【作者】 李明成；

【导师】 李凡；

【作者基本信息】 吉林大学 ， 病原生物学， 2006， 博士

【摘要】 产志贺毒素大肠埃希菌(Shiga toxin-producing E.coli,STEC)是世界范围内最严重的食源性致病菌。STEC耐药性产生与传播已受到世界广泛关注。为探讨STEC多重耐药性产生和传播机制,本研究采用PCR、接合与转化、克隆测序、Southern blot和酶切图谱分析等技术,在测定抗菌药物敏感性的基础上,对STEC分离株携带整合子、R质粒和耐药基因进行研究分析。结果表明多重耐药STEC染色体、R质粒携带1500bp ~750bp第1类整合子和dfrA1、aadA1基因盒及sul1、tem、tet、erm等耐药基因,传递对磺胺类、氨基糖苷类、β-内酰胺类、四环素类和红霉素类等抗菌药物的耐药性。发现了染色体上一种缺陷型及编码宋内志贺菌(S.sonnei)跨膜转运蛋白基因的第1类整合子。证明了STEC多重耐药菌株的出现和流行与农业、畜牧业将磺胺类、氨基糖苷类等抗菌药物作为动物生长促进剂长期使用有关。STEC在抗菌药物的选择压力下不断进化,通过整合子与R质粒促进多重耐药性的产生和扩散。本研究结果揭示了STEC在耐药性表达及耐药性传递方面的作用机制,为进一步更好地控制STEC基因水平的耐药性传播提供重要的科学依据。更多还原

【Abstract】 Foodborne disease is the most general public health problem in the world.Everyone is in the danger of foodborne illness during his daily life. Harm ofmicroorganisms is the most important cause of foodborne illness. Among allthe foodborne diseases, hemorrhagic colitis (HC), hemolytic uremic syndrome(HUS) and thrombotic thrombocytopenic purpura (TTP) caused by Shigatoxin-producing E.coli (STEC) are the most serious. Main serotype of STEC isEnterohaemorrhagic E.coli (EHEC) O157:H7.The emergence of antibiotic resistance among STEC isolates is closelyassociated with the abuse of all kinds of antibiotics. In recent years, since theantibiotics have been administered in agriculture and animal husbandryincluding almost all the antibiotics used in human, varieties of pathogenicbacteria and normal flora colonized in animal intestinal tract have producedantibiotic resistance. These drug resistance bacteria can spread to humandirectly through soil, air, water and food contaminated by stool. Theunder-dosage and residuals resulted in the emergence and dissemination ofantimicrobial resistance among strains and furthermore to humans throughfood chain. At the same time, in clinical and community medical treatment,with the great quantity and long-time use of β-lactamases, aminoglycosidesand quinolones, the number of foodborne drug resistance bacteria is on therise and the spectrum of resistance also is expanding continuously.It has been proved that gene cassettes-integron systems played animportant role in the acquisition and dissemination of antibiotic resistanceamong foodborne pathogens.Integrons are mobile DNA elements located in genomic or plasmid DNAconsisting of two highly conserved nucleotide sequences (3’CS and 5’CS)flanking variable region containing cassettes. Six classes of integrons havebeen identified to date. The majority of integrons identified among differentisolates belong to class 1 type. In class 1 integrons, the 5’conserved segmentencodes a site-specific recominase (integrase, int I), specific recombinationsite (attI) and variable gene cassette in length and number. Gene cassettesusually contain a single open reading frame (ORF) coding resistance tospecific antimicrobial and a recognition site for integrase attC (known as the59-base element before).However, their consensus motifs have littlehomologous sequences, varying in 57~147 bp, their two regions have highlyconserved sequences as invers core site, RYYYAAC, and the other as core site,GTTRRRY. Expression of gene cassette relies on the promoter in the upstreamof 5’CS. The 3’CS carries qacΔE, which specifies resistance to antisepticsand disinfectants;sul-1 gene, which confers sulfonamide resistance;and anopen reading frame of unknown fuction. Only a few cassettes have their ownpromoters. Currently, more than 70 distinct cassettes have been identifiedamong integrons. These gene cassettes encode proteins involved in resistanceto almost all kinds of antibiotics used in clinic frequently and widely now.There are some gene cassettes encoding toxin or other distinct proteins.This study was initiated to isolate and identify 245 E.coli strains fromdifferent specimen. Isolates were characterized for STEC-associated virulencegenes using specific primers of stx1, stx2, eaeA and hlyA. 8 STEC isolateswere identified by PCR amplification. A total of 50 STEC isolates includingserotypes O157 and non-O157 strains were used in this study.The antimicrobial susceptibilities of 16 different antibiotics weredetermined using K-B test recommended by WHO. The results showed thatSTEC isolates are highly resistant to sulfamethoxazole, ampicillin,streptomycin, tetracycline, erythromycin and ciprofloxacin. However, theresistance to tetracycline, erythromycin and ciprofloxacin are different inChinese and USA isolates. Especially, the resistance to sulfamethoxazole is100%. There are one strain from China and America respectively, which areeven resistant to third and fourth generation cephalosporin and are identifiedto be ESBLs through Double disk test.The emergence of multi-resistant STEC was associated with thewidespread use of antimicrobial agents, sulfamethoxazole, erythromycin,ampicillin, tetracycline, aminoglycosides as animal growth promotion (AGP).The selection pressure of antibiotics would promote the development ofantibiotic-resistance of foodborne pathogens. Data suggested that selectivepressure imposed by the use of antibiotics drived the development ofantimicrobial resistance in STEC.In this study, the presence of class 1 and 2 integrons was detected byPCR and multiple PCR assay using the specific primers of class 1, 2 integrasegenes and consensus sequence of integrons. Bacterial total DNA andtransconjugants were used as template for detecting class 1、2 integons byPCR technique respectively. The finds showed that class 1 integrons wereidentified among 13 isolates. In STEC isolates from America, five isolatescontained a 1000bp amplicon and one contained a 750bp amplicon. In fiveChina isolates, the amplified fragments were 1000bp in size and one containedtwo integrons of 1500bp and 500bp. The detection rates of class 1 integronsfrom China and U.S.A were 30 %(6/20) and 26.7 %(8/30)respectively andthere was no significant difference. Comparing the resistance rate of integronpositive strains with the negative strains, the results demonstrated that allpositive strains were resistant to three types of antimicrobial agents at least,especially, the difference of resistance to aminoglycosides was significant(P<0.01)between positive and negative strains.To testify the possible insert cassettes, PCR-RFLP, PCR-mapping,sequencing and searching the GenBank database of the National Center forBiotechnology Information via the BLAST network service, the resultsdemonstrated that 10 strains of STEC harbored 1000bp integrons with aadA1cassette, one strain harbored 1500bp integron with dfrA1 and aadA1 cassettesconferring resistance to aminoglycosides and sulfamethoxazole respectively,two strains harbored 750bp with dfrA1 cassette conferring resistance tosulfamethoxazole. Although they were simple, all cassettes had the completeintegrons carrying integrase and sul1 gene, and they had the potential abilityof capturing, recombinating other cassettes. In the meantime, two kinds ofspecial class 1 integrons had been detected by molecular method, one was1500bp which had a defective 3’conserved segment, the other was 500bpwhich encoded transporting protein of S.sonnei within the chromosome.To determine the dissemination mechanism of drug-resistance andlocation of integrons, the plasmids in bacteria with integrons were conveyedto the engineering bacteria via transconjugantion, and plasmid, transconjugantDNA profile was analyzed. It proved that 14 STEC strains had differentplasmids from 4200bp to 8500bp, which carried the resistance tosulfamethoxazole, erythromycin and aminoglycosides identified through theresistant phenotype. Integrons were detected by PCR assay as template oftransconjugants, only 11 strains with integrons were discovered and 1500bpintegron hadn’t been detected. It suggested that the integron might be locatedon the chromosome.To test the hypothesis, southern blot hybridizations were performed todetermine the locatin of identified class 1 integrons. Genomic and plasmidDNA were extracted from integron-positive STEC and genomic DNAdigested with EcoR I. The class 1 integrase gene was used as a DNA probeand labeled with the digoxigenin-11-dUTP label and detection kit. Thefindings demonstrated that all integrons were located on the transferableplasmids except two strains with 1500bp integrons. It was worth to mentionthat the bigger plasmid like p O157 hadn’t been extracted from all STECs.The reasons lied in the fact that the plasmids missed naturally, or disappearedduring experimenting. However, the resistant STEC would improve theirtoxigenicity once they acquired the other big plasmids and the food bornediseases would emerge and spread.To detect the drug-resiatant gene and their location, their associatedprimers were designed and detected by PCR as template of the total andtransconjugants DNA. The gene tem, sul1, tet and erm conferred theresistance to ampicillin, sulfamethoxazole, streptomycin, tetracycline anderythromycin had been detected. The phenotypes and inherit properties provedthe integron-positive STEC have multiple antimicrobial resistance.The test proved that class 1 integron among STEC had an associationwith their multiple antimicrobial resistance, and played a role in spreading anddissemination of drug-resistant gene. The integon and R plasmid couldprovide the quite efficient mechanism of new drug-resistant genes inacquisition and dissemination.However, during experiment, some strains without integrons showed thesame the resistance as other strains with integrons, some strains’resistantgenes hadn’t been detected even though they were resistant to drug;somestrains without 1 integrons might possess other kinds of integrons. All theseshowed that the antimicrobial resistant mechanisms weren’t simple but theyresulted from many intricate mechanisms together.Many kinds of factors affect the distribution and dissemination ofbacterial drug-resistance. The resistant gene and phenotype vary with differentcountry, district, city and community. It implys that we should pay a greatattention to studying on resistant gene cloning, expressing, and make an effortto find the strategies to block the resistant gene in order to prolong the lifetimeof antibiotics. The study will give us a new idea and direction to investigatethe mechanism of capture and dissemination of antimicrobial resistant genethrough integrons furthermore, and consider integrase and recombination siteas the inhibitory site of new drug.更多还原