牛源大肠杆菌毒素基因、黏附素基因、O血清型及耐药性调查

发布时间：2018-11-05 16:14

【摘要】：大肠杆菌是大肠杆菌科、埃希氏菌属的成员,是Escherich在1885年发现的,一直被认为是肠道中的正常菌群,不会致病。到了20世纪中叶,发现在动物和人,特别是幼龄动物和婴儿,大肠杆菌的一些血清型有一定的致病性,往往会引起严重腹泻和败血症,无论在发达国家还是在发展中国家,大肠杆菌导致的腹泻疾病仍然是一个严重威胁人类健康和导致病畜生长发育迟缓,生产性能降低,甚至死亡,严重损害养殖业的重要因素。本研究针对黑龙江省不同地区的不同牛场,共采集108头犊牛腹泻粪便病料。通过麦康凯培养基与伊红美蓝培养基的联合培养,利用16S r RNA通用引物进行PCR检测与序列分析,并结合传统大肠杆菌生化试验鉴定方法,共分离、鉴定出88株大肠杆菌,并对其进行了毒素基因(Eae+Stx1+Stx2+bfp A+LT+Sta+Stb+)与黏附素基因(k88+k99+F17+F6+F18+F41+)检测、O血清型筛选以及耐药性分析。研究结果表明:共有38株大肠杆菌检测出毒素基因,检出率为43.18%,可分为11种毒素类型,以eae、sta、stb、bfp A4种为主要类型,分别为13株、8株、15株和16株。以基因型为基础,可将这些大肠杆菌分为5种类型:ETEC、非典型EPEC、典型EPEC、ETEC/EPEC以及ETEC/EHEC。其中ETEC共检出15株,占毒素基因阳性菌株的39.47%。共有60株菌株表达一种或多种黏附素,检出率为68.18%。本研究共检出11种黏附素类型,其中以F18和K88黏附素类型为主,分别占黏附素阳性菌株的53.33%和20%。采用血清凝集实验进行了19种常见致病性O血清型的检测,共检出14种血清型,优势血清型为O2,O125和O128。通过药敏实验可知分离菌株呈多重耐药,对三种或更多抗生素产生耐药性,其中,对利福平(96.6%)、阿莫西林(97.7%)和氨苄西林(97.7%)表示强耐药性,对庆大霉素(30.7%)、氧氟沙星(30.7%)、环丙沙星(33.0%)和萘啶酸(37.5%)较敏感。本研究表明,黑龙江地区导致犊牛腹泻的大肠杆菌类型多样化,其中主要以ETEC,EPEC为主,O血清型分布散在,无集中趋势,且分离株存在严重的耐药性。
[Abstract]:Escherichia coli, a member of the family Escherichia coli, was discovered by Escherich in 1885. By the middle of the 20th century, it was found that in animals and humans, especially young animals and infants, some of the serotypes of E. coli were pathogenicity, often leading to severe diarrhoea and septicemia, both in developed and developing countries. The diarrhea disease caused by Escherichia coli is still a serious threat to human health and lead to the growth retardation of diseased animals, production performance decline, even death, serious damage to the aquaculture industry an important factor. In this study, 108 calves were collected from different cattle farms in Heilongjiang province. In this paper, 88 strains of Escherichia coli were isolated by co-culture of wheat Kang Kai medium and Ihloromethylene blue medium, using 16s r RNA universal primer for PCR detection and sequence analysis, and combining with traditional methods of biochemical identification of Escherichia coli. The toxin gene (Eae Stx1 Stx2 bfp A LT Sta Stb) and adhesion gene (k88k99 F 17 F 6 F 18 F 41) were detected, O serotype screening and drug resistance analysis were performed. The results showed that a total of 38 strains of Escherichia coli were detected, and the detection rate was 43.18, which could be divided into 11 types, including 13 strains, 8 strains, 15 strains and 16 strains of eae,sta,stb,bfp A. Based on genotypes, these Escherichia coli can be classified into five types: ETEC, atypical EPEC, typical EPEC,ETEC/EPEC and ETEC/EHEC. Among them, 15 strains were detected by ETEC, accounting for 39.47% of toxin gene positive strains. A total of 60 strains expressed one or more adhesives, and the detection rate was 68. 18%. In this study, 11 types of adhesion were detected, of which F18 and K88 were the main types, accounting for 53.33% and 20% of the positive strains, respectively. Serum agglutination test was used to detect 19 common pathogenic O serotypes. A total of 14 serotypes were detected. The dominant serotypes were O _ 2O _ (125) and O _ (128). The results showed that the isolates were multidrug resistant and resistant to three or more antibiotics, including rifampicin (96.6%), amoxicillin (97.7%) and ampicillin (97.7%). It was sensitive to gentamicin (30.7%), ofloxacin (30.7%), ciprofloxacin (33.0%) and naphthalic acid (37.5%). The results showed that the type of Escherichia coli caused by diarrhea of calves in Heilongjiang province was diversified, in which ETEC,EPEC was the main type, O serotype was scattered, there was no tendency of concentration, and the isolates had serious drug resistance.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

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