Abstract
Horizontal gene transfer (HGT) is the major mechanism responsible for spread of antibiotic resistance. Antibiotic treatment has been suggested to promote HGT, either by directly affecting the conjugation process itself or by selecting for conjugations subsequent to DNA transfer. However, recent research suggests that the effect of antibiotic treatment on plasmid conjugation frequencies, and hence the spread of resistance plasmids, may have been overestimated. We addressed the question by quantifying transfer proteins and conjugation frequencies of a blaCTX−M−1 encoding IncI1 resistance plasmid in Escherichia coli MG1655 in the presence and absence of therapeutically relevant concentrations of cefotaxime (CTX). Analysis of the proteome by iTRAQ labeling and liquid chromatography tandem mass spectrometry revealed that Tra proteins were significantly up-regulated in the presence of CTX. The up-regulation of the transfer machinery was confirmed at the transcriptional level for five selected genes. The CTX treatment did not cause induction of the SOS-response as revealed by absence of significantly regulated SOS associated proteins in the proteome and no significant up-regulation of recA and sfiA genes. The frequency of plasmid conjugation, measured in an antibiotic free environment, increased significantly when the donor was pre-grown in broth containing CTX compared to growth without this drug, regardless of whether blaCTX-M-1 was located on the plasmid or in trans on the chromosome. The results shows that antibiotic treatment can affect expression of a plasmid conjugation machinery and subsequent DNA transfer.
Highlights
Plasmid conjugation contributes significantly to horizontal spread of antibiotic resistance, because antibiotic resistance genes are frequently located on plasmids (Zatyka and Thomas, 1998; Sorensen et al, 2005; Thomas and Nielsen, 2005; Bennett, 2008; Norman et al, 2009)
The results showed that Tra-proteins and other conjugation related proteins were significantly upregulated in the presence of high levels of CTX (One-way ANOVA, 0.0001 < P < 0.05 depending on the protein), while bacteria grown at the low concentrations of CTX showed insignificant differences in the proteome compared to the untreated control (One-Way ANOVA with correction for multiple comparison, p > 0.05)
No proteins associated with the SOS response, such as LexA or RecA were significantly upregulate in the proteome of MG1655/pTF2 grown in the presence of high concentrations of CTX when compared to growth without CTX. This was further supported by RT-qPCR analysis showing that expression of the typical SOS-responsive genes, recA and sfiA, were not affected significantly by growth in the presence of CTX (Supplementary Figure S3). It is well-established that conjugation plays a major role in the global spread of antibiotic resistance, and evidence implicating antibiotic treatment as a stimulating factor for conjugation transfer has been presented (Aminov, 2011; Andersson and Hughes, 2014)
Summary
Plasmid conjugation contributes significantly to horizontal spread of antibiotic resistance, because antibiotic resistance genes are frequently located on plasmids (Zatyka and Thomas, 1998; Sorensen et al, 2005; Thomas and Nielsen, 2005; Bennett, 2008; Norman et al, 2009). This report concluded that antimicrobials only serve to select for transconjugants, and that sub-lethal concentrations of antibiotics from the most widely used antimicrobial classes do not significantly increase the conjugation efficiency (Lopatkin et al, 2016a). Treatment with sub-inhibitory concentrations of several antibiotics has been shown to induce the expression of conjugative transfer genes from an R-plasmid in Aeromonas hydrophila (Cantas et al, 2012), and low concentrations of tetracycline has been reported to increase transfer of conjugative transposons in strains of Bacteroides by a factor 10–100 (Whittle et al, 2002)
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