Selective culture enrichment and sequencing of feces to enhance detection of antimicrobial resistance genes in third-generation cephalosporin resistant Enterobacteriaceae

Metagenomic sequencing of fecal DNA can usefully characterise an individual’s intestinal resistome but is limited by its inability to detect important pathogens that may be present at low abundance, such as carbapenemase or extended-spectrum beta-lactamase producing Enterobacteriaceae. Here we aimed to develop a hybrid protocol to improve detection of resistance genes in Enterobacteriaceae by using a short period of culture enrichment prior to sequencing of DNA extracted directly from the enriched sample. Volunteer feces were spiked with carbapenemase-producing Enterobacteriaceae and incubated in selective broth culture for 6 hours before sequencing. Different DNA extraction methods were compared, including a plasmid extraction protocol to increase the detection of plasmid-associated resistance genes. Although enrichment prior to sequencing increased the detection of carbapenemase genes, the differing growth characteristics of the spike organisms precluded accurate quantification of their concentration prior to culture. Plasmid extraction increased detection of resistance genes present on plasmids, but the effects were heterogeneous and dependent on plasmid size. Our results demonstrate methods of improving the limit of detection of selected resistance mechanisms in a fecal resistome assay, but they also highlight the difficulties in using these techniques for accurate quantification and should inform future efforts to achieve this goa

Compartmentalized Culture of Perivascular Stroma and Endothelial Cells in a Microfluidic Model of the Human Endometrium

The endometrium is the inner lining of the uterus. Following specific cyclic hormonal stimulation, endometrial stromal fibroblasts (stroma) and vascular endothelial cells exhibit morphological and biochemical changes to support embryo implantation and regulate vascular function, respectively. Herein, we integrated a resin-based porous membrane in a dual chamber microfluidic device in polydimethylsiloxane that allows long term in vitro co-culture of human endometrial stromal and endothelial cells. This transparent, 2-m porous membrane separates the two chambers, allows for the diffusion of small molecules and enables high resolution bright field and fluorescent imaging. Within our primary human co-culture model of stromal and endothelial cells, we simulated the temporal hormone changes occurring during an idealized 28-day menstrual cycle. We observed the successful differentiation of stroma into functional decidual cells, determined by morphology as well as biochemically as measured by increased production of prolactin. By controlling the microfluidic properties of the device, we additionally found that shear stress forces promoted cytoskeleton alignment and tight junction formation in the endothelial layer. Finally, we demonstrated that the endometrial perivascular stroma model was sustainable for up to 4 weeks, remained sensitive to steroids and is suitable for quantitative biochemical analysis. Future utilization of this device will allow the direct evaluation of paracrine and endocrine crosstalk between these two cell types as well as studies of immunological events associated with normal versus disease-related endometrial microenvironments

A qualitative study exploring the experiences of parents of children admitted to seven Dutch pediatric intensive care units

Purpose: To explore parents' experiences during the admission of their children to a pediatric intensive care unit (PICU). Method: Qualitative method using in-depth interviews. Thematic analysis was applied to capture parents' experiences. Thirty-nine mothers and 25 fathers of 41 children admitted to seven of the eight PICUs in university medical centers in The Netherlands were interviewed. Results: Parents were interviewed within 1 month after their child's discharge from a PICU. Thematic analysis identified 1,514 quotations that were coded into 63 subthemes. The subthemes were categorized into six major themes: attitude of the professionals; coordination of care; emotional intensity; information management; environmental factors; parent participation. Most themes had an overarching relationship representing the array of experiences encountered by parents when their child was staying in a PICU. The theme of emotional intensity was in particular associated with all the other themes. Conclusions: The findings provided a range of themes and subthemes describing the complexity of the parental experiences of a PICU admission. The subthemes presen

Regulation of human endometrial function: mechanisms relevant to uterine bleeding

This review focuses on the complex events that occur in the endometrium after progesterone is withdrawn (or blocked) and menstrual bleeding ensues. A detailed understanding of these local mechanisms will enhance our knowledge of disturbed endometrial/uterine function – including problems with excessively heavy menstrual bleeding, endometriosis and breakthrough bleeding with progestin only contraception. The development of novel strategies to manage these clinically significant problems depends on such new understanding as does the development of new contraceptives which avoid the endometrial side effect of breakthrough bleeding

Effects of tibolone and its metabolites on prolactin and insulin-like growth factor binding protein-1 expression in human endometrial stromal cells

The effects of the postmenopausal replacement steroid tibolone and its 3 alpha-, 3 beta-OH and Delta-4 tibolone metabolites were evaluated on progesterone receptor-mediated classic decidualization markers insulin-like growth factor binding protein-1 (IGFBP-1) and prolactin expression in human endometrial stromal cells (HESCs). Supernatants of conditioned medium or erxtracted RNA from experimental cell incubations of confluent HESCs were subjected to ELISAs, Western blot analysis and RT/PCR, and results were statisically assesed. Over 21 days, specific ELISAs observed linear increases in secreted IGFBP-1 and prolactin levels elicited by tibolone and its metabolites. Cultured HESCs were refractory to E2 and dexamethasone, whereas tibolone and each metabolite exceeded medroxyprogesterone acetate in significantly elevating IGFBP-1 and prolactin output. Anti-progestins eliminated IGFBP-1 and prolactin induction by tibolone and its metabolites. lmmunoblotting and RT/PCR confirmed ELISA results. These observations of IGFBP-1 and prolactin expression: (a) indicate the relevance of cultured HESCs in evaluating the chronic effects of tibolone administration to women; (b) are consistent with PR-mediated endometrial atrophy and protection against endometrial bleeding despite the persistence of circulating ER-binding, but not PR-binding metabolites following tibolone administration to women