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 resistance against β-lactam antibiotics.

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 pEVP3^ciaH was constructed for the deletion of ciaH gene and then transformed into the ATCC6306 strain by using the CaCl₂ method. The mutant strain of S. pneumonia strain ATCC6306 with ciaH gene-knockout (ΔciaH) was generated 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 the ΔciaH mutant strain and the wild type strain. The differences between the MICs were further analyzed. 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).

The ciaH gene in the genomic DNA of the generated ΔciaH mutant strain was inactivated by insertion as indicated by PCR and sequencing analysis. Results of the immunofluorescence assay showed that the ΔciaH mutant strain did not express the CiaH protein. The MICs of PCN and CTX against the ΔciaH mutant strain were 32 μg/ml and 64 μg/ml, respectively, which were significantly higher than that of the wild type strain (0.06 μg/ml and 1 μg/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).

The CiaH protein in the CiaH/CiaR two-component signaling system is involved in the resistance of S. pneumoniae against β-lactam antibiotics.

Streptococcus pneumoniae; Two-component signaling system; CiaH protein; β-lactam antibiotics; Drug resistance