Abstract：
Cryptic conjugative plasmids lack antibiotic-resistance genes (ARGs). These plasmids can capture ARGs from the vast pool of the environmental metagenome, but the mechanism to recruit ARGs remains to be elucidated. To investigate the recruitment of ARGs by a cryptic plasmid, we sequenced and conducted mating experiments with Escherichia coli SW4848 (collected from a lake) that has a cryptic IncX (IncX4) plasmid and an IncF (IncFII/IncFIIB) plasmid with five genes that confer resistance to aminoglycosides (strA and strB), sulfonamides (sul2), tetracycline [tet(A)], and trimethoprim (dfrA5). In a conjugation experiment, a novel hybrid Tn21/Tn1721 transposon of 22,570 bp (designated Tn7714) carrying the five ARG mobilized spontaneously from the IncF plasmid to the cryptic IncX plasmid. The IncF plasmid was found to be conjugative when it was electroporated into E. coli DH10B (without the IncX plasmid). Two parallel conjugations with the IncF and the new IncX (carrying the novel Tn7714 transposon) plasmids in two separate E. coli DH10B as donors and E. coli J53 as the recipient revealed that the conjugation rate of the new IncX plasmid (with the novel Tn7714 transposon and five ARGs) is more than two orders of magnitude larger than the IncF plasmid. For the first time, this study shows experimental evidence that cryptic environmental plasmids can capture and transfer transposons with ARGs to other bacteria, creating novel multidrug-resistant conjugative plasmids with higher dispersion potential.
OBJECTIVE: Cryptic conjugative plasmids are extrachromosomal DNA molecules without antibiotic-resistance genes (ARGs). Environmental bacteria carrying cryptic plasmids with a high conjugation rate threaten public health because they can capture clinically relevant ARGs and rapidly spread them to pathogenic bacteria. However, the mechanism to recruit ARG by cryptic conjugative plasmids in environmental bacteria has not been observed experimentally. Here, we document the first translocation of a transposon with multiple clinically relevant ARGs to a cryptic environmental conjugative plasmid. The new multidrug-resistant conjugative plasmid has a conjugation rate that is two orders of magnitude higher than the original plasmid that carries the ARG (i.e., the new plasmid from the environment can spread ARG more than two orders of magnitude faster). Our work illustrates the importance of studying the mobilization of ARGs in environmental bacteria. It sheds light on how cryptic conjugative plasmids recruit ARGs, a phenomenon at the root of the antibiotic crisis.
OBJECTIVE: Cryptic conjugative plasmids are extrachromosomal DNA molecules without antibiotic-resistance genes (ARGs). Environmental bacteria carrying cryptic plasmids with a high conjugation rate threaten public health because they can capture clinically relevant ARGs and rapidly spread them to pathogenic bacteria. However, the mechanism to recruit ARG by cryptic conjugative plasmids in environmental bacteria has not been observed experimentally. Here, we document the first translocation of a transposon with multiple clinically relevant ARGs to a cryptic environmental conjugative plasmid. The new multidrug-resistant conjugative plasmid has a conjugation rate that is two orders of magnitude higher than the original plasmid that carries the ARG (i.e., the new plasmid from the environment can spread ARG more than two orders of magnitude faster). Our work illustrates the importance of studying the mobilization of ARGs in environmental bacteria. It sheds light on how cryptic conjugative plasmids recruit ARGs, a phenomenon at the root of the antibiotic crisis.
摘要：
隐性接合质粒缺乏抗生素抗性基因(ARGs)。这些质粒可以从巨大的环境宏基因组中捕获ARGs，但是招募ARGs的机制仍有待阐明。为了研究隐藏质粒对ARGs的募集，我们对大肠杆菌SW4848（从湖中收集）进行了测序并进行了交配实验，该大肠杆菌具有隐匿性IncX（IncX4）质粒和IncF（IncFII/IncFIIB）质粒，该质粒具有五个赋予氨基糖苷（strA和strB）抗性的基因，磺胺类(sul2)，四环素[tet（A）]，和甲氧苄啶（dfrA5）。在共轭实验中，携带五个ARG的22,570bp的新型杂种Tn21/Tn1721转座子（命名为Tn7714）自发地从IncF质粒转移到隐秘的IncX质粒。当将其电穿孔到大肠杆菌DH10B(没有IncX质粒)中时,发现IncF质粒是接合的。在作为供体的两个单独的大肠杆菌DH10B和作为受体的大肠杆菌J53中,与IncF和新的IncX(携带新的Tn7714转座子)质粒的两个平行接合表明,新的IncX质粒(具有新的Tn7714转座子和五个ARG)的接合率比IncF质粒大两个数量级以上。第一次,这项研究表明，实验证据表明，隐蔽的环境质粒可以捕获和转移转座子与ARGs到其他细菌，创建具有更高分散潜力的新型多药耐药共轭质粒。
目的：隐性接合质粒是不含抗生素抗性基因（ARGs）的染色体外DNA分子。携带具有高结合率的隐性质粒的环境细菌威胁公众健康，因为它们可以捕获临床相关的ARG并将其迅速传播给病原菌。然而,尚未通过实验观察到通过环境细菌中的隐性接合质粒招募ARG的机制。这里,我们记录了具有多个临床相关ARG的转座子首次易位到隐蔽的环境接合质粒。新的多药耐药接合质粒的接合率比携带ARG的原始质粒高两个数量级（即，来自环境的新质粒可以更快地传播ARG两个数量级以上）。我们的工作说明了研究环境细菌中ARGs动员的重要性。它揭示了神秘的共轭质粒如何招募ARGs，抗生素危机的根源。
目的：隐性接合质粒是不含抗生素抗性基因（ARGs）的染色体外DNA分子。携带具有高结合率的隐性质粒的环境细菌威胁公众健康，因为它们可以捕获临床相关的ARG并将其迅速传播给病原菌。然而,尚未通过实验观察到通过环境细菌中的隐性接合质粒招募ARG的机制。这里,我们记录了具有多个临床相关ARG的转座子首次易位到隐蔽的环境接合质粒。新的多药耐药接合质粒的接合率比携带ARG的原始质粒高两个数量级（即，来自环境的新质粒可以更快地传播ARG两个数量级以上）。我们的工作说明了研究环境细菌中ARGs动员的重要性。它揭示了神秘的共轭质粒如何招募ARGs，抗生素危机的根源。
