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Study on the VEB-1 Gene Cassette in Clinical Pan-resistant Pseudomonas Aeruginosa and the Impact of Its Structure on the Integration of Sub-frequency Capture

Author: ChenXiaoYun
Tutor: LvYuan；JiangXiaoFei；LiMin；GuanMing
School: Fudan University
Course: Clinical Laboratory Science
Keywords: Pseudomonas aeruginosa Resistance VEB-1 gene cassette Integron IS1999 insertion sequence Recombinant Lateral gene transfer
CLC: R378
Type: Master's thesis
Year: 2010
Downloads: 90
Quote: 1
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Abstract

In recent years due to the misuse of antibiotics in humans, animals, plants, pathogens, antibiotic resistance is becoming more severe, the treatment of infectious diseases every year becomes more challenging, especially caused by the opportunistic pathogen Pseudomonas aeruginosa infection, it can rapidly generate multiple drug resistance, so that a plurality of antibiotics lost clinical utility. Although mobile elements caused by drug-resistant mechanism has always been one of the issues we care about, but how we face the most difficult challenge is rapidly induced resistance of Pseudomonas aeruginosa in the treatment of the infection process. Research on the mechanisms of bacterial antibiotic resistance, leading to transposons and conjugated plasmid including many natural movement of the components found by a comparative analysis of the sequences of these elements, and ultimately found integron (integron) exist. Integron initially in the mobile element of the pathogen was found and identified integron is now considered to be an old structure and can exist in different strains, strains has now been identified by sequencing, about 9% containing integron. Overall, the gene cassette has a very high diversity, suggesting that integration - cassette system in to improve most bacteria have played a significant role in the environmental adaptability. Integron located in the chromosome of Gram-negative bacilli, transposon or plasmid with a broad host integron resistance gene is a newly discovered horizontally transfer DNA elements. Integron by site-specific recombination in bacteria to capture gene cassette, and to ensure that these resistant gene expression by promoter. Integron can be described with the plasmid, or transposon itself around a part of and transfer of resistance genes occurs disseminated. The study by clinical multidrug-resistant Pseudomonas aeruginosa integration in the sub-VEB-1 gene cassette screening and structure analysis, and the use of a simple real-time quantitative PCR method to study the structure of the VEB-1 gene cassette characteristics and some structure to explain the phenomenon of clinical resistance gene cassette disseminated integron capture efficiency, so as to analyze. Part I: clinical isolates with VEB-1 gene cassette integron screening VEB-1 gene cassette present in the integron and encoded extended spectrum lactamase VEB-1 (Vietnamese extended-spectrum b-lactamase). VEB-1 gene cassette is initially in Vietnam one of the clinical isolates of E. coli strain was found, and then also in Vietnamese 2 clinical isolates of Pseudomonas aeruginosa was detected and identified. Pseudomonas aeruginosa in Southeast Asia, such as Thailand, China also detected this type of ESBL. This study selected the 6 to 11 months of 2004, Huashan Hospital microbiology laboratory identified by Merieux instrument collection and identification of 37 wholly-resistant Pseudomonas aeruginosa strains, primers were designed VEB-1 gene cassette amplification containing strain, and then take advantage of the high-resolution sensitive curve HRM genotyping to identify VEB gene cassette VEB-1 and VEB-3 gene cassette. The design the primer amplification the clinical isolates VEB-1 gene cassette 5cs end, to understand its gene-environment, and sequencing analysis. Finally, we have 37 full-resistant Pseudomonas aeruginosa, found 35 VEB gene exists, VEB-type gene cassette multidrug-resistant bacteria is very widely distributed. After the hrm typing and sequencing to determine the type, VEB-1 gene cassette 9. After PCR amplification and sequencing and Blast 9 bacteria learned the eight 5cs containing IS1999 insertion sequence. ERIC PCR identification and then after only three of the same type, and other several strains of different type to each other. Prompts the IS1999 are widely distributed in multi-drug resistant Pseudomonas aeruginosa VEB-1 gene cassette upstream. Part II: study VEB-1 Raw IS1999 capture efficiency of the structure of the integrated sub according to the first part of the study, all VEB-1 gene cassette is located on the integrated gene cassette, and integration for the first one, the other has a long lkb around the insertion sequence widespread in multidrug-resistant Pseudomonas aeruginosa in VEB-1 gene cassette upstream aroused our interest in this structure and integration of sub-integration efficiency. Previous studies have shown that the the IS1999 transposase encoding 402 amino acid protein itself, which has 71% amino acid homology with IS10, so it belongs to the IS4 family of insertion sequence. IS1999 fragment a total of 1,328 bp, imperfect repeats of 21-bp fragment located on both sides, and may be generated after the transposition repeating a 9bp target fragment, mediated transfection horizontal movement of the transposon and the transposase fragment. This study with long-primer method build lacZα gene cassette, and connected to the vector pACYC184 plasmid named pac-Lac. From clinical isolates of P. aeruginosa strain No. 684 was amplified containing IS1999 the inserted sequences attI loci VEB-1 gene cassette fragment was amplified from the No. 961 strain Raw ordinary attI VEB-1 gene loci cassette fragment. Build these two fragments enter pAC-lac, using relative quantitative PCR method to compare the two different gene cassettes captured frequency. Was found, containing IS1999 fragment integron capture gene cassettes declined significantly compared to before VEB-1 gene cassette inserted into the ability and integration of sub-structure, which means that a the IS1999 structure blocking the way of effective integration, prevent integron capture other gene cassette, thereby maintaining the advantages of the location of the VEB-1 integron. Part III: compare different attC sites structure VEB-1 gene cassette integration efficiency class Ⅰ integron including the the integrase gene coding (IntI) and adjacent to a recombinant sites (attI). Gene cassette is not necessary integron components, but once they are integrated, they become part of the integron dependent on the integron promoter (Part) to express the promoter is also part of the integration of sub-5 'conservative interval . In addition, the integration of the sub-sequence (such as the 3 'end) can be conservative, but not all integron homology. The gene cassette contains a coding sequence. At the 3 'terminus exist for some nucleotides, referred to as 59 - nucleotide sequence or attC locus. This study compared the VEB-1 and VIM-2 both clinical common spread is not the same degree of gene cassette, and build the VEB-1 gene cassette attC sites replaced VIM-2 attC sites gene cassette, and then quantitative PCR method using a simple, rational design primers to amplify the gene cassette integrated, thereby measuring the difference between different gene cassette integrated frequency; in which part of the gene cassette for integration and can be analyzed efficiency. And take into account experimental strains before this study are at nutritious ideal environment, this does not match with the survival of the clinical strains, so we detected the control strains MIC, and then take the antibiotic concentrations below the MIC, the induction observe the frequency of their integration also whether there is a difference, can basically remove the effect of the choice of antibiotics. The results showed that the VEB-1 integration frequency in case no antibiotic is smaller than the gene cassettes of the VIM-2 and the replacement attC sites 59VIM-VEB gene cassette described the integration efficiency and attC presence of certain relationship with the encoded sequence relationships obvious. Join Amp1μg/ml, other variables controlled conditions, the integration between these three efficiency difference disappeared. Gene cassette attC elements as well as the structural gene sequence is different, but after the addition of antibiotics, the bacteria itself to adjust the trigger the role of other self-help system, which affects the integration process, this part of the reason also need some follow-up studies to analyze .

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