耐药基因岛在我国部分地区动物源和人源弯曲菌中的流行和传播

发布时间：2018-03-13 03:08

  本文选题：弯曲菌　切入点：ermB基因　出处：《中国农业大学》2016年博士论文　论文类型：学位论文

【摘要】：空肠弯曲菌(C. jejuni)和结肠弯曲菌(C. coli)是重要的食源性病原菌。畜禽是弯曲菌的重要宿主和天然储库,抗菌药物在畜牧生产中大量应用使耐药的弯曲菌被筛选出来,并有可能通过食物链传播给人,这将给人类健康和食品安全构成潜在威胁。耐药基因岛因同时介导数种甚至数类抗菌药物的耐药,而引起人们关注。国内外对耐药基因岛在弯曲菌中的流行病学和遗传背景方面的研究尚属空白。本研究将调查我国部分地区弯曲菌的耐药性情况,在此基础上探讨耐药基因岛在食品动物源和腹泻病人来源弯曲菌中流行及传播机制的异同。本研究在山东、宁夏和广东地区采集到的3083份畜禽样本中分离到763株弯曲菌(分离率为24.7%),包括679株C. coli和84株C. jejuni。465株猪源弯曲菌中,99.1%的菌株是C. coli, C. coli依然是猪源弯曲菌的优势菌种；而298株禽源(鸡和鸭)弯曲菌中,73.2%的菌株是C.coli, C.coli正取代C. jejuni成为我国禽源弯曲菌中的优势菌种。药敏试验显示C. coli对红霉素、克林霉素和庆大霉素的耐药率显著高于C. jejuni。弯曲菌的多重耐药情况严峻,多重耐药率达88.3%,且C. coli的多重耐药率(90.4%)远高于C.jejuni的多重耐药率(71.4%)。对分离自我国多地区的1555株畜禽源及人源弯曲菌PCR检测多药耐药基因ermB后获得59株阳性菌(阳性率为3.8%；58株C. coli和1株C. jejuni),包括33株猪源、14株鸡源、10株人源和2株鸭源,禽源、人源弯曲菌和C. jejuni中发现ermB尚属首次。随后对ermB的侧翼结构测定分析后发现,28株阳性菌携带的多重耐药基因岛(MDRGI)可分为7个类型。其中,type Ⅲ MDRGI同时确证分离于不同年份不同地区不同宿主的16株C. coli中；在type Ⅱ MDRGI中发现新型磷霉素耐药基因fosXCC。此外,PFGE和MLST分型提示ermB阳性菌在山东和上海地区发生了克隆传播；并且携带MDRGIs的弯曲菌有通过食物链传播给人的较大可能性。自然转化试验表明7种MDRGIs及其耐药表型均可跨弯曲菌亚种进行传播。另外,ermB的表达调控试验显示弯曲菌DZB40和86c的ermB是诱导型耐药表达,其诱导机制与ermB的mRNA二级结构的碱基组成相关；而27株弯曲菌的ermB则是固有型耐药表达,其表达与ermB调控区的变化有关。对山东畜禽源和上海腹泻病人来源的110株C. coli PCR检测耐药基因簇aadE-sat4-aphA-3后获得14株阳性菌(12.7%),包括7株人源、6株猪源和1株鸭源。对基因簇的侧翼结构测定分析后发现,10株阳性菌携带的氨基糖苷耐药基因岛(ARGI)可分为3个类型。其中, type Ⅰ ARGI同时发现在分离于畜禽源和人源的C. coli中；仅发现在一株人源C. coli的type Ⅲ ARGI中包含全新的IS-lide元件。随后分子分型发现分离自腹泻病人的C. coli的ARGI有可能是经食物链从携带ARGI的畜禽源C. coli中获得。稳定性试验显示ARGIs在没有抗生素压力的条件下能够稳定存在,并介导传代子与原代菌相同水平的耐药性。耐药基因岛在64株弯曲菌中的整合位点分别有①：整合至cj068c与sodB之间,只发现在1株携带MDRGI的鸡源C. jejuni中；②：直接插入到cj1063中,发现在7株人源的C. coli中,包括2种MDRGIs;③:取代cj1477c后整合至cj1476c和cadF之间,发现在畜禽源和人源的55株C. coli中,包括4种MDRGIs和2种ARGIs;④：直接插入到cj1500中,仅发现在一株携带ARGI的人源C. coli中。推测位点③是耐药基因岛在弯曲菌的染色体DNA中的整合热点。综上所述,携带氨基糖苷耐药基因岛的弯曲菌对临床上用于治疗弯曲菌的急性或全身性感染时使用的氨基糖苷类药物均耐药；携带多重耐药基因岛的弯曲菌对用于治疗弯曲菌病的抗菌药物(包括大环内酯类、氟喹诺酮类、氨基糖苷类、四环素类、林可胺类)均耐药。7种MDRGIs和3种ARGIs在我国畜禽源及人源弯曲菌中已有流行的趋势,均可跨弯曲菌亚种进行传播,而且携带MDRGIs或ARGIs的弯曲菌都有通过食物链传播给人的潜在风险。另外,我国畜牧业长期亚剂量的用药,将会对携带诱导型耐药表达ermB的弯曲菌的耐药性诱导产生提供便利,并且促使诱导型耐药的ermB进化为固有型耐药表达的ermB。
[Abstract]:Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli) is an important food borne pathogen. Livestock and poultry is an important host of Campylobacter and natural antibacterial drug reservoir, Campylobacter widely used in livestock production in the resistant were screened and may spread to people through the food chain, which will a potential threat to human health and food safety. At the same time because of resistance Island resistance gene mediates a variety of even a few antibiotics, and attracted attention. The domestic and foreign research on epidemiology and genetic background in Campylobacter in resistance gene island is still blank. This study will investigate the situation of drug resistance of Campylobacter in parts of China in China, based on the similarities and differences of resistance gene island in the animal food source and source of Campylobacter in patients with diarrhea prevalence and transmission mechanism. The research in Shandong, 3083 livestock and poultry to sample collected from Ningxia and Guangdong area Isolated 763 strains of Campylobacter in (the isolation rate was 24.7%), including 679 strains of C. coli and C. jejuni.465 84 strains from swine Campylobacter, 99.1% strains are C. coli, C. coli is still the dominant strain of swine Campylobacter; and 298 strains of avian (chicken and duck) in Campylobacter 73.2%, the strain is C.coli C.coli, replacing C. jejuni as the dominant strain of avian Campylobacter in China. Drug sensitivity test showed that C. coli of erythromycin, clindamycin and gentamicin multi drug resistance rate was significantly higher than that of C. jejuni. of Campylobacter grim, multi drug resistance rate was 88.3%, and the multi drug resistant C. coli the rate of multi drug resistance (90.4%) is much higher than the rate of C.jejuni (71.4%). Of 1555 strains of livestock and poultry sources and human Campylobacter PCR detection region isolated from the multidrug resistance gene ermB obtained 59 positive bacteria (positive rate was 3.8%; 58 strains and 1 strains of C. coli C. jejuni), including 33 strains swine, 14 From chicken, 10 strains and 2 strains of duck origin source, avian, human Campylobacter and C. found in jejuni ermB for the first time. Then the flank structure determination of ermB analysis showed that 28 strains of Gram-positive bacteria carrying multidrug resistance gene Island (MDRGI) can be divided into 7 types. Among them, type MDRGI also confirmed the separation of III in different regions of different year and host of the 16 strains of C. coli; hormone resistant gene fosXCC. model fosfomycin also found in type II MDRGI, PFGE and MLST type ermB positive bacteria in Shandong and Shanghai area has undergone clonal propagation; Campylobacter and MDRGIs are more likely to carry through the food chain spread to people. The natural transformation test showed that 7 kinds of MDRGIs and its drug-resistant phenotype can be spread across Campylobacter subspecies. In addition, expression regulation test of ermB display DZB40 and 86C of Campylobacter ermB is inducible resistance expression, its mechanism of induction and ermB MRNA two level structure of base composition; and 27 strains of Campylobacter ermB is the expression of innate resistance, its expression is related with the change of ermB regulatory region of aadE-sat4-aphA-3. 110 strains of C. coli PCR detection of drug resistance gene cluster of livestock and poultry in Shandong and Shanghai from the source of patients with diarrhea after 14 strains of positive bacteria (12.7%). Including 7 strains of human source, 6 strains of swine and 1 strains of duck origin. Determination and analysis of flanking structure of the gene cluster found 10 positive bacteria carrying the aminoglycoside resistance gene Island (ARGI) can be divided into 3 types. Among them, type I ARGI also found in isolated from livestock and poultry sources and human. C. coli; found only contains IS-lide element in a new strain of human C. coli type 3 ARGI. Then the molecular type was isolated from diarrhea patients C. coli ARGI may be through the food chain from animal source C. carrying ARGI in coli. The stability test showed that ARGIs in the There are no antibiotics can stabilize the pressure condition, and drug resistance mediated by primary and passage sub level. The same bacteria resistance gene Island integration sites in 64 strains of Campylobacter in respectively: integration to cj068c and sodB, only to find the chicken C. jejuni carrying MDRGI in strain 1; II: insert into cj1063, found in 7 strains of human origin C. coli, including 2 MDRGIs; 3: substitution of cj1477c to cj1476c and cadF integration between the 55 strains of C. coli, found in animal sources and human sources, including 4 MDRGIs and 2 ARGIs; 4: direct insertion into the cj1500. Only found human C. coli carrying ARGI in a line. That is the integration of the site of chromosome DNA hot resistance gene island in Campylobacter in summary, Campylobacter with aminoglycoside resistant gene island of clinically used in the treatment of acute Campylobacter or systemic infection with ammonia Glucoside type of drug resistance; multidrug resistance gene carrying Campylobacter island for antimicrobial treatment of Campylobacter diseases (including macrolide and fluoroquinolones, aminoglycosides, tetracyclines, lincosamide) were resistant.7 MDRGIs and 3 ARGIs existing in livestock and poultry sources in China and the source of bending from the popularity of the trend, can cross the spread of Campylobacter subspecies, and Campylobacter carrying MDRGIs or ARGIs have the potential risk of food chain spread to people. In addition, long-term mild dose of animal husbandry industry in China, will carry on inducible resistance resistance of Campylobacter table up to ermB induced facilitation ermB, and prompted the evolution of inducible resistance for the expression of innate resistance ermB.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.61
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本文编号：1604518