ABSTRACT
Objective:To investigate the relationship between ciaH, eno, pykF genes and fluoride resistance through determining the differential expressions of ciaH, eno and pykF genes of fluoride-resistant Streptococcus mutans cultivated in fluoride environment. Methods:The cultured Streptococcus mutans and their fluoride-resistant strains were divided into UA (Streptococcus mutans subcultured in BHI without NaF), FR (fluoride-resistant Streptococcus mutans subcultured in BHI without NaF) and FFR (fluoride-resistant Streptococcus mutans subcultured in BHI containing 1 g·L-1 NaF) groups.After 11 h (logarithmic phase) and 20 h (platform stage) cultivation, the expression levels of ciaH, eno and pykF mRNA were detected by RT-PCR method.Results:Compared with FR group, the expression levels of ciaH, eno and pykF mRNA in FFR group were increased both in the logarithmic phase and the platform stage(P0.05), but it was increased in the platform stage (P<0.01).Conclusion:Fluoride can increase the expression levels of ciaH, eno and pykF genes in fluoride-resistant Streptococcus mutans, indicating that these genes are related to the production of fluoride resistance.
ABSTRACT
Objective To construct a mutant strain of Streptococcus pneumoniae ( S.pneumoniae) with ciaH gene-knockout (ΔciaH) and to analyze the correlation between the ciaH gene and the bacterial re-sistance against β-lactam antibiotics.Methods The ciaH gene segament of S.pneumoniae strain ATCC6306 was amplified by PCR.The PCR product was sequenced after T-A cloning.A suicide plasmid pEVP3ciaH was constructed for the deletion of ciaH gene and then transformed into the ATCC 6306 strain by using the CaCl2 method .The mutant strain of S.pneumonia strain ATCC6306 with ciaH gene-knockout (ΔciaH) was genera-ted through homologous recombination , insertion inactivation and amphemycin screening , which was further identified by PCR , sequencing analysis and laser confocal microscopy .Double agar dilution method was used to detect the minimal inhibitory concentrations ( MICs ) of penicillin G ( PCN ) and cefotaxime ( CTX ) against theΔciaH mutant strain and the wild type strain .The differences between the MICs were further ana-lyzed.The changes of ciaH gene expression at mRNA level after treatment with 1/4 MIC of PCN or CTX were detected by real-time fluorescent quantitative RT-PCR ( qRT-PCR ) .Results The ciaH gene in the genomic DNA of the generated ΔciaH mutant strain was inactivated by insertion as indicated by PCR and se-quencing analysis .Results of the immunofluorescence assay showed that the ΔciaH mutant strain did not ex-press the CiaH protein .The MICs of PCN and CTX against the ΔciaH mutant strain were 32 μg/ml and 64μg/ml, respectively, which were significantly higher than that of the wild type strain (0.06 μg/ml and 1μg/ml) (P<0.01).The expression of ciaH gene at mRNA level was significantly elevated after treatment S.pneumoniae ATCC6306 strain with 1/4 MIC PCN or CTX (P<0.01).Conclusion The CiaH protein in the CiaH/CiaR two-component signaling system is involved in the resistance of S.pneumoniae against β-lac-tam antibiotics.