| Objective:1.To investigate the distribution and characteristics of multi-drug resistant Klebsiella pneumonia which was isolated clinically from First Affiliated Hospital of Nanchang University between 2002 to 2013.2.To study the distribution and characteristics of the plasmid-mediated quinolone resistance genes of multi-drug resistant Klebsiella pneumoniae isolated continuously of 2012-2013.3. To Research the relationship between the resistance genes and the mobile genetic elements,and analyze the correlation with the different antimicrobial drug resistance.Methods:1. Collected 40 multi-drug resistant Klebsiella pneumoniae(MDR-KPN) strains from different clinical specimens from the first Affiliated Hospital of Nanchang University. The minimum bacteriostatic concentration(MIC) method is used to determine antimicrobial drug sensitivity of bacteria. The results of susceptibility testing were interpreted according to Clinical and Laboratory Standards Institute guidelines(CLSI) 2012. ERIC-PCR and MLST analysis were used to determine the cloning spread.2. PCR assay was used to determine the plasmid-mediated fluoroquinolone resistance genes(PMQR), inculding qnr genes, drug efflux pump protein qep A and aminoglycoside acetyltransferase gene variation aac(6 ’)- Ib-cr. The PCR products was determined by the DNA sequencing and compared with sequences available on the Genbank. All the PMQR positive strains were conjugated by the conjugation studies and mating was carried out on blood agar medium without selection.Azide-resistant E. coli J53 was used as the recipient.3. All the mobile genetic elements were determined by the PCR amplification,including insertion sequence IS26, IS2, ISCR1, ISEcp1, integrated child int I1, int I2,int I3, et al. By the agarose gel electrophoresis, we selected the PCR products to sequence and compare. PCR-mapping was used to determine the relationship between the mobile genetic elements and the PMQR genes and analyze the correlation with the multi-drug resistance.Result:1. In this study, we collected 40 multi-drug resistant Klebsiella pneumoniae during 2012 to 2013 in our hosptial. The susceptibility results showed that the resistance rate of ampicillin was the highest, accounting for 100%. However, the resistance rates of cefazolin, ceftazidime, ceftriaxone, aztreonam, ertapenem,gentamicin were 80% ~ 97.5%, respectively. The resistance rate of imipenem was77.5%. Although the resistance rate of levofloxacin was the lowest, but also reach to55%. ERIC-PCR and MLST results show that the vast majority of strains were clonning, and the main clone was the ST11 / A type, followed by ST340/A type,ST437/A type, ST147/J type and other major clone.2.Among the 40 multi-drug resistant Klebsiella pneumoniae, there were 37(92.5%) which carried the qnr genes, including 33 qnr A which exhibit the qnr A1 type;12 qnr B which exhibit the qnr B4 type; 16 qnr S exhibit the qnr S1 type. In addition,there were four strains carried more than three qnr genes at the same time. qnr C, qnr D and qep A genes were not detected in this study. Twenty-eight strains carried the aac(6 ’)- Ib gene. By the sequence comparative analysis, we found there were 12 aac(6 ’)- Ib-cr gene positive strains. However, among the 40 MDR-KPN strains, there were only 8 isolates which were successfully conjugated. Compared with the recipient strains, we found that the MIC value of fluoroquinolone in all the recipients were increased by 2 to 32 mg/m L. In addition, some qnr genes were also detected in the recipients.3.Among the 40 MDR-KPN, we detected 30 strains which were positive to ISCR1 gene, 40 strains positive to ISEcp1 gene, 32 strains positive to intl1 gene, 16 strains positive to IS2 gene, and 10 positive to IS26 gene. PCR-mappin amplification and sequence analysis showed that the ISCR1 gene was connected with qnr A1 in 15 strains, while ISCR1 connected with qnr B4 and qnr S1 only in 2 and 4 strains,respectively. In additionm, the ISEcp1 connected with qnr B4 in nine isolates, whileconnected qnr A1 and qnr S1 in 4 and 1 isolate, respectively. IS2 gene connected with qnr S1 in eight isolates.conclusion:1. The resistance was serious in all the 40 MDR-KP isolates. The resistant rates of all the common antimicrobial drugs were more than 55%. It forced us to strengthen the monitoring the drug resistance in Klebsiella pneumoniae and standarded the use of antibiotics in the clinic.2. In our hospital, the qnr gene was the common PMQR gene in the klebsiella pneumoniae, followed by the aac(6 ’)- Ibr gene. The qnr A was the major qnr gene,followed by the qnr S and qnr B. We detected the subtypes including qnr A1, qnr B4,qnr S1 and aac(6 ’)- Ib-cr. However, the qnr C, qnr D and qep A were not detected.3. The carried rates of mobile genetic elements which detected in the MDR-KPN was high in our hosptial, and co-existed in a variety ways. The qnr A was connected at the downstream of ISCR1, while the qnr B4 was connected at the downstream of ISECP1 and the qnr S1 was mainly connected at the downstream of IS2. |
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