Genotypes Of Extended-Spectrum β-Lactamases In Clinical Isolates Of Enterobacteriaceae

OBJECTIVE:Clinical isolates of Enterobacteriaceae are the most common members in hospitals and they have been associated with human disease. Extended-spectrum beta-lactamases(ESBLs) are considered to be one of the most important antibiotic resistance mechanism. Nucleotide sequencing is the golden standard and can detect all variants. In the study,the main genotypes and the epidemic characteristics of ESBLs isolates in the third hospital of Wuhan city are investigated. These data are important for implementing appropriate antimicrobial therapy and effective infection control measures.METHOD:The ESBLs phenotype of333clinical isolates of Enterobacteria-ceae was primarily screened and the isolates were collected in the third hospital of Wuhan city from January2011to December2011.The ESBLs phenotype of clinical isolates were confirmed by CLSI phenotypic confirmatory test and according to guidelines of CLSI(medicine sensitive paper of ceftazidime, ceftazidime/CA and cefotaxime, cefotaxime/CA for detection ESBLs). ESBLs phenotype-positive clinical isolates using primers specific for TEM, CTX-M and SHV respectively,and the polymerase chain reaction (PCR) products were purified and sequenced.The sequenced fragments will be blasted with NCBI gene bank for ESBLs genotypes.RESULTS:333ESBLs producing clinical isolates mainly come from the wound secretion samples, accounting for36.6%of all specimens, followed by sputum and urine samples.283strains are ESBLs producing positive strains by confirmatory test,including Escherichia coil(67.1%),Klebsierlla pneumoni-ae(23.3%),proteus mirabilis(3.5%),proteus vulgaris(2.2%) and Enterobacter cloacae(3.9%).The positive rate of PCR results for CTX-M and TEM,two of them in the ESBLs phenotype positive clinical isolates of Enterobacteriaceae is77.39%(219/283).CTX-M accounted for38.17%(108/283), TEM type accounted for23.32%(66/283),45(15.90%) are both blaTEM genes and balCTx-M genes,22.61%(64/283)of the ESBLs phenotype positive clinical isolates has not been amplificated to be positive strip. The PCR products for CTX-M and TEM were further indentified as CTX-M-15,CTX-M-3,CTX-M-14and TEM-3. SHV genotype was not found.CONCLUSION:The experimental results show the consistency is as high as77.39%between the double disc synergy test for phenotype confirmatory test and polymerase chain reaction (PCR)based amplification of the specific genes; The department of burn in the ward with the highest ESBLs-producting rate accounted for24.4%in the third hospital of Wuhan city. The most common isolates were Escherichia coil and Klebsierlla pneumoniae;The highest prevalence resistance genetype is blaCTX-M-15gene and blaTEM-3gene. The sequenced fragtment was blasted to NCBI gene bank, homology is above99%. Local epidemiological data are important for implementing appropriate antimicrobial therapy and effective infection control measures.