Study On Resistant Mechanism To β-lactam Antibiotics Of ESBL And PAmpC β-lactamase Producing K.pneumoniae

Antibiotic is one of the best achievements in medicine which improve human health level and ensure people life. However, due to misuse, abuse and overuse of antibiotic, it become resistant thus, threatened human health due to failure of clinical treatment and the break of nosocomial infection. P-lactam antibiotic is one of the most commonly used antibiotics, especially the third generation cephalosporin because of its strong powerful antibacterial activity, broad antimicrobial spectrum and low toxicity, plays an important role in the treatment of gram negative bacterial infection and has been the most popular antibiotics in clinical. Recently, the heavy and increasing use of third generation cephalosporin such as ceftazidime, cefotaxime and ceftriaxone undoutbly drove the selection of mutant enzymes, which is called extended spectrum beta lactamase (ESBL).ESBLs have capability of hydrolyzing the oxyimino -cephalosporins (cefpodoxime, ceftriaxone, cefotaxime, and ceftazidime), monobactam (aztreonam) and inhibited by P-lactamase inhibitor such as clavulanic acid. Since first described in 1983, ESBLs have been reported at almost everywhere in the world. There are many reports about the break of nosocomial infection caused by ESBLs producing bacteria. Klebsiella pneumonia, an important cause of nosocomial infections, is among those organisms which produce ESBLs. The studies have addressed that the more than75% ESBL production is related to infection with Klebsiella pneumonia. Studies have also shown that ESBL and pAmpC P-lactamase producing Klebsiella pneumonia have rapidly spread worldwide a pose a serious threat for health care-associated infections. Increasingly the ESBL Klebsiella pneumonia are also showing co-resistance to quinolone and aminoglycoside antibiotics may be due to presence of plasmids which can transferred by conjugation to other strains.The plasmid mediated AmpC P-lactamase is derivative of the chromosomally, encoded AmpC P-lactamase of several member of the family Enterobacteriaceae, including Enterobacter, Citrobacter freundi, Morganella morganii and Seratia marcescens etc. and it has been detected in bacterial species that lack chromosomal AmpCβ-lactamase enzyme such as Klebsiella pneumoniae, Escherichia coli, Salmonella species and Proteus mirabilis. The pAmpC P-lactamase have ability of hydrolyzing the oxyimino -cephalosporins such as, cefpodoxime, ceftriaxone, cefotaxime, ceftazidime and monobactam such as aztreonam along with cephamycin (cefoxitin,cefotetan) and unlike ESBL, it is not inhibited by P-lactamase inhibitor such as clavulanic acid. Thus, resistance to broad spectrumβ-lactam, mediated by extended spectrum (3-lactamase (ESBL) and pAmpCβ-lactamase enzymes is an increasing worldwide problem because when genes encoding both ESBLs and pAmpCβ-lactamase are usually located on large multidrug resistant plasmids. Presence of these in clinical infections can result in treatment failure if one of the second- or third generation cephalosporin is used. Therefore, it is recommended that any ESBL producing organism according to the National committee for clinical laboratory (NCCL) criteria can be reported as resistant to all extended spectrum P-lactam antibiotics regardless of the susceptibility test results.The aim of this study was to detect and determine the genes encoding the ESBLs including bla TEM, SHV, CTX-M and pAmpC (3-lactamase including DHA, FOX,CMY,MOX among the Klebsiella pnumoniae isolated from clinical sample at First hospital of Jilin University by Polymerase chain reaction(PCR) and characterized them by direct sequencing of PCR products. A total of 227 bacterial strains were isolated from patients at different ward from March 2009 to August 2010. They were identified as Klebsiella pnumoniae using different bio-chemical tests. The susceptibility of isolates to 17 different antimicrobial agents was determined using agar disk diffusion (Kirby Bauer) method. The phenotyping screening test was used as expanded spectrum cephalosporin and cephamycin to screen the isolates for production of ESBLs and plasmid mediated AmpC (3-lactamase. The phenotyping confirmatory test was used as modified boronic acid method to confirm the isolates for ESBL or pAmpC P-lactamase or both. To amplify the ESBLs and pAmpC P-lactamase, the DNA was extracted by boiling method. The extracted DNA used as template in PCR for detection of bla TEM, bla SHV, blaCTX-M and pAmpCβ-lactamase genes with respective primer. One of each P-lactamase PCR product was sequenced and analyzed.The P-lactamase producing strains were more resistant to most antibiotics than non-β-lactamase producing strains. The coexistence of both ESBL and pAmpC P-lactamase were shown more resistant to both oxyimino -cephalosporins and 7-a-methoxy cephalosporins than alone pAmpCβ-lactamase or ESBLs producing strains. The phenotyping confirmatory test detect 51 isolates as ESBL 34(66.6%), pAmpCβ-lactamase17 (33.3%) and co-existence ESBL and pAmpCβ-lactamase 6 (11.7%) producing K. pneumonia. The PCR amplified gene encoding ESBLs and pAmpC P-lactamase as following bla TEM positivel5(29.4%), bla SHV positive 14 (27.4%), bla CTX-M positive 19 (37.2%), and blaDHA-1 positive 23 (45.0%) and among them, some strains showed multiple ESBLs and coexistence with plasmid mediated AmpCβ-lactamase. The multiple ESBLs as blaTEM+SHV 3(5.9%) blaTEM+CTX-M 3(5.9%) blaSHV+CtX-M (3.9%) and ESBL co-existence with AmpCβ-lactamase blaDHA+TEM 2(3.9%) blaDHA+SHV 3(5.9%) blaDHA+CTX-M 1(1.9%). The CMY, MOX and FOX were not detected. The sequencing analysis was shown that the most frequently identified ESBLs genes in this group were bla TEM-1, bla SHV-1, and blaCTX-M-15.