Research Of Resistance Mechanism Of ISCR And Integron In 1800 Gram-negative Bacterias

Background and objectiveThe drug resistance has become more and more serious, because of the general use of the antibiotics. Our country is one of the most severe areas in which the antibiotics are misused. The problems of the resistance in the bacterias have taken the attention of the people. At present, the rate of drug resistance in the bacterias which come from the clinic is upgrading year by year. The bacterias product more and more multidrug resistance become of the hingh selection pressure. Therefore, it is very necessary and urgent to research the mechanism of drug resistance in the bacterias.The mechanism of drug resistance is very complex. Bacterias can product drug resistance because of alteration of target site, change of pervasiveness, inactivation of drug, enhanced drug efflux, biological membrane and so on. To study the mechanism of drug resistance from the site of molecular biology, we focus on gene mutation and gene transfer. Gene transfer is the cause of fast diffusion of the resistance. There are plasmid, transposon, temperate phage, integron and insertion sequences common regions (ISCR), which can get and transfer the resistance genes. Resistance genes can be transferred by conjugation, transformation, transduction and transposition. Many resistance genes in integron, transposon and insertion sequences common regions can integrate in one plasmid by transposition, which is the important reason leading to multidrug resistance. ISCR comes from "insertion sequences" (ISs) and "common regions" (CRs), which has the feature of insertion sequences and common regions. Insertion sequences are the transposable elements which can move frequently, and the small DNA fragments which can insert one or more fragments to the genome and remove the adjacent genes. Common regions were first discovered and reported in the early 1990s as a DNA sequence of 2,154 bp that was found in two complex class 1 integrons, In6 and In7. CR sequences have two key features of IS91-like elements that are similarly located. CRs lack terminal inverted repeats (IRs) and are thought to transpose by a mechanism termed rolling-circle (RC) transposition. And CR sequences have another feature, terIS, which is a replication terminator. Further, the numbers presently used to distinguish CRs are retained to distinguish different ISCRs. Therefore, CR1 becomes ISCR1, CR2 becomes ISCR2, etc.ISCRs have an ORF, orf513, encoding a putative product of 513 amino acids. The use of orf513 is similar to the transposase, which can transfer the gene cassettes. In the complex class 1 integrons, downstream of orf513 can have one or more resistance gene cassettes, and qac/sul3'-CS.There are 6 frequent types of ISCR, and 1-4 types are researched in major studies. ISCR elements are notable for their close association with a wide variety of antibiotic resistance genes, and the ISCR1 is the most important one. ISCR1 can mediate transposition, and it can transfer resistance gene cassettes with difference length, such as the genes of catA2,dfrA,qnr and blaCMY and so on. ISCR1 plays an important role on the transmission of the resistance.Integron is also an important gene transfer element. Integron is genetic fragments that can recognize and mobile gene cassettes by site-specific recombination. Most of the gene cassettes in the integrons relate to resistance, and it is very important to the genome evolution and the resistance. According to the kinds of integrase, there are main four kinds of integrons. Among the total, class 1 integron is the most common and the first discovered, and the movement of the gene cassettes has be confirmed. Class 1 integron can lead to the multidrug resistance in the Gram-negative bacterias by the integration of one or more resistance gene cassettes. Class 1 integron includes 5'-CS,3'-CS and the gene cassettes.In the 5'-CS, there are genes of intll integrase, recombination site attll and promoter. In the 3'-CS, there are three ORFs (qacEΔ1, sull and ORF5). Some integrons have no 3'-CS. Class 1 integron is widespread in the Gram-negative bacterias, such as Escherichia, Klebsierlla, Pseudomonas and Acinetobacter and so on. Class 1 integron can transfer many kinds of resistance gene cassettes, such asβ- lactam, aminoglycosides, amphemycin, phytomycin, Quinolone and quaternary amine, and they are important in the transmission of resistance gene cassettes.ISCR1 usually exist in the complex class 1 integrons. The upstream sequences of ISCR1 are the same, and the sequences in junction point between ISCR1and class 1 integron are not change, that indicate that ISCR1 consistent with the feature of complex class 1 integrons. The complex class 1 integrons with ISCR and integron, and ones with two ISCRs, are relating to resistance. They have potentiality to transfer the resistance genes. Multidrug resistance is not negligible, and the structure can offer more pathway and direction to the studies of multidrug resistance.In China, antibiotics are abused acutely. The resistance of bacterias is more serious than other courtries and bacterias can bring more kinds of resistance genes, which is an affluent resource to researchs. If we can take a good use of these bacterias, it makes sure that we can get much message about resistance genes and it is profit for the cure to the bacterias infection.ISCR1 and class 1 integron are important transposable elements, and they are correlated with resistance genes. In abroad, there are many researches of ISCR1about kinds of bacterias, but in China no one. At the same time, although there are many studies about class 1 integron, in China no study with lots of kinds of bacterias.Therefore, in our research, we screen the resistance genes in ISCR1and class 1 integron in the Gram-negative bacterias by PCR, and then we can get the gene arrangements, so that we can understand the mechanism and the mutual relationship of ISCR1 and class 1 integron.Methods1. The choice of Gram-negative bacterias and extraction of the genome DNA Escherichia coli and Klebsierlla peumoniae strains were from 2005 to 2009, which are multidrug resistance and not reduplicative. Other strains were from 2008 to 2009, such as Enterobcter cloacae, Enterobcter aerogenes, Proteus vulgaris, Proteus mirabilis, Serratia marcescens, Serratia fonticola, Pseudomonas aeruginosa, Acinetobacter baumannii, Pseudomonas maltophilia, Burkholderia cepacia, Pseudomonas putica and so on. There were total 1800 strains, and we used the method of SDS- protease K- alcohol phenyl-trichlormethane to extract the genome DNA.2. The detection of genes of orf513 and gene cassettes in ISCR1 Used the methods which had been used internal or overseas to detect genes of orf513,then detected the gene cassettes, and then used two incision enzymes to cut the gene cassettes, classified the kinds of gene cassettes, and got some gene sequences to make sequencing, at last analyzed the sequences and submitted to the GeneBank.3. The detection of genes of integrase and gene cassettes in class 1 integron Used the methods which had been used internal or overseas to detect genes of integrase, then detected the gene cassettes, and then used two incision enzymes to cut the gene cassettes, classified the kinds of gene cassettes, got some gene sequences to make sequencing, at last analyzed the sequences and submitted to the GeneBank.4. Initial validation of the tandem relationship of ISCR1 and class 1 integron We assumed that ISCR1 and class 1 integron were connection in series.Then we used the PCR primer to detect.Results1. Some resistance genes were most frequently associated with ISCR1 and some new resistance gene arrays were discovered in different bacteriums The genes of quinolone(qnr),β-Lactamase and quaternary amine(qacEA) were most frequently associated with ISCR1 in our study.And there were 10 new resistance gene arrays in ISCR1 in the world, such as orf513+sapA-like+qnrB2+qacEdeltal,orf513+short chain dehydrogenase/reductase+qnrB6+qacEdeltal (Escherichia coli); orf513+short chain dehydrogenase/reductase+qnrB6+qacEdeltal,orf513+blaCTX-M-9+insB+ qacEdeltal(Enterobcter cloacae); orf513+ampC+ampR+qacEdeltal(Enterobcter aerogenes); orf513+short chain dehydrogenase/reductase+qnrB6+qacEdeltal (Serratia fonticola); orf513+qnrA1+ampR+qacEdeltal,orf513+blaPER-1+GST-like pseudogene+qacEdeltal(Acinetobacter baumannii); orf513+qnrA1+ampR+qacEdeltal (Pseudomonas aeruginosa); orf513+qnrA1+ampR+qacEdeltal(Pseudomonas maltophilia).And there were 2 new resistance gene arrays in China in different bacteriums, such as orf513+sapA-like+qnrB2+qacEdeltal (Klebsierlla peumoniae),orf513+qnrA1+ampR+qacEdeltal(Enterobcter cloacae).2. Some resistance genes were most frequently associated with class 1 integron and some new resistance gene arrays were discovered in different bacteriumsThe genes of aminoglycosides,β-Lactamase and trimethoprim were most frequently associated with class 1 integron in our study.And there were 16 new resistance gene arrays in different bacteriums in the world, such as aacA4+catB8+aadA1 (Escherichia coli); dfrA1+aadA5,dfrA1+orfC,dfrA16+aadA2(Klebsierlla peumoniae); aacA4+catB8+aadA1,aadB+aadA2,catB8+aadA1,dfrA1+aadA5,dfrA12+orfF+aadA2+ORFⅡ+ORFⅢ,dfrA12+orfF+aadA2 (Enterobcter cloacae); aacA4+catB8+aadA1,ant(3")-Ih-aac(6')-Iid+catB8,dfrA12+orfF+aadA2 (Enterobcter aerogenes); dfrA12+orfF+aadA2(Proteus mirabilis); aacA4+blaIMP-25+ blaOXA-30+blacatB3(Pseudomonas maltophilia); blaIMP-9+aacA4+blaOXA-10+aadA2 (Pseudomonas putica).And there were 6 new resistance gene arrays in different bacteriums in China, such as aadB+aadA2(Escherichia coli); aacC4+cmlA1,aacA4 +blaOXA-4+aadA2,aacC1/aacCA1+orfP+orfQ+aadA1,acc(6')-Ⅱ/aac(6')-Ⅱ,arr-3+ereC+aadA1+cmlA 7(Klebsierlla peumoniae).3. Initial validation of the tandem relationship of ISCR1 and class 1 integronThe results of PCR showed that ISCR1 and class 1 integron could connect in series. It was very important to the studues on the ISCR1and class 1 integron in the fucture.Conclusion 1. There were different kinds and amount of resistance gene cassettes in different bacteriums.2. In ISCR1 there were 10 new resistance gene arrays in the world, and 2 new resistance gene arrays in China in different bacteriums; in class 1 integron there were 16 new resistance gene arrays in the world, and 6 new resistance gene arrays in China in different bacteriums.3. The bacteriums of same kind could bring different resistance gene arrays, and ones of different kinds could bring same arrays, in ISCR1 or class 1 integron.4. The genes of quinolone(qnr),β-Lactamase and quaternary amine(qacEΔ) were most frequently associated with ISCR1 in our study.5. The genes of aminoglycosides,trimethoprim (dfr) andβ-Lactamase were most frequently associated with class 1 integron in our study.6. Class 1 integron and ISCR1 could connect in series.