利用MLST技术探讨不同地区致病性耐药鸡源大肠杆菌的遗传进化关系

Genetic evolutionary relationship of drug-resistant and pathogenic chicken originated Escherichia coli strains from different regions with MLST

选取来自山东等10省698株鸡源大肠杆菌为研究对象,分别利用PCR、微量肉汤稀释法、MLST技术方法进行系统进化群分群、耐药表型检测、分子分型,并采用Bio Numerics v4.0软件和SPSS20.0软件进行数据处理分析。结果显示,低致病群B_1群为48.85%(341/698)和非致病群A群24.64%(172/698)分布较多,高致病群D群20.20%(141/698)和B_2群为6.31%(44/698)。鸡源大肠杆菌耐药严重,12种药物的耐药率约在50%以上。B_2和D群菌株对8种抗菌药的耐药率高于A和B_1群菌株,其中对庆大霉素、大观霉素、头孢噻呋、氨苄西林和奥格门丁5种药物极显著性差异(P<0.01),对氟苯尼考和复方新诺明显著性差异(P<0.05)。135株高致病菌株经MLST分型得到48个ST型,ST117(12株)和ST354(12株)最多,其次是ST115(8株),ST2309(8株),ST10型(7株),ST69(6株),ST1011(5株),ST1158(5株)。有29个ST型各1株,18株菌株未分出序列型。不同地区大肠杆菌ST型分布存在差异,ST69和ST354型分布最为广泛(4个省),其次是ST115、ST117和ST2309型(3个省),ST10、ST48、ST57、ST362、ST371、ST1011和ST1158(2个省),其余36个ST型菌株仅在1个省市有检出。135株菌株分为35个聚类簇,其中23个聚类簇的遗传相似性在40%以下,但有6个聚类簇菌株间的遗传相似性均在80%以上。48个ST型菌株共获得了76种耐药谱。我国各地健康鸡群隐性携带相对较高比例的高致病菌株,且耐药程度相对严重,耐药谱较广。鸡源大肠杆菌菌株分布具有多态性和地域性,ST型与耐药谱之间无相关性。

Abstract：To investigate the phylogenetic group distribution, and reveal the relationship between drug-resistance and the genetic evolutionary of Escherichia coli （E. coli） strains originated from chicken in different regions with a view of providing evidence for molecular tracing and control of this bacteria. A total of 698 E. coli strains were isolated from Shandong and other 9 provinces in China. PCR technology was adopted to detect the phylogenetic clustering, and micro broth dilution method was used to detect drug-resistance phenotype,while MLST technique was applied to carry out molecular type,and Bio Numerics v4.0 and SPSS20.0 software were employed to analyze the experimental data. Low pathogenic group B1 and the nonpathogenic group A distributed widely,ac- counting for 48. 85%（341/698） and 24. 64% （172/698）, respectively. Next to them were highlypathogenic group D making up 20.20%（172/698） and group B2 making up 6.31% （44/698）. The resistance of chicken originated E. colt was serious and the resistant rate of 12 kinds of drugs was all more than 50.0 %. The resistant rate of group B2 and D strains for 8 kinds of drugs was higher than that of A and B1 strains. For the two categories of strains, there was extremely significant difference for 5 kinds of drugs such as gentamicin, spectinomycin, ceftiofur, ampicillin, augmentin （P〈0.01） ;and significant differences for florfenicol, sulfamethoxazole （P〈0.05）. 48 STs were obtained from 135 highly pathogenic strains by MLST. STl17 and ST354 were most popular,both from 12 strains, then followed by ST115 （8）,ST2309 （8）,ST10 （7）,ST69 （6）,ST1011 （5）, ST1158 （5） 29 ST types were from 1 strain,respectively. And ST types of 18 strains were uncer- tain. There was difference of E. coli ST type distribution in different areas. ST69 and ST354 dis- tributed most widely （4 provinces）, and then STl15, ST117 and ST2309 （3 provinces）, next were ST10,ST48,ST57,ST362,ST371,ST1011 and ST1158 （2 provinces） , the remaining 36 ST types strains were detected only in one province. 135 strains were divided into 35 clustering groups. A- mong them,the genetic similarity of 23 clustering groups was under 400//00 ,but the genetic similari- ty of 6 clustering groups strains were above 80%. 76 kinds of drug-resistant spectrum were ob- tained from these 48 ST types of strains. Healthy chickens in our country silently carry a relatively high proportion of highly pathogenic strains, and their drug resistance problem is serious, with a wide drug-resistant spectrum. The distribution of E. coli strains originated from chicken is quiet diversified and has distinct regional features. There is no correlation between ST types and drug- resistant spectrums.