Study On The Nosocomical Klebsiella Pneumoniae Resistant To Fluoroquinolones

Klebsiella pneumoniae(Kpn) is a major opportunistic gram negative pathogen and thecause of about over 10% of the nosocomial infections. In the recent years the nosocomialinfections caused by antimicrobial-resistant Kpn strains have been increasing continuously,especially for the infections due to the fluoroquinolones (FQNLs)-resistant Kpn dramaticalincrease. FQNLs are a kind of synthetic drugs with broad antimicrobial spectrum and strongeffects and become the major agents to treat infections ever since being introduced in clinicbecause of their simple structures, the convenience and no cross-resistance to other speciesof antimicrobial agents.Till now the comsuption of FQNLs in the hospital accounts forabout 15% of the total amount of antimicrobial agents used and still increasing, so it is surethat the resistance of Kpn to FQNLs will seriously influences the use of this kind of agentsin clinic. Since the FQNLs-resistance of Kpn and its epidemiology have been verified to beclosely related with various treatmental procedures and nursing intervention ,therefore, it istheoretically and clinically important to study and monitor the resistance to FQNLs in Kpnstrains so as to supervise and improve the appropriate use of FQNLs in clinics, tostrengthen and improve the treatmental and nursing measures to reduce the nosocomialinfections. Previous studies showed that the target of FQNLs was DNA topoisomerase, includingDNA gyrase and topoisomerase Ⅳ (Topo Ⅳ). Usually the mechanism of bacteria resistantto FQNLs is due to the alterations in the genes of topoisomerase, especially in the subunit Aof DNA gyrase (GyrA) and subunit C of Topo Ⅳ (ParC). So far it is not very clear if theresistance levels of Kpn to different hydrophilic and hydrophobic FQNLs were related withthe differences in the alterations in the topoisomerase . In this study, 68 clinical strains ofKpn isolated from nosocomial infections from April, 2002 to March, 2003 in the FirstCollege of Clinical Medicine of the Third Millitary Medical University were as the target ofinvestigation. Based on their susceptibility to antibiotics, 10 strains of Kpn resistant to bothciprofloxacin and ofloxacin at different resistance level and 1 susceptible strain, and the invitro resistant Kpn mutants selected with hydrophilic ciprofloxacin and hydrophobic 4第三军医大学硕士研究生论文sparfloxacin, respectively, were employed in the further study. The quinolone resistancedetermining region (QRDR) of these strains were analyzed by polymerase chain reaction(PCR), then the products of PCR were sequenced and the point mutations in gyrA and parCwere analyzed to discover if the genetic alterations were related with the resistance toFQNLs in Kpn and if there were any differences in targets of the hydrophilic andhydrophobic FQNLs. The study was divided into three parts: (1) the epidemiological survey on theresistance to antimicrobial agents in clinical Kpn isolates. (2) study on the alterations inQRDR of clinical Kpn isolates resistant to FQNLs. (3) sequence analyses of QRDR inin-vitro FQNLs-selected resistant Kpn mutants .1.Methods1.1 Susceptibility of Kpn to antimicrobial agents The susceptibility of the clinical Kpnisolates to 16 antimicrobial agents were determined by Kirby-Bauer disc diffusion test.Strains producing extended spectrum β -lactamases (ESBLs) were determined byphenotypic confirmatory testing.1.2 Alterations in gyrA and parC of FQNLs-resistant clinical Kpn strains Minimuminhibitory concentration (MIC) of 10 clinical Kpn strains resistant to FQNLs at differentresistant level and a susceptible strains were determined by the twofold agar dilutionmethod. Specific primers corresponding to QRDR in gyrA and parC sequences weredesigned. With the chromosomes of the 11 clinical isolates being template, PCR wasperformed and PCR products were sequenced to analyze the alterations in gyrA and parC ofthe Kpn strains. The relatio...