【作者】 王磊；

【导师】 佟盼盼；

【作者基本信息】 新疆农业大学 ， 兽医学， 2025， 硕士

【摘要】 目的:本研究对2015～2023年分离出的新疆牛源非O157产志贺毒素大肠杆菌(Shiga toxin-producing Escherichia coli,STEC)耐药性和耐药基因进行了系统分析,针对氟苯尼考和阿奇霉素耐药的菌株,探究耐药基因floR和mph(A)的流行特点及floR和mph(A)基因共存质粒的适应性代价,以评价其传播风险及带来的公共卫生威胁。方法:2015～2023年,采集新疆9个地区25个牛场和2家屠宰场的直肠拭子、粪便、鼻拭子、环境样本共计4 181份。分离得到367株非O157 STEC通过药敏试验检测其耐药性,并采用PCR检测floR和mph(A)基因的携带情况,分析携带floR和/或mph(A)基因的非O157 STEC的流行特征。对携带floR和/或mph(A)基因的菌株进行质粒复制子分型、接合试验及最小抑菌浓度(MIC)测定,以评估耐药基因的水平传播能力。通过全基因组测序(WGS)和系统发育分析,探究携带floR和/或mph(A)基因菌株间的亲源关系及质粒结构和遗传背景等。利用生长曲线、体外竞争试验评估floR和mph(A)基因共存质粒对宿主菌适应性的影响。结果:药敏试验结果显示,16.3%(60株)的耐药菌对四环素(10.6%)、氨苄西林(9.5%)、头孢噻肟(9.3%)等抗生素表现出较高的耐药性,对氟苯尼考和阿奇霉素的耐药率分别为7.9%和5.4%,其中33株(9.1%)的耐药菌株具有多重耐药性。PCR检测发现31株携带floR和/或mph(A)基因,其中18株同时携带floR和mph(A)基因(4.90%),检出率总体呈上升趋势,博乐、喀什和伊犁地区检出率较高。质粒复制子类型主要为Inc FIB、Inc HI2和Inc A/C,接合试验表明floR和mph(A)基因可同时发生水平转移。系统发育分析表明,多数菌株属于ST443型,且O178:H19血清型占优势。WGS结果揭示floR和mph(A)基因共同位于携带IS26和IS6100移动转移元件的Inc A/C型质粒上。质粒适应性变化实验表明,在无抗生素选择压力下,携带floR和mph(A)基因的Inc A/C质粒和耐药基因易丢失,而在有抗的选择压力下可稳定遗传。此外,质粒携带会对宿主菌造成适应性代价,但随着传代,其适应性逐步提升。结论:新疆牛源非O157 STEC的耐药性逐渐增强,其中对氟苯尼考和阿奇霉素耐共耐药菌株的检出率攀升,耐药基因floR和mph(A)共同位于Inc A/C型质粒上,可发生水平转移,该质粒可在抗生素压力下稳定存在,需要持续监测。以上研究对理解氟苯尼考和阿奇霉素耐药菌株的扩散机制及防控策略具有重要的科学意义。更多还原

【Abstract】 Objective:This study conducted a systematic analysis of antimicrobial resistance and resistance genes in non-O157 Shiga toxin-producing Escherichia coli(STEC)isolated from cattle in Xinjiang between 2015and 2023.It focused on isolates resistant to florfenicol and azithromycin,investigating the epidemiological characteristics of the resistance genes floR and mph(A),as well as the fitness cost of plasmids carrying both genes.The aim was to assess the transmission risk and potential public health threat posed by these resistant isolates.Methods:From 2015 to 2023,a total of 4,181 samples-including rectal swabs,feces,nasal swabs,and environmental samples-were collected from 25 cattle farms and 2 slaughterhouses across 9 regions in Xinjiang.A total of 367 non-O157 STEC isolates were obtained and subjected to antimicrobial susceptibility testing.PCR was used to detect the presence of floR and mph(A)genes,and to analyze the epidemiological characteristics of non-O157 STEC isolates carrying these genes.Isolates carrying floR and/or mph(A)were further analyzed through plasmid replicon typing,conjugation experiments,and minimum inhibitory concentration(MIC)testing to evaluate the potential for horizontal gene transfer.Whole-genome sequencing(WGS)and phylogenetic analysis were performed to investigate genetic relationships among the isolates and to characterize plasmid structure and genetic backgrounds.The impact of floR and mph(A)co-carrying plasmids on host bacterial fitness was assessed using growth curves and in vitro competition assays.Results:Antimicrobial susceptibility testing showed that 16.3%(60 isolates)of resistant isolates exhibited high resistance to antibiotics such as tetracycline(10.6%),ampicillin(9.5%),and cefotaxime(9.3%).Resistance rates to florfenicol and azithromycin were 7.9%and 5.4%,respectively.Among these,33isolates(9.1%)were identified as multidrug-resistant.PCR analysis detected floR and/or mph(A)genes in31 isolates,with 18 isolates(4.90%)carrying both genes.The detection rate is generally on the rise,with higher separation rates in Bole,Kashgar,and Yili regions.The dominant plasmid replicon types were Inc FIB,Inc HI2,and Inc A/C.Conjugation experiments confirmed that floR and mph(A)can be co-transferred horizontally.Phylogenetic analysis indicated that most isolates belonged to sequence type ST443,with the O178:H19 serotype being predominant.Whole-genome sequencing(WGS)revealed that floR and mph(A)were co-located on Inc A/C-type plasmids carrying the mobile elements IS26 and IS6100.Plasmid adaptation experiments showed that,without antibiotic selection pressure,the Inc A/C plasmid carrying floR and mph(A),along with the resistance genes,was prone to loss.Under antibiotic pressure,however,the plasmid was stably maintained.Although the plasmid imposed a fitness cost on the host bacteria,this cost decreased over time with serial passage.Conclusion:The antimicrobial resistance of non-O157 STEC from cattle in Xinjiang is increasing.In particular,the detection rate of isolates co-resistant to florfenicol and azithromycin is rising.The resistance genes floR and mph(A)are located together on an Inc A/C-type plasmid,which can be horizontally transferred and remains stable under antibiotic pressure.Continuous monitoring is necessary.This study provides important scientific insight into the dissemination mechanisms of florfenicol and azithromycin-resistant isolates and the development of control strategies.更多还原