【作者】 杨保伟；

【导师】 孟江洪； 曲东；

【作者基本信息】 西北农林科技大学 ， 微生物学， 2010， 博士

【摘要】 本文研究了陕西省西安、杨凌和宝鸡地区零售鸡肉、猪肉、牛肉、羊肉和即食凉拌菜中沙门氏菌的污染状况,沙门氏菌分离株的血清型、药敏性、基因型、Ⅰ类整合子、Ⅰ型沙门氏菌基因岛和部分耐药基因的检出状况等基本特性。同时,基于从陕西省和美国马里兰州等地分离的食源性沙门氏菌,探讨了Ⅰ类整合子、沙门氏菌基因岛、部分耐药基因、氟喹诺酮类抗生素靶标基因和甲基错配修复系统基因突变及质粒对食源性沙门氏菌耐药性产生的贡献和机制。得出以下主要结论:(1)陕西部分地区超级市场和农贸市场中零售肉和即食凉拌菜存在严重的沙门氏菌污染现象,沙门氏菌阳性鸡肉、猪肉、牛肉、羊肉和即食凉拌菜的平均检出率分别为69.9%、19.4%、30.1%、55.3%和9.6%,肉类食品被沙门氏菌污染的平均比例明显高于即食凉拌菜。此外,在零售肉和即食凉拌菜中除有沙门氏菌检出外,也检出了致病性大肠杆菌和金黄色葡萄球菌。(2)食源性沙门氏菌的血清型和基因型表现为多样性。研究共从陕西省分离到沙门氏菌359株,涵盖24个血清型,最常见的血清型为Salmonella Enteritidis(31.5%),其次为S. Typhimurium(13.4%),S. Shubra (10.0%)和S. Indiana (9.7%)等。检出了Salmonella Rissen、S. Brancaster、S. Braenderup、S. Litchfield、S. Pakistan和S. Bsilla等我国沙门氏菌的稀有血清型。检出的鸡肉源沙门氏菌涵盖了21个血清型,猪肉源、牛肉源和羊肉源沙门氏菌分别涵盖了8、11和9个血清型。从西安、杨凌和宝鸡3地采集的零售肉中分离出的沙门氏菌分别涵盖20、15和3种血清型。使用XbaⅠ酶切、脉冲场凝胶电泳对沙门氏菌分型后,测定血清型内沙门氏菌株呈现出相同或不同的基因型,表明在某一特定的血清型内,沙门氏菌株既具有较高的基因同源性,又具有明显的多样性。(3)食源性沙门氏菌对大部分常用抗生素产生抗性,多重耐药沙门氏菌的出现已经成为普遍现象。359株食源性沙门氏菌对磺胺甲恶唑的耐药率已高达67%,对四环素、卡那霉素、萘啶酮酸、氨苄西林、阿莫西林/克拉维酸和头孢曲松的耐药率分别为56%、37%、35%、33%、32%和16%。其中,189株(52.6%)沙门氏菌可抗至少4种以上抗生素,93株(25.9%)可抗10种以上的抗生素,33株(9.2%)可抗11种抗生素,25株(7.0%)可抗12种抗生素,17株(4.7%)可抗13种抗生素,9株(2.5%)可抗高达14种抗生素。产生多重耐药的沙门氏菌常见血清型主要为SalmonellaⅡ、S. Rideau、S. Saintpaul、S. Shubra、S. Indiana、S. Typhimurium和S. Infantis。(4)Ⅰ类整合子和Ⅰ型沙门氏菌基因岛等基因元件以及blaTEM和/或blaCMY-2基因编码的超广谱β-内酰胺酶介导的耐药性是食源性沙门氏菌产生多重耐药的重要机制。在陕西省和美国马里兰州等地分离的食源性沙门氏菌中,整合子的检出率分别为16%和17.3%,Ⅰ型沙门氏菌基因岛的检出率分别为13.6%和7.4%。整合子均存在于多重耐药的沙门氏菌之中,整合子携带的耐药基因主要有aadA1、aadA2、aadA5、tetR、blaPSE-1、blaDHA-1、blaVEB-1、dhfr1、dhfrⅤ、dhfrⅦ和dhfr17等。携带有Ⅰ型沙门氏菌基因岛的菌株一般都具有比较宽的耐药谱,除可以同时对氨苄西林、头孢曲松、头孢哌酮等青霉素类和头孢类抗生素产生抗性外,还对其它种类的抗生素如氨基糖苷类抗生素庆大霉素、卡那霉素、阿米卡星和链霉素,喹诺酮类抗生素萘啶酮酸和氟喹诺酮类抗生素环丙沙星,氯霉素等产生抗性。359株沙门氏菌中,有32株(8.9%)检出blaTEM基因,35株(9.7%)检出blaCMY-2基因,这些菌株除均对氨苄西林耐药外,也表现出对头孢曲松和/头孢哌酮和/或头孢西丁的耐药现象,表明blaTEM和/或blaCMY-2基因编码的超广谱β-内酰胺酶是介导沙门氏菌产生抗生素抗性的因素之一。(5)解旋酶和拓扑异构酶基因突变在沙门氏菌对氟喹诺酮类抗生素药敏性降低及多重耐药性的形成过程起着非常重要的作用。30株多重耐药沙门氏菌中,4株(13.3%)对7种氟喹诺酮抗生素、16株(53.3%)对6种抗生素、8株(26.6%)对3种抗生素产生抗性。除未在gyrB基因中检出突变外,在gyrA、parC和parE基因中共检出68个点突变。GyrA基因中Ser83→Phe、Asp87→Gly和Asp87→Asn为最常见的突变,parC基因中常见突变类型为Ser80→Arg。4株菌的parE基因发生了突变,突变类型分别为Lys428→Gln、Lys441→Ile、Asp494→Asn和Gly442→Ser。GyrA基因突变只可以赋予沙门氏菌低水平的氟喹诺酮抗生素抗性,如伴随着gyrA基因上其它位点的突变和parC和/或parE基因上的突变,则会介导高水平的氟喹诺酮类抗生素抗性。(6)MMR系统基因突变是介导沙门氏菌多重耐药的机制之一,但自然界发生MMR系统基因突变的沙门氏菌超级突变子检出率却很低。研究从分离于美国马里兰州等地的390株食源性沙门氏菌中筛选得到5株疑似突变子,它们分别为Salmonella Typhimurium ST20751、S. Heidelberg 22396、S. Enteritidis 17929、17929N和17929R。野生型mutH、mutL、mutS和uvrD基因互补实验和DNA序列测定结果证明5株突变子均未发生MMR系统基因的显性突变或缺失,沙门氏菌染色体MMR系统发生基因突变而导致多重耐药超级突变子产生的几率很低。(7)质粒编码的抗生素抗性在沙门氏菌耐药性形成和传递过程起着至关重要的作用。MDR沙门氏菌中质粒的酶切图谱和聚类分析结果表明,质粒作为一种常见的可移动基因元件,可以通过不同的宿主和基质作为载体,在不同的地域和食品中间进行传播,使其编码的抗生素抗性进行水平传递。质粒通过接合作用将抗性基因转移到受体菌EC1003(E. Coli)和17929N(S. Enteritidis)的接合效率分别在2.4×10-4到5.6×10-1之间,接合后绝大部分接合子均获得了供体菌质粒编码的相应抗性表型,耐药基因在接合过程也发生了转移,赋予被接合细菌新的耐药性。更多还原

【Abstract】 Salmonella is one of the most important foodborne pathogens worldwide. Foods of animal origin are frequently contaminated with the pathogen and can serve as vehicles in transmitting salmonellosis in humans. A survey on Salmonella in retail meats and salads was conducted to determine the prevalence, and phenotypic and genotypic characteristics including serotype, antimicrobial susceptibility, genomic subtype and possession of integronsⅠ, Salmonella Gene IslandⅠ(SGIⅠ) and antimicrobial resistance genes. The food products including chicken, pork, beef, lamb and read to eat salad were collected in Xian, Yangling and Baoji in Shaanxi Province. Additionally, the contributions of several genetic elements such as integronsⅠ, SGIⅠ, plasmids and antimicrobial resistance genes, as well as gene mutation, methyl-directed mismatch repair (MMR) system mutation and defective, to the antimicrobial resistance of Salmonella were investigated. Major findings and conclusions were summarized in the following:(1) Salmonella contamination in chicken, pork, beef, lamb and read to eat salad in supermarket and freemarket in several districts of Shaanxi province was common. The comtamination rates ranged from 9.6% in salad to 69.9% in chicken. The pathogen was also identified in pork (19.4%), beef (30.1%) and lamb (55.3%). In addition, pathogenic Escherichia coli and Staphyloccocus aureus were also detected in these products.(2) The serotypes and genetypes of foodborne Salmonella were diverse. Among 359 Salmonella isolates recovered in the study, 24 serotypes were identified with S.Enteritidis (31.5%) being most common, followed by S. Typhimurium (13.4%), S. Shubra (10.0%) and S. Indiana (9.7%). Several rare serotypes, such as S. Rissen, S. Brancaster, S. Braenderup, S. Litchfield, S. Pakistan and S. Bsilla, were identified as well. ??? of pattens were generated when the chromosome DNA of Salmonella was digested with restriction endonuclease XbalⅠa nd analyzed using pluse-field gel electrophoresis (PFGE). Serotyping and PFGE results were in agreement although PFGE was much discriminatory than serotyping.(3)The Salmonella isolates also exhibited resistance to antimicrobials. Multidrug resistance (MDR) was common. Of the 359 isolates, 67% was resistant to sulfamethoxazole, 56% to tetracycline, 37% to kanamycin, 35% to nalidixic acid, 33% to ampicillin, 32% to amoxicillin / clavulanic acid and 16% to ceftriaxone. One hundred and eighty nine (52.6%) isolates were resistant to at least four antimicrobials, 93 (25.9%) to≥10 or more, 33 (9.2%) to≥11 and 25 (7.0%) to≥12, 17 (4.7%) to≥13, and 9 (2.5%) to≥14 antimicrobials. The common serotypes of MDR Salmonella included SalmonellaⅡ, S. Rideau, S. Saintpaul, S. Shubra, S. Indiana, S. Typhimurium and S. Infantis.(4) Antimicrobial resistance encoded by mobile genetic elements, such as integronsⅠand SGIⅠ, were important in mediating MDR of Salmonella.Of 359 Salmonella isolates recovered from Shaanxi, China and 390 from U.S.A, integronsⅠw ere found in 16.0% and 17.3% isolates, and SGIⅠin 13.6% and 7.4%, respectively. All integrons were detected in MDR Salmonella isolates. Genes carried in integronsⅠgene cassettes included aadA1, aadA2, aadA5, tetR, blaPSE-1, blaDHA-1, blaVEB-1, dhfr1, dhfrⅤ, dhfrⅦand dhfr17. Isolates with SGIⅠexhibited relatively extended antimicrobial resistance spectrum, and showed resistant to not only penicillin and cephalosporin, such as ampicillin, ceftriaxone and cefoperazone, but also aminoglycosides and fluoroquinolones, such as gentamicin, kanamycin, amikacin, streptomycin and ciprofloxacin, as well as resistance to chloramphenicol.BlaTEM gene was detected in 32 (8.9%) and blaCMY-2 in 35 (9.7%) Salmonella isolates that were resistant to ampicillin, and to ceftriaxone and/or cefoperazone, and/or cefoxitin, indicating that ESBLs encoded by blaTEM and/or blaCMY-2 were responsible for the resistance.(5) Mutations in Salmonella gyrase and topoisomerase genes cause decreased susceptibility to fluouoquinolones and multidrug resistance. Of 30 MDR Salmonella isolates, 4 (13.3%) exhibited resistance to all seven fluoroquinolones tested, 16 (53.3%) to 6, 8 (26.6%) to 3 fluoroquinolones. A total of 68 mutations in gyrA, parC and parE were found in the 30 Samonella isolates, whereas no gyrB mutation was detected. The most common mutations were Ser83→Phe, Asp87→Gly and Asp87→Asn in gyrA, and Ser80→Arg in parC gene. Four of the isolates had novel mutations in parE, including Lys427→Gln, Lys441→Ile, Gly442→Ser and Asp494→Asn. Isolates with single gyrA mutations were less resistant to fluoroquinolones than those together with an additional parC and/or parE mutation in Salmonella.(6) MMR system gene mutation in Salmonella is an important mechanism of antimicrobial resistance; however, the incidence of hypermutator due to defective in MMR in Salmonella seems rare. In this study, five presumptive hypermutators, S. Typhimurium ST20751, S. Heidelberg 22396, S. Enteritidis 17929, 17929N and 17929R, were identified after screening 390 Salmonella isolated from U.S.A. Wild-type mutH, mutL, mutS and uvrD complementation and DNA sequencing study demonstrated no defective or sense mutation occurred in MMR genes, and the probability of hypermutation in the chromosome DNA that resulted in MDR in Salmonella were rare.(7) Plasmid plays an important role development and dissemination of antimicrobial resistance. Endonuclease digested plasmid profiles and clustering analysis indicated that antimicrobial resistance encoded by plasmid was able to be transmitted to different bacteria among through horizontal gene transfer. The conjugation ratios of resistance gene transfer to the recipients (EC1003 and 17929N) were from 2.4×10-4 to 5.6×10-4. After conjugation, most transconjugants acquired the corresponding antimicrobial resistant phenotypes. The antimicrobial genes transferred during the conjugation were verified by PCR, and conferred the transconjugant new antimicrobial resistance phenotype.更多还原