The Study Of DHA-1 Type Plasmid-Mediated AmpC β-LACTAMASE

Objective: The aim of this project is to study our clinical isolates that produce DHA-1-type plasmid-mediated AmpCβ-lactamase in terms of antibiotics susceptibility, prevalence and inpatients clinical features. The results would provide valuable information for resistant spectrum, prevalence control of antibiotic resistance, and clinical significance caused by these DHA-1 and CMY-2 pathogens.The regμlatory genes are also investigated for better understanding of regμlation mechanisms and its correlation with induction phenomenon.Methods: 1. Collecting the clinical data of inpatients infected by DHA-1 and CMY-2 producing E.coli and K.pneumoniae, and comparing the risk factors of different groups. 2. MIC test, polymerase chain reaction (PCR) amplification and sequencing to determine the antibiotic susceptibility, plasmid transfer, the DHA-1 gene and other ESBL genes. 3.Random amplified polymorphic DNA (RAPD) was used to investigate the origin of different DHA-1 isolates. 4. Conjugation and plasmid profiles further describe the dissemination of plasmid carrying DHA-1 among wards. 5. Southern blotting was taken to locate the DHA-1 gene and the band size where it locates. 6. PCR amplification and sequencing of the main regμlatory genes blaampR and blaampD, then clone both blaampR and blaampC to plasmid pUC19 and test the recombinant's MIC.Results: To further describe the clinical features of the 32 DHA-1 and 10 CMY-2 infectious inpatients from January 2003 to July 2005, they were classified to two comparing groups: only AmpC and AmpC with ESBLs group, DHA-1 and CMY-2 group. There was no significant difference between these groups in terms of risk factors, sites of infection and underlying disease with one exception that CMY-2 producer infections were more correlated with diabetes (P=0.036). Prior use of the third cephalosporins (P=0.02) and fluoroquinolones (P=0.008) showed statistical difference between the AmpC (only), AmpC and ESBLs group. RAPD analysis indicates that the prevalence of DHA-1 isolates was due to the colonial spread and plasmid transfer. The Southern blot hybridization studies confirmed that DHA-1 gene was located on plasmids. After sequencing the ampR regulatory gene and then comparing the AmpR protein with its origin Morganella morganii, two alterations were discovered, 229(L-Q) and 273(A-T). Conclusions: There is statistically significant difference with respect to prior use of antibiotics comparing the AmpC only and AmpC with ESBL group. The prevalence of DHA-1 was high and the colonial and plasmid transfer both contribute to its prevalence.