Seasonality in carriage of extended-spectrum ß-lactamase-producing Escherichia coli and Klebsiella pneumoniae in the general population: a pooled analysis of nationwide cross-sectional studies.

Infections due to extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-E) are often preceded by asymptomatic carriage. Higher incidences in enteric infectious diseases during summer have been reported. Here, we assessed whether the presence of seasonality in intestinal ESBL-Escherichia coli/Klebsiella pneumoniae (ESBL-E/K) carriage in the general Dutch population exists. From 2014 to 2017, the faecal carriage of ESBL-E/K in healthy individuals was determined in three cross-sectional studies in the Netherlands, including 5985 subjects. Results were pooled to identify seasonal trends in prevalence (by month of sampling). Multivariate logistic regression analysis was used to calculate pooled odds ratios and 95% confidence intervals. Results were adjusted for age, sex, antibiotic use and travel. Overall prevalence of ESBL-E/K carriage was 4.3% (n = 260 ESBL-E/K-positive), with differences between months ranging from 2.6% to 7.4%. Compared to January, the monthly prevalence of ESBL-E carriage was highest in August (OR 1.88, 95% CI 1.02-3.49) and September (OR 2.25, 95% CI 1.30-3.89). The observed monthly differences in ESBL-E/K carriage rates suggest that there is seasonal variation in exposure to ESBL-E/K other than due to travelling and antibiotic use. This should be taken into account in designing future ESBL-E prevalence studies in temperate regions.

Faecal carriage, risk factors, acquisition and persistence of ESBL-producing Enterobacteriaceae in dogs and cats and co-carriage with humans belonging to the same household.

BACKGROUND: ESBL-producing Enterobacteriaceae (ESBL-E) are observed in many reservoirs. Pets might play an important role in the dissemination of ESBL-E to humans since they live closely together. OBJECTIVES: To identify prevalence, risk factors, molecular characteristics, persistence and acquisition of ESBL-E in dogs and cats, and co-carriage in human-pet pairs belonging to the same household. METHODS: In a nationwide study, one person per household was randomly invited to complete a questionnaire and to submit a faecal sample. Dog and cat owners were invited to also submit a faecal sample from their pet. Repeated sampling after 1 and 6 months was performed in a subset. ESBL-E were obtained through selective culture and characterized by WGS. Logistic regression analyses and random forest models were performed to identify risk factors. RESULTS: The prevalence of ESBL-E carriage in these cohorts was 3.8% (95% CI: 2.7%-5.4%) for human participants (n=550), 10.7% (95% CI: 8.3%-13.7%) for dogs (n=555) and 1.4% (95% CI: 0.5%-3.8%) for cats (n=285). Among animals, blaCTX-M-1 was most abundant, followed by blaCTX-M-15. In dogs, persistence of carriage was 57.1% at 1 month and 42.9% at 6 months. Eating raw meat [OR: 8.8, 95% CI: 4.7-16.4; population attributable risk (PAR): 46.5%, 95% CI: 41.3%-49.3%] and dry food (OR: 0.2, 95% CI: 0.1-0.5; PAR: 56.5%, 95% CI: 33.2%-66.6%) were predictors for ESBL-E carriage in dogs. Human-dog co-carriage was demonstrated in five households. Human-cat co-carriage was not observed. CONCLUSIONS: ESBL-E prevalence was higher in dogs than in humans and lowest in cats. The main risk factor for ESBL-E carriage was eating raw meat. Co-carriage in dogs and household members was uncommon.

Intestinal carriage of ampicillin- and vancomycin-resistant Enterococcus faecium in humans, dogs and cats in the Netherlands.

Background: The prevalence of ampicillin- and/or vancomycin-resistant Enterococcus faecium (AREf and VREf) has increased in hospitalized patients in the Netherlands. Objectives: To quantify the prevalence, risk factors and co-carriage of AREf and VREf in humans, cats and dogs in the Dutch population. Methods: From 2014 to 2015, ∼2000 inhabitants of the Netherlands each month were randomly invited to complete a questionnaire and provide a faecal sample. Subjects owning pets were also asked to submit one dog or cat sample. Faecal samples were screened for AREf and VREf. The genetic relatedness of isolates was determined using core genome MLST. Logistic regression analysis was used to determine risk factors. Results: Of 25 365 subjects, 4721 (18.6%) completed the questionnaire and 1992 (42.2%) human, 277 dog and 118 cat samples were submitted. AREf was detected in 29 human (1.5%), 71 dog (25.6%) and 6 cat (5.1%) samples. VREf (vanA) was detected in one human and one dog. AREf/VREf co-carriage was not detected in 388 paired samples. The use of antibiotics (OR 4.2, 95% CI 1.7-11.2) and proton pump inhibitors (OR 2.7, 95% CI 1.1-6.3) were risk factors for AREf carriage in humans. In dogs, these were the use of antibiotics (OR 2.3, 95% CI 1.1-4.6) and eating raw meat (OR 3.2, 95% CI 1.4-6.6). Core genome MLST-based phylogenetic linkage indicated clonal relatedness for a minority of human (16.7%) and pet AREf isolates (23.8%) in three clusters. Conclusions: Intestinal carriage with AREf or VREf is rare in the Dutch general population. Although AREf carriage is high in dogs, phylogenetic linkage between human and pet AREf isolates was limited.

ESBL/AmpC-producing Enterobacteriaceae in households with children of preschool age: prevalence, risk factors and co-carriage.

OBJECTIVES: ESBL/AmpC-producing Enterobacteriaceae are an emerging public health concern. As households with preschool children may substantially contribute to the community burden of antimicrobial resistance, we determined the prevalence, risk factors and co-carriage of ESBL/AmpC-producing bacteria in preschool children and their parents. METHODS: From April 2013 to January 2015, each month 2000 preschool children were randomly selected from Dutch population registries. The parents were invited to complete an epidemiological questionnaire and to obtain and send a faecal sample from the selected child and from one parent. Samples were tested for ESBL/AmpC-producing bacteria. Logistic regression was used to identify risk factors for ESBL/AmpC carriage in children and parents, and findings were internally validated by bootstrapping. RESULTS: In total, 1016 families were included and ESBL/AmpC prevalence was 4.0% (95% CI 3.2%-5.0%); 3.5% (95% CI 2.5%-4.8%) in children and 4.5% (95% CI 3.4%-6.0%) in parents. Attending a daycare centre (DCC) was the only significant risk factor for children (OR 2.1, 95% CI 1.0-4.3). For parents, the only significant risk factor was having one or more children attending DCCs (OR 2.2, 95% CI 1.2-4.8). For parents of ESBL/AmpC-positive children the OR for ESBL/AmpC carriage was 19.7 (95% CI 9.2-42.4). Co-carriage of specific ESBL/AmpC genotypes in child and parent occurred more often than expected by chance (14.6% versus 1.1%, P < 0.001). CONCLUSIONS: In this study, intestinal carriage with ESBL/AmpCs was detected in ∼4% of households with preschool children. DCC attendance was a risk factor in both children and parents and co-carriage of specific genotypes frequently occurred in child-parent pairs. These findings suggest household transmission or/and family-specific exposure to common sources of ESBL/AmpC-producing bacteria.

Cathepsin B launches an apoptotic exit effort upon cell death-associated disruption of lysosomes.

The release of cathepsin proteases from disrupted lysosomes results in lethal cellular autodigestion. Lysosomal disruption-related cell death is highly variable, showing both apoptotic and necrotic outcomes. As the substrate spectrum of lysosomal proteases encompasses the apoptosis-regulating proteins of the Bcl-2 family, their degradation could influence the cell death outcome upon lysosomal disruption. We used Förster resonance energy transfer (FRET)-based biosensors to image the real-time degradation of the Bcl-2-family members, Bcl-xl, Bax and Bid, in living cells undergoing lysosomal lysis and identified an early chain of proteolytic events, initiated by the release of cathepsin B, which directs cells toward apoptosis. In this apoptotic exit strategy, cathepsin B's proteolytic activity results in apoptosis-inducing Bid and removes apoptosis-preventing Bcl-xl. Cathepsin B furthermore appears to degrade a cystein protease that would otherwise have eliminated apoptosis-supporting Bax, indirectly keeping cellular levels of the Bax protein up. The concerted effort of these three early events shifts the balance of cell fate away from necrosis and toward apoptosis.

The role of social cohesion among residents in a therapeutic community.

According to the Therapeutic Community (TC) treatment approach, social affiliation with the drug-free peer community is the basis for patients initiating therapeutic change. A total of 322 TC residents were assessed with regard to social affiliation, acceptance of TC philosophy, perceived benefit of program components, level of depression, and length of time in treatment. Residents exhibited a higher level of affiliation with TC members than with untreated substance abusers they knew outside the program. Being female, and separated from a spouse, were each associated with a higher level of TC member affiliation. After controlling for sociodemographic characteristics, two variables emerged as unique significant correlates of affiliation: perceived benefit for recovery of TC treatment was associated with greater TC member affiliation, whereas level of depression was inversely correlated. These findings are interpreted in relation to the goals of the TC process and to implications for treatment.

Regulation of cytosolic phospholipase A(2) in a new perspective: recruitment of active monomers from an inactive clustered pool.

cPLA(2) plays a key role in many signal transduction cascades by hydrolyzing arachidonic acid from membrane phospholipids. Tight control of cPLA(2) activity by a number of regulatory mechanisms is essential to its cellular function. We recently described the localization of cPLA(2) in clusters in fibroblasts and now propose that these clusters reflect a localized inactive pool from which active monomers can be recruited to keep cPLA(2) activity under control on the subcellular level. Using an electron microscopic in vitro approach, we show that cPLA(2) monomers, but not the clusters, bind to membranes in a Ca(2+)-dependent manner. This binding is accompanied by hydrolytic activity. The present data combined with our previous observation of a relative abundance of clusters over monomers in fixed fibroblasts [Bunt, G., de Wit, J., van den Bosch, H., Verkleij, A., and Boonstra, J. (1997) J. Cell Sci. 110, 2449-2459] gives rise to a concept of cPLA(2) regulation in which small amounts of active monomers are recruited to fulfill their function upon stimulation. This is in contrast to processes described for inflammatory cells, where a substantial part of the cytoplasmically localized cPLA(2) translocates to the perinuclear region upon stimulation to become active. Small-scale regulation of cPLA(2) by the proposed cluster-monomer cycle allows local and strictly confined control of cPLA(2) activity, apparently necessary for its cellular role in fibroblasts.

Ultrastructural localization of cPLA2 in unstimulated and EGF/A23187-stimulated fibroblasts.

The 85 kDa cytosolic phospholipase A2 is the key enzyme in the release of arachidonic acid. To gain insight into cytosolic phospholipase A2 action in mitogen-activated cells, the localization of the phospholipase was investigated in fibroblasts upon stimulation with epidermal growth factor and the calcium ionophore A23187. By the use of indirect immunofluorescence microscopy, staining of endogenous cytosolic phospholipase A2 resulted in a punctate labeling pattern randomly distributed throughout the cytoplasm of the cell. Immunogold electron microscopy revealed that this punctate labeling pattern exhibited the presence of the 85 kDa phospholipase A2 in small clusters. These clusters were found in the cytosol in the vicinity of all organellar membranes, except for the Golgi system. The enzyme showed no preference for the nuclear envelope, the endoplasmic reticulum or the plasma membrane. Stimulation of cells with epidermal growth factor or A23187 or both did not change the punctate immunofluorescence labeling pattern. Furthermore, a similar labeling pattern was observed by the artificial introduction of extremely low or high intracellular calcium concentrations. Even by electron microscopy, translocation of cytosolic phospholipase A2 to membranes was not observed after stimulation of cells with epidermal growth factor and A23187. From these results it is concluded that cytosolic phospholipase A2 is localized in clusters close to membranes in stimulated as well as unstimulated fibroblasts, without preference for a specific organellar membrane.

Maximal epidermal growth-factor-induced cytosolic phospholipase A2 activation in vivo requires phosphorylation followed by an increased intracellular calcium concentration.

The 85 kDa cytosolic phospholipase A2 (cPLA2) preferentially catalyses the hydrolysis of arachidonic acid from the sn-2 position of phospholipids. cPLA2 can be activated by extracellular stimuli such as thrombin, platelet-derived growth factor and epidermal growth factor (EGF): A full activation of cPLA2 requires an increase of intracellular Ca2+ concentration and phosphorylation on Ser-505 by mitogen-activated protein (MAP) kinase. Because EGF can provoke an increase in intracellular [Ca2+] ([Ca2+]i) and activation of MAP kinase, we investigated the role of these pathways in EGF-induced activation of cPLA2. Characterization of two cell lines expressing different numbers of EGF receptors (HERc13 and HER14) revealed that both were activating MAP kinase in response to EGF, but only HER14 responded with an increase in [Ca2+]i. In this study we used both cell lines as a tool to clarify the role of each pathway in cPLA2 activation. We show that EGF stimulates cPLA2 activity in both cell lines in vitro as measured in cytosolic fractions, but only in HER14 in vivo as measured by 3H release from cells prelabelled with [3H]arachidonic acid. This latter activation can be restored in HERc13 cells by the addition of the ionophore A23187. Interestingly, this effect is only observed when EGF stimulation precedes A23187 addition. The phosphorylation of MAP kinase, however, was identical under identical conditions. We conclude that a maximal cPLA2 activation by EGF requires both, and in this order: MAP kinase activation followed by a rise in [Ca2+]i concentration.

Arabidopsis mutants with a reduced seed dormancy.

The development of seed dormancy is an aspect of seed maturation, the last stage of seed development. To isolate mutants of Arabidopsis thaliana that are affected in this process, we selected directly for the absence of dormancy among freshly harvested M2 seeds. The screen yielded two mutants exhibiting a reduced dormancy, rdo1 and rdo2, that are specifically affected in dormancy determined by the embryo. The rdo1 and rdo2 mutants show normal levels of abscisic acid and the same sensitivity to abscisic acid, ethylene, auxin, and cytokinin as the wild type. The rdo2 mutant but not the rdo1 mutant has a reduced sensitivity to the gibberellin biosynthesis inhibitor tetcyclacis. Double-mutant analysis suggested that the RDO1 and RDO2 genes are involved in separate pathways leading to the development of dormancy. We assume that the RDO2 gene controls a step in the induction of dormancy that is most likely induced by abscisic acid and is expressed as an increase of the gibberellin requirement for germination.

Enhanced Mg(2+)-ATPase activity in ghosts from HS erythrocytes and in normal ghosts stripped of membrane skeletal proteins may reflect enhanced aminophospholipid translocase activity.

Hereditary spherocytosis (HS) is a congenital haemolytic anaemia which is characterized by a great variety of structural defects in the red cell's membrane skeleton and/or deficiencies in particular membrane (skeletal) proteins. Enhanced (Mg2+)-dependent adenosine triphosphatase (Mg(2+)-ATPase) activities, varying from 115% to 160%, were invariably found in erythrocyte ghosts derived from 13 HS patients. Similarly, an enhancement of Mg(2+)-ATPase activity by 30% is observed in normal red cell ghosts that have been stripped of the greater part of their membrane skeletal proteins by treatment with a low ionic strength buffer. Reassociation of those stripped ghosts with spectrin reduces the enhanced Mg(2+)-ATPase activity to its original level. Since in both cases, HS ghosts and stripped normal ghosts, the stabilizing effects that the membrane skeleton exerts on the maintenance of an endofacial localization of the aminophospholipids are impaired, the enhanced Mg(2+)-ATPase activity is interpreted to reflect an increased activity of the aminophospholipid translocase. The present observations therefore support a role of the membrane skeleton in the stabilization of phospholipid asymmetry in the red cell membrane and consequently in reducing the energy consumption of the translocase.

New perspectives in the legal psychiatry of cocaine-related crimes.

The legal criteria for the insanity defense as it applies to cocaine-related crimes remains elusive because of cocaine's unique spectrum of effects on human thought and action. This paper discusses the literature relevant to cocaine and forensic psychiatry/psychology, and summarizes the results of a survey of forensic psychiatrists on the topic of drug-induced psychosis. A conceptual framework is posited for the expert witness to distinguish the separable effects of cocaine on human behavior and to clarify their relationship to criminal responsibility.

Cocaine/"crack" dependence among psychiatric inpatients.

The authors studied 40 cocaine-dependent subjects admitted to psychiatric inpatient wards of a metropolitan hospital because of general psychiatric symptoms. The results indicate that the predominant form of cocaine administration (88%) was freebasing "crack." DSM-III-R cluster B personality disorders (N = 17) and schizophrenia (N = 13) constituted the diagnoses for 75% of the sample. Compared to the schizophrenic patients in this cohort, the patients with cluster B personality disorders used cocaine in greater quantities and more frequently and began abuse of the drug at an earlier age. The escalation in urban areas of psychiatric hospitalizations attributed to use of crack may be largely related to psychiatric symptoms in cocaine-dependent patients with personality disorders as well as cocaine-induced psychopathology in schizophrenic patients.