Klebsiella Pneumoniae Drug Resistance And The Class 1 Integrase Expression In The Biofilm

Objective:Klebsiella pneumoniae is an important opportunistic pathogen of human with high drug resistance. To understand the Klebsiella pneumoniae strains presents situation and levels of the drug resistance,to provide the basis for the reasonable clinical application of the antibiotics;Gene cassettes are small mobile elements, consisting of more than a single gene and recombination site, which are captured by larger elements called integrons. Integrons are elements that participate in a powerful site-specific recombination system and play a major role in spreading antibiotic resistance genes in a clinical setting. In this study we investigated the occurrence and distribution of class 1 integrese gene. Biofilms are communities of microorganisms attached to a surface. Bacterial biofilms are formed when unicellular organisms come together to form a community that is attached to a solid surface and encased in an exopolysaccharide matrix. Biofilms can be made up of single or multiple bacterial species. It has become clear that biofilm-grown cells express properties distinct from planktonic cells, one of which is an increased resistance to antimicrobial agents. Recent studies suggest that biofilm formation may also be an important virulence factor for K. pneumoniae. In order to understand the role of integron, reverse transcription-polymerase chain reaction (RT-PCR) was developed to measure the changes in int I 1 gene expression of Klebsiella pneumoniae biofilm and planktonic cells.Methods:One hundred and twenty three clinical isolates of Klebsiella pneumoniae were isolated.Antibiotic susceptibility testing was performed by disc diffusion method.PCR were performed to amplified integrase and variable region of class 1 integron with specific primers. PCR product of the variable region was futher identified by sequencing. The clinical strains of Klebsiella pneumoniae with intll gene were cultured in planktonic cells and biofilm cells. The total RNA of these cultured bacteria were extracted by the conventional method. The RT-PCR was developed to measure the changes in intll mRNA expression of the Klebsiella pneumoniae with bacterial 16s rRNA as an internal control.Result:1.Resistant rate of clinical Klebsiella pneumoniae to beta-lactams, quinolones and sulfonamides were high (PRL100%, CRO76%, LEV60%,TOB60%and SXT63%), however, Meropenem showed good antimicrobial activity amony all these clinical strains.2.The class 1 integrons were found to be extensively existed in clinical isolates of Klebsiella pneumoniae.47 of 123 isolates were identified as being positive for integrase 1 gene,positive rate was 38.2%. The resistant rate is diffierent for 8 antibiotics between integrase-positive strains and integrase-negative in statistic. Among the strains with class 1 integrase gene, there were 23 strains including gene-cassette in the variable regions of integrons in Klebsiella pneumoniae strains. The length of the amplicons ranged from 0.6kb to 2.4kb. The analysis of DNA sequencing revealed that variable regions of class 1 integrons contained trimethoprim resistance gene(dfrA12),streptomycin and spectinomycin resistance gene(aadA2) and open reading frames(orfF) with unknown functions. DfrA and aadA cassettes were predominant and cassette combination dfrA12,orfF and aadA2 was most frequently found.3.The clinical strains of Klebsiella pneumoniae expressed int I 1 mRNA in both biofilm and planktonic cells, but with different levels. Klebsiella pneumoniae strains class 1 integrase mRNA expressed in their biofilm cells were higher than their planktonic cultured cells. The gene expression of intIl in the planktonic cells is 0.08±0.05,but the gene expression of intIl in the biofilm cells is 0.28±0.04. The expression level of Klebsiella pneumoniae strains in the biofilm is about 3 times higher than the planktonic cells.Conclusion:Resistant rate of clinical Klebsiella pneumoniae to beta-lactams, quinolones and sulfonamides were high. The class 1 integrons were found to be extensively existed in clinical isolates of Klebsiella pneumoniae. There was a good correlation between integron-carrying and muti-drug resistance. The intIl gene expression of Klebsiella pneumoniae in the biofilm is up-regulated at mRNA level. The Klebsiella pneumoniae biofilm formation may lead to spread integrons and integrated drug resistance genes. The integron may capture and accumulate drug resistance gene cassettes more effectively in the biofilm condition.