Antimicrobial Resistance And Genotype Identification Of Extend-Spectrum β-Lactamases Produced In Biofilm-Forming Klebsiella Pneumoniae

Objective:To investigate biofilms in vitro formed by clinical isolated of Klebsiella Pneumonia(eKP)and detect the extended- spectrumβ-lactamases (ESBLs) production in biofilm- forming KP, while analyzing the drug resistance and genotyping in that condition.Methods:1. The in vitro model of Klebsiella Pneumoniae bacterial biofilm was formed by modified plating method. Biofilm was identified through light microscope and scanning electron microscope, and quantitatively analysis the biofilm production with crystal violet stain. Categories of BF-formed strains were compared using the chi-square test.2. Three dimensional test were used to detect biofilm- forming Klebsiella Pneumoniae which produced extended- spectrumβ-lactamases.3. Biofilm-forming strains which produced extended- spectrumβ-lactamases were group B and planktonic bacteria were group A. Antimicrobial susceptibility testing was performed using Kirby-Bauer method.4. The blaSHV,blaTEM and blaCTX-M genes of ESBLs were amplified by PCR, then sequenced and analyzed after cloned into pMD18-T vector respectively.Results:1. 46 of 60 (76.7%)ESBLs negative Klebsiella pneumoniae strains had formed biofilm. 7(15.2%) strains were weak biofilm producers, 15(32.6%)strains were moderate biofilm producers and 24(52.2%)strains were strong biofilm producers. The chi-square test showed that different categories of strains had the similar rate in BF formation, no significance was found within groups.2. 9 of 46 (19.6%) biofilm-positive strains observed ESBLs phenotype by three dimensional test.3. The strains of group A were higher resistance to cefuroxime and ceftazidime than cefotaxim, but sensitive to ceftriaxone and aztreonam. However, the strains of group B were high resistance to all above.4. PCR gene amplification results showed that all the 9 strains (100%) in B group carried SHV gene, among which 4 strains (44.5%) carried TEM gene while none carried CTX-M gene.5. The PCR products of group B were purified and then cloned into PMD-18 T vector. The inserted fragments were further confirmed by DNA sequencing. Sequence alignment analyses were performed on-line using the BLAST program. The 9 SHV genes belonged to SHV-5, SHV-12 and SHV-28 respectively, while all the 4 TEM genes belonged to TEM-1.Conclusion:1. The majority of clinical isolates of ESBLs-negative Klebsiella pneumoniae had biofilm-forming ability and most were strong biofilm producers.2. Biofilm-forming Klebsiella pneumoniae strains tend to produce ESBLs more than planktonic bacteria.3. One of the main reasons that Klebsiella pneumoniae was highly resistant to antibiotics was the synergetic effect of ESBLs and biofilm.4. Can BF induce the mutation of SHV-1 need to be studied in the future.