A major mechanism for bacterial resistance to antibiotics is through the acquisition of a plasmid coding for resistance-mediating proteins. Described herein is a strategy to eliminate these plasmids from bacteria, thus resensitizing the bacteria to antibiotics. This approach involves mimicking a natural mechanism for plasmid elimination, known as plasmid incompatibility. The compound apramycin was identified as a tight binder to SLI RNA (Kd = 93 nM), the in vivo target of the plasmid incompatibility determinate RNA I, and footprinting/mutagenesis studies indicate apramycin binds SLI in the important regulatory region that dictates plasmid replication control and incompatibility. In vivo studies demonstrate that this compound causes significant plasmid loss and resensitizes bacteria to conventional antibiotics. The demonstration that a small molecule can mimic incompatibility, cause plasmid elimination, and resensitize bacteria to antibiotics opens up new targets for antibacterial research.

Bacteria routinely become resistant to antibiotics through the uptake of plasmids that encode resistance-mediating proteins. Such plasmid-based resistance is seen extensively in clinical settings and has been documented for a wide variety of bacterial infections from both Gram-positive and Gram-negative organisms. We recently reported that a small molecule could be used to mimic a natural process of plasmid elimination, called plasmid incompatibility, and that the addition of this compound causes elimination of an IncB plasmid from E. coli and a subsequent resensitization to antibiotics [DeNap, Thomas, Musk, and Hergenrother (2004) J. Am. Chem. Soc. 126, 15402-15404]. Described herein is a further substantiation and validation of the notion that plasmid incompatibility can be mimicked with small molecules that bind to important RNA targets controlling plasmid replication. In this study, the dissociation constant and stoichiometry of RNA binding are determined for 12 aminoglycosides with stem-loop I (SLI) of the IncB replication machinery. Importantly, it is found that compounds that do not bind to this RNA replication control element fail to induce plasmid loss in vivo, whereas those that do bind to the RNA typically cause measurable plasmid loss. These results highlight the potential for targeting key RNA regions for induction of plasmid loss and the subsequent resensitization of bacteria to antibiotics.

It is now common for bacterial infections to resist the preferred antibiotic treatment. In particular, hospital-acquired infections that are refractory to multiple antibiotics and ultimately result in death of the patient are prevalent. Many of the bacteria causing these infections have become resistant to antibiotics through the process of lateral gene transfer, with the newly acquired genes encoding a variety of resistance-mediating proteins. These foreign genes often enter the bacteria on plasmids, which are small, circular, extrachromosomal pieces of DNA. This plasmid-encoded resistance has been observed for virtually all classes of antibiotics and in a wide variety of Gram-positive and Gram-negative organisms; many antibiotics are no longer effective due to such plasmid-encoded resistance. The systematic removal of these resistance-mediating plasmids from the bacteria would re-sensitize bacteria to standard antibiotics. As such, plasmids offer novel targets that have heretofore been unexploited clinically. This Perspective details the role of plasmids in multi-drug resistant bacteria, the mechanisms used by plasmids to control their replication, and the potential for small molecules to disrupt plasmid replication and re-sensitize bacteria to antibiotics. An emphasis is placed on plasmid replication that is mediated by small counter-transcript RNAs, and the "plasmid addiction" systems that employ toxins and antitoxins.

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