NEW METHODS FOR DETECTION, SUSCEPTIBILITY TESTING AND BIOFILM ERADICATION OF DIFFICULT ORGANISMS

Aujourd'hui, les problèmes des maladies infectieuses concernent l'émergence d'infections difficiles à traiter, telles que les infections associées aux implants et les infections fongiques invasives chez les patients immunodéprimés. L'objectif de cette thèse était de développer des stratégies pour l'éradication des biofilms bactériens (partie 1), ainsi que d'étudier des méthodes innovantes pour la détection microbienne, pour l'établissement de nouveaux tests de sensibilité (partie 2). Le traitement des infections associées aux implants est difficile car les biofilms bactériens peuvent résister à des niveaux élevés d'antibiotiques. A ce jour, il n'y a pas de traitement optimal défini contre des infections causées par des bactéries de prévalence moindre telles que Enterococcus faecalis ou Propionibacterium acnés. Dans un premier temps, nous avons démontré une excellente activité in vitro de la gentamicine sur une souche de E. faecalis en phase stationnaire de croissance Nous avons ensuite confirmé l'activité de la gentamicine sur un biofilm précoce en modèle expérimental animal à corps étranger avec un taux de guérison de 50%. De plus, les courbes de bactéricidie ainsi que les résultats de calorimétrie ont prouvé que l'ajout de gentamicine améliorait l'activité in vitro de la daptomycine, ainsi que celle de la vancomycine. In vivo, le schéma thérapeutique le plus efficace était l'association daptomycine/gentamicine avec un taux de guérison de 55%. En établissant une nouvelle méthode pour l'évaluation de l'activité des antimicrobiens vis-à-vis de micro-organismes en biofilm, nous avons démontré que le meilleur antibiotique actif sur les biofilms à P. acnés était la rifampicine, suivi par la penicilline G, la daptomycine et la ceftriaxone. Les études conduites en modèle expérimental animal ont confirmé l'activité de la rifampicine seule avec un taux de guérison 36%. Le meilleur schéma thérapeutique était au final l'association rifampicine/daptomycine avec un taux de guérison 63%. Les associations de rifampicine avec la vancomycine ou la levofloxacine présentaient des taux de guérisons respectivement de 46% et 25%. Nous avons ensuite étudié l'émergence in vitro de la résistance à la rifampicine chez P. acnés. Nous avons observé un taux de mutations de 10"9. La caractérisation moléculaire de la résistance chez les mutant-résistants a mis en évidence l'implication de 5 mutations ponctuelles dans les domaines I et II du gène rpoB. Ce type de mutations a déjà été décrit au préalable chez d'autres espèces bactériennes, corroborant ainsi la validité de nos résultats. La deuxième partie de cette thèse décrit une nouvelle méthode d'évaluation de l'efficacité des antifongiques basée sur des mesures de microcalorimétrie isotherme. En utilisant un microcalorimètre, la chaleur produite par la croissance microbienne peut être-mesurée en temps réel, très précisément. Nous avons évalué l'activité de l'amphotéricine B, des triazolés et des échinocandines sur différentes souches de Aspergillus spp. par microcalorimétrie. La présence d'amphotéricine Β ou de triazole retardait la production de chaleur de manière concentration-dépendante. En revanche, pour les échinochandines, seule une diminution le pic de « flux de chaleur » a été observé. La concordance entre la concentration minimale inhibitrice de chaleur (CMIC) et la CMI ou CEM (définie par CLSI M38A), avec une marge de 2 dilutions, était de 90% pour l'amphotéricine B, 100% pour le voriconazole, 90% pour le pozoconazole et 70% pour la caspofongine. La méthode a été utilisée pour définir la sensibilité aux antifongiques pour d'autres types de champignons filamenteux. Par détermination microcalorimétrique, l'amphotéricine B s'est avéré être l'agent le plus actif contre les Mucorales et les Fusarium spp.. et le voriconazole le plus actif contre les Scedosporium spp. Finalement, nous avons évalué l'activité d'associations d'antifongiques vis-à-vis de Aspergillus spp. Une meilleure activité antifongique était retrouvée avec l'amphotéricine B ou le voriconazole lorsque ces derniers étaient associés aux échinocandines vis-à-vis de A. fumigatus. L'association échinocandine/amphotéricine B a démontré une activité antifongique synergique vis-à-vis de A. terreus, contrairement à l'association échinocandine/voriconazole qui ne démontrait aucune amélioration significative de l'activité antifongique. - The diagnosis and treatment of infectious diseases are today increasingly challenged by the emergence of difficult-to-manage situations, such as infections associated with medical devices and invasive fungal infections, especially in immunocompromised patients. The aim of this thesis was to address these challenges by developing new strategies for eradication of biofilms of difficult-to-treat microorganisms (treatment, part 1) and investigating innovative methods for microbial detection and antimicrobial susceptibility testing (diagnosis, part 2). The first part of the thesis investigates antimicrobial treatment strategies for infections caused by two less investigated microorganisms, Enterococcus faecalis and Propionibacterium acnes, which are important pathogens causing implant-associated infections. The treatment of implant-associated infections is difficult in general due to reduced susceptibility of bacteria when present in biofilms. We demonstrated an excellent in vitro activity of gentamicin against E. faecalis in stationary growth- phase and were able to confirm the activity against "young" biofilms (3 hours) in an experimental foreign-body infection model (cure rate 50%). The addition of gentamicin improved the activity of daptomycin and vancomycin in vitro, as determined by time-kill curves and microcalorimetry. In vivo, the most efficient combination regimen was daptomycin plus gentamicin (cure rate 55%). Despite a short duration of infection, the cure rates were low, highlighting that enterococcal biofilms remain difficult to treat despite administration of newer antibiotics, such as daptomycin. By establishing a novel in vitro assay for evaluation of anti-biofilm activity (microcalorimetry), we demonstrated that rifampin was the most active antimicrobial against P. acnes biofilms, followed by penicillin G, daptomycin and ceftriaxone. In animal studies we confirmed the anti-biofilm activity of rifampin (cure rate 36% when administered alone), as well as in combination with daptomycin (cure rate 63%), whereas in combination with vancomycin or levofloxacin it showed lower cure rates (46% and 25%, respectively). We further investigated the emergence of rifampin resistance in P. acnes in vitro. Rifampin resistance progressively emerged during exposure to rifampin, if the bacterial concentration was high (108 cfu/ml) with a mutation rate of 10"9. In resistant isolates, five point mutations of the rpoB gene were found in cluster I and II, as previously described for staphylococci and other bacterial species. The second part of the thesis describes a novel real-time method for evaluation of antifungals against molds, based on measurements of the growth-related heat production by isothermal microcalorimetry. Current methods for evaluation of antifungal agents against molds, have several limitations, especially when combinations of antifungals are investigated. We evaluated the activity of amphotericin B, triazoles (voriconazole, posaconazole) and echinocandins (caspofungin and anidulafungin) against Aspergillus spp. by microcalorimetry. The presence of amphotericin Β or a triazole delayed the heat production in a concentration-dependent manner and the minimal heat inhibition concentration (MHIC) was determined as the lowest concentration inhibiting 50% of the heat produced at 48 h. Due to the different mechanism of action echinocandins, the MHIC for this antifungal class was determined as the lowest concentration lowering the heat-flow peak with 50%. Agreement within two 2-fold dilutions between MHIC and MIC or MEC (determined by CLSI M38A) was 90% for amphotericin B, 100% for voriconazole, 90% for posaconazole and 70% for caspofungin. We further evaluated our assay for antifungal susceptibility testing of non-Aspergillus molds. As determined by microcalorimetry, amphotericin Β was the most active agent against Mucorales and Fusarium spp., whereas voriconazole was the most active agent against Scedosporium spp. Finally, we evaluated the activity of antifungal combinations against Aspergillus spp. Against A. jumigatus, an improved activity of amphotericin Β and voriconazole was observed when combined with an echinocandin. Against A. terreus, an echinocandin showed a synergistic activity with amphotericin B, whereas in combination with voriconazole, no considerable improved activity was observed.

Prognostication of neurologic outcome in cardiac arrest patients after mild therapeutic hypothermia: a meta-analysis of the current literature.

PURPOSE: To assess the sensitivity and false positive rate (FPR) of neurological examination and somatosensory evoked potentials (SSEPs) to predict poor outcome in adult patients treated with therapeutic hypothermia after cardiopulmonary resuscitation (CPR). METHODS: MEDLINE and EMBASE were searched for cohort studies describing the association of clinical neurological examination or SSEPs after return of spontaneous circulation with neurological outcome. Poor outcome was defined as severe disability, vegetative state and death. Sensitivity and FPR were determined. RESULTS: A total of 1,153 patients from ten studies were included. The FPR of a bilaterally absent cortical N20 response of the SSEP could be calculated from nine studies including 492 patients. The SSEP had an FPR of 0.007 (confidence interval, CI, 0.001-0.047) to predict poor outcome. The Glasgow coma score (GCS) motor response was assessed in 811 patients from nine studies. A GCS motor score of 1-2 at 72 h had a high FPR of 0.21 (CI 0.08-0.43). Corneal reflex and pupillary reactivity at 72 h after the arrest were available in 429 and 566 patients, respectively. Bilaterally absent corneal reflexes had an FPR of 0.02 (CI 0.002-0.13). Bilaterally absent pupillary reflexes had an FPR of 0.004 (CI 0.001-0.03). CONCLUSIONS: At 72 h after the arrest the motor response to painful stimuli and the corneal reflexes are not a reliable tool for the early prediction of poor outcome in patients treated with hypothermia. The reliability of the pupillary response to light and the SSEP is comparable to that in patients not treated with hypothermia.

The Stagnation Regime of the New Keynesian Model and Current US Policy

In Evans, Guse, and Honkapohja (2008) the intended steady state is locally but not globally stable under adaptive learning, and unstable deflationary paths can arise after large pessimistic shocks to expectations. In the current paper a modified model is presented that includes a locally stable stagnation regime as a possible outcome arising from large expectation shocks. Policy implications are examined. Sufficiently large temporary increases in government spending can dislodge the economy from the stagnation regime and restore the natural stabilizing dynamics. More specific policy proposals are presented and discussed.

New insights into the phylogenetics and biogeography of Arum (Araceae): unravelling its evolutionary history

The heat- and odour-producing genus Arum (Araceae) has interested scientists for centuries. This long-term interest has allowed a deep knowledge of some complex processes, such as the physiology and dynamics of its characteristic lure-and-trap pollination system, to be built up. However, mainly because of its large distributional range and high degree of morphological variation, species' limits and relationships are still under discussion. Today, the genus comprises 28 species subdivided into two subgenera, two sections and six subsections. In this study, the phylogeny of the genus is inferred on the basis of four plastid regions, and the evolution of several morphological characters is investigated. Our phylogenetic hypothesis is not in agreement with the current infrageneric classification of the genus and challenges the monophyly of several species. This demonstrates the need for a new infrageneric classification based on characters reflecting the evolution of this enigmatic genus. To investigate the biogeography of Arum deeply, further spatiotemporal analyses were performed, addressing the importance of the Mediterranean basin in the diversification of Arum. Our results suggest that its centre of origin was the European-Aegean region, and that major diversification happened during the last 10 Myr.

Macrophages from Crohn's disease patients exhibit deficient pro-repair functions

BACKGROUND: We previously reported that myeloid cells can induce mucosal healing in a mouse model of acute colitis. Promotion of mucosal repair is becoming a major goal in the treatment of Crohn's disease. Our aim in this study is to investigate the pro-repair function of myeloid cells in healthy donor (HD) and Crohn's disease patients (CD). METHODS: Peripheral blood mononuclear cells (PBMC) from HD and CD patients were isolated from blood samples by Ficoll density gradient. Monocytic CD14+ cells were positively selected by Macs procedure and then differentiated ex-vivo into macrophages (Mφ). The repair function of PBMC, CD14+ monocytic cells and macrophages were evaluated in an in vitro wound healing assay. RESULTS: PBMC and CD14+ myeloid cells from HD and CD were not able to repair at any tested cell concentration. Remarkably, HD Mφ were able to induce wound healing only at high concentration (105 added Mφ), but, if activated with heat killed bacteria, they were able to repair even at very low concentration. On the contrary, not activated CD Mφ were not able to promote healing at any rate, but this function was restored upon activation. CONCLUSION: We showed that CD Mφ in their steady state, unlike HD Mφ, are defective in promoting wound healing. Our results are in keeping with the current theory of CD as an innate immunodeficiency. Defective Mφ may be responsible to the mucosal repair defects in CD patients and to the subsequent chronic activation of the adaptive immune response.

Current status of biotechnology in China

There has been increasing interest over past decade in exploring the possibility of using new biotechinology to produce new products and to improve the old productive process. The researches and applications of genetic engineering, cell fusion, mutagenesis, cell and enzyme immobilization in enzyme, antibiotic, vitamine, steroid, amino acid, organic acid, solvent, food and brewage industries is reviewed.

Quantifying rates of landscape evolution and tectonic processes by thermochronology and numerical modeling of crustal heat transport using PECUBE

PECUBE is a three-dimensional thermal-kinematic code capable of solving the heat production-diffusion-advection equation under a temporally varying surface boundary condition. It was initially developed to assess the effects of time-varying surface topography (relief) on low-temperature thermochronological datasets. Thermochronometric ages are predicted by tracking the time-temperature histories of rock-particles ending up at the surface and by combining these with various age-prediction models. In the decade since its inception, the PECUBE code has been under continuous development as its use became wider and addressed different tectonic-geomorphic problems. This paper describes several major recent improvements in the code, including its integration with an inverse-modeling package based on the Neighborhood Algorithm, the incorporation of fault-controlled kinematics, several different ways to address topographic and drainage change through time, the ability to predict subsurface (tunnel or borehole) data, prediction of detrital thermochronology data and a method to compare these with observations, and the coupling with landscape-evolution (or surface-process) models. Each new development is described together with one or several applications, so that the reader and potential user can clearly assess and make use of the capabilities of PECUBE. We end with describing some developments that are currently underway or should take place in the foreseeable future. (C) 2012 Elsevier B.V. All rights reserved.

The current molecular phylogeny of Eutherian mammals challenges previous interpretations of placental evolution.

Based on histology, the placentae of eutherians are currently grouped in epitheliochorial, endotheliochorial and haemochorial placentae. In a haeckelian sense, the epitheliochorial contact with marked histiotrophic feeding by uterine milk is generally considered as primitive, especially since similar contacts exist in Marsupials. In contrast, the more intimate endotheliochorial and haemochorial contact, facilitating haemotrophic nutrition, is interpreted as a derived state. A cladistic analysis based on the phylogenetic relationships established by molecular analyses reveals that the basic clades are all characterized by an endotheliochorial or haemochorial placenta, and that the epitheliochorial placenta evolved at least three times in a convergent manner. This evolution may be explained by the fact that the epitheliochorial placenta in eutherians is more efficient in nutritional transfer (flow rate by exchange surface). Moreover, this arrangement may confer an advantage to the mother who can probably reduce the degree of manipulation by a genetically imprinted embryo.

On the current existence of a coccidial line of development in the malaria parasites: a theory

Most opinion favors the origin of the malaria parasites from a coccidial ancestor. It is assumed that whatever the process through which the coccidia differentiated into a Plasmodium this phenomenon very probably occured millions of year ago, and during that differentiation process the original coccidia vanished. Therefore it has never repeated. At the light of some experiments the existence, at the present time, of a coccidial cycle of development in the malaria parasites, is proposed. The conection routes and mechanisms through which the malaria parasite changes to a coccidial life, and the routes in reverse are exposed. Transmission of the malaria-coccidial forms is suggested.

Current Molecular Imaging of Spinal Tumors in Clinical Practice.

Energy metabolism measurements in spinal cord tumors, as well as in osseous spinal tumors/metastasis in vivo, are rarely performed only with molecular imaging (MI) by positron emission tomography (PET). This imaging modality developed from a small number of basic clinical science investigations followed by subsequent work that influenced and enhanced the research of others. Apart from precise anatomical localization by coregistration of morphological imaging and quantification, the most intriguing advantage of this imaging is the opportunity to investigate the time course (dynamics) of disease-specific molecular events in the intact organism. Most importantly, MI represents one of the key technologies in translational molecular neuroscience research, helping to develop experimental protocols that may later be applied to human patients. PET may help monitor a patient at the vertebral level after surgery and during adjuvant treatment for recurrent or progressive disease. Common clinical indications for MI of primary or secondary CNS spinal tumors are: (i) tumor diagnosis, (ii) identification of the metabolically active tumor compartments (differentiation of viable tumor tissue from necrosis) and (iii) prediction of treatment response by measurement of tumor perfusion or ischemia. While spinal PET has been used under specific circumstances, a question remains as to whether the magnitude of biochemical alterations observed by MI in CNS tumors in general (specifically spinal tumors) can reveal any prognostic value with respect to survival. MI may be able to better identify early disease and to differentiate benign from malignant lesions than more traditional methods. Moreover, an adequate identification of treatment effectiveness may influence patient management. MI probes could be developed to image the function of targets without disturbing them or as treatment to modify the target's function. MI therefore closes the gap between in vitro and in vivo integrative biology of disease. At the spinal level, MI may help to detect progression or recurrence of metastatic disease after surgical treatment. In cases of nonsurgical treatments such as chemo-, hormone- or radiotherapy, it may better assess biological efficiency than conventional imaging modalities coupled with blood tumor markers. In fact, PET provides a unique possibility to correlate topography and specific metabolic activity, but it requires additional clinical and experimental experience and research to find new indications for primary or secondary spinal tumors.

Optimal exponent heat balance and refined integral methods applied to Stefan problems

When using a polynomial approximating function the most contentious aspect of the Heat Balance Integral Method is the choice of power of the highest order term. In this paper we employ a method recently developed for thermal problems, where the exponent is determined during the solution process, to analyse Stefan problems. This is achieved by minimising an error function. The solution requires no knowledge of an exact solution and generally produces significantly better results than all previous HBI models. The method is illustrated by first applying it to standard thermal problems. A Stefan problem with an analytical solution is then discussed and results compared to the approximate solution. An ablation problem is also analysed and results compared against a numerical solution. In both examples the agreement is excellent. A Stefan problem where the boundary temperature increases exponentially is analysed. This highlights the difficulties that can be encountered with a time dependent boundary condition. Finally, melting with a time-dependent flux is briefly analysed without applying analytical or numerical results to assess the accuracy.

Application of standard and refined heat balance integral methods to one-dimensional Stefan problems

The work in this paper concerns the study of conventional and refined heat balance integral methods for a number of phase change problems. These include standard test problems, both with one and two phase changes, which have exact solutions to enable us to test the accuracy of the approximate solutions. We also consider situations where no analytical solution is available and compare these to numerical solutions. It is popular to use a quadratic profile as an approximation of the temperature, but we show that a cubic profile, seldom considered in the literature, is far more accurate in most circumstances. In addition, the refined integral method can give greater improvement still and we develop a variation on this method which turns out to be optimal in some cases. We assess which integral method is better for various problems, showing that it is largely dependent on the specified boundary conditions.

Current concepts of snail control

Schistosomiasis control was impossible without effective tools. Synthetic molluscicides developed in the 1950s spearheaded community level control. Snail eradication proved impossible but repeated mollusciciding to manage natural snail populations could eliminate transmission. Escalating costs, logistical complexity, its labour-intensive nature and possible environmental effects caused some concern. The arrival of safe, effective, single-dose drugs in the 1970s offered an apparently better alternative but experience revealed the need for repeated treatments to minimise reinfection in programmes relying on drugs alone. Combining treatment with mollusciciding was more successful, but broke down if mollusciciding was withdrawn to save money. The provision of sanitation and safe water to prevent transmission is too expensive in poor rural areas where schistosomiasis is endemic; rendering ineffective public health education linked to primary health care. In the tropics, moreover, children (the key group in maintaining transmission) will always play in water. Large scale destruction of natural snail habitats remains impossibly expensive (although proper design could render many new man-made habitats unsuitable for snails). Neither biological control agents nor plant molluscicides have proved satisfactory alternatives to synthetic molluscicides. Biologists can develop effective strategies for using synthetic molluscicides in different epidemiological situations if only, like drugs, their price can be reduced.

Current advances on the study of snail-snail interactions, with special emphasis on competition process

Field work research on population dynamic of snails from the regions of Belo Horizonte and Lagoa Santa give much information about interactions among two or more species of mollusks: Pomacea haustrum, Biomphalaria glabrata, B. tenagophila, B. straminea and Melanoides tuberculata. Data ranging from two years to several decades ago suggest that the Pampulha reservoir is like a cemetery of B. glabrata and B. straminea, species that coexist for more than 14 years in a small part of a stream, whereas only B. glabrata lives in all the streams of the basin. In the last ten to twenty years B. tenagophila has coexisted with P. haustrum and M. tuberculata in the Serra Verde ponds and in the Pampulha dam. However these species have not settled in any of the brooks, except temporarily. The data suggest that the kind of biotope and the habitat conditions are decisive factors for the permanence of each species in its preferencial biotope. B. glabrata, natural from streams and riverheads, quickly disappears from the reservoirs and ponds where it coexists with other species for a short time, independently of the competitive process. Competition needs to be better studied, since in Central America and Caribean islands this kind of study has favored the biological control of planorbid species.