ABSTRACT

The incidence and transmission of Klebsiella pneumoniae carbapenemase (KPC) producing plasmids have been well documented. However, the evolutionary dynamics of KPC plasmids and their fitness costs are not well characterized. Here, two carbapenemase-producing plasmids from Proteus mirabilis, pT18 and pT211 (both carrying bla _KPC-2), were characterized through whole genome sequencing. pT211 is a 24.2 kbp N-type plasmid that contains bla _KPC-2 and a single copy of the IS6-family insertion sequence IS26. pT18 is a 59 kbp cointegrate plasmid comprised of sequences derived from three different plasmids: a close relative of pT211 (containing bla _KPC-2), an FII-33 plasmid (bla _TEM-1B, bla _CTX-M-65, rmtB and fosA3) and a rolling-circle plasmid. The segments of pT18 derived from each of the different plasmids are separated by copies of IS26, and sequence analysis indicated that pT18 was likely generated by both conservative and replicative IS26-mediated cointegrate formation. pT18 and pT211 were transferred into Escherichia coli DH5α separately to assess the impact of plasmids on host fitness. Only DH5α harbouring pT18 grew slower than the wild type in antibiotic-free media. However, in sub-inhibitory concentrations of fosfomycin and amikacin, cells containing pT18 grew faster than the wild type, and the minimum concentrations of fosfomycin and amikacin required to observe an advantage for plasmid-carrying cells were 1/3 and 1/20 the DH5α MIC, respectively. This study highlights the importance of the role of cointegrate plasmids in the dissemination of antibiotic resistance genes between pathogenic bacterial species, and highlights the importance of sub-inhibitory concentrations of antibiotics to the persistence of such plasmids.

摘要

肺炎克雷伯氏菌碳青霉烯酶（KPC）产生质粒的发生和传播已得到充分证明。但是，KPC质粒的进化动力学及其适应性成本尚未得到很好的表征。在这里，通过全基因组测序表征了来自变形杆菌（Proteus mirabilis）的两个产生碳青霉烯酶的质粒pT18和pT211（均携带bla _KPC-2）。pT211是24.2 kbp的N型质粒，包含bla _KPC-2和IS 6家族插入序列IS 26的单拷贝。pT18是一个59 kbp的共整合质粒，由三个不同质粒衍生的序列组成：pT211的近亲（包含bla _KPC-2），FII-33质粒（bla _TEM-1B，bla _CTX-M-65，rmtB和fosA3）和滚环质粒。来源于每个不同质粒的pT18的片段被IS 26的拷贝分开，并且序列分析表明pT18可能是由IS 26介导的保守和复制性共整合形成的。将pT18和pT211转移到大肠杆菌中分别用DH5α评估质粒对宿主适应性的影响。在不含抗生素的培养基中，只有带有pT18的DH5α比野生型慢。然而，在磷霉素和阿米卡星的亚抑制浓度下，含有pT18的细胞比野生型细胞生长快，观察携带质粒的细胞的优势所需的磷霉素和阿米卡星的最低浓度为DH5α的1/3和1/20。 MIC。这项研究突出了共整合质粒在致病细菌物种之间传播抗生素抗性基因中的作用的重要性，并强调了亚抑制浓度的抗生素对此类质粒的持久性的重要性。