The mechanism of resistance in Escherichia coli to ridinilazole and other antibacterial head-to-head bis-benzimidazole compounds

Abstract

The appY gene has been characterised as conferring resistance to a novel series of antimicrobial benzimidazole derivatives in E. coli MC1061 cells when expressed in high copy-number. A microarray approach was used to identify genes involved in the mechanism of appY-mediated antibacterial resistance, that were up- or down-regulated following induction of the gene in the appY knockout strain JW0553. In total, expression of 90 genes was induced and 48 repressed greater than 2.5-fold (P < 0.05), 45 min after appY induction. Over half the genes up-regulated following appY expression had confirmed or putative roles in acid resistance (AR) and response to oxidative and antibiotic stresses. These included the genes for MdtE and MdtF, which form a multi-drug transporter with TolC and have been implicated in resistance to several antibiotics including erythromycin. Amongst the acid resistance genes were gadAB and adiAC encoding the glutamate-dependant (AR2) and arginine-dependant (AR3) acid resistance systems respectively, in addition to the transcriptional activators of these systems gadE and gadX. In agreement with earlier studies, appA, appCB and hyaA-F were also up-regulated following induction of appY. This study has also confirmed that over-expression of mdtEF confers resistance to these antibacterial benzimidazoles, indicating that the observation of appY conferring resistance to these compounds, proceeds through an appY-mediated up-regulation of this efflux transporter. To assess the importance of the AppY enzyme to acid stress responses, the percentage survival of bacteria in acidified media (pH ≤ 2) was measured. From an initial input of 1 × 106 CFU/ml, the wild-type strain MG1655 showed 7.29% and 0.46% survival after 2 and 4 h, respectively. In contrast, strain JW0553 in which appY is deleted was completely killed by the treatment. Transformation of JW0553 with a plasmid carrying appY returned survival to wild-type levels (7.85% and 1.03% survival at 2 and 4 h). Further dissection of the response by prior induction of each of the three AR systems has revealed that AR1 and AR3 were most affected by the absence of appY. This work highlights an important and previously unidentified role for the AppY enzyme in mediating the responses to several stress conditions. It is likely that the appY gene fits into a complex transcriptional regulatory network involving σS and gadE and gadX. Further work to pinpoint its position in such a hierarchy and to assess the contribution of appY to oxidative stress responses should help determine its full significance. This work is also consistent with recent studies in C. difficile showing that the mechanism of action of ridinilazole involves AT-rich DNA minor groove binding.

The Novel DNA Binding Mechanism of Ridinilazole, a Precision Clostridiodes difficile Antibiotic

“…5A ), returning MICs lower than the values observed against C. difficile , while the wild-type BW25113 parental strain was not susceptible. These data complement recent observations that overexpression of an efflux pump ( mdtEF ) restored ridinilazole resistance to a permeable E. coli strain ( 18 ). We also demonstrated that the enhanced fluorescence of ridinilazole upon DNA binding in vitro was apparent, and comparable when using either E. coli or C. difficile genomic DNA ( Fig.…”

“…Of the top 60 upregulated genes, eight comprise the entirety of the glycine reductase operon ( grdX , trxB3 , trxA2 , grdEABCD ) (Table S1) ( 17 ). Glycine reductase is a key enzyme in the Stickland pathway which couples the oxidation and reduction of amino acid pairs to the formation of ATP and is the major anaerobic mechanism by which C. difficile generates energy ( 18 ). The most efficient Stickland electron acceptors are glycine, proline and hydroxyproline ( 17 ) and we observed that genes in the proline reductase operon were among the most downregulated genes ( prdABDE prdE2 prdF ).…”