To assess toxicokinetics of polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs), oral intake and fecal excretion were measured in two breast-fed infants and one formula-fed infant during the 1st y of life. The intake of these compounds was up to 50 times higher in the breast-fed infants. In these children, fecal excretion of the main tetra- to hexachlorinated congeners was less than 9% of the intake at age of 1 and 5 mo, indicating almost complete intestinal absorption during breast-feeding. In contrast, distinctly higher fecal excretion rates were observed for the hepta- and octachlorinated compounds. Despite much lower PCDD/PCDF intake after weaning, concentrations in stool fat did not decrease substantially. We conclude that concentrations in fecal fat more or less reflect those in body fat. Additionally, PCDD/PCDF concentrations were measured in blood fat of all infants (and in a second formula-fed baby) at the age of 11 mo. International toxicity equivalent (I-TEq) concentrations in the formula-fed infants were less than 25% of maternal values and about 10 times lower than in the infants breast-fed for 6-7 mo. In the latter, a distinct accumulation was found for the tetra- to hexachlorinated congeners compared with maternal concentrations. We conclude that accumulation of PCDDs and PCDFs in infants is as high as expected on the basis of intake data and assuming complete absorption and negligible elimination during the 1st y of life.
A highly porous inorganic material (Anopore) was shown to be an effective support for culturing and imaging a wide range of microorganisms. An inert barrier grid was printed on the rigid surface of Anopore to create a "living chip" of 336 miniaturized compartments (200/cm 2 ) with broad applications in microbial culture.There is a pressing need in microbiology for the development of automated, miniaturized, and multiplexed growth formats that improve on the petri dish and the microtiter plate (1, 3). One approach to microbial culture has been to use flexible organic membranes as a support, permitting growth and imaging in situ (2, 10). We suggest in this work that an inorganic and rigid porous surface may be a better choice for many applications. Anopore is an inert ceramic, an aluminum oxide that is formed in sheets by a high-pressure and etching technique (4,6,8), creating a uniquely porous planar material; up to 50% of the volume is pores. Anopore has been identified as a superior surface for the imaging of microorganisms compared to flexible organic membranes (8, 9), in part due to its flatness and limited autofluorescence. The motivation for this work was to test the suitability of Anopore in the creation of versatile new growth formats or "living chips." Anopore strips (8 by 36 mm; 60 m thick; 0.2-m-diameter pores; 3 ϫ 10 9 pores cm Ϫ2 ) were a gift from PamGene International ('s-Hertogenbosch, The Netherlands). A latex solution (masking fluid 052; Royal Talens, The Netherlands) was applied to one surface in 1-mm-thick lines using a mapping pen and allowed to polymerize at room temperature for 20 min. The strips were then washed with distilled water and twice with 96% (vol/vol) ethanol and air dried. The grid formed a surface barrier 0.5 mm wide and 0.4 mm high that delineated eight culture areas of 7 by 4 mm. The polymerized latex was not strongly autofluorescent: illumination in the 515-to 730-nm range required exposures of Ͼ5 seconds to saturate the chargecoupled device camera. Anopore strips were placed on an appropriate nutrient agar base (5), inoculated on the upper surface at a density of 100 to 2,000 CFU/mm 2 , and then incubated. Microcolonies were stained by transfer of these strips right-side up to a microscope slide covered with a 1-mm-thick film of 1% (wt/vol) solidified low-melting-point agarose (Sigma, The Netherlands) containing 10 M Syto-9 dye plus 40 M propidium iodide (PI) or 5 M hexidium iodide (HI) for bacteria and 20 M Fun-1 for yeasts (7) (all dyes were from Invitrogen, The Netherlands). Staining was for 20 min at room temperature for bacteria and 30°C for yeasts. These procedures allowed staining of the organisms on the Anopore surface through the pores without disruption of the microcolonies. Strips were then imaged directly (without coverslip, immersion oil, or fixative) using an Olympus BX-41 fluorescence microscope equipped with U-MWIBA filters (excitation spectrum of 460 to 490 nm, dichroic mirror splitting at 505 nm, and an emission spectrum of 515 to 550 nm, used for Syto9 an...
A porous inorganic material (Anopore) was employed as a microbial culture and microcolony imaging support. Rapid Anopore-based antibiotic sensitivity testing (AST) methods were developed to assess the growth of clinical isolates, with the primary focus on testing the response of the Enterobacteriaceae to trimethoprim, but with the method supporting a wider applicability in terms of strains and antibiotics. It was possible to detect the growth of Enterobacter aerogenes after 25 min culture and to distinguish a trimethoprim-sensitive from a trimethoprim-resistant strain with 40 min incubation. MIC 90 determinations were made on Anopore; these were in good agreement with the results from the Vitek 2 and E-test methods. The Anopore method correctly identified sensitive (40/40) and resistant (17/17) strains of the Enterobacteriaceae and other Gram-negative rods within only 2-3 h culture. Additionally, a trimethoprim-resistant subpopulation (10 % of population) could be detected by microcolony formation within 2 h, and a smaller subpopulation (1 %) after 3?5 h. These results suggest that this is a viable approach for the rapid AST of purified strains, and that it may be able to deal with mixed populations. The microscopic examination of microcolonies during AST is an advantage of this method which revealed additional information. Filamentation triggered by trimethoprim was discovered in many species of the Enterobacteriaceae for which this phenomenon has not previously been reported. Filamentation was characterized by heterogeneity in terms of cell length, and also uneven nucleic acid distribution and flattening of damaged cells. The development and application of Anopore-based AST within clinical diagnostics is discussed.
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