impairment of vasorelaxation properties due to reduced nitric-oxide production through L-arginine plays a major role in the pathogeny of this disease. Exercise training is beneficial for the treatment and prevention of cardiovascular diseases, notably by potentiating of nitric oxide pathway. Surprisingly, very few clinical studies incorporated exercise sessions into the therapy of patients with PHT, we hypothesized that combining exercise training and L-arginine supplementation would limit PHT by potentiating nitric oxid-related relaxation. Therefore, we investigated the effect of chronic aerobic exercise training in rats with hypoxia-induced PHT, by measuring in vivo pulmonary artery blood pressure and ex-vivo the vasomotor properties of isolated pulmonary arterial rings. Pulmonary arterial pressure was 18% lower in exercise-trained than in sedentary PHT rats. Pulmonary artery rings of PHT rats showed impaired vasorelaxation and potentiated vasoconstriction with depressed NO production and decreased smooth muscle cell sensibilité to NO. L-arginine addition to th organ bath improved vasorelaxation in pulmonary artery rings of PHT trained but not of untrained rats, pointing to the involment of reduced L-arginine use and/or bioavailability in the endothelial dysfunction. These results might be clinically relevant for the treatment of PHT as synergistic effects of chronic exercise and L-arginine supplementation might be expectedAVIGNON-BU Centrale (840072102) / SudocSudocFranceF

Aortic vasoconstriction related to smooth muscle cells ET-A and ET-B receptors is not involved in hypoxia-induced sustained systemic arterial hypertension in rats.

International audienceOBJECTIVES: We report in the present study the role of endothelin (ET-1) and ET-1 receptors in the sustained hypoxia-induced systemic hypertension. METHODS: Wistar rats were randomly assigned to live continuously in hypobaric hypoxia (CH rats) or normoxia (N rats). At the end of hypoxic stress exposure (5 weeks at 450 mm Hg), measurements of mean systemic arterial pressure were done. The effects of ET-1 in the presence or not of the endothelium and/or of specific ET-A inhibitors (BQ-123) or ET-B inhibitors (BQ-788), have been investigated in an isolated model of rat thoracic aorta. Finally, plasmatic ET-1 concentrations have been determined by assay procedure. RESULTS: Following five weeks of chronic hypoxic stress, CH rats presented a significant increase of mean systemic arterial pressure (N: 129.1+/-6.8 mm Hg vs CH: 152.5+/-3.4 mm Hg; P<0.05). Despite of this hypoxia-induced hypertension, ET-1 plasmatic concentration was not different between N and CH rats. Finally, CH rats presented a reduce response to ET-1 when compared to N rats. This phenomenon seems to be associated to the ET-A vascular smooth muscle cell receptors, since difference between N and CH rats was still present in endothelium denuded aortic rings in the presence or not of the specific ET-B inhibitors (BQ-788). In addition, in the presence of the specific ET-A inhibitor (BQ-123) response to ET-1 was abolished in N and CH rats to the same extent (N:-98%; CH:-99%). CONCLUSION: This work clearly suggests that, following long term exposure to hypoxia, ET-1 and ET-1 receptors are not involved in the persistence of systemic hypertension in a rat model, and that chronic exposure to severe hypoxic stress was associated with a downregulation of the ET-A receptors response to ET-1

Myostatin up-regulation is associated with the skeletal muscle response to hypoxic stimuli

Myostatin and hypoxia signalling pathways are able to induce skeletal muscle atrophy, but whether a relationship between these two pathways exists is currently unknown. Here, we tested the hypothesis that a potential mechanism for hypoxia effect on skeletal muscle may be through regulation of myostatin. We reported an induction of myostatin expression in muscles of rats exposed to chronic hypoxia. Interestingly, we also demonstrated increased skeletal muscle myostatin protein expression in skeletal muscle of hypoxemic patients with severe chronic obstructive pulmonary disease (COPD). Parallel studies in human skeletal muscle cell cultures showed that induction of myostatin expression in myotubes treated with hypoxia-mimicking agent such as cobalt chloride (CoCl2) is associated with myotube atrophy. Furthermore, we demonstrated that inhibition of myostatin by means of genetic deletion of myostatin or treatment with blocking antimyostatin antibodies inhibits the CoCl2-induced atrophy in muscle cells. Finally, addition of recombinant myostatin restored the CoCl2-induced atrophy in myostatin deficient myotubes. These results strongly suggest that myostatin can play an essential role in the adaptation of skeletal muscle to hypoxic environmen

Main characteristics of the <i>E. coli</i> clinical isolates used in this study.

<p>Main characteristics of the <i>E. coli</i> clinical isolates used in this study.</p

Virulence of ST131 B2 group <i>Escherichia coli</i> strains in zebrafish embryos compared to other non-ST131 B2 group strains.

<p>A. Survival probability of zebrafish embryos following infection with three ST131 strains and two non-ST131 strains. Thirty hour post fertilization (hpf) embryos were microinjected with an average inoculum size of 1.9×10³.CFU. The data represent the average of 5 independent experiments (n = 20 for each strain per experiment). NEC20 and NECS81153 were analysed twice. A Mantel-Cox test was performed (between strains p<0.001). NECS81153 and NEC20 did not differ significantly (p = 0.08) from each other in virulence. The non-ST131 group B2 strains NEC20 and NECS81153 induced a significantly higher rate of embryo mortality than the two ST131 strains MECB5 (p<0.001) and FV7563 (p = 0.002). MECB5 was significantly less virulent than S250 (p<0.001), but did not differ from FV7563 (p = 0.73). Although S250 and NECS81153 did not differ in virulence (p = 0.82), S250 was less virulent than NEC20 (p = 0.009). In this model, NEC20 was significantly (p<0.05) more virulent than all the other group B2 strains except for NECS81153(p = 0.08). B. Survival probability of zebrafish embryos following injection of ST131 strain MECB5 compared with group A strain NEC3, and non-ST131 group B2 control strains CFT073, 536 and ED1a. Thirty hour post fertilization (hpf) embryos were microinjected with an average inoculum size of 1.4×10³.CFU. The data represent the average of 2 independent experiments (n = 20 for each strain per experiment) in this context, but each strain was analysed at least 5 times. A Mantel-Cox test was performed. MECB5 was significantly less virulent than the strains CFT073, 536 and NEC3 (p<0.0001). ED1a was significantly less virulent than MECB5 (p = 0.047). Within the group of fast killing strains, strain 536 was less virulent than CFT073 (p = 0.011) and NEC3 (p = 0.020), whereas NEC3 and CFT073 were not significantly different (p = 0.079). C. Infection kinetics after microinjection with the following inocula: FV7563 (300 CFU), S250 (520 CFU), MECB5 (1760 CFU), NECS81153 (2200 CFU) and NEC20 (880 CFU). Bacterial counts were performed in 5 individual embryos per time point per strain (geometric mean ± SEM, n = 5), however, dead embryos were not included in the counts as explained in the text. Shown is a typical experiment. The experiment was repeated 5 times. D. Bright field and fluorescence overlay of a NEC20 infected embryo, 48 hpi, showing a close up of bacteria in a phagocytic cell. Inset shows an enlargement of the phagocytic cell (arrow). n = notochord, arrowhead marks an erythrocyte. E. Fluorescent image of an embryo, 24 hpi, infected with NEC20-DSRed showing bacteria have multiplied in the vasculature. A non-infected control embryo does not show any fluorescence with similar camera settings (not shown). Inset: corresponding bright field image. F. Embryo, 24 hpi, microinjected with MECB5-DSRed. Fluorescence and bright field overlay image of central nervous system infection.</p

Map similarity cluster using unweighted-pair group method using arithmetic averages (UPGMA) tree of optical maps performed on different <i>Escherichia coli</i> strains.

<p>The scale indicates the percentage of genetic difference.</p

Evaluation of CFU in the <i>C. elegans</i> digestive tract obtained from 3 experiments for each strain.

<p>Evaluation of CFU in the <i>C. elegans</i> digestive tract obtained from 3 experiments for each strain.</p

50% Lethal Time (LT50) of <i>Caenorhabditis elegans</i> infected by the <i>E. coli</i> ST131 strains and non-ST131 strains.

<p>The results are representative of at least five independent trials for each group of strains. p: Pairwise comparison using a log rank tests. NS: non significant.</p

Overview of optical maps of the <i>E. coli</i> ST131 strains S250, FV7563 and MECB5, and reference UPEC CFT073.

<p>Alignment software highlights <i>Nco</i>I restricted fragment polymorphisms between strains by using white color and homologous regions by using blue color. Vertical lines in each bacterial genome represent matching <i>Nco</i>I restriction sites. PAIs present in the genome of strain CFT073 are represented by different colors: PAI I: green, PAI II: pink, PAI III: red, PAI IV: yellow. Red lines under the genome of S250, FV7563 and MECB5 indicate four insertions only observed in this strain. Red frame on the genome of strain MECB5 indicates the insertion only observed in this strain. A close up view of this region is shown under the genome optical maps.</p