AUTHOR=Sun Lingyan , Zhang Ping , Qu Tingting , Chen Yan , Hua Xiaoting , Shi Keren , Yu Yunsong 

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

JOURNAL=Frontiers in Microbiology

VOLUME=8

YEAR=2017

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2017.01541

DOI=10.3389/fmicb.2017.01541

ISSN=1664-302X

ABSTRACT=<p>To further characterize the <italic>fosB</italic>-carrying plasmids of 19 vancomycin-resistant enterococci, the complete sequences of the <italic>fosB</italic>- and <italic>vanA</italic>-containing plasmids of <italic>Enterococcus faecium</italic> (pEMA120) and <italic>E. avium</italic> (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 <italic>fosB</italic>-negative <italic>E. faecium</italic> plasmid, pZB18. There is a <italic>traE</italic> disrupted in the transfer region of pEMA120, in comparison to pZB18 with an intact <italic>traE</italic>. The difference of their transfer frequencies between pEMA120 and pZB18 suggests this interruption of <italic>traE</italic> might affect conjugative transfer. Two copies of the <italic>fosB</italic> gene linked to a <italic>tnpA</italic> gene, forming an IS<italic>L3</italic>-like transposon, were found at separate locations within pEMA120, which had not been reported previously. These two <italic>fosB</italic>-carrying transposons were confirmed to form circular intermediates by inverse PCR. The hybridization of plasmid DNA digested by <italic>Bsa</italic>I, having restriction site within the <italic>fosB</italic> sequence, demonstrated that the presence of multiple copies of <italic>fosB</italic> per plasmid is common. The total copy number of the <italic>fosB</italic> 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 <italic>fosB</italic> 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 <italic>fosB</italic> in VRE suggests that the <italic>fosB</italic> gene can transfer to multiple loci of plasmids by the IS<italic>L3</italic> family transposase TnpA, possibly in the form of circular intermediates, leading to the dissemination of high fosfomycin resistance in VRE.</p>