Promising activity of etomidate against mixed biofilms of fluconazole-resistant Candida albicans and methicillin-resistant Staphylococcus aureus.

Introduction. Candida albicans and Staphylococcus aureus are recognized for their development of resistance and biofilm formation. New therapeutic alternatives are necessary in this context.Hypothesis. Etomidate shows potential application in catheters against mixed biofilms of fluconazole-resistant C. albicans and methicillin-resistant S. aureus (MRSA).Aim. The present study aimed to evaluate the activity of etomidate against mixed biofilms of fluconazole-resistant C. albicans and MRSA.Methodology. The action of etomidate against mature biofilms was verified through the evaluation of biomass and cell viability, and its ability to prevent biofilm formation in peripheral venous catheters was determined based on counts of colony forming units (c.f.u.) and confirmed by morphological analysis through scanning electron microscopy (SEM).Results. Etomidate generated a reduction (P<0.05) in biomass and cell viability starting from a concentration of 250 µg ml-1. In addition, it showed significant ability to prevent the formation of mixed biofilms in a peripheral venous catheter, as shown by a reduction in c.f.u. SEM revealed that treatment with etomidate caused substantial damage to the fungal cells.Conclusion. The results showed the potential of etomidate against polymicrobial biofilms of fluconazole-resistant C. albicans and MRSA.

Analysis of possible pathways on the mechanism of action of minocycline and doxycycline against strains of Candida spp. resistant to fluconazole.

Species of the genus Candida, characterized as commensals of the human microbiota, are opportunistic pathogens capable of generating various types of infections with high associated costs. Considering the limited pharmacological arsenal and the emergence of antifungal-resistant strains, the repositioning of drugs is a strategy used to search for new therapeutic alternatives, in which minocycline and doxycycline have been evaluated as potential candidates. Thus, the objective was to evaluate the in vitro antifungal activity of two tetracyclines, minocycline and doxycycline, and their possible mechanism of action against fluconazole-resistant strains of Candida spp. The sensitivity test for antimicrobials was performed using the broth microdilution technique, and the pharmacological interaction with fluconazole was also analysed using the checkerboard method. To analyse the possible mechanisms of action, flow cytometry assays were performed. The minimum inhibitory concentration obtained was 4-427 µg ml-1 for minocycline and 128-512 µg ml-1 for doxycycline, and mostly indifferent and additive interactions with fluconazole were observed. These tetracyclines were found to promote cellular alterations that generated death by apoptosis, with concentration-dependent reactive oxygen species production and reduced cell viability. Therefore, minocycline and doxycycline present themselves as promising study molecules against Candida spp.

Evaluation of the antifungal effect of chlorogenic acid against strains of Candida spp. resistant to fluconazole: apoptosis induction and in silico analysis of the possible mechanisms of action.

Introduction. Candida spp. are commensal fungal pathogens of humans, but when there is an imbalance in the microbiota, or weak host immunity, these yeasts can become pathogenic, generating high medical costs.Gap Statement. With the increase in resistance to conventional antifungals, the development of new therapeutic strategies is necessary. This study evaluated the in vitro antifungal activity of chlorogenic acid against fluconazole-resistant strains of Candida spp. Mechanism of action through flow cytometry and in silico analyses, as well as molecular docking assays with ALS3 and SAP5, important proteins in the pathogenesis of Candida albicans associated with the adhesion process and biofilm formation.Results. The chlorogenic acid showed in vitro antifungal activity against the strains tested, causing reduced cell viability, increased potential for mitochondrial depolarization and production of reactive oxygen species, DNA fragmentation and phosphatidylserine externalization, indicating an apoptotic process. Concerning the analysis through docking, the complexes formed between chlorogenic acid and the targets Thymidylate Kinase, CYP51, 1Yeast Cytochrome BC1 Complex e Exo-B-(1,3)-glucanase demonstrated more favourable binding energy. In addition, chlorogenic acid presented significant interactions with the ALS3 active site residues of C. albicans, important in the adhesion process and resistance to fluconazole. Regarding molecular docking with SAP5, no significant interactions were found between chlorogenic acid and the active site of the enzyme.Conclusion. We concluded that chlorogenic acid has potential use as an adjuvant in antifungal therapies, due to its anti-Candida activity and ability to interact with important drug targets.

Adult rats are more sensitive to the vascular effects induced by hyperhomocysteinemia than young rats.

We aimed to investigate the vascular effects of hyperhomocysteinemia (HHcy) on carotid arteries from young and adult rats. With this purpose young and adult rats received a solution of DL-homocysteine-thiolactone (1 g/kg body weight/day) in the drinking water for 7, 14 and 28 days. Increase on plasma homocysteine occurred in young and adult rats treated with DL-homocysteine-thiolactone in all periods. Vascular reactivity experiments using standard muscle bath procedures showed that HHcy enhanced the contractile response of endothelium-intact, carotid rings to phenylephrine in both young and adult rats. However, in young rats, the increased phenylephrine-induced contraction was observed after hyperhomocysteinemia for 14 and 28 days, whereas in adult rats this response was already apparent after 7 day treatment. HHcy impaired acetylcholine-induced relaxation in arteries from adult but not young rats. The contraction induced by phenylephrine in carotid arteries in the presence of Y-27632 was reversed to control values in arteries from young but not adult rats with hyperhomocysteinemia. HHcy did not alter the contraction induced by CaCl(2) in carotid arteries from young rats, but enhanced CaCl(2)-induced contraction in the arteries from adult rats. HHcy increased the basal levels of superoxide anion in arteries from both groups. Finally, HHcy decreased the basal levels of nitrite in arteries from adult but not young rats. The major new finding of the present work is that arteries from young rats are more resistant to vascular changes evoked by HHcy than arteries from adult rats. Also, we verified that the enhanced vascular response to phenylephrine observed in carotid arteries of DL-homocysteine thiolactone-treated rats is mediated by different mechanisms in young and adult rats.

Hyperhomocysteinemia induced by feeding rats diets rich in DL-homocysteine thiolactone promotes alterations on carotid reactivity independent of arterial structure.

We aimed to investigate whether hyperhomocysteinemia (HHcy) interferes on carotid vascular reactivity, and how morphological and functional aspects are related. With this purpose male Wistar rats received a solution of dl-homocysteine-thiolactone (1g/kg body weight/day) in the drinking water for 4, 15 and 30 days. Lipid profile, carotid artery-morphology and -responsiveness to acetylcholine, phenylephrine and endothelin-1 were analyzed. Similar increase on homocysteine plasmatic levels occurred in rats treated for 4, 15 and 30 days. High levels of serum cholesterol and triglycerides were observed after HHcy 30 days. Vascular reactivity experiments using standard muscle bath procedures showed that HHcy induced a time-dependent reduction on acetylcholine-induced-relaxation at 4, 15 and 30 days. HHcy enhanced the contractile response of endothelium-intact, but not denuded carotid rings to phenylephrine and endothelin-1, despite the treatment time. Morphometric analysis showed that intimal/medial area ratio was enhanced only at 30 days of HHcy, despite its reduced cell density. The major new finding of the present study is that it establishes a time-course relationship for the events involved on vascular effects associated with HHcy. We demonstrated that alterations on vascular responsiveness precede alterations on arterial structure. Based on such findings it is possible to suggest that vascular dysfunction occurs in early stages while alterations on vessel morphology take place in latest stages of HHcy.

Aging and total stenosis triggers differential responses of carotid and basilar arteries to endothelin-1 and phenylephrine.

Our aim was to investigate the effects of ageing on the vascular contractility of carotid and basilar arteries from guinea-pigs, in a model of total stenosis. Moreover, we attempted to identify whether total stenosis of the left common carotid (stenosed) in adult guinea-pigs, would affect the contractions of contralateral carotid (intact) and basilar arteries to different vasoconstrictors. With this purpose, the left carotid was occluded with a silk thread at a position close to its origin. Vascular reactivity experiments using standard muscle bath were performed 7, 15, 30, and 90 days after carotid occlusion. Reactivity of carotid and basilar arteries to endothelin-1, phenylephrine and KCl was reduced with ageing in naive guinea-pigs. The endothelin-1 and KCl-induced contractions were unaltered in arteries from SHAM-operated animals. Moreover, phenylephrine-induced contractions were reduced in both carotids from 7 days SHAM-operated guinea-pigs, when compared to naive group. Stenosis induced progressive reduction in the contraction induced by endothelin-1, phenylephrine and KCl in the stenosed carotid, when compared to their respective age-matched naive and SHAM groups. Interestingly, an increased contractile-response to vasoconstrictor agents in all the contralateral carotids was observed. Stenosis (30 and 90 days) also induced an increase in the contractions induced by endothelin-1 in the basilar artery. Conversely, phenylephrine and KCl-induced contractions were reduced in basilar arteries 7, 15, 30 and 90 days after stenosis. These results showed that stenosis in adult guinea-pigs induce alterations of vascular reactivity in arteries distant from the site of injury. Thus, in spite of the common use of contralateral carotid as control, it must be aware of the potential alteration induced by stenosis in the vascular motility of such vessels. Additionally, it was verified a relationship between the period of stenosis and the alterations in the vascular reactivity to these vasoconstrictors.

Total stenosis triggers compensatory responsiveness of carotid and basilar arteries to endothelin-1 and phenylephrine.

The aim of this study was to investigate whether total stenosis of the common carotid artery (ipsilateral) would affect the vascular responsiveness of the contralateral carotid as well as the basilar artery from guinea pigs. With this purpose, the carotid artery was occluded with a silk thread at a position close to its origin. Vascular reactivity experiments using standard muscle bath were performed 7, 15, 30, and 90 days after carotid occlusion. Stenosis induced a progressive reduction in the contraction induced by endothelin-1, phenylephrine and KCl in the ipsilateral carotid, when compared to their respective age-matched SHAM groups. Endothelial removal or incubation of endothelium-intact ipsilateral carotids with L-NAME, a nitric oxide synthase inhibitor, did not alter the response to endothelin-1 or phenylephrine, when compared to the endothelium-intact ipsilateral carotid in the absence of the inhibitor. Interestingly, an increased contractile response to endothelin-1, phenylephrine and KCl was observed in the contralateral carotid. Indomethacin, a non-selective cyclooxygenase inhibitor, prevented the increased contraction to endothelin-1 in the contralateral carotid. Stenosis also induced an increase in the contractile response to endothelin-1 in the basilar artery while the contractile response to phenylephrine and KCl were reduced. Indomethacin, but not L-NAME, prevented the increased contraction to endothelin-1 in the basilar artery. Morphometric analysis showed no differences in the medial area (wall thickness) of carotid or basilar arteries from the stenosis group when compared to their respective age-matched SHAM groups. The present study confirms the importance of adaptation to stenosis on the vascular reactivity of the stenosed artery to different vasoconstrictor agents. Moreover, our results show that stenosis induces alterations of the vascular reactivity on arteries distant from the site of injury. The increased response to endothelin-1 in the contralateral carotid artery and basilar artery seems to involve the release of vasoconstrictor prostanoids from endothelial origin.

Alpha1D-adrenoceptor-induced relaxation on rat carotid artery is impaired during the endothelial dysfunction evoked in the early stages of hyperhomocysteinemia.

Hyperhomocysteinemia is a known risk factor for cardiovascular diseases, but the underlying mechanisms of this pathology are complex. We aimed to evaluate the effect of hyperhomocysteinemia in vasorelaxations induced by alpha(1D)-adrenoceptor agonists. Vascular reactivity of rat carotid artery to the alpha-adrenoceptor agonist, phenylephrine, was enhanced in hyperhomocysteinemia. Mechanical removal of endothelium did not modify the carotid responsiveness to phenylephrine, compared to control. Phenylephrine induces endothelium-dependent relaxation, in the presence of 5-methyl urapidil (alpha(1A)-adrenoceptor antagonist). We hypothesised that endothelial-relaxant alpha(1)-adrenoceptors are impaired by hyperhomocysteinemia. Incubation with prazosin (selective alpha(1)-adrenoceptor antagonist) or BMY7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]decane-7, 9-dione dihydrochloride) (selective alpha(1D)-adrenoceptor antagonist), similarly inhibited phenylephrine-induced relaxations in both control and hyperhomocysteinemic carotids. Immunohistochemistry showed enhanced immunoreactivity for eNOS and iNOS in hyperhomocysteinemic rats. In carotid arteries from hyperhomocysteinemic rats there was a decrease in superoxide dismutase activity and enhanced superoxide anion production. We conclude that alpha(1D)-adrenoceptors mediate endothelium-dependent relaxation triggered by phenylephrine in rat carotid artery and affect the final tone. Furthermore, the enhanced phenylephrine-induced contraction in carotid artery due to hyperhomocysteinemia is endothelium-dependent and involves a loss of the inhibitory effect of relaxant alpha(1D)-adrenoceptors by reducing NO biodisponibility.