Identification of Novel Conjugative Plasmids with Multiple Copies of fosB that Confer High-Level Fosfomycin Resistance to Vancomycin-Resistant Enterococci

• Published in: Frontiers in microbiology 2017-08, Vol.8, p.1541-1541
• Main Authors: Sun, Lingyan, Zhang, Ping, Qu, Tingting, Chen, Yan, Hua, Xiaoting, Shi, Keren, Yu, Yunsong
• Format: Article
• Language: English
• Subjects: copy number; fosB; fosfomycin resistance; Microbiology; single-molecule real-time sequencing; vancomycin resistant enterococci
• ISSN: 1664-302X; 1664-302X
• DOI: 10.3389/fmicb.2017.01541

To further characterize the -carrying plasmids of 19 vancomycin-resistant enterococci, the complete sequences of the - and -containing plasmids of (pEMA120) and (pEA19081) were obtained by single-molecule, real-time sequencing. We found that these two plasmids are essentially identical (99.99% nucleotide sequence identity), which proved the possibility of interspecies transmission. Comparative analysis of the plasmids revealed that the backbone of pEMA120 is 99% similar to a conjugative -negative plasmid, pZB18. There is a disrupted in the transfer region of pEMA120, in comparison to pZB18 with an intact . The difference of their transfer frequencies between pEMA120 and pZB18 suggests this interruption of might affect conjugative transfer. Two copies of the gene linked to a gene, forming an IS -like transposon, were found at separate locations within pEMA120, which had not been reported previously. These two -carrying transposons were confirmed to form circular intermediates by inverse PCR. The hybridization of plasmid DNA digested by I, having restriction site within the sequence, demonstrated that the presence of multiple copies of per plasmid is common. The total copy number of the gene as revealed by qRT-PCR did not correlate with fosfomycin MICs or growth rates at sub-MICs of fosfomycin in different transconjugants. From susceptibility tests, the gene, regardless of the copy number, conferred high fosfomycin MICs that ranged from 16384 to 65536 μg/ml. This first complete nucleotide sequence of a plasmid carrying two copies of in VRE suggests that the gene can transfer to multiple loci of plasmids by the IS family transposase TnpA, possibly in the form of circular intermediates, leading to the dissemination of high fosfomycin resistance in VRE.