Abstract
Quinoxaline 1, 4-dioxides (QdNOs) has been used in animals as antimicrobial agents and growth promoters for decades. However, the resistance to QdNOs in pathogenic bacteria raises worldwide concern but it is barely known. To explore the molecular mechanism involved in development of QdNOs resistance in Escherichia coli, 6 strains selected by QdNOs in vitro and 21 strains isolated from QdNOs-used swine farm were subjected to MIC determination and PCR amplification of oqxA gene. A conjugative transfer was carried out to evaluate the transfer risk of QdNOs resistant determinant. Furthermore, the transcriptional profile of a QdNOs-resistant E. coli (79O4-2) selected in vitro with its parent strain 79–161 was assayed with a prokaryotic suppression subtractive hybridization (SSH) PCR cDNA subtraction. The result showed that more than 95% (20/21) clinical isolates were oqxA positive, while all the 6 induced QdNOs-resistant strains carried no oqxA gene and exhibited low frequency of conjugation. 44 fragments were identified by SSH PCR subtraction in the QdNOs-resistant strain 79O4-2. 18 cDNAs were involved in biosynthesis of Fe-S cluster (narH), protein (rpoA, trmD, truA, glyS, ileS, rplFCX, rpsH, fusA), lipoate (lipA), lipid A (lpxC), trehalose (otsA), CTP(pyrG) and others molecular. The 11 cDNAs were related to metabolism or degradation of glycolysis (gpmA and pgi) and proteins (clpX, clpA, pepN and fkpB). The atpADG and ubiB genes were associated with ATP biosynthesis and electron transport chain. The pathway of the functional genes revealed that E. coli may adapt the stress generated by QdNOs or develop specific QdNOs-resistance by activation of antioxidative agents biosynthesis (lipoate and trehalose), protein biosynthesis, glycolysis and oxidative phosphorylation. This study initially reveals the possible molecular mechanism involved in the development of QdNOs-resistance in E. coli, providing with novel insights in prediction and assessment of the emergency and horizontal transfer of QdNOs-resistance in E. coli.
Highlights
Quinoxaline-1,4-dioxides (QdNOs) have been introduced into animal production as potent antibacterial agents against most gram-positive and gram–negative pathogens since 1970s [1,2,3]
The homology search results showed that QdNOs-resistant E. coli alters the expression of 18 genes involved in biosynthesis, 11 genes associated with metabolism, 3 genes encoding transporters, 3 genes involved in flagellar assembly, 1 gene encoding mobilization protein and 5 genes associated with hypothetical proteins
The overexpression of genes involved in biosynthesis of lipoic acid and trehalose (13-lipA and ostA) may eliminate superoxide formation and hydroxyl radical production
Summary
Quinoxaline-1,4-dioxides (QdNOs) have been introduced into animal production as potent antibacterial agents against most gram-positive and gram–negative pathogens since 1970s [1,2,3] These drugs have been widely used as growth promoters for decades [4,5]. For the mechanism of QdNOs resistance, it is only known that plasmid-encoded multidrug efflux pump OqxAB confers resistance to ampicillin, chloramphenicol, ciprofloxacin and olaquindox in E. coli [14,15]. This OqxAB located plasmid has a high frequency of transfer among enterobacterial pathogens During the development of QdNOs resistance, it is an urgent need to explore the novel molecular mechanisms involved in the emergency and transfer of QdNO’s resistance in pathogenic bacteria especially E. coli
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