suis infection that induces meningitis and brain damage [18–20]. The septicemic phase of S. suis infections is characterized by depression, rough hair coat, swollen eyes, weakness, and death during the first 48 h post-infection. If animals survive this critical step of the disease, they may still develop central nervous system damage and meningitis, with the sudden appearance of nervous signs beginning 3-4 days post-infection, including hyperexcitation, episthotonus, opisthotonus, bending of the head toward one side, and walking in circles [18]. Clinical signs of infection and survival selleck compound were recorded on a daily basis

post-infection for 14 days as previously described [18]. Mice exhibiting extreme lethargy or neurological signs were considered moribund and were humanely euthanized. All experiments involving mice were conducted in accordance with the guidelines and policies of the Canadian Council on Animal Care and the principles set out in the Guide for the Care and Use of Laboratory Animals, and were approved by the Animal Welfare Committee of Université de Montréal. Overall survival rates for the various

groups were calculated using Kaplan-Meier plots. Survival curves were compared using the log-rank test with the Holm-Sidak method used to analyze multiple curves. A p < 0.05 was considered statistically significant. All analyses were performed using the Sigma Plot System (v.11; Systat Software, San Jose, see more CA, USA). Results The S. suis mutant library created Mirabegron by insertion of Tn917 transposon (1,200 mutants) was screened for degradation of the chromogenic substrate N-succinyl-Ala-Ala-Pro-Phe-pNa. Three mutants (G6G, J9G, and M3G) were found to be devoid of activity (A415 < 0.05) compared to parental strain (A415 = 0.85). With the objective to show that only one transposon insertion was present in mutants, chromosomal DNA was analyzed by Southern blotting using a DIG-labeled probe Selleck PKC412 specific for the erm gene

in the Tn917 transposon. As shown in Figure 1, only one Tn917 insertion occurred in the G6G and M3G mutants. Since the J9G mutant had two insertions, we only used the G6G and M3G mutants for further experiments. The mutations were highly stable, with G6G and M3G still unable to degrade the chromogenic substrate after 35 serial transfers in liquid medium (erythromycin-free). Figure 1 Southern blot of S. suis P1/7 and the Tn 917 mutants. Chromosomal DNA was digested with HindIII restriction endonuclease and hybridized with a DIG-labeled probe specific for the erm gene. Lane 1, wild-type parent strain P1/7; lane 2, mutant J9G; lane 3, mutant M3G; lane 4, G6G. To identify which gene was inactivated in mutants, the Tn917 insertion sites in G6G and M3G were sequenced. The affected gene corresponded to a gene coding for the SSU0757 protein in the genome of S. suis P1/7 based on a comparison of the sequence with those of the S. suis Sequencing Group at Sanger Institute.