Plates were then incubated anaerobically at 37 °C for 48–72 h. Transformants were cultivated on cMRS
supplemented with chloramphenicol at a final concentration of 3 μg mL−1. DNA was extracted from colonies using GeneReleaser (BioVentures), and the AZD8055 ic50 presence of pNZ8048 in transformants was confirmed by PCR using the primers pNZFw (5′-TTTGCAGCGAAGATGTTGTC-3′) and pNZRv (5′-CTATAGCTAACGCCGCAACC-3′) targeting DNA regions on this plasmid. The transformation efficiency was calculated according to the following formula: Transformation experiments were performed in triplicate. Transformants were inoculated into fresh broth in the presence of chloramphenicol and grown for 24 h. These cultures were then screened for plasmid Selleckchem BI 6727 content prior to the start of the experiment to ensure that plasmid pNZ8048 was present.
Cultures were then diluted (1%) in fresh broth without chloramphenicol, followed by continuous subcultivation for 15 days by dilution into fresh broth every 24 h in the absence of antibiotic selection. To determine plasmid stability, at least 50 colonies from each tested transformant were transferred to cMRS agar plates with or without chloramphenicol (3 μg mL−1). Growth of these colonies was monitored following 24 h of incubation, and plasmid extractions were performed where relevant. All animals used in this study were cared for in compliance with guidelines established by the Italian Ministry of Health. All procedures were approved by the University of Parma, as executed by the Institutional Animal Care and Use Committee (Dipartimento per la Sanità Pubblica Veterinaria, la Nutrizione e la Sicurezza degli Alimenti Direzione Generale della Sanità Animale e del Farmaco Veterinario). Two groups, each containing six animals of 3-month-old
female BALB/c mice, were orally inoculated with bacteria or with water. Bacterial colonization was established by five consecutive daily administrations whereby each animal received 20 μL of 109 mL−1 of cells using a micropipette AMP deaminase tip placed immediately behind the incisors (Sleator et al., 2001). Bifidobacterial inocula were prepared by growing B. bifidum PRL2010 containing pNZ8048 anaerobically overnight at 37 °C in cMRS broth containing 3 μg mL−1 chloramphenicol. Cultures were harvested by centrifugation (950 g for 8 min), washed, and resuspended in 100 μL of water. The viable count of each inoculum was determined by retrospective plating on cMRS containing the antibiotic. To estimate the number of B. bifidum PRL2010 cells per gram of feces, individual fecal samples were weighed and followed by serial dilution and culturing on selective cMRS agar with chloramphenicol. Following enumeration of B.