In vivo survival of teicoplanin-resistant Staphylococcus aureus and fitness cost of teicoplanin resistance.

Glycopeptide resistance, in a set of in vitro step-selected teicoplanin-resistant mutants derived from susceptible Staphylococcus aureus SA113, was associated with slower growth, thickening of the bacterial cell wall, increased N-acetylglucosamine incorporation, and decreased hemolysis. Differential transcriptome analysis showed that as resistance increased, some virulence-associated genes became downregulated. In a mouse tissue cage infection model, an inoculum of 10(4) CFU of strain SA113 rapidly produced a high-bacterial-load infection, which triggered MIP-2 release, leukocyte infiltration, and reduced leukocyte viability. In contrast, with the same inoculum of the isogenic glycopeptide-resistant derivative NM67, CFU initially decreased, resulting in the elimination of the mutant in three out of seven cages. In the four cages in which NM67 survived, it partially regained wild-type characteristics, including thinning of the cell wall, reduced N-acetylglucosamine uptake, and increased hemolysis; however, the survivors also became teicoplanin hypersusceptible. The elimination of the teicoplanin-resistant mutants and selection of teicoplanin-hypersusceptible survivors in the tissue cages indicated that glycopeptide resistance imposes a fitness burden on S. aureus and is selected against in vivo, with restoration of fitness incurring the price of resistance loss.

Role of gluconic acid production in the regulation of biocontrol traits of Pseudomonas fluorescens CHA0.

The rhizobacterium Pseudomonas fluorescens CHA0 promotes the growth of various crop plants and protects them against root diseases caused by pathogenic fungi. The main mechanism of disease suppression by this strain is the production of the antifungal compounds 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin (PLT). Direct plant growth promotion can be achieved through solubilization of inorganic phosphates by the production of organic acids, mainly gluconic acid, which is one of the principal acids produced by Pseudomonas spp. The aim of this study was to elucidate the role of gluconic acid production in CHA0. Therefore, mutants were created with deletions in the genes encoding glucose dehydrogenase (gcd) and gluconate dehydrogenase (gad), required for the conversion of glucose to gluconic acid and gluconic acid to 2-ketogluconate, respectively. These enzymes should be of predominant importance for rhizosphere-colonizing biocontrol bacteria, as major carbon sources provided by plant root exudates are made up of glucose. Our results show that the ability of strain CHA0 to acidify its environment and to solubilize mineral phosphate is strongly dependent on its ability to produce gluconic acid. Moreover, we provide evidence that the formation of gluconic acid by CHA0 completely inhibits the production of PLT and partially inhibits that of DAPG. In the Deltagcd mutant, which does not produce gluconic acid, the enhanced production of antifungal compounds was associated with improved biocontrol activity against take-all disease of wheat, caused by Gaeumannomyces graminis var. tritici. This study provides new evidence for a close association of gluconic acid metabolism with antifungal compound production and biocontrol activity in P. fluorescens CHA0.

Efficacy of levofloxacin in the treatment of experimental endocarditis caused by viridans group streptococci.

Levofloxacin was investigated against viridans group streptococci in vitro and in rats with experimental aortic endocarditis. The MIC(90)s of levofloxacin and ciprofloxacin for 20 independent isolates of such bacteria were 1 and 8 mg/L, respectively. Rats were infected with two types of organism: either fully susceptible to levofloxacin MIC < or = 0.5 mg/L) or borderline susceptible (MIC 1-2 mg/L). Fully levofloxacin-susceptible bacteria comprised one penicillin-susceptible (MIC 0.004 mg/L) Streptococcus gordonii, and one penicillin-tolerant as well as one intermediate penicillin-resistant (MIC 0.125 mg/L) isogenic strains. Borderline levofloxacin-susceptible bacteria comprised one penicillin-susceptible Streptococcus sanguis and one highly penicillin-resistant Streptococcus mitis (MIC 2 mg/L). Rats were treated for 5 days with drug dosages simulating the following treatments in humans: (i) levofloxacin 500 mg orally once a day (q24 h), (ii) levofloxacin 500 mg orally twice a day (q12 h), (iii) levofloxacin 1 g orally q24 h, (iv) ciprofloxacin 750 mg orally q12 h, and (v) ceftriaxone 2 g iv q24 h. Levofloxacin was equivalent or superior to ceftriaxone, and was successful in treating experimental endocarditis irrespective of penicillin resistance. Nevertheless, standard levofloxacin treatment equivalent to 500 mg q24 h in human was less effective than twice daily 500 mg or once daily 1 g doses against borderline-susceptible organisms. Ciprofloxacin, used as a negative control, was ineffective and selected for resistant isolates. This underlines the importance of MIC determinations when treating severe streptococcal infection with quinolones. In the case of borderline-susceptible pathogens, total daily doses of 1 g of levofloxacin should be considered.

Mutations in the epithelial Na+ channel ENaC outer pore disrupt amiloride block by increasing its dissociation rate.

The epithelial Na+ channel ENaC mediates transepithelial Na+ transport in the distal kidney, the colon, and the lung and is a key element for the maintenance of Na+ balance and the regulation of blood pressure. Mutagenesis studies have identified residues alphaS583 and the homologous betaG525 and gammaG537 in the outer pore entrance that are critical for ENaC block by the K+-sparing diuretic amiloride. The aim of the present study was to determine first, whether these residues are part of the amiloride binding site, and second, whether they are general determinants of ENaC block by amiloride and its derivatives. Kinetic analysis of the association and dissociation rates of amiloride and benzamil to ENaC showed that mutation of residue alphaS583C and the homologous betaG525C increased the dissociation rate of the drugs from the binding site, with little changes in their association rate. Thus, these mutations destabilize the binding interaction between the blockers and the receptor on the channel, favoring the unbinding of the ligand. This strongly suggests that they are part of the binding site. Because mutations of alphaS583, betaG525, and gammaG537 have similar effects on amiloride, benzamil, and triamterene block, we conclude that these three ENaC blockers share a common receptor within the ion channel pore.

Increased renal responsiveness to vasopressin and enhanced V2 receptor signaling in RGS2-/- mice.

The antidiuretic effect of vasopressin is mediated by V2 receptors (V2R) that are located in kidney connecting tubules and collecting ducts. This study provides evidence that V2R signaling is negatively regulated by regulator of G protein signaling 2 (RGS2), a member of the family of RGS proteins. This study demonstrates that (1) RGS2 expression in the kidney is restricted to the vasopressin-sensitive part of the nephron (thick ascending limb, connecting tubule, and collecting duct); (2) expression of RGS2 is rapidly upregulated by vasopressin; (3) the vasopressin-dependent accumulation of cAMP, the principal messenger of V2R signaling, is significantly higher in collecting ducts that are microdissected from the RGS2(-/-) mice compared with their wild-type littermates; and (4) analysis of urine output of mice that were exposed to water restriction followed by acute water loading revealed that RGS2(-/-) mice exhibit an increased renal responsiveness to vasopressin. It is proposed that RGS2 is involved in negative feedback regulation of V2R signaling.

Functional interaction between the estrogen receptor and CTF1: analysis of the vitellogenin gene B1 promoter in yeast.

Eukaryotic gene expression depends on a complex interplay between the transcriptional apparatus and chromatin structure. We report here a yeast model system for investigating the functional interaction between the human estrogen receptor (hER) and CTF1, a member of the CTF/NFI transcription factor family. We show that a CTF1-fusion protein and the hER transactivate a synthetic promoter in yeast in a synergistic manner. This interaction requires the proline-rich transactivation domain of CTF1. When the natural estrogen-dependent vitellogenin B1 promoter is tested in yeast, CTF1 and CTF1-fusion proteins are unable to activate transcription, and no synergy is observed between hER, which activates the B1 promoter, and these factors. Chromatin structure analysis on this promoter reveals positioned nucleosomes at -430 to -270 (+/-20 bp) and at -270 to - 100 (+/-20 bp) relative to the start site of transcription. The positions of the nucleosomes remain unchanged upon hormone-dependent transcriptional activation of the promoter, and the more proximal nucleosome appears to mask the CTF/NFI site located at - 101 to -114. We conclude that a functional interaction of hER with the estrogen response element located upstream of a basal promoter occurs in yeast despite the nucleosomal organization of this promoter, whereas the interaction of CTF1 with its target site is apparently precluded by a nucleosome.

Performance of microcalorimetry for early detection of methicillin resistance in clinical isolates of Staphylococcus aureus.

We describe a calorimetric assay for the detection of methicillin-resistant Staphylococcus aureus (MRSA) within 5 h. Microbial heat was calculated in culture with and without cefoxitin. Among 30 genetically distinct clinical isolates, 19/20 MRSA (95%) and 10/10 methicillin-susceptible Staphylococcus aureus (100%) were correctly identified. Microcalorimetry may be useful for rapid MRSA screening.

Cost-effective experimental design to support modeling of concentration-response functions

Modeling concentration-response function became extremely popular in ecotoxicology during the last decade. Indeed, modeling allows determining the total response pattern of a given substance. However, reliable modeling is consuming in term of data, which is in contradiction with the current trend in ecotoxicology, which aims to reduce, for cost and ethical reasons, the number of data produced during an experiment. It is therefore crucial to determine experimental design in a cost-effective manner. In this paper, we propose to use the theory of locally D-optimal designs to determine the set of concentrations to be tested so that the parameters of the concentration-response function can be estimated with high precision. We illustrated this approach by determining the locally D-optimal designs to estimate the toxicity of the herbicide dinoseb on daphnids and algae. The results show that the number of concentrations to be tested is often equal to the number of parameters and often related to the their meaning, i.e. they are located close to the parameters. Furthermore, the results show that the locally D-optimal design often has the minimal number of support points and is not much sensitive to small changes in nominal values of the parameters. In order to reduce the experimental cost and the use of test organisms, especially in case of long-term studies, reliable nominal values may therefore be fixed based on prior knowledge and literature research instead of on preliminary experiments

Preserved capillary density of dorsal finger skin in treated hypertensive patients with or without type 2 diabetes.

OBJECTIVES: Capillary rarefaction is a hallmark of untreated hypertension. Recent data indicate that rarefaction may be reversed by antihypertensive treatment in nondiabetic hypertensive patients. Despite the frequent association of diabetes with hypertension, nothing is known on the capillary density of treated diabetic patients with hypertension. METHODS: We enrolled 21 normotensive healthy, 25 hypertensive only, and 21 diabetic (type 2) hypertensive subjects. All hypertensive patients were treated with a blocker of the renin-angiotensin system, and a majority had a home blood pressure ≤135/85 mmHg. Capillary density was assessed with videomicroscopy on dorsal finger skin and with laser Doppler imaging on forearm skin (maximal vasodilation elicited by local heating). RESULTS: There was no difference between any of the study groups in either dorsal finger skin capillary density (controls 101 ± 11 capillaries/mm(2) , nondiabetic hypertensive 99 ± 16, diabetic hypertensive 96 ± 18, p > 0.5) or maximal blood flow in forearm skin (controls 666 ± 114 perfusion units, nondiabetic hypertensive 612 ± 126, diabetic hypertensive 620 ± 103, p > 0.5). CONCLUSIONS: Irrespective of the presence or not of type 2 diabetes, capillary density is normal in hypertensive patients with reasonable control of blood pressure achieved with a blocker of the renin-angiotensin system.

Role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro.

Doxorubicin (DOX) is a potent available antitumor agent; however, its clinical use is limited because of its cardiotoxicity. Cell death is a key component in DOX-induced cardiotoxicity, but its mechanisms are elusive. Here, we explore the role of superoxide, nitric oxide (NO), and peroxynitrite in DOX-induced cell death using both in vivo and in vitro models of cardiotoxicity. Western blot analysis, real-time PCR, immunohistochemistry, flow cytometry, fluorescent microscopy, and biochemical assays were used to determine the markers of apoptosis/necrosis and sources of NO and superoxide and their production. Left ventricular function was measured by a pressure-volume system. We demonstrated increases in myocardial apoptosis (caspase-3 cleavage/activity, cytochrome c release, and TUNEL), inducible NO synthase (iNOS) expression, mitochondrial superoxide generation, 3-nitrotyrosine (NT) formation, matrix metalloproteinase (MMP)-2/MMP-9 gene expression, poly(ADP-ribose) polymerase activation [without major changes in NAD(P)H oxidase isoform 1, NAD(P)H oxidase isoform 2, p22(phox), p40(phox), p47(phox), p67(phox), xanthine oxidase, endothelial NOS, and neuronal NOS expression] and decreases in myocardial contractility, catalase, and glutathione peroxidase activities 5 days after DOX treatment to mice. All these effects of DOX were markedly attenuated by peroxynitrite scavengers. Doxorubicin dose dependently increased mitochondrial superoxide and NT generation and apoptosis/necrosis in cardiac-derived H9c2 cells. DOX- or peroxynitrite-induced apoptosis/necrosis positively correlated with intracellular NT formation and could be abolished by peroxynitrite scavengers. DOX-induced cell death and NT formation were also attenuated by selective iNOS inhibitors or in iNOS knockout mice. Various NO donors when coadministered with DOX but not alone dramatically enhanced DOX-induced cell death with concomitant increased NT formation. DOX-induced cell death was also attenuated by cell-permeable SOD but not by cell-permeable catalase, the xanthine oxidase inhibitor allopurinol, or the NADPH oxidase inhibitors apocynine or diphenylene iodonium. Thus, peroxynitrite is a major trigger of DOX-induced cell death both in vivo and in vivo, and the modulation of the pathways leading to its generation or its effective neutralization can be of significant therapeutic benefit.

Isothermal microcalorimetry: a novel method for real-time determination of antifungal susceptibility of Aspergillus species.

We evaluated microcalorimetry for real-time susceptibility testing of Aspergillus spp. based on growth-related heat production. The minimal heat inhibitory concentration (MHIC) for A. fumigatus ATCC 204305 was 1 mg/L for amphotericin B, 0.25 mg/L for voriconazole, 0.06 mg/L for posaconazole, 0.125 mg/L for caspofungin and 0.03 mg/L for anidulafungin. Agreement within two 2-fold dilutions between MHIC (determined by microcalorimetry) and MIC or MEC (determined by CLSI M38A) was 90% for amphotericin B, 100% for voriconazole, 90% for posaconazole and 70% for caspofungin. This proof-of-concept study demonstrated the potential of isothermal microcalorimetry for growth evaluation of Aspergillus spp. and real-time antifungal susceptibility testing.

Unsatisfactory outcomes in myasthenia gravis: influence by care providers.

Myasthenia gravis (MG) can be difficult to treat despite an available therapeutic armamentarium. Our aim was to analyze the factors leading to unsatisfactory outcome (UO). To this end we used the Myasthenia Gravis Foundation of America classification system. Forty one patients with autoimmune MG were followed prospectively from January 2003 to December 2007. Outcomes were assessed throughout follow-up and at a final visit. 'Unchanged', 'worse', 'exacerbation' and 'died of MG' post-intervention status were considered UOs. During follow-up, UO rates reached 54% and were related to undertreatment (41%), poor treatment compliance (23%), infections (23%), and adverse drug effects (13%). The UO rate at final study assessment was 20%. UO during follow-up was significantly (P = 0.004) predictive of UOs at final assessment. When care was provided by neuromuscular (NM) specialists, patients had significantly better follow-up scores (P = 0.01). At final assessment UO rates were 7% and significantly better in patients treated by NM specialists, compared to other physicians where UO rates reached 27%. UO was a frequent finding occurring in more than half our patients during follow-up. Nearly two-thirds of the UOs could have been prevented by appropriate therapeutic adjustments and improved compliance. The differential UO rates at follow-up, their dependency on the degree to which the management was specialized and their correlation with final outcomes suggest that specialized MG care improves outcomes.

Effect of captopril on renal vascular tone in patients with essential hypertension.

1. The effect of acute inhibition of angiotensin-converting enzyme by captopril (50 mg) on renal haemodynamics and function was assessed in nine patients with essential hypertension on unrestricted sodium intake (n = 8) or low sodium diet (n = 1). 2. Captopril induced a rapid and significant decrease in arterial pressure, which was maximal within 60 min. 3. Effective renal plasma flow (ERPF) increased, glomerular filtration rate (GFR) did not change and filtration fraction (FF) decreased after captopril. No change in sodium excretion and a decrease in urinary potassium occurred. 4. In the patient on low sodium diet, captopril induced striking increases in GFR and ERPF (64 and 106% respectively). 5. The logarithm of baseline plasma renin activity was positvely correlated with the change in ERPF and negatively correlated with changes in FF and renal resistance. 6. The results indicate that in patients with essential hypertension angiotensin participates actively in the maintenance of renal vascular tone at the efferent arteriolar level. A possible influence of kinins remains to be defined.

Effect of elective antibiotic therapy on resting energy expenditure and inflammation in cystic fibrosis.

Cystic fibrosis (CF) patients often present with malnutrition which may partly be due to increased resting energy expenditure (REE) secondary to inflammation. Both REE and tumour necrosis factor-alpha (TNF-alpha), as other markers of inflammation, are elevated during respiratory exacerbations and decrease after antibiotic treatment. However, the effect of antibiotic therapy on REE and inflammation in patients without respiratory exacerbation is not known. The aim of our study was to determine the effect of such an elective antibiotic therapy on REE, TNF-alpha, and other serum markers of inflammation. Twelve CF patients 5F/7M, age 15.9 +/- 6.1 years, weight for height ratio 89 +/- 8% without clinically obvious exacerbation and treated by intravenous antibiotics were studied. Both before (D0) and after (D14) treatment, pulmonary function tests were performed. REE was measured by indirect calorimetry and blood taken to measure inflammation parameters. Body weight increased by 1.1 kg from D0 to D14 (P < 0.001), composed of 0.3 kg fat mass and 0.8 kg fat-free mass (FFM). The forced expiratory volume at 1 s increased from 43 +/- 15% of predicted at D0 to 51 +/- 15% of predicted at D14 (P < 0.01). Mean REE was 41.1 +/- 7.6 kcal/kg FFM per day at D0 and did not change significantly at D14 (40.6 +/- 8.5 kcal/kg FFM per day). Serum markers of inflammation decreased from D0 to D14: C-reactive protein 17 +/- 17 mg/l to 4 +/- 7 mg/l (P < 0.05), elastase 62 +/- 29 microg/l to 45 +/- 18 microg/l (P < 0.02), orosomucoid acid 1.25 +/- 0.11 g/l to 0.80 +/- 0.15 g/l (P < 0.001), and TNF-alpha 37 +/- 14 pg/ml to 29 +/- 6 pg/ml (P = 0.05). Individual values showed a correlation between changes in REE and in TNF-alpha (P < 0.02). The contribution of inflammation to energy expenditure is possible but appears to be minimal in cystic fibrosis patients treated by antibiotics on a regular basis in the absence of clinically obvious exacerbation.