Adherent bacteria were quantified by plating serial dilutions onto TSA plates and counting resultant colonies. Also the inoculum was plated to determine viable counts. The assay was performed simultaneously in 3 separate wells in duplicate and repeated on 3 different days. Mice Specific pathogen-free 10-week-old female C57BL/6 Selleckchem HM781-36B mice (14 mice in total) were purchased from Harlan Sprague-Dawley (Horst, The Netherlands). The animals were housed in individual cages in rooms with a controlled temperature and a 12-h light-dark cycle. They were acclimatized for 1 week prior to usage, and received
standard rodent chow and water ad libitum. The Animal Care and Use Committee of the University of Amsterdam approved all experiments. Induction of intestinal colonization Mice were administered subcutaneous injections of ceftriaxone
(Roche, Woerden, The Netherlands; 100 μl per injection, 12 mg/ml) 2 times a day, starting 2 days before inoculation of bacteria and continuing for the duration of the experiment. Two days after the initiation of the antibiotic treatment 2 × 109 CFU of E1162 or E1162Δesp in 300 μl TH broth was inoculated by orogastric inoculation using an 18-gauge stainless animal feeding tube. In addition, selleck products in one experiment mice were administered a mixture of an equal amount (1.5 × 109 CFU) of E1162 and E1162Δesp simultaneously. For all experiments, plate-grown bacteria were inoculated in TH broth and grown at 37°C to an OD620 1.0, while shaking. The inoculum was plated to determine viable counts. Mice were sacrificed after 10 days of colonization. Seven mice per group were examined. Collection of samples Stool samples were collected from naive mice, 2 days after antibiotic treatment and 1, 3, 6 and 10 days after bacterial inoculation. Per mice, 2 Adenosine triphosphate stool pellets were collected, pooled, weighed (50–129 mg), and 1 ml of sterile saline was added. After 10 days of colonization mice were anesthetized with Hypnorm® (Janssen Pharmaceutica, Beerse, Belgium; active ingredients fentanyl citrate and fluanisone)
and midazolam (Roche, Meidrecht, The Netherlands), blood was drawn by cardiac puncture and transferred to heparin-gel vacutainer tubes. Mesenteric lymph nodes (MLN) were excised, weighed and collected in 4 volumes of sterile saline. Subsequently, the intestines were excised, opened and fecal contents of small bowel, cecum, and colon were weighed and 1 ml of sterile saline was added. Determination of bacterial outgrowth The number of E. faecium CFU was determined in stool, MLN, blood, and fecal contents of small bowel, cecum, and colon. Stool, MLN, and fecal contents were homogenized at 4°C using a tissue homogenizer (Biospec Products, Bartlesville, UK). CFU were determined from serial dilutions of the homogenates and undiluted blood.
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