Identification of Genes Essential for Antibiotic-Induced Up-Regulation of Plasmid-Transfer-Genes in Cephalosporin Resistant Escherichia coli

Abstract

Bacterial conjugation is one of the most important mechanisms for spread of antibiotic resistance among bacteria. We have previously demonstrated that cefotaxime (CTX) exposure up-regulates expression of Type-IV conjugation transfer genes, and that this leads to increased transfer of a bla _{CTX-M- 1} encoding IncI1 resistance plasmid pTF2 in Escherichia coli. To elucidate the underlying mechanisms, a search for genes that are essential for the up-regulated expression of the transfer (tra) genes in the presence of CTX was undertaken. We constructed a reporter gene-fusion strain MG1655/pTF2 ΔtraF:lacZ where the promoter region of the traF-gene of the plasmid pTF2 was fused with a lacZ on the native plasmid. Random mutagenesis mediated by Tn5 transposon was carried out in the strain, and seven genes (rfaH, yhiN, waaP, waaQ, gnd, pgl, and ISEcp1) were identified where insertion prevented CTX-induced up regulation of traF. Site-specific mutagenesis was carried out, and for all seven mutants, gene deletions abolished the CTX induced up-regulation of traF, and the increased conjugation transfer of the plasmid in the presence of CTX was no longer observed. In addition, the deletion of the genes also abolished CTX induced expression of the bla _{CTX-M- 1} gene. Our results suggested that through CTX induced induction of the identified genes, bla _{CTX-M- 1} expression increased, which led to up-regulation of traF and plasmid transfer. These data reveal that a number of chromosomally encoded genes contribute to the antibiotic induced up-regulation of the conjugation machinery of plasmids, and such genes may be future targets to prevent antibiotic induced spread of resistance plasmids.

Keywords: Escherichia coli; antibiotic induced conjugation; blaCTX–M–1 resistance plasmid; cefotaxime; transfer genes.

FIGURE 1

β-galactosidase activity increases in MG1655/pTF2 ΔtraF:lacZ during CTX exposure, but not in the transposon-mutants. (A) LacZ expression of MG1655/pTF2 ΔtraF:lacZ with (square) and without (Circle) CTX during growth. (B) LacZ expression of MG1655/pTF2 ΔtraF:lacZ (WT) and the seven transposon-mutants without (squared bars) and with 1/2 MIC CTX (black bars) exposure at OD₆₀₀ = 0.5. All experiments were carried out in triplicate, and each value is presented as the average plus standard deviation. The star indicates statistical significance between antibiotic treated and untreated at level: ^∗P ≤ 0.05.

FIGURE 2

Growth phenotypes of MG1655/pTF2 and its mutants with and without CTX. All strains were grown without and with CTX (1/2 MIC of the corresponding strain). Three independent replicates were performed and the data shown represent the mean and dots represent standard deviations.

FIGURE 3

CTX induced expression of the traF gene in WT (MG1655/pTF2) and deletion mutants. Strains were grown with 1/2 MIC CTX or without. Data are presented as fold change in CTX induced expression relative to expression levels without CTX. Three independent replicates including two technical replicates each were performed. The data shown represents the mean and the error bars represent standard deviations. The expression data was normalized to gapA and nusG. The stars indicate statistical significance between the two conditions at level: ^∗P ≤ 0.05.

FIGURE 4

CTX induced expression of bla_CTX–M–1 in WT (MG1655/pTF2) and deletion mutants. Strains were grown with 1/2MIC CTX or without. Data are presented as fold change in CTX induced expression relative to expression levels without CTX. Two independent replicates including two technical replicates each were performed. The data shown represents the mean and the error bars represent standard deviations. The expression data was normalized to two validated reference genes, gapA and nusG. The stars indicate statistical significance at level: ^∗P ≤ 0.05.

FIGURE 5

CTX induces expression of six genes involved in antibiotic induced traF expression in MG1655/pTF2. MG1655/pTF2 (Black bars) and the ΔrfaH (squared bars) were grown with 1/2 MIC CTX or without. Data are presented as fold change in CTX induced expression relative to expression levels without CTX. Three independent replicates including two technical replicates each were performed. The data shown represents the mean and the error bars represent standard deviations. The expression data was normalized to gapA and nusG. The stars indicate statistical significance at different levels: ^∗P ≤ 0.05, ^∗∗P ≤ 0.01.