| The status of drug resistance caused by unreasonable application of antibiotics was becoming severer and severer. It was a worldwide difficult problem in therapy in infections. One of the methods to deal with this problem was that the treatment should be based on the data of drug resistance caused by infectious agents. Detection of drug resistance was still based on traditional susceptibility test. The most disadvantage was that the cycle of diagnose for treatment was so long that doctors could not make the correct choices at the beginning of infections. Drug resistant gene could be detected by hybridization by gene chip directly. It was an efficient technology to determine the drug resistance of pathogen quickly and sensitively, which should provide evidences to choose the reasonable antibiotics.β-lactam antibiotics were used to treat infections caused by Gram-Negative bacillus, which were the familiar pathogenic bacterium now.β-lactamase was mostly produced by gram-negative clinical isolates, responding to resistance to β-lactam antibiotics. It bad a great variety, including AmpC enzyme, ESBLs and so on. AmpC enzyme was belonging to group I of Bush and group C of Ambler and was encoded by ampC gene. We found that the sequences of ampC gene were remarkable different in different genera of strains. Objective To analyze the relationship between the status of drug resistance and the distribution of ampC gene in gram-negative clinical isolates. On this basis, the methods should be established initially to detect ampC gene by multi-PCR and hybridization by oligonucleotide chip. Methods 1. The collection of specimens were from department of clinical laboratory of first affiliated hospital , Third Military Medical University and the Children's Hospital, from October in 2003 to May in 2004. 2. AmpC gene was amplified by polymerase chain reaction (PCR) in 389 gram-negative clinical isolates. 3. AmpC enzyme was examined by Cefoxitin in three dimensional test. 4. Microscan walkaway-4.0 was used for detecting the susceptibility of antimicrobial agents against clinical isolates. 5. To establish the system of multi-PCR to detect ampC gene in 7 genera of strains. 6. The method should be established to detect ampC gene based on the hybridization by PCR amplicons labeled by Cy3 and oligonucleotide chip. Results 1. Among 389 gram-negative clinical isolates, positive rate of ampC gene was 51.7%. The distribution of ampC gene was imcompatible in different genera of strains: 34 strains of Enterobacter cloacae accounted for 79.1%, 93 strains of Escherichia accounted for 79.5%, 14 strains of Klebsiella accounted for 1.22%, 41 strains of Pseudomonas accounted for 61.2% and 14 strains of Acinetobacter accounted for 50.0%. 2. 34 strains of 201 clinical isolates with ampC gene had produced AmpC enzyme accounted for 16.9%. 3. The sensitivity rates of 389 strains to IPM, AK, CIP, FEP, CN, SXT, CAZ , ATM, CRO, PIP were 92.5%, 77.4%, 72.5%, 49.6%, 48.6%,42.7%, 36.5%, 32.4%, 30.6%, 28.8%, respectively. The status of drug resistance in strains with ampC gene was more severer than those without ampC gene in PIP, CIP, SXT; there should be statistically significant between them. The status of drug resistance in strains producing AmpC enzyme was more severer than those non-producing AmpC enzyme in CAZ, CRO, PIP, ATM; there should also be statistically significant between them. 4. The method of multi-PCR was established to detect the ampC gene at the same time in 7 genera of strains by optimizing the system of PCR. 5. The positive signal of hybridization could be catched in 7 genera of strains including Escherichia, Klebsiella, Acinetobacter, Enterobacter cloacae, Enterobacter aerogenes, Pseudomonas and Serratia, by using the PCR amplicons labelled by Cy3 after gragmentation by UDG. Conclusions These experiments confirmed that the status of drug resistance in strains with ampC gene was more severer than those without ampC gene in PIP, CIP, SXT; the method was established initially to detect ampC gene by multi-PCR and hybridization by oligonucleotide chip.
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