青岛地区产ESBLs鸡源大肠杆菌耐药性调查与优势基因型分析

Antimicrobial Resistance Investigation and Dominant Genotype Analysis of ESBLs- Producing Escherichia coli Strains from Chicken in Qingdao

【目的】通过抗菌药物敏感性测定试验以及超广谱β-内酰胺酶(ESBLs)表型和基因型检测试验,了解青岛地区肉鸡养殖场产ESBLs大肠杆菌菌株的分布情况及耐药特征,分析ESBLs流行基因型和基因亚型,为指导临床合理使用抗菌药物、有效控制产ESBLs菌株的感染和流行提供依据。【方法】采用微量肉汤稀释法对249株大肠杆菌菌株进行抗菌药物敏感性测定试验,采用CLSI推荐方法进行ESBLs表型检测和确证实验,采用PCR方法、序列测定以及生物学分析软件进行ESBLs耐药质粒的DNA扩展和基因型分析,利用SPSS19.0软件对产ESBLs菌株与非产ESBLs菌株的耐药情况进行差异显著性分析。【结果】83.13%(207/249)的鸡源大肠杆菌菌株产ESBLs;产ESBLs菌株对7种抗菌药的耐药率高于非产ESBLs菌株,其中对庆大霉素、大观霉素、氨苄西林、阿莫西林/克拉维酸、头孢噻呋等5种药物差异显著(P<0.05),而产ESBLs菌株对四环素和氟苯尼考2种抗菌药的耐药率显著低于非产ESBLs菌株;产ESBLs菌株多重耐药程度显著高于非产ESBLs菌株,其多重耐药率分别为99.03%和92.86%(P=0.035);CTX-M型、TEM型和OXA型ESBLs菌株的检出率分别为100.00%、99.52%和47.83%,未检测出SHV型ESBLs菌株;产酶菌株分属于10个基因亚型,TEM-1型、CTX-M-65型和CTX-M-55型、OXA-1型是优势基因亚型,并首次从健康家禽中检测到基因重组嵌合体CTX-M-123和CTX-M-64。【结论】产ESBLs鸡源大肠杆菌菌株在青岛地区广泛流行和传播;产ESBLs菌株耐药相对非产ESBLs菌株严重;相比国内其它地区,CTX-M型和TEM型同样成为青岛地区产ESBLs鸡源大肠杆菌菌株的流行基因型,但基因亚型存在差异,TEM-1型、CTX-M-65型和CTX-M-55型、OXA-1型分别是各基因型的优势基因亚型。

[Objective] By using antimicrobial susceptibility testing and detection of phenotype and genotype in extended spectrum beta-lactamases （ESBLs）, this study aims to find out the distribution and drug-resistance of ESBLs-producing E.coli strains on broiler farms in Qingdao, and then analyze genotype and gene subtype of ESBLs, so as to provide a basis for guiding rational clinical use of antimicrobial agents and effective control of infection and spread of ESBLs-producing strains.[Method]The antimicrobial susceptibility of 249 E. coli strains was determined by micro broth dilution, the phenotype of ESBLs was identified and determined by CLSI standard method, DNA was amplified and the genotype of drug resistance plasmid of ESBLs was analyzed＆amp;nbsp;by PCR, sequencing and biological software, and the significant difference between ESBLs-producing and non-producing strains was analyzed with SPSS19.0 software.[Result]There were 83.13% of E.coli strains produced ESBLs. The resistance rate of ESBLs-producing strains to 7 kinds of drugs was higher than that of non-ESBLs-producing ones, and there was a significant difference between them （P＆lt;0.05） to GM, SPT, AM, A/C and CEF. But the resistance rate of ESBLs-producing strains to tetracycline and florfenicol was significantly lower than those in non-ESBLs-producing ones. The multi-drug resistance rate of ESBLs producing and non-producing strains was 99.03%and 92.86%, respectively （P=0.035）. The detection rate of CTX-M, TEM and OXA genotype was 100%, 99.52%and 47.83%, respectively, and SHV genotype wasn’t detected. The enzyme producing strains belong to 10 gene subtypes, the dominant ones of which were TEM-1, CTX-M-65, CTX-M-55, and OXA-1. CTX-M-123 and CTX-M-64, the recombinant chimeras, were found from healthy poultry for the first time. [Conclusion]The chicken origin ESBLs-producing E.coli strains are widely spread and transmitted in Qingdao. The antimicrobial resistance of ESBLs-producing strains are more serious than that of non-ESBLs-producing strains. Compared to other areas in China, CTX-M and TEM are also popular genotypes in Qingdao, but with different gene subtypes, TEM-1, CTX-M-65, CTX-M-55 and OXA-1 are dominant gene subtypes of each genotype, respectively.