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

With the abuse of antibiotics in human, animals and plants, treatment of infectious diseases becomes more challenging with each passing year. This is especially true for infections caused by the opportunistic pathogen Pseudomonas aeruginosa, with its ability to rapidly develop resistance to multiple classes of antibiotics. Although the import of resistance mechanisms on mobile genetic elements is always a concern, the most difficult challenge we face with P. aeruginosa is its ability to rapidly develop resistance during the course of treating an infection. The study of bacterial antibiotic resistance mechanisms results in discovery of many natural moving elements, including transposon and plasmid conjugation, and by comparative sequence analysis of these components, the existence of integron was eventally found.Integrons were originally identified on mobile elements from pathogenic bacteria and were found to be a major reservoir of antibiotic-resistance genes. Integrons are now known to be ancient structures that are phylogenetically diverse and, to date, have been found in approximately 9% of sequenced bacterial genomes. Overall, gene diversity in cassettes is extraordinarily high, suggesting that the integron/gene cassette system has a broad role in adaptation rather than being confined to simply conferring resistance to antibiotics. Integron was located on the chromosome of Gram-negative bacteria, transposon, or a broad host plasmid, which was found in recent years a new mobile DNA element that can horizontal transfer the resistance gene. By capturing exogenous gene cassettes through site-specific recombination and ensuring the expression of genes contained in gene cassettes, integrons played important roles in the horizontal dissemination of drug-resistance genes in bacteria.Through this study, VEB-1 gene cassette was screened in the clinical pan-resistant Pseudomonas aeruginosa and process a series of structural Analysis. A new and simple method through real-time quantitative PCR was used to study the VEB-1 gene cassette and the structural characteristics of it on the efficiency of integration, so as to explain some clinical resistance gene cassette in the spread phenomenon.PartⅠ:Screening and identifying of the clinical isolates contained VEB-1 gene cassettesThe integron-borne blaVEB-1 gene encodes the extendedspectrumβ-lactamase VEB-1 (Vietnamese extended-spectrumβ-lactamase) found initially in an Escherichia coli clinical isolate from Vietnam. Subsequently, the veb-1 gene cassette was identified in two Pseudomonas aeruginosa clinical isolates from Thailand. This type of ESBL was also detected in Pseudomonas aeruginosa in Thailand, China and other Southeast Asian countries.This study selected clinical strains from June to November in 2004 in Huashan Hospital microbiology laboratory.37 Pan-resistant Pseudomonas aeruginosa strains was collected from the strains identified by the BioMerieux. Primers was designed to amplified the strains containing VEB-1 gene cassette, and then using high-resolution curve (HRM) to identify gene type, tellng VEB-1 from VEB-3 gene cassette.5cs fragment in VEB-1 gene cassette of clinical isolates was amplified with specific primers to explor their genetic environment and then sequenced. Finally, we have 37 pan-resistant Pseudomonas aeruginosa and found that 35 isolates haboring VEB-type genes, indicating the VEB-type gene cassette are widely distributed in multi-drug resistant bacteria. After HRM typing, and sequenced to determine type, we got nine strains harboring VEB-1 gene cassette. After PCR amplification and sequencing with Blast comparison, we learned that eight out of nine contained IS 1999 insertion sequence in 5cs region. After another ERIC PCR identified, only 3 of them were the same type. Other several strains have mutually different types. It indicated that IS 1999 is widely distributed upstream of VEB-1 gene cassette in the Pan-resistant Pseudomonas aeruginosaPartⅡ:IS1999 upstream of VEB-1 in the integron influence the integration efficiencyAccording to the first part of the study, there was a long insertion sequence of about lkb widespread in the pan-resistant Pseudomonas aeruginosa habouring the VEB-1 gene cassette, which cause our interest about this structure and its influence about integration efficiency. A previous study shows that IS 1999 itself encoded 402 amino acid protein of transposon activity, which has 71% amino acid homology with the IS 10. So it is a member of IS4 family of insertion sequences. IS1999 is an element of a total of 1,328 bp, a 21-bp imperfect repeat sequence located on both sides, and can generate a 9bp transposable repeat after the target fragment, mediated the horizontal movement of transposons and transposon fragments.This study constructed lacZa gene cassette with long primers, and connect it into the vector pACYC184 plasmid. Then name it pAC-lac. And the IS1999 insertion sequence in the attI site of VEB-1 gene cassette was amplified from clinical isolates of P. aeruginosa strain No.684; and the same to strain No.961. Then we introduced these two fragments into the pAC-lac, using relative quantitative PCR method to compare the integration frequency of these two integrons with different attI sites. The results showed that comparing to that of the original structure of integron, the integration frequency of the one containing IS 1999 sequence declined significantly. It can concluded that IS 1999 can block effective integration of gene cassettes, which means prevent the capture of other integron gene cassette before VEB-1, thereby maintaining the VEB-1 advantage in the integration of sub-location.PartⅢ:Comparison the integration efficiency of different attC sites structure of the VEB-1 gene cassetteClass 1 integrons include the gene for an integrase (int) and an adjacent recombination site (attI). Gene cassettes are not necessarily part of the integron, but when integrated, they become part of the integron. Expression of the integron relies on the promotor in the integron (PANT) and thus the promotor is part of the 5'-conserved segment of the integron. In addition, other sequences may be conserved between some integrons, but not all.Gene cassettes consist of one coding sequence. At the 3'end of this sequence, a so-called 59-base element or attC site is located.We compared the VEB-1 and VIM-2 gene cassettes which have two different level of clinical disseminated in this study, and construct a new VEB-1 gene cassette with the replaced attC site that from VIM-2 sites. Then use a simple quantitative PCR method, to amplify the gene cassette integration with primers designed, which couold measured the frequency of different gene cassette; and can analyze in which part of the gene cassette has been affected the integration efficiency. And the environmental factors were taken into account. The experimental strains are in an ideal nutritional environment, which is does not match the clinical strain. So we carried out MIC for the control strains, and then use the antibiotics of concentration below the MIC to induce the integration. It can essentially remove the choice effect of antibiotic.Results show that in the case of antibiotics absence, VEB-1 integration frequency is less than VIM-2 gene cassette and that of replacement attC sites 59VIM-VEB gene cassette, which could shows there are certain relationships between attC site and integration efficiency, but not obvious with the coding sequence. After adding Amp1μg/ml, with other variables conditions under controlled, the integration between the three efficiency difference disappeared. Although these gene cassettes of attC element and structural of gene sequences are different, maybe the presence of antibiotics arouse other self regulating system of the bacteria, thereby affecting the integration process. This part needs further experiments to study and analyze.