Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) system, a genome editing technology, was shown to be versatile in treating several antibiotic-resistant bacteria. In the present study, we applied the CRISPR/ Cas9 technology to kill extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli. ESBL bacteria are mostly multidrug resistant (MDR), and have plasmid-mediated antibiotic resistance genes that can be easily transferred to other members of the bacterial community by horizontal gene transfer. To restore sensitivity to antibiotics in these bacteria, we searched for a CRISPR/Cas9 target sequence that was conserved among >1,000 ESBL mutants. There was only one target sequence for each TEM- and SHV-type ESBL, with each of these sequences found in ~200 ESBL strains of each type. Furthermore, we showed that these target sequences can be exploited to re-sensitize MDR cells in which resistance is mediated by genes that are not the target of the CRISPR/Cas9 system, but by genes that are present on the same plasmid as target genes. We believe our Re-Sensitization to Antibiotics from Resistance (ReSAFR) technology, which enhances the practical value of the CRISPR/Cas9 system, will be an effective method of treatment against plasmid-carrying MDR bacteria.

g Bacterial persisters are a small fraction of quiescent cells that survive in the presence of lethal concentrations of antibiotics. They can regrow to give rise to a new population that has the same vulnerability to the antibiotics as did the parental population. Although formation of bacterial persisters in the presence of various antibiotics has been documented, the molecular mechanisms by which these persisters tolerate the antibiotics are still controversial. We found that amplification of the fumarate reductase operon (FRD) in Escherichia coli led to a higher frequency of persister formation. The persister frequency of E. coli was increased when the cells contained elevated levels of intracellular fumarate. Genetic perturbations of the electron transport chain (ETC), a metabolite supplementation assay, and even the toxin-antitoxin-related hipA7 mutation indicated that surplus fumarate markedly elevated the E. coli persister frequency. An E. coli strain lacking succinate dehydrogenase (SDH), thereby showing a lower intracellular fumarate concentration, was killed ϳ1,000-fold more effectively than the wild-type strain in the stationary phase. It appears that SDH and FRD represent a paired system that gives rise to and maintains E. coli persisters by producing and utilizing fumarate, respectively. Bacterial persisters are phenotypic variants that are tolerant even to supralethal concentrations of multiple antibiotics (1-3). Reseeding of the persisters yields a bacterial population with a frequency of antibiotic-tolerant cells that is similar to that of the parental population (4, 5). Persisters are distinct from antibioticresistant cells because the ability to tolerate antibiotics is neither genetically determined nor inherited. Persisters showing tolerance of different classes of antibiotics are observed in most microbial species and have been implicated in chronic and recurrent infections (1). Furthermore, it is highly probable that persisters are a potential reservoir for the development of drug resistance in pathogenic bacteria (6, 7).Despite the discovery of bacterial persisters more than 70 years ago (4), the mechanisms that underlie noninheritable persistence phenotypes remain unclear. Various researchers recently identified a number of genes and pathways that lead to persister formation or survival upon antibiotic treatments. These include toxinantitoxin (TA) modules, a stringent response, phosphate metabolism, alternative energy production, and antioxidative defense (8-13). Because nongrowing or slow-growing bacteria are less sensitive to antibiotics, dormancy has been proposed to be the mechanism of last resort in many of these persistence studies. Thus, many recent mechanistic studies have focused on how bacterial cells reach the dormant state (8-15). Nonetheless, the prevailing hypothesis that persisters might survive solely because of dormancy is being challenged. A lack of significant growth or metabolic activity does not guarantee persistence, and dormancy is neither necessary nor sufficient fo...

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