8 −2.0 * Decrease in the expression of nanI in NCTRR and increase of its expression in 13124R was confirmed by qRT-PCR. All of the data are the means of three different experiments. Validation of DNA microarray data by qRT-PCR To verify that fluoroquinolone resistance selection indeed had different effects on the expression of some of the genes in C. perfringens, the transcription of the genes that were generally Rho inhibitor upregulated or unchanged in NCTRR and downregulated in 13124R was measured by qRT-PCR (Table 1). Real-time PCR verified the upregulation of all of the genes that were tested in NCTRR and HIF inhibitor downregulation of a majority of the genes that were downregulated in 13124R. qRT-PCR

was also performed on the genes that are reported to have regulatory functions (Table 4). virR, virS, vrr, virX and others were all upregulated in NCTRR by at least twofold. In strain 13124R, virX was downregulated more than twofold, but vrr also was substantially downregulated. Among the genes whose expression was altered by fluoroquinolone resistance selection were phospholipase C (PLC), perfringolysin O (PFO), α-clostripain, hemolysin III, and collagenase. Both microarray analysis and qRT-PCR showed upregulation of these genes in NCTRR

and downregulation in 13124R. Both microarray and qRT-PCR showed downregulation of the sialidase gene, Captisol nanI, in NCTRR and upregulation of this gene in 13124R. Table 4 Results of qRT-PCR for the C. perfringens regulatory genes in the wild types and mutants Gene ID and name Regulatory function qRT-PCR fold       change (mt/wt)       NCTR ATCC13124 CPE_1501 CPF_1752 (virR) DNA binding

response regulator, VirR 7.4 1.3 CPE_1500 CPF_1751 (virS) sensor histidine kinase, VirS 9.7 0.3 CPE_0646 CPF_0627 (virX) conserved hypothetical protein 2.2 −3.0 CPE_0957 CPF_1204 (vrr) VR-RNA 2.0 −158.5 CPE_1701 CPF_1955 (codY) GTP-sensing transcriptional pleiotropic repressor CodY 6.9 −1.8 CPE_0073 CPF_0069 Transcription antiterminator 1.5 −116.5 CPE_0642 CPF_0623 (RevR) DNA binding response regulator 2 −2 Toxin production in the mutants and wild types The quantities of several enzymes that Metalloexopeptidase are implicated in bacterial virulence were measured for each absorbance unit of cells of wild types and mutants of both strains (Figures 1 and 2). The production of phospholipase C (PLC), perfringolysin O (PFO), collagenase, clostripain, and sialidase were all affected in the resistant mutant. Strain 13124R produced less PLC and PFO than the wild type. In contrast, as previously reported [30], the production of both enzymes increased in NCTRR. Collagenase and clostripain production also were similarly affected by fluoroquinolone resistance selection, but the most dramatic effect was for perfringolysin O (PFO) in ATCC 13214, which was totally inhibited in 13124R. However, sialidase had increased in 13124R but decreased in NCTRR. Hyaluronidase was not significantly affected.