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Antibiotic action and cell responses to antibiotics are very complex and include not only the direct target of an antibiotic but also secondary targets, signalling effects and indirect effects such as antibiotic-induced generation of hydroxyl radicals or changes in gene expression and cell metabolism. Identifying genes important for antibiotic action in the cell or antibiotic resistance would uncover new targets for potentiating existing antibiotics or for discovering new ones. Several approaches have been used to identify these genes, including studying antibiotic-resistant strains of clinical origin or selected in the laboratory and, more recently, screening the entire Keio collection of 4000 single-gene knockouts of Escherichia coli for increased antibiotic susceptibility . Such approaches have been successful but have limitations.
Here we propose a new approach to identify genes affecting antibiotic resistance or susceptibility based on screening genes that have functional interactions with other genes already known to be important for drug resistance. Such an approach was suggested by recent studies  with the multiple antibiotic resistance regulator MarA, a transcription factor in E. coli that upregulates multidrug efflux and downregulates membrane permeability . The effect of inactivation of 24 unrelated chromosomal genes using the EZ-Tn5™ <Kan-2> transposome (Epicentre, Madison, WI) on antibiotic resistance mediated by MarA was studied . Fifteen of these genes affected MarA-mediated antibiotic resistance, whereas nine of them did not. Here we examine whether these 24 inactivated genes affect antibiotic susceptibility per se in the absence of MarA.
Antibiotic susceptibility to cefoxitin, norfloxacin, chloramphenicol and minocycline of the parental strain CR2000 (E. coli BW25113 ΔmarRA ) and of its 24 gene-inactivated mutant derivatives was determined by Etest as described previously . Briefly, the different strains were grown overnight in Luria–Bertani (LB) medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl) at 37 °C with agitation and their minimum inhibitory concentrations were determined using Mueller–Hinton plates and Etest strips (AB BIODISK, Solna, Sweden) according to the manufacturer’s specifications following incubation for 20 h at 37 °C. The results for each mutant were compared with those of the parental strain by t-test (two independent samples with equal variance, two-tailed distribution, performed using Microsoft® Excel 2003 software) to determine statistically significant differences.
Nine (60%) of the fifteen genes functionally related to MarA-mediated antibiotic resistance also affected antibiotic susceptibility/resistance independently from MarA. Such a double role might help cells to have a more co-ordinated response to antibiotics. In contrast, only two (22%) of the nine genes that do not affect MarA-mediated antibiotic resistance affected antibiotic susceptibility/resistance (Table 1).
Those genes that affect both antibiotic resistance mediated by MarA and antibiotic susceptibility/resistance independently from MarA were as follows. First, acrA, acrB and tolC, whose inactivation strongly increased susceptibility to all the antibiotics tested (Table 1), as expected . Second, crp, cyaA, hns and degP, whose inactivation increased resistance to cefoxitin (crp and hns) or norfloxacin (crp and cyaA), increased susceptibility to cefoxitin (degP) and strongly increased susceptibility to chloramphenicol and minocycline (hns) (Table 1). Inactivation of crp and cyaA was previously shown to produce resistance to some β-lactams  and fosfomycin . Inactivation of hns or degP was found to increase susceptibility to rifampicin, sulphonamide and vancomycin (hns), or rifampicin and sulfamethoxazole (degP) in the Keio collection screen . And finally, pcnB and nikD, two genes with no previous known role in antibiotic resistance whose inactivation increased resistance to cefoxitin (pcnB) or minocycline (nikD) (Table 1).
Only two genes that did not affect MarA-mediated antibiotic resistance (ompR and metL) affected antibiotic susceptibility/resistance (Table 1). Inactivation of ompR affected resistance to cefoxitin (in agreement with previous studies [3,5]) and norfloxacin. Inactivation of metL, a gene with no previous known role in antibiotic susceptibility, produced a small but statistically significant increase in susceptibility to cefoxitin (Table 1).
We have found that genes known to affect MarA-mediated antibiotic resistance were three times more likely to affect antibiotic susceptibility per se than genes that do not affect MarA-mediated antibiotic resistance. Moreover, these experiments have allowed us to confirm and expand the role of crp, cyaA, hns, degP and ompR in antibiotic susceptibility in E. coli and to identify three new genes (pcnB, nikD and metL) previously not known to affect antibiotic susceptibility in any bacterium. These findings support our proposed approach of studying genes that have functional interactions with other genes associated with drug resistance as a method to identify genes that independently affect antibiotic susceptibility.
This work was supported by a United States Public Health Service grant (AI56021) from the National Institutes of Health to SBL.
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