| Today, because antibiotic agents are wildely used to treat infection in our daily life caused the bacteria resistance problem and the multidrug resistance (MDR) phenomenon. The abuse of antibiotic agents was not only the way of bacteria multidrug resistance dissemination but also the important factor for bacteria evolution of multidrug resistance, which provided a selection pressure making human body and the natural environment to be a reservoir for the bacteria to evolve into multi-drug resistant strains. Integron is a mobile element which is referred the recombinase and a recombination site. The recombinase recognizes the specific recombination site, then excises or captures the gene cassettes which can be expressed by a promoter of integron and encoding a variety of hydrolytic enzymes or modifying enzymes, efflux pumps and bacterial resistance related genes.Clinical pathogenic bacteria with different resistance genes released into the environment by many ways and the resistance mechanism can be transferred by daughter colony and the other bacteria can obtain the resistance genes through the horizontal gene transfer. Mutations or recombinations were occurred frequently in the gene transfer process to form the new resistant genes, and novel arrays of multiple drug resistance genes were formed by recombination involving integrases. Consequently, these new resistance factors may be transferred to the clinical pathogenic bacteria from the environment and through air, food or water to infect the human body to form new resistance factors in clinical and threat to human health. Integron can be transferred horizentally by transposon and conjugation plasmids, especially under the aquatic environment with antibiotic selection pressures, which is one of the mechanisms for competition and adaption in bacteria and the most important way for resistance scattering.This study, we investigate the integrons dissemination and the related resistance gene cassette array structures in the wastewater nearby hospital to explore and identify the novel integron genetic structures. We analysis the bacteria resistance mechanisms and the related gene transfer mechanisms by genetic level to illustrate the risk of antibiotic abuse and guide the proper use of antibiotics to prevent the overuse antibiotics. As well, we could develope a number of new antibacterial drugs according to the mechanisms of bacterial resistance. The main methods and results are as follows:1. Bacteria were selected from contaminated water in Jinan by mebrane filter method, all the resistant isolates were also investigated by polymerase chain reactions of class 1,2 and class 3 integrasesFifteen different water samples were collected from 2008 to 2009, including the waste water nearby Thousand Buddha hospital, Qilu hospital, the XiaoQing River, a pigpen wastewater in Qilu pharmaceutical factory, water sample pollutated by petroleum, Waste Water Treatment Plant, and the river nearby the Shandong Traditional Chinese Medicine Hospital, the spring of Heihu, and the Xi Lake at different times.391 different resistant Enterobacteriaceae were selected and the antibiotics susceptibility test was used to distinguish the bacteria resistance profiles by CLSI criterion. More than 85% isolates were resistant to ampicillin, trimethoprim and sulfisoxazole. According to the conserved sequences of class 1, class 2 and class 3 integrase, primers intI1F/R, intI2F/R and intI3F/R were designed to investigate the prevalence of integron and 59.3% belonged to class 1 integron,9.2% identified to class 2 integron but no class 3 integron were detected. Comparing the susceptibility between integron bacteria and non-integron bacteria, the ampicillin, trimethoprim and sulfisoxazole resistances are more than 90% in integron bacteria and 87.2% in non-integron bacteria. In all, except the fosmycin, all the other antibiotics in containing integron structure are 10%-20% more than the resistance in non-integron bacteria.2. We analyzed the structure of class 1 integron by PCR amplification and the RFLP type by EcoRⅡAccording to class 1 integron 5'CS and 3'CS, the variable region were obtained by primers hep58 and hep59. The sizes of PCR production were from 0.2kb to 6.3kb, and the related profiles of EcoRⅡrestriction RFLP map, we found 34 different restriction maps, sequencing results showed that 35 different gene cassette arrays with 42 kinds of gene cassettes. Nine of 35 different gene cassette arrays were belonged to the novel rearragement.16S rRNA sequencing results showed the nine isolates were belonged to Aeromonas sp., Klebsiela sp., Escherichia sp., Comanionas sp., Providencia sp. and Proteus sp. The nine novel gene cassette arrays are showed as following:orfI; arr3—dfrA27；aadA△16—acc(6')-Ⅱ; aac(6')-Ib-cr—blaoxA-1—catB3；aacA4—orfun—aacA4—catB3; dhfrV—aac(6)-Ⅱ—nitl—nit2—catB3—blaOXA-1; aac(6')-Ib-cr—arr3—dfrA27—aadA16—IS26; aadB—cat—blaoxA-10—aadA1—dfrA1—aacA4; aadB—cat—blaoxA-10—aadA1—orfll. The gene cassette array about dfrAl7—aadA5 was the most prevelance in our study, contained 43.5%, and followed arrays was 19.3% of dfrA12—orfF—aadA2.3. Thirty six isolates were positive to the class 2 integrons and the structures of class 2 integrons were analysised to five different gene cassette arrays. The transformation and cojugation expriments were conducted to analysist the horizontal gene transferPCR products of class 2 variable regions were carried out by the conservative primers hep74 and hep51 and show five different sizes,4.3kb,3.0kb,2.5kb and 2.2kb, as well as a 2.5kb amplicon either negative to the primers or appeared a weaker band. Sequencing results showed the arrays are linF—dfrA1—aadA1—orf441; dfrA1—catB2—sat2—aadA1; estXvr—sat—aadA1; dfrA1—sat2—aadA1 and the hybrid array dfrA1—sat2—aadA1—qacH. Rep-PCR results showed that the 36 bacteria containing 15 different profiles,16S rRNA identification results showed that 19 isolates are Proteus spp., Escherichia spp.,and Providencia spp. are both 7 isolates and Citrobacter freundii, Shieglla sp., and Acinetobacter sp. are only one isolate, respectively. Cojugation and transformation experiment, PFGE and southern hybrid results were showed that except the hybrid class 2 integron, E.coli C4, the other novel class 2 integrons were chromosomally located. The specific primers about tnsD and tnsE were used to identify the Tn7 family. As a result, all the class 2 integrons except the hybrid class 2 integrons were the members of typical Tn7 family. The elements about IS26, tnp440 and sul3 were located downstream of the hybrid class 2 integrons.4. The new variant of quinolone resistance gene qnrVC4 was identified in Aeromonas puctata isolates and anlaysis of the mechanisms of quinolone resistanceThe 3.3 kb aacA4—orfun—aacA4—catB3 array was identified in class 1 gene cassette array and orfun was a lkb gene cassette encoded an open reading frame of 218aa which was 45%-81% similarities with QnrB6, QnrAl, QnrS1, QnrC, QnrVCl, and QnrVC3 by GenBank Blast suit analysis. According to the similarity of nucleotides, amino acids and the attC recombination site, the gene was named qnrVC4, and its protein was designated QnrVC4 based on the qnr nomenclature. Conjugative transfer and transformation expriments were both unsuccessful. The location of qnr VC4 was carried out by southern bolting with probes of qnr VC4 gene and 23 S rRNA, respectively. Both hybridizations were carried out using plasmids isolated by alkaline lysis, followed by agarose gel electrophoresis and total genetic material digested with S1 nuclease and I-CeuI, followed by pulsed-field gel electrophoresis (PFGE). The results indicated that qnrVC4 was located on a large plasmid. The qnrVC4 gene and qnrVC4 with the promoter of integron and aacA4 gene were both subcloned in plasmid pBC KS (+), both of which expressed by Lac promoter or promoter of integron (PcWTGN-10)-The minimum inhibitory concentrations (MICs) of ciprofloxicin, gatifloxacin, and nalidixic acid were 0.032, 0.047 and 4μg/mL with the recombinant pBCKS-qnrVC4 and 0.008,0.006 and 2μg/mL in the recombinant pBCKS-Pt-qnrVC4, but the MIC of the original isolate Aeromonas punctata was 0.38,0.19 and 96μg/mL, respectively. The analysis of the GyrA, GyrB, and ParC about the quinolone resistance determining regions (QRDR) showed the GyrA mutation at 83 Ser to Ile was the main facter of quinolone resistance.5. Quinolone resistances genes qnrA, qnrB, qnrS were detected in the 391 isolatesBecause of qnrA, qnrB and qnrS gene were reported on plasmids, which provided the easier way of low quinolone resistance transfer and the possibility to form the high quinolone resistance. Eventhough they make little contribute to the quinolone resistance in quinolone resistance isolates but many researchers are more trendency to research the dissemination of these genes and their variants. In 391 isolates,2 isolates were positive qnr A,17 isolates contained qnrB gene,9 qnrS isolates and 3 isolates were positive to both qnrB and qnrS. The PCR with two specific primers and the PCR-RFLP method to analysis the qnrB variants were successful to identify the qnrB variant and which were identical to the sequencing results. The aac(6')-Ib-cr gene identified with qnr gene can promote the quinolone resistance, and 12 isolates were both contained aac(6')-Ib-cr and one kind of qnr gene. The efflux pump gene qepA related quinolone resistance was also detected in one qnrB isolate and four qnrS isolates.18 isolates of qnr gene can be transferred by horizontal gene transfer.6. ISCR1 element of the complex class 1 integron was detected in 391 isolates and the related resistance mechanisms were analysisedThe ISCR1 element was detected in the 391 resistance isolates,41 isolates were showed with ISCR1, including all the qnrA and qnrB isolates. Primers of conserved regions (513BF, qac△EB and SEFA-PCR primers) were designed to analysis the downstream genetic structures of ISCR1 element.11 different kinds of genetic structures were illustrated, including ISC1—qnrA—ampR—qac△E—sull, ISCR1—qnrB2—qac△E—sull, ISCRl—qnrB6—qac△E—sull, ISCR1—dfrA10—qac△E—sull, ISCR1—tnpU—armA, ISCR1—dfrL—qac△E—sull, ISCR1—dfrM—qac△E—sull, ISCR1—dfrN—qac△E—sull, ISCR1—dfrA19—int11—catB3—aadB—qac△E—sull, ISCR1—blaPER-1—gst—ABC transporter—orf252—qac△E, ISCR1—IS630—blaPER-1—gst——ABC transporter—orf252—qac△E. Three dihydrofolate reductase (DfrL, DfrM and DfrN) were analysised the MICs of trimethoprom by the mimic genetic structure with ISCR1, and showed the high resistance more than 1024μg/mL。... |
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