Chatterjee Somdatta, Mondal Ayan, Mitra Shravani, Basu Sulagna
Division of Bacteriology, National Institute of Cholera and Enteric Diseases, P33, CIT Road, Scheme XM, Beliaghata, Kolkata 700010, India.
Division of Pathophysiology, National Institute of Cholera and Enteric Diseases, P33, CIT Road, Scheme XM, Beliaghata, Kolkata 700010, India.
J Antimicrob Chemother. 2017 Aug 1;72(8):2201-2207. doi: 10.1093/jac/dkx131.
To investigate the transmission of the gene encoding New Delhi metallo-β-lactamase-1 ( bla NDM-1 ) through outer membrane vesicles (OMVs) released from an Acinetobacter baumannii strain (A_115).
Isolation and purification of OMVs by density gradient from a carbapenem-resistant clinical strain of A. baumannii harbouring plasmid-mediated bla NDM-1 and aac(6')-Ib-cr genes was performed. DNA was purified from the OMVs and used for PCR and dot-blot analysis. Vesicles treated with DNase I and proteinase K were used to transform A. baumannii ATCC 19606 and Escherichia coli JM109 strains. MIC values for the transformants were determined, followed by PCR and restriction digestion of plasmids. PFGE was done for A_115 and transformants of ATCC 19606 and JM109.
The A. baumannii strain (ST 1462) released vesicles (25-100 nm) during in vitro growth at late log phase. PCR and dot-blot analysis confirmed the presence of bla NDM-1 and aac(6')-Ib-cr genes in intravesicular DNA. bla NDM-1 and aac(6')-Ib-cr genes were transferred to both the A. baumannii ATCC 19606 and E. coli JM109 recipient cells. The transformation frequency of the purified OMVs was in the range of 10 -5 -10 -6 and gradually reduced with storage of OMVs. The sizes of the plasmids in the transformants and their restriction digestion patterns were identical to the plasmid in A_115. The transformants showed elevated MIC values of the β-lactam group of antibiotics, which confirmed the presence of a bla NDM-1 -harbouring plasmid.
This is the first experimental evidence of intra- and inter-species transfer of a plasmid harbouring a bla NDM-1 gene in A. baumannii via OMVs with high transformation frequency.
研究编码新德里金属β-内酰胺酶-1（blaNDM-1）的基因通过鲍曼不动杆菌菌株（A_115）释放的外膜囊泡（OMV）进行传播的情况。
从携带质粒介导的blaNDM-1和aac(6')-Ib-cr基因的耐碳青霉烯类鲍曼不动杆菌临床菌株中，通过密度梯度法分离和纯化OMV。从OMV中纯化DNA并用于PCR和斑点印迹分析。用DNase I和蛋白酶K处理的囊泡用于转化鲍曼不动杆菌ATCC 19606和大肠杆菌JM109菌株。测定转化体的最低抑菌浓度（MIC）值，随后对质粒进行PCR和限制性消化。对A_115以及ATCC 19606和JM109的转化体进行脉冲场凝胶电泳（PFGE）。
鲍曼不动杆菌菌株（ST 1462）在对数生长后期的体外生长过程中释放囊泡（25 - 100nm）。PCR和斑点印迹分析证实囊泡内DNA中存在blaNDM-1和aac(6')-Ib-cr基因。blaNDM-1和aac(6')-Ib-cr基因被转移到鲍曼不动杆菌ATCC 19606和大肠杆菌JM109受体细胞中。纯化的OMV的转化频率在10^-5 - 10^-6范围内，并且随着OMV的储存逐渐降低。转化体中质粒的大小及其限制性消化模式与A_115中的质粒相同。转化体显示β-内酰胺类抗生素的MIC值升高，这证实了携带blaNDM-1的质粒的存在。
这是首次通过具有高转化频率的OMV在鲍曼不动杆菌中进行携带blaNDM-1基因的质粒种内和种间转移的实验证据。
