Study On Resistance Of Citrobacter To β-Lactams And Its Correlated Resistant Genes

OBJECTIVE:Study the resistance of Citrobacter clinical isolates to beta-lactams, identify the ESBLs-producing Citrobacter and their correlated resistant genes, and analyze the structural features of CTX-M type ESBLs.METHODS:1.The minimal inhibitory concentrations (MIC) of beta-lactams against 24 Citrobacter clinical isolates in vitro were detected with double broth dilution method. ESBLs-containing strains were determined by using disc diffusion method. The beta-lactamases from the ESBLs-containing strains were extracted and the enzyme activities were detected.2. Three pairs of oligonucleotide primers targeted blaTEM, blaSHV, and blaCTX-M were designed. The genotype of the ESBLs-producing strains were determined by using Polymerase Chain Reaction (PCR).3. S240G mutants of extended-spectrumβ-lactamase CTX-M were produced by site-directed mutagenesis. The enzyme activities before and after mutagenesis were detected and compared. RESULTS:1. The resistance rates of 24 Citrobacter clinical isolates were as followings: AMP 100%, CEF 100%, FAM 87.50%, CFP83.33%, CFZ 75%, PIP 70.83%,CTX66.67%,FEP 62.5%,SAM 58.33%,CAZ 54.17%,CAZ/SB 50%, CRO 33.33%, TZP 12.5%,FD 4.17%, IMI 0%. 12 of 24 isolates expressed ESBLs.2. PCR amplification showed that eight Citrobacter isolates had blaCTX-M, three had blaTEM and two had blaSHV. One Citrobacter isolate expressed both blaTEM and blaCTX-M.3. S240G mutants of extended-spectrumβ-lactamase CTX-M were constructed.β-lactamase extracted from JM109 transformed with recombinant plasmid expressing wild CTX-M showed high enzyme activity, however,β-lactamase extracted from JM109 transformed with recombinant plasmid expressing mutant CTX-M showed no enzyme activity.CONCLUSIONS:1. Imipenem is the most powerful antibacterial agent against Citrobacter infection in our area.2. blaCTX-M is the main genotype of Citrobacter clinical isolates in our hospital.3. S240G mutants of extended-spectrumβ-lactamase CTX-M can be constucted by using PCR based on site-directed mutagenesis. Ser240 is critical in maitaining the enzyme activity of the extended-spectrumβ-lactamase CTX-M.