两株blaNDM-5基因介导的碳青霉烯耐药禽源大肠杆菌ST10和ST354耐药性

Two carbapenem-resistant avian Escherichia coli strains ST10 and ST354 mediated by blaNDM-5 gene

摘要:

【背景】近年来，对碳青霉烯类抗生素耐药的肠杆科细菌的出现对公众的健康构成了严重的威胁。【目的】为了解家禽养殖中肠道菌对碳青霉烯类抗生素耐药情况，对陕西西安和浙江海宁两地的鸡和鸭养殖场粪便进行耐药菌筛选并分析。【方法】在含美罗培南的LB琼脂平板上筛选可疑菌落并利用基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption/ionization time of flight mass spectrometry, MALDI-TOF MS)鉴定。通过微量肉汤稀释法进行最小抑菌浓度(minimum inhibitory concentration，MIC)测定。通过Illumina HiSeq平台进行全基因组测序。使用ResFinder数据库预测获得性耐药基因。利用S1酶切后脉冲场电泳(pulsed-field gel electrophoresis，PFGE)和Southern blot杂交进行质粒和碳青霉烯类耐药基因的确认。【结果】分离出两株碳青霉烯类耐药的大肠杆菌HN1-26和XN3-1，均对头孢他啶、头孢噻呋、氨苄西林、奥格门丁、复方新诺明、磺胺异恶唑、恩诺沙星、氧氟沙星、美罗培南、四环素耐药。此外，XN3-1对氟苯尼考和大观霉素耐药。菌株HN1-26和XN3-1的多位点序列分型(multilocus sequence typing，MLST)分别为ST354和ST10。两株菌对美罗培南的MIC值均为64 μg/mL，但是在含美罗培南的LB平板上，菌株HN1-26比菌株XN3-1生长得更快。两株菌都存在携带blaNDM-5基因的IncX3型质粒。两个质粒的序列完全一致，大小为46 161 bp，包含接合转移元件，能够以大肠杆菌作为受体细胞进行转移。【结论】IncX3型质粒是转移blaNDM-5基因的重要载体，在我国畜禽养殖中具有扩散风险。

Abstract:

[Background] Carbapenem-resistant Enterobacteriaceae is now becoming a globally serious threat to public health. [Objective] To study the carbapenem resistance of intestinal bacteria isolated from poultry farms, feces of chicken and duck were screened at Xi’an of Shaanxi and Haining of Zhejiang Provinces. [Methods] The potential bacterial colonies on LB agar plate containing meropenem were picked and further characterized by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Minimum inhibitory concentration (MIC) was determined by the microbroth dilution method. Whole genome sequencing was performed by the Illumina HiSeq platform. Acquired resistance genes were predicted by the online ResFinder database analysis. In addition, plasmids and carbapenem resistance genes were confirmed by S1-pulsed-field gel electrophoresis (S1-PFGE) and Southern blot hybridization. [Results] Two carbapenem-resistant Escherichia coli strains HN1-26 and XN3-1 were isolated. Both of these strains were resistant to ceftazidime, ceftiofur, ampicillin, amoxicillin/clavulanate, trimethoprim/sulfamethoxazole, sulfisoxazole, enrofloxacin, ofloxacin, meropenem and tetracycline. Besides, XN3-1 was also shown to be resistant to florfenicol and spectinomycin. Multilocus sequence typing (MLST) results revealed that HN1-26 and XN3-1 were ST354 and ST10, respectively. The MIC values of these two strains to meropenem were 64 μg/mL, while HN1-26 grew faster than XN3-1 on LB plate with meropenem. Furthermore, these two strains contained the identical IncX3 type plasmid carrying antibiotic resistance gene blaNDM-5, with a size of 46 161 bp. This IncX3 type plasmid contained complete conjugative elements and were able to transfer to E. coli recipient cells. [Conclusion] Our results suggested that the IncX3 plasmid is an important vehicle for transferring blaNDM-5 gene, which has the risk of spreading in livestock and poultry in China.