Colonies were Trichostatin A chemical structure counted, tested by PCR to confirm species identity, and corrected for the dilution factor to calculate CFU per gram of stool/MLN/fecal contents. MLVA was performed
to confirm strain identity. PCR analysis to confirm species Stool samples from naïve mice and from mice treated for 2 days with ceftriaxone were examined for presence of E. faecium. The lowest dilutions of stool homogenates that contained well-separated selleck inhibitor colonies were chosen and each colony of that dilution (12–24 CFU/20 μl diluted stool homogenate) was tested by PCR for presence of the housekeeping gene ddl (encoding D-alanine, D-alanine ligase) using the E. faecium specific primers ddlF (5′-GAG ACA TTG AAT ATG CCT) and ddlR (5′-AAA AAG AAA TCG CAC CG) . The colonies were directly diluted in 25-μl-volumes with HotStarTaq Master Mix (QIAQEN Inc., Valencia, CA). PCR’s were performed with a 9800 Fast Thermal Cycler (Applied GSK1838705A mw Biosystems, Foster City, CA) and the PCR amplification conditions were as follows: initial denaturation at 95°C for 15 min, followed by 10 touchdown cycles starting at 94°C for 30 s, 60°C for 30 s, and 72°C (the time depended on the size of the PCR product) with the annealing temperature decreasing by 1°C per cycle, followed by 25 cycles with an annealing temperature of 52°C. All primers used in this study were purchased from Isogen Life Science (IJselstijn, The Netherlands).
For mono infection, colonies obtained from stool (1, 3, 6, and 10 days after bacterial inoculation), MLN, and fecal contents from small bowel, cecum, and colon were examined to confirm species identity. Colonies were randomly picked and presence MycoClean Mycoplasma Removal Kit of the ddl gene, in case E1162 was inoculated, or the cat gene, in case E1162Δesp was inoculated, was assessed by PCR using primer pairs ddlF – ddlR and CmF (5′-GAA TGA CTT CAA AGA GTT TTA TG) – CmR (5′-AAA GCA TTT TCA GGT ATA GGT G) , respectively. When both strains
were inoculated simultaneously, all colonies from the lowest dilution with well-separated colonies were picked (3–28 CFU/20 μl diluted homogenate). Species identity and the number of E1162 and E1162Δesp were determined by multiplex PCR using primer pairs ddlF – ddlR and CmF – CmR. In PCR’s, a colony of E1162 and E1162Δesp was used as positive control and a colony of E. faecalis V583  was used as negative control. MLVA to confirm strain identity For both mono infection and mixed infection, colonies obtained from stool (1, 3, 6, and 10 days after bacterial inoculation), MLN, and fecal contents from small bowel, cecum, and colon were randomly picked and MLVA was performed to confirm strain identity. MLVA was performed as described previously . Histological examination Small bowel, cecum and colon tissue were fixed in 4% buffered formalin and embedded in paraffin. Four-micrometer-thick sections were stained with hematoxylin-eosin and analyzed.