New methods for detecting antibiotic resistant bacteria and preventing their spread

Methicillin resistant Staphylococcus aureus (MRSA) bacteria have emerged in the early 1980's in numerous health care institutions around the world. The main transmission mechanism within hospitals and healthcare facilities is through the hands of health care workers. Resistant to several antibiotics, the MRSA is one of the most feared pathogens in the hospital setting since it is very difficult to eradicate with the standard treatments. There are still a limited number of anti-MRSA antibiotics but the first cases of resistance to these compounds have already been reported and their frequency is likely to increase in the coming years. Every year, the MRSA infections result in major human and financial costs, due to the high associated mortality and expenses related to the required care. Measures towards a faster detection of resistant bacteria and establishment of appropriate antibiotic treatment parameters are fundamental. Also as part as infection prevention, diminution of bacteria present on the commonly touched surfaces could also limit the spread and selection of antibiotic resistant bacteria. During my thesis, projects were developed around MRSA and antibiotic resistance investigation using innovative technologies. The thesis was subdivided in three main parts with the use of atomic force microscopy AFM for antibiotic resistance detection in part 1, the importance of the bacterial inoculum size in the selection of antibiotic resistance in part 2 and the testing of antimicrobial surfaces creating by sputtering copper onto polyester in part 3. In part 1 the AFM was used two different ways, first for the measurement of stiffness (elasticity) of bacteria and second as a nanosensor for antibiotic susceptibility testing. The stiffness of MRSA with different susceptibility profiles to vancomycin was investigated using the stiffness tomography mode of the AFM and results have demonstrated and increased stiffness in the vancomycin resistant strains that also paralleled with increased thickness of the bacterial cell wall. Parts of the AFM were also used to build a new antibiotic susceptibility-testing device. This nano sensor was able to measure vibrations emitted from living bacteria that ceased definitively upon antibiotic exposure to which they were susceptible but restarted after antibiotic removal to which they were resistant, allowing in a matter of minute the assessment of antibiotic susceptibility determination. In part 2 the inoculum effect (IE) of vancomycin, daptomycin and linezolid and its importance in antibiotic resistance selection was investigated with MRSA during a 15 days of cycling experiment. Results indicated that a high bacterial inoculum and a prolonged antibiotic exposure were two key factors in the in vitro antibiotic resistance selection in MRSA and should be taken into consideration when choosing the drug treatment. Finally in part 3 bactericidal textile surfaces were investigated against MRSA. Polyesters coated after 160 seconds of copper sputtering have demonstrated a high bactericidal activity reducing the bacterial load of at least 3 logio after one hour of contact. -- Au cours des dernières décennies, des bactéries multirésistantes aux antibiotiques (BMR) ont émergé dans les hôpitaux du monde entier. Depuis lors, le nombre de BMR et la prévalence des infections liées aux soins (IAS) continuent de croître et sont associés à une augmentation des taux de morbidité et de mortalité ainsi qu'à des coûts élevés. De plus, le nombre de résistance à différentes classes d'antibiotiques a également augmenté parmi les BMR, limitant ainsi les options thérapeutiques disponibles lorsqu'elles ont liées a des infections. Des mesures visant une détection plus rapide des bactéries résistantes ainsi que l'établissement des paramètres de traitement antibiotiques adéquats sont primordiales lors d'infections déjà présentes. Dans une optique de prévention, la diminution des bactéries présentes sur les surfaces communément touchées pourrait aussi freiner la dissémination et l'évolution des bactéries résistantes. Durant ma thèse, différents projets incluant des nouvelles technologies et évoluant autour de la résistance antibiotique ont été traités. Des nouvelles technologies telles que le microscope à force atomique (AFM) et la pulvérisation cathodique de cuivre (PCC) ont été utilisées, et le Staphylococcus aureus résistant à la méticilline (SARM) a été la principale BMR étudiée. Deux grandes lignes de recherche ont été développées; la première visant à détecter la résistance antibiotique plus rapidement avec l'AFM et la seconde visant à prévenir la dissémination des BMR avec des surfaces crées grâce à la PCC. L'AFM a tout d'abord été utilisé en tant que microscope à sonde locale afin d'investiguer la résistance à la vancomycine chez les SARMs. Les résultats ont démontré que la rigidité de la paroi augmentait avec la résistance à la vancomycine et que celle-ci corrélait aussi avec une augmentation de l'épaisseur des parois, vérifiée grâce à la microscopie électronique. Des parties d'un AFM ont été ensuite utilisées afin de créer un nouveau dispositif de test de sensibilité aux antibiotiques, un nanocapteur. Ce nanocapteur mesure des vibrations produites par les bactéries vivantes. Après l'ajout d'antibiotique, les vibrations cessent définitivement chez les bactéries sensibles à l'antibiotique. En revanche pour les bactéries résistantes, les vibrations reprennent après le retrait de l'antibiotique dans le milieu permettant ainsi, en l'espace de minutes de détecter la sensibilité de la bactérie à un antibiotique. La PCC a été utilisée afin de créer des surfaces bactéricides pour la prévention de la viabilité des BMR sur des surfaces inertes. Des polyesters finement recouverts de cuivre (Cu), connu pour ses propriétés bactéricides, ont été produits et testés contre des SARMs. Une méthode de détection de viabilité des bactéries sur ces surfaces a été mise au point, et les polyesters obtenus après 160 secondes de pulvérisation au Cu ont démontré une excellente activité bactéricide, diminuant la charge bactérienne d'au moins 3 logio après une heure de contact. En conclusion, l'utilisation de nouvelles technologies nous a permis d'évoluer vers de méthodes de détection de la résistance antibiotique plus rapides ainsi que vers le développement d'un nouveau type de surface bactéricide, dans le but d'améliorer le diagnostic et la gestion des BMR.

Women's decision making and experience of subsequent pregnancy following stillbirth.

INTRODUCTION: This study sought to increase understanding of women's thoughts and feelings about decision making and the experience of subsequent pregnancy following stillbirth (intrauterine death after 24 weeks' gestation). METHODS: Eleven women were interviewed, 8 of whom were pregnant at the time of the interview. Modified grounded theory was used to guide the research methodology and to analyze the data. RESULTS: A model was developed to illustrate women's experiences of decision making in relation to subsequent pregnancy and of subsequent pregnancy itself. DISCUSSION: The results of the current study have significant implications for women who have experienced stillbirth and the health professionals who work with them. Based on the model, women may find it helpful to discuss their beliefs in relation to healing and health professionals to provide support with this in mind. Women and their partners may also benefit from explanations and support about the potentially conflicting emotions they may experience during this time.

Role of intracellular signaling pathways activated in fibroblasts in response to lipoproteins

Summary The best described physiological function of low-density lipoproteins (LDL) is to transport cholesterol to target tissues. LDL deliver their cholesterol cargo to cells following their interaction with the LDL receptor. LDL, when their vascular concentrations increase, have also been implicated in pathologies such as atherosclerosis. Among the cell types that are found in blood vessels, endothelial and smooth muscle cells have dominated cellular research on atherosclerotic mechanisms and LDL activation of signaling pathways, while very little is known about adventitial fibroblast activation caused by elevated lipoprotein levels. Since fibroblasts participate in wound repair and since it has recently been recognized that fibroblasts may play pivotal roles in vascular remodeling and repair of injury, we assessed whether lipoproteins affect fibroblast function. We have found that LDL specifically mediate the activation of a class of mitogen-activated protein kinases (MAPKs): the p38 MAPKs. The activation of this pathway in turn modulates cell shape by promoting lamellipodia formation and extensive cell spreading. This is of particular interest because it provides a mechanism by which LDL can promote wound healing or vessel wall remodeling as observed during the development of atherosclerosis. In order to understand the molecular mechanisms by which LDL induce p38 activation we searched for the component in the LDL particle responsible for the induction of this pathway. We found that cholesterol is the major component of lipoprotein particles that mediates their ability to stimulate the p38 MAPK pathway. Furthermore, we investigated the cellular mechanisms underlying the ability of LDL to induce cell shape changes and whether this could participate in wound repair. Our recent data demonstrates that the capacity of LDL to induce fibroblast spreading relies on their ability to stimulate IL-8 secretion, which in turn leads to accelerated wound healing. LDL-induced IL-8 production and subsequent wound closure are impaired upon inhibition of the p38 MAPK pathway indicating that the LDL-induced spreading and accelerated wound sealing rely on the ability of LDL to stimulate IL-8 secretion in a p38 MAPK-dependent manner. Therefore, regulation of fibroblast shape and migration by lipoproteins may be relevant to atherosclerosis that is characterized by increased LDL-cholesterol levels, IL-8 production and extensive remodeling of the vessel wall. Résumé: La fonction physiologique des lipoprotéines à faible densité (LDL) la mieux décrite est celle du transport du cholestérol aux tissus cibles. Les LDL livrent leur cargaison de cholestérol aux cellules après leur interaction avec le récepteur au LDL. Une concentration vasculaire des LDL augmenté est également impliquée dans le développement de l'athérosclérose. Parmi les types de cellule présents dans les vaisseaux sanguins, les cellules endothéliales et les cellules du muscle lisse ont dominé la recherche cellulaire sur les mécanismes athérosclérotiques et sur l'activation par les LDL des voies de signalisation intracellulaire. A l'inverse peu de choses sont connues sur l'activation des fibroblastes de l'adventice par les lipoprotéines. Puisqu'il a été récemment reconnu que les fibroblastes peuvent jouer un rôle central dans la remodélisation vasculaire et la réparation tissulaire, nous avons étudié si les lipoprotéines affectent la fonction des fibroblastes. Nous avons constaté que les LDL activent spécifiquement une classe de protéines kinases: les p38 MAPK (mitogen-activated protein kinases). L'activation de cette voie module à son tour la forme de la cellule en favorisant la formation de lamellipodes et l'agrandissement des cellules. Cela a un intérêt particulier car il fournit un mécanisme par lequel les LDL peuvent promouvoir la cicatrisation ou la remodélisation des parois vasculaires comme observés lors du développement de l'athérosclérose. Pour comprendre les mécanismes moléculaires par lesquels les LDL provoquent l'activation des p38 MAPK, nous avons cherché à identifier les composants dans la particule de LDL responsables de l'induction de cette voie. Nous avons constaté que le cholestérol est l'élément principal des particules de lipoprotéine qui contrôle leur capacité à stimuler la voie des p38 MAPK. En outre, nous avons examiné les mécanismes cellulaires responsables de la capacité des LDL à induire des changements dans la forme des cellules. Nos données récentes démontrent que la capacité des LDL à induire l'agrandissement des cellules, ainsi que leur aptitude à favoriser la cicatrisation, reposant sur leur capacité à stimuler la sécrétiond'IL-8. La production d'IL-8 induite par les LDL est bloquée par l'inhibition de la voie p38 MAPK, ce qui indique que l'étalement des cellules induit par les LDL ainsi que l'accélération de la cicatrisation sont liés à la capacité des LDL à stimuler la sécrétion d'IL8 via l'activation des p38 MAPK. La régulation de la forme et de la migration des fibroblastes par les lipoprotéines peuvent donc participer au développement de l'athérosclérose qui est caractérisée par l'augmentation des niveaux de production de LDL-cholestérol et d'IL-8 ainsi que par une remodélisation augmentée de la paroi du vaisseau.

Ambulatory blood pressure measurement and antihypertensive therapy.

The traditional basis for assessing the effect of antihypertensive therapy is the blood pressure reading taken by a physician. However, several recent trials have been designed to evaluate the blood pressure lowering effect of various therapeutic agents during the patients' normal daytime activities, using a portable, semi-automatic blood pressure recorder. The results have shown that in a given patient, blood pressure measured at the physician's office often differs greatly from that prevailing during the rest of the day. This is true both in treated and untreated hypertensive patients. The difference between office and ambulatory recorded pressures cannot be predicted from blood pressure levels measured by the physician. Therefore, a prospective study was carried out in patients with diastolic blood pressures that were uncontrolled at the physician's office despite antihypertensive therapy. The purpose was to evaluate the response of recorded ambulatory blood pressure to treatment adjustments aimed at reducing office blood pressure below a pre-set target level. Only patients with high ambulatory blood pressures at the outset appeared to benefit from further changes in therapy. Thus, ambulatory blood pressure monitoring can be used to identify those patients who remain hypertensive only when facing the physician, despite antihypertensive therapy. Ambulatory monitoring could thus help to evaluate the efficacy of antihypertensive therapy and allow individual treatment.

Transcatheter stent-valve implantation in a stenotic pulmonary conduit via a sub-xyphoidian access

Patients who develop a severe stenosis in biological pulmonary conduits previously implanted for pulmonary outflow trunk reconstructions are treated either by surgical re-replacement, or by transcatheter stent-valve implantation through a femoral vein access. A catheter-based sub-xyphoidian access through the right ventricle for stent-valve positioning in a pulmonary conduit has rarely been proposed. We describe the case of a 20-year-old man who underwent a pulmonary trunk reconstruction for a congenital pulmonary valve dysplasia and a few years later developed a stenosis in the pulmonary conduit. He was successfully treated with a 23 mm Edwards Sapien stent-valve implantation in pulmonary position, through an unusual right ventricular, sub-xyphoidian access and without contrast medium injections and pleura opening.

Molecular variation at a candidate gene implicated in the regulation of fire ant social behavior

The fire ant Solenopsis invicta and its close relatives display an important social polymorphism involving differences in colony queen number. Colonies are headed by either a single reproductive queen (monogyne form) or multiple queens (polygyne form). This variation in social organization is associated with variation at the gene Gp-9, with monogyne colonies harboring only B-like allelic variants and polygyne colonies always containing b-like variants as well. We describe naturally occurring variation at Gp-9 in fire ants based on 185 full-length sequences, 136 of which were obtained from S. invicta collected over much of its native range. While there is little overall differentiation between most of the numerous alleles observed, a surprising amount is found in the coding regions of the gene, with such substitutions usually causing amino acid replacements. This elevated coding-region variation may result from a lack of negative selection acting to constrain amino acid replacements over much of the protein, different mutation rates or biases in coding and non-coding sequences, negative selection acting with greater strength on non-coding than coding regions, and/or positive selection acting on the protein. Formal selection analyses provide evidence that the latter force played an important role in the basal b-like lineages coincident with the emergence of polygyny. While our data set reveals considerable paraphyly and polyphyly of S. invicta sequences with respect to those of other fire ant species, the b-like alleles of the socially polymorphic species are monophyletic. An expanded analysis of colonies containing alleles of this clade confirmed the invariant link between their presence and expression of polygyny. Finally, our discovery of several unique alleles bearing various combinations of b-like and B-like codons allows us to conclude that no single b-like residue is completely predictive of polygyne behavior and, thus, potentially causally involved in its expression. Rather, all three typical b-like residues appear to be necessary.

Epidemiological traits of the malaria-like parasite Polychromophilus murinus in the Daubenton¿s bat Myotis daubentonii.

BackgroundThe great diversity of bat haemosporidians is being uncovered with the help of molecular tools. Yet most of these studies provide only snapshots in time of the parasites discovered. Polychromophilus murinus, a malaria-like blood parasite, specialised on temperate-zone bats is a species that is being `rediscovered¿. This study describes the infection dynamics over time and between host sex and age classes.MethodsFor three years we followed the members of three breeding colonies of Myotis daubentonii in Western Switzerland and screened them for the prevalence and parasitemia of P. murinus using both molecular tools and traditional microscopy. In order to identify more susceptible classes of hosts, we measured, sexed and aged all individuals. During one year, we additionally measured body temperature and haematocrit values.ResultsJuvenile bats demonstrated much higher parasitemia than any other age class sampled, suggesting that first exposure to the parasite is very early in life during which infections are also at their most intense. Moreover, in subadults there was a clear negative correlation between body condition and intensity of infection, whereas a weak positive correlation was observed in adults. Neither body temperature, nor haematocrit, two proxies used for pathology, could be linked to intensities of infection.ConclusionIf both weaker condition and younger age are associated with higher infection intensity, then the highest selection pressure exerted by P. murinus should be at the juvenile stage. Confusion over the identities and nomenclature of malarial-like parasites requires that molecular barcodes are coupled to accurate morphological descriptions.

Enjeux ethiques de la psychiatrie sous contrainte [Ethical issues in psychiatry under coercion].

Psychiatry is now subject to two apparently contradictory movements. On the one hand, the need to respect the autonomy and rights of patients is reinforced and coercive measures are strictly defined and limited. On the other hand, security concerns in our society leads to prosecution of psychiatric disorders, especially when accompanied by behavioral problems or criminal acts. In these situations of compulsory treatment or care provided in prisons, a number of dilemmas emerge. The place of the healthcare professional in treatments ordered by the Justice and problems related to administrative detention are discussed in more detail.

Female plumage spottiness and parasite resistance in the barn owl (Tyto alba)

The hypothesis that extravagant ornaments signal parasite resistance has received support in several species for ornamented males but more rarely for ornamented females. However, recent theories have proposed that females should often be under sexual selection, and therefore females may signal the heritable capacity to resist parasites. We investigated this hypothesis in the socially monogamous barn owl, Tyto alba, in which females exhibit on average more and larger black spots on the plumage than males, and in which males were suggested to choose a mate with respect to female plumage spottiness. We hypothesized that the proportion of the plumage surface covered by black spots signals parasite resistance. In line with this hypothesis, we found that the ectoparasitic fly, Carnus hemapterus, was less abundant on young raised by more heavily spotted females and those flies were less fecund. In an experiment, where entire clutches were cross-fostered between nests, we found that the fecundity of the flies collected on nestlings was negatively correlated with the genetic mother's plumage spottiness. These results suggest that the ability to resist parasites covaries with the extent of female plumage spottiness. Among females collected dead along roads, those with a lot of black spots had a small bursa of Fabricius. Given that parasites bigger the development of this immune organ, this observation further suggests that more spotted females are usually less parasitized. The same analyses performed on male plumage spottiness all provided non-significant results. To our knowledge, this study is the first one showing that a heritable secondary sexual characteristics displayed by females reflects parasite resistance.