Determination of Antibiotic Hypersensitivity among 4,000 Single-Gene-Knockout Mutants of Escherichia coli

Abstract

We have tested the entire Keio collection of close to 4,000 single-gene knockouts in Escherichia coli for increased susceptibility to one of seven different antibiotics (ciprofloxacin, rifampin, vancomycin, ampicillin, sulfamethoxazole, gentamicin, or metronidazole). We used high-throughput screening of several subinhibitory concentrations of each antibiotic and reduced more than 65,000 data points to a set of 140 strains that display significantly increased sensitivities to at least one of the antibiotics, determining the MIC in each case. These data provide targets for the design of "codrugs" that can potentiate existing antibiotics. We have made a number of double mutants with greatly increased sensitivity to ciprofloxacin, and these overcome the resistance generated by certain gyrA mutations. Many of the gene knockouts in E. coli are hypersensitive to more than one antibiotic. Together, all of these data allow us to outline the cell's "intrinsic resistome," which provides innate resistance to antibiotics.Antibiotics have had a major impact over the past 6 decades in the fight against infectious diseases (for a review by Davies, see reference 11). However, the spread of antibiotic-resistant microorganisms has reached an alarming point (1,11,35), prompting renewed efforts to find new antibiotics by detecting new targets through genomics, altering existing antibiotics, screening chemical (e.g., see reference 9) or peptide (21, 31) libraries for specific inhibitors (e.g., see reference 9), or finding new sources of antibiotics via metagenomics (e.g., see reference 53). While these lines of investigation show great promise, additional approaches are constantly being sought to yield a new generation of useful antimicrobial compounds. For instance, focusing on species-specific antibiotics rather than broad-spectrum antibiotics can result in important new agents (38), as could targeting bacterial transcription factors (5) or different processes, such as Holliday junction processing (21, 31) and quorum sensing (24), or even targeting host factors that support pathogen growth (33). Another approach, examined here, involves potentiating existing antibiotics by identifying targets for increasing susceptibility to specific antimicrobials. There are precedents for using such combinational therapy. For example, inhibitors of ␤-lactamase have been used together with ␤-lactam antibiotics (for a review by Buynak, see reference 7) and inhibitors of efflux pumps together with tetracycline in Escherichia coli (46) and with levofloxacin in Pseudomonas aeruginosa (37). In the case of chemotherapeutics, zebularine, a cytosine analog (41) and mutagen (34) that is converted in vivo to an inhibitor of cytosine deaminase (41), is used in combination with certain cytosine deaminase-susceptible cytosine-based drugs (14, 40). With regard to finding new targets for this type of approach, a number of genes that increase the sensitivities of microorganisms to different antibiotics have been identified (e.g., for a review by Drlica and Zhao, ...

Recruitment of genes and enzymes conferring resistance to the nonnatural toxin bromoacetate

“…Class 6 encompasses genes involved in protein synthesis/degradation and class 7 genes are of unknown function. Eighteen of the hypersensitive mutants we identified have also been shown to be sensitive to at least 1 of 22 clinically used antibiotics (16,17). These coding sequences most likely represent a common framework of genes crucial to resisting the adverse effects of toxic molecules (Table S1).…”

“…To this end, we screened the entire Keio collection of approximately 4,000 E. coli single-gene knockouts (15) for mutants hypersensitive to bromoacetate. The Keio collection has previously been used to identify mutants hypersensitive to at least 1 of 22 different antibiotics (16,17). Similarly, screening of transposon libraries in Acinetobacter baylyi and P. aeruginosa has identified genes whose disruption hypersensitizes these organisms to a variety of antibiotics (18)(19)(20).…”

Parallel evolution and local differentiation in quinolone resistance in Pseudomonas aeruginosa

“…Recent genetic screens in E. coli (Tamae et al, 2008) and P. aeruginosa (Breidenstein et al, 2008) have identified genes whose knockouts either reduce or increase sensitivity to ciprofloxacin, with .100 and 40 genes, respectively, contributing to resistance. Most of these mutations have not been genotyped in clinical samples, so that their contributions to clinical resistance cannot yet be fully determined.…”

How to Determine the Role of the Microbiome in Drug Disposition

“…Concerning gut bacteria, these methods are mostly limited to the model gut bacteria Escherichia and Bacteroides spp. but have been valuable in understanding microbial interactions with drugs in vitro (Tamae et al, 2008) and in the host (García-González et al, 2017;Scott et al, 2017).…”