Preparation of a blood culture pellet for rapid bacterial identification and antibiotic susceptibility testing.

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

Synthesis and antiprotozoal activity of oligomethylene- and p-phenylene-bis(methylene)-linked bis(+)-huprines

We have synthesized a series of dimers of (+)-(7R,11R)-huprine Y and evaluated their activity against Trypanosoma brucei, Plasmodium falciparum, rat myoblast L6 cells and human acetylcholinesterase (hAChE), and their brain permeability. Most dimers have more potent and selective trypanocidal activity than huprine Y and are brain permeable, but they are devoid of antimalarial activity and remain active against hAChE. Lead optimization will focus on identifying compounds with a more favourable trypanocidal/anticholinesterase activity ratio.

Synthesis and antiprotozoal activity of oligomethylene- and p-phenylene-bis(methylene)-linked bis(+)-huprines

We have synthesized a series of dimers of (+)-(7R,11R)-huprine Y and evaluated their activity against Trypanosoma brucei, Plasmodium falciparum, rat myoblast L6 cells and human acetylcholinesterase (hAChE), and their brain permeability. Most dimers have more potent and selective trypanocidal activity than huprine Y and are brain permeable, but they are devoid of antimalarial activity and remain active against hAChE. Lead optimization will focus on identifying compounds with a more favourable trypanocidal/anticholinesterase activity ratio.

A espectrometria atômica e a determinação de elementos metálicos em material polimérico

Polymeric materials are widely used in the chemical industry and are part of our daily lives. Inorganic species may be added to them as additives, anti-oxidizing agents, stabilizers, plasticizers, colorants and catalysts and may be present in a wide range of concentrations. Their determination demands the development of analytical methods considering different kinds of polymeric materials, their composition and the final use of the material. Although many different analytical techniques may be used, this review emphasizes those based on atomic absorption and emission spectrometry. Solid sampling techniques and digestion methods are described and discussed and compared considering published results.

Ocorrência e diversidade estrutural de metabólitos fúngicos com atividade antibiótica

Several reasons motivated the development of new generations of antibiotics, such as their high ability to develop resistance to virtually all kinds of anti-infective agents and the crescent market demand for new drugs to treat special demanding patients. After penicillin discovery, several antibiotics were developed from fungal metabolites, since antibacterial secondary metabolites consists on a fungal endogenous protective mechanism against natural competitors. The aim of this review is to present the structural diversity of antibacterial and antifungal metabolites produced by fungi, mentioning sources of fungal isolates, cultivation process and details on the scope of their antibiotic activity.

HIV - recentes avanços na pesquisa de fármacos

The development of new antiretroviral drugs is a dynamic process that is continuously fueled by identification of new molecular targets and new compounds for know targets. The current available drugs can be classified into five categories: nucleoside analogues reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors, integrase inhibitors and entry inhibitors (fusion inhibitors and CCR5 antagonist). In addition, the maturation inhibitors may be considered as potential target for chemotherapeutic intervention. This review presents some anti-HIV agents that have already gone through the advance development process for final approval for the treatment of AIDS.

Incidence of uterine post abortion infection at Hospital de Clínicas de Porto Alegre. Is prophylactic antibiotic necessary?

OBJECTIVE: To identify the incidence of pelvic infection after miscarriage undergoing uterine evacuation in a tertiary hospital in southern Brazil and to compare with the international literature.METHODS: we reviewed electronic medical records of the Hospital de Clinicas de Porto Alegre of all patients who underwent uterine evacuation for miscarriage between August 2008 and January 2012 were reviewed. We included all patients submitted to uterine curettage due to abortion and who had outpatient visits for review after the procedure. We calculated emographic and laboratory data of the study population, number needed for treatment (NNT) and number needed to harm (NNH).RESULTS: of the 857 revised electronic medical records, 377 patients were subjected to uterine evacuation for miscarriage; 55 cases were lost to follow-up, leaving 322 cases that were classified as not infected abortion on admission. The majority of the population was white (79%); HIV prevalence and positive VDRL was 0.3% and 2%, respectively. By following these 322 cases for a minimum of seven days, it was found that the incidence of post-procedure infection was 1.8% (95% CI 0.8 to 4). The NNT and NNH calculated for 42 months were 63 and 39, respectively.CONCLUSION: The incidence of post-abortion infection between August 2008 to January 2012 was 1.8% (0.8 to 4).

Effects of low-level laser therapy on wound healing

OBJECTIVE: To gather and clarify the actual effects of low-level laser therapy on wound healing and its most effective ways of application in human and veterinary medicine.METHODS: We searched original articles published in journals between the years 2000 and 2011, in Spanish, English, French and Portuguese languages, belonging to the following databases: Lilacs, Medline, PubMed and Bireme; Tey should contain the methodological description of the experimental design and parameters used.RESULTS: doses ranging from 3 to 6 J/cm2 appear to be more effective and doses 10 above J/cm2 are associated with deleterious effects. The wavelengths ranging from 632.8 to 1000 nm remain as those that provide more satisfactory results in the wound healing process.CONCLUSION: Low-level laser can be safely applied to accelerate the resolution of cutaneous wounds, although this fact is closely related to the election of parameters such as dose, time of exposure and wavelength.

The cost of excessive postoperative use of antimicrobials: the context of a public hospital

OBJECTIVE: To evaluate the improper use of antimicrobials during the postoperative period and its economic impact.METHODS: We conducted a prospective cohort study by collecting data from medical records of 237 patients operated on between 01/11/08 and 31/12/08.RESULTS: from the 237 patients with the information collected, 217 (91.56%) received antimicrobials. During the postoperative period, 125 (57.7%) patients received more than two antimicrobials. On average, 1.7 ± 0.6 antimicrobials were prescribed to patients, the most commonly prescribed antibiotic being cephalothin, in 41.5% (154) of cases. The direct cost of antimicrobial therapy accounted for 63.78% of all drug therapy, this large percentage being attributed in part to the extended antimicrobial prophylaxis. In the case of clean operations, where there was a mean duration of 5.2 days of antibiotics, antimicrobials represented 44.3% of the total therapy cost.CONCLUSION: The data illustrate the impact of overuse of antimicrobials, with questionable indications, creating situations that compromise patient safety and increasing costs in the assessed hospital.

Histopathological analysis of rat mesentery as a method for evaluating neutrophil migration: differential effects of dexamethasone and pertussis toxin

In the present study, histopathological analysis of rat mesentery was used to quantify the effect of two anti-inflammatory agents, dexamethasone (Dex) and pertussis toxin (Ptx), on leukocyte migration. The intravenous injection of Dex (1 mg/kg) and Ptx (1,200 ng) 1 h prior to the intraperitoneal injection of the inflammatory stimuli lipopolysaccharide (LPS) or formyl-methionyl-leucyl-phenylalanine (fMLP) significantly reduced the neutrophil diapedesis (LPS: Ptx = 0.86 ± 0.19 and Dex = 0.35 ± 0.13 vs saline (S) = 2.85 ± 0.59; fMLP: Ptx = 0.43 ± 0.09 and Dex 0.01 ± 0.01 vs S = 1.08 ± 0.15 neutrophil diapedesis/field) and infiltration (LPS: Ptx = 6.29 ± 1.4 and Dex = 3.06 ± 0.76 vs S = 15.94 ± 3.97; fMLP: Ptx = 3.85 ± 0.56 and Dex = 0.40 ± 0.16 vs S = 7.15 ± 1.17 neutrophils/field) induced by the two agonists in the rat mesentery. The inhibitory effect of Dex and Ptx was clearly visible in the fields nearest the venule (up to 200 µm), demonstrating that these anti-inflammatory agents act preferentially in the transmigration of neutrophils from the vascular lumen into the interstitial space, but not in cell movement in response to a haptotactic gradient. The mesentery of rats pretreated with Dex showed a decreased number of neutrophils within the venules (LPS: Dex = 1.50 ± 0.38 vs S = 4.20 ± 1.01; fMLP: Dex = 0.25 ± 0.11 vs S = 2.20 ± 0.34 neutrophils in the lumen/field), suggesting that this inhibitor may be acting at a step that precedes neutrophil arrival in the inflamed tissue. In contrast to that observed with Dex treatment, the number of neutrophils found in mesenteric venules was significantly elevated in animals pretreated with Ptx (LPS: Ptx = 9.85 ± 2.25 vs S = 4.20 ± 1.01; fMLP: Ptx = 4.66 ± 1.24 vs S = 2.20 ± 0.34 neutrophils in the lumen/field). This discrepancy shows that Ptx and Dex act via different mechanisms and suggests that Ptx prevents locomotion of neutrophils from the vascular lumen to the interstitial space. In conclusion, the method described here is useful for quantifying the inflammatory and anti-inflammatory effect of different substances. The advantage of this histopathological approach is that it provides additional information about the steps involved in leucocyte migration.

Schwann cells for spinal cord repair

The complex nature of spinal cord injury appears to demand a multifactorial repair strategy. One of the components that will likely be included is an implant that will fill the area of lost nervous tissue and provide a growth substrate for injured axons. Here we will discuss the role of Schwann cells (SCs) in cell-based, surgical repair strategies of the injured adult spinal cord. We will review key studies that showed that intraspinal SC grafts limit injury-induced tissue loss and promote axonal regeneration and myelination, and that this response can be improved by adding neurotrophic factors or anti-inflammatory agents. These results will be compared with several other approaches to the repair of the spinal cord. A general concern with repair strategies is the limited functional recovery, which is in large part due to the failure of axons to grow across the scar tissue at the distal graft-spinal cord interface. Consequently, new synaptic connections with spinal neurons involved in motor function are not formed. We will highlight repair approaches that did result in growth across the scar and discuss the necessity for more studies involving larger, clinically relevant types of injuries, addressing this specific issue. Finally, this review will reflect on the prospect of SCs for repair strategies in the clinic.

Vaahtoamisen estäminen vesiohenteisissa sisäseinämaaleissa

Maalit ovat hyvin komplekseja seoksia, jotka sisältävät erilaisia pinta-aktiivisia aineita. Erilaisia pinta-aktiivisia apuaineita käytetään varsinkin vesiohenteisissa maaleissa veden korkean pintajännityksen takia. Nämä maalin komponentit saat-tavat kuitenkin aiheuttaa maaliin vaahtoamisongelmia. Maalien vaahtoaminen aiheuttaa esimerkiksi erilaisia pintavikoja maalikalvossa ja epäotpimaalista tuo-tantosäiliöiden ja pakkausten täyttöä. Joissain tapauksissa myös maalin funktio-naalisuus saattaa häiriintyä. Maalien vaahtoamista pyritään vähentämään pääasiassa vaahdonestäjiksi kutsu-tuilla apuaineilla. Tärkeimmät vesipohjaisissa maaleissa käytettävät vaahdonestäjät ovat mineraaliöljyjä sekä silikonipohjaisia vaahdonestäjiä. Tässä työssä tutkitaan kolmen eri vesiohenteisen sisäseinämaalin vaahtoamista ja mahdollisia keinoja vähentää sitä. Laboratoriossa kokeillaan nykyisten vaahdonestäjien tilalle mahdollisia korvaavia apuaineita. Lisäksi kullekin maalille luodaan koesuunni-telma, jonka mukaan toteutetaan tehtaalla sarja koeajoja, joilla pyritään löytämään sopiva vaahdonestäjän annostus sekä lisäystapa kullekin maalille, siten että maalien vaahtoaminen vähenee. Laboratoriokokeiden perusteella korvaavaa vaahdonestäjää nykyisten tilalle ei löydetty. Valmistusprosessin yhteydessä sen sijaan havaittiin, että vaahdonestäjän lisäystapaa muuttamalla voisi olla mahdollista vähentää maalien vaahtoamista merkittävästi lisäämättä käytettävää vaahdonestäjän annostusta.

Pharmacology and toxicology of diphenyl diselenide in several biological models

The pharmacology of synthetic organoselenium compounds indicates that they can be used as antioxidants, enzyme inhibitors, neuroprotectors, anti-tumor and anti-infectious agents, and immunomodulators. In this review, we focus on the effects of diphenyl diselenide (DPDS) in various biological model organisms. DPDS possesses antioxidant activity, confirmed in several in vitro and in vivo systems, and thus has a protective effect against hepatic, renal and gastric injuries, in addition to its neuroprotective activity. The activity of the compound on the central nervous system has been studied since DPDS has lipophilic characteristics, increasing adenylyl cyclase activity and inhibiting glutamate and MK-801 binding to rat synaptic membranes. Systemic administration facilitates the formation of long-term object recognition memory in mice and has a protective effect against brain ischemia and on reserpine-induced orofacial dyskinesia in rats. On the other hand, DPDS may be toxic, mainly because of its interaction with thiol groups. In the yeast Saccharomyces cerevisiae, the molecule acts as a pro-oxidant by depleting free glutathione. Administration to mice during cadmium intoxication has the opposite effect, reducing oxidative stress in various tissues. DPDS is a potent inhibitor of d-aminolevulinate dehydratase and chronic exposure to high doses of this compound has central effects on mouse brain, as well as liver and renal toxicity. Genotoxicity of this compound has been assessed in bacteria, haploid and diploid yeast and in a tumor cell line.

Étude structure-fonction des fructose-1,6-bisphosphate aldolases métallo-dépendantes : mécanisme catalytique et développement d’antimicrobiens

Les fructose-1,6-bisphosphate aldolases (FBPA) sont des enzymes glycolytiques (EC 4.1.2.13) qui catalysent la transformation réversible du fructose-1,6-bisphosphate (FBP) en deux trioses-phosphates, le glycéraldéhyde-3-phosphate (G3P) et le dihydroxyacétone phosphate (DHAP). Il existe deux classes de FBPA qui diffèrent au niveau de leur mécanisme catalytique. Les classes I passent par la formation d’un intermédiaire covalent de type iminium alors que les classes II, métallodépendantes, utilisent généralement un zinc catalytique. Contrairement au mécanisme des classes I qui a été très étudié, de nombreuses interrogations subsistent au sujet de celui des classes II. Nous avons donc entrepris une analyse détaillée de leur mécanisme réactionnel en nous basant principalement sur la résolution de structures cristallographiques. De nombreux complexes à haute résolution furent obtenus et ont permis de détailler le rôle de plusieurs résidus du site actif de l’enzyme. Nous avons ainsi corrigé l’identification du résidu responsable de l’abstraction du proton de l’O4 du FBP, une étape cruciale du mécanisme. Ce rôle, faussement attribué à l’Asp82 (chez Helicobacter pylori), est en fait rempli par l’His180, un des résidus coordonant le zinc. L’Asp82 n’en demeure pas moins essentiel car il oriente, active et stabilise les substrats. Enfin, notre étude met en évidence le caractère dynamique de notre enzyme dont la catalyse nécessite la relocalisation du zinc et de nombreux résidus. La dynamique de la protéine ne permet pas d’étudier tous les aspects du mécanisme uniquement par l’approche cristallographique. En particulier, le résidu effectuant le transfert stéréospécifique du proton pro(S) sur le carbone 3 (C3) du DHAP est situé sur une boucle qui n’est visible dans aucune de nos structures. Nous avons donc développé un protocole de dynamique moléculaire afin d’étudier sa dynamique. Validé par l’étude d’inhibiteurs de la classe I, l’application de notre protocole aux FBPA de classe II a confirmé l’identification du résidu responsable de cette abstraction chez Escherichia coli (Glu182) mais pointe vers un résidu diffèrent chez H. pylori (Glu149 au lieu de Glu142). Nos validations expérimentales confirment ces observations et seront consolidées dans le futur. Les FBPA de classe II sont absentes du protéome humain mais sont retrouvées chez de nombreux pathogènes, pouvant même s'y révéler essentielles. Elles apparaissent donc comme étant une cible idéale pour le développement de nouveaux agents anti-microbiens. L’obtention de nouveaux analogues des substrats pour ces enzymes a donc un double intérêt, obtenir de nouveaux outils d’étude du mécanisme mais aussi développer des molécules à visée pharmacologique. En collaboration avec un groupe de chimistes, nous avons optimisé le seul inhibiteur connu des FBPA de classe II. Les composés obtenus, à la fois plus spécifiques et plus puissants, permettent d’envisager une utilisation pharmacologique. En somme, c’est par l’utilisation de techniques complémentaires que de nouveaux détails moléculaires de la catalyse des FBPA de classe II ont pu être étudiés. Ces techniques permettront d’approfondir la compréhension fine du mécanisme catalytique de l’enzyme et offrent aussi de nouvelles perspectives thérapeutiques.

Le récepteur CD36 : implication dans le développement de l'athérosclérose et dans le recrutement des leucocytes aux sites inflammatoires

Le CD36 est un récepteur éboueur de classe B exprimé par plusieurs types cellulaires dont les macrophages et les cellules endothéliales de la microvasculature. Le CD36 présente une haute affinité de liaison pour les ligands lipidiques tels que les lipoprotéines oxydées de basse densité (LDLox). De part sa capacité à internaliser les LDLox au niveau des macrophages et de son implication dans la formation des cellules spumeuses, le CD36 joue un rôle critique dans le développement des lésions athérosclérotiques. Nous avons testé l'hypothèse selon laquelle le EP 80317, un ligand synthétique sélectif du CD36, exerce des effets anti-athérosclérotiques chez les souris déficientes en apolipoprotéine E. Un traitement prolongé (12 semaines) avec le EP 80317 réduit fortement (de 51%) la surface des lésions athérosclérotiques par comparaison aux souris témoins. L'effet anti-athérosclérotique est associé à une diminution des taux de cholestérol plasmatique, à une réduction de l’internalisation des LDLox au niveau des macrophages et à une augmentation de l’expression des protéines impliquées dans le transport inverse du cholestérol. De plus, un traitement par le EP 80317 est également associé une diminution de l’expression aortique et plasmatique de protéines pro-inflammatoires. Nos études ont aussi montré un rôle pour le CD36 dans le recrutement des phagocytes mononucléés au niveau des lésions athérosclérotiques, tel que démontré par une réduction de l’accumulation des phagocytes mononucléés radiomarqués CD36–/– par rapport aux cellules CD36+/+. À l’échelle moléculaire, nous avons montré que les phospholipides oxydés induisent la phosphorylation de la kinase Pyk2 des podosomes des monocytes/macrophages de manière dépendante de l’expression du CD36 et de Src. Cette phosphorylation est atténuée par un traitement par le EP80317. Nos résultats appuient le rôle important du CD36 dans l’athérosclérose et suggèrent que les ligands synthétiques qui modulent la fonction du CD36 représentent potentiellement une nouvelle classe d'agents anti-athérosclérotiques. Le CD36 exprimé par les cellules endothéliales de la microvasculature est un récepteur de l’hétérodimère protéique S100A8/A9. Ces protéines s’associent à l’acide arachidonique intracellulaire (AA) des neutrophiles polymorphonucléaires (PMN) et le complexe S100A8/A9/AA peut être sécrété par les PMN activés au contact de l’endothélium. Nous avons vérifié l’hypothèse selon laquelle le CD36 exprimé par la microvasculature est impliqué dans le métabolisme transcellulaire de l’AA par la liaison du complexe S100A8/A9/AA et la réponse inflammatoire. Chez deux modèles murins d'inflammation aiguë (ischémie/reperfusion des membres inférieurs et poche d’air dorsale), nous avons observé que la réponse inflammatoire, notamment l’accumulation des PMN au niveau des sites inflammatoires, est diminuée en moyenne de 63% chez les souris CD36-/-. De même, un traitement par le EP 80317 ou par les anticorps anti-S100A8/A9 diminue chacun de 60% en moyenne l’extravasation des PMN vers les tissus inflammatoires. L’administration simultanée des deux traitements n’a aucun effet supplémentaire, et ces traitements n’exercent aucun effet chez les souris CD36-/-. Nos résultats appuient le rôle du récepteur CD36 de la microvasculature dans la régulation de la réponse inflammatoire. L’utilisation des ligands synthétiques du CD36 pourrait représenter une nouvelle avenue thérapeutique dans le traitement des réponses inflammatoires aiguës.