We examined the mutagenic specificity of the widely used antibiotic ciprofloxacin (CPR), which displays weak to moderate mutagenic activity in several bacteria and generates short in-frame deletions in rpoB in Staphylococcus aureus. To determine the spectrum of mutations in a system where any gene knockout would result in a recovered mutant, including frameshifts and both short and long deletions, we examined CPR-induced mutations in the thymidylate synthase-encoding thyA gene. Here, any mutation resulting in loss of thymidylate synthase activity generates trimethoprim (Trm) resistance. We found that deletions and insertions in all three reading frames predominated in the spectrum. They tend to be short deletions and cluster in two regions, one being a GC-rich region with potential extensive secondary structures. We also exploited the well-characterized rpoB-Rif r system in Escherichia coli to determine that cells grown in the presence of sublethal doses of CPR not only induced short inframe deletions in rpoB, but also generated base substitution mutations resulting from induction of the SOS system. Some of the specific point mutations prominent in the spectrum of a strain that overproduces the dinB-encoded Pol IV were also present after growth in CPR. However, these mutations disappeared in CPR-treated dinB mutants, whereas the deletions remained. Moreover, CPR-induced deletions also occurred in a strain lacking all three SOS-induced polymerases. We discuss the implications of these findings for the consequences of overuse of CPR and other antibiotics. Several bactericidal antibiotics have been reported to have low to moderate mutagenic activity when used at subinhibitory or sublethal concentrations (1-14), usually ranging from 3-to 8-fold over background levels of spontaneous mutations (with one exception [7]). In particular, ciprofloxacin (CPR) and its close derivative norfloxacin (NOR) display mutagenic activity in different detector systems (1-14). The mutagenic properties can result in an increase in the appearance of resistant mutants (1, 2, 4, 5). A previous study of CPR-induced mutations in the rpoB gene of Staphylococcus aureus detected short in-frame deletions (5). However, the rpoB system cannot detect out-of-frame deletions or insertions or in-frame deletions or insertions of more than 21 bp, since the integrity of the RNA polymerase must remain intact. Here, we have undertaken a study of the types of mutations resulting from treatment with CPR in Escherichia coli using the thyATrm r system, which detects trimethoprim (Trm)-resistant mutants that result from any mutations inactivating the thyA gene (15), including large or small deletions or additions, both in frame and out of frame. We also used the E. coli rpoB-Rif r system, which monitors mutations leading to rifampin (Rif) resistance (16-23), since the system is so extensively characterized that the spectra of different mutagenic pathways leave telltale fingerprints. This allows us to separate effects of CPR itself from those emanating from the ...

We tested pairwise combinations of classical base analog mutagens in Escherichia coli to study possible mutagen synergies. We examined the cytidine analogs zebularine (ZEB) and 5-azacytidine (5AZ), the adenine analog 2-aminopurine (2AP), and the uridine/thymidine analog 5-bromodeoxyuridine (5BrdU). We detected a striking synergy with the 2AP plus ZEB combination, resulting in hypermutability, a 35-fold increase in mutation frequency (to 53,000 ؋ 10 ؊8 ) in the rpoB gene over that with either mutagen alone. A weak synergy was also detected with 2AP plus 5AZ and with 5BrdU plus ZEB. The pairing of 2AP and 5BrdU resulted in suppression, lowering the mutation frequency of 5BrdU alone by 6.5-fold. Sequencing the mutations from the 2AP plus ZEB combination showed the predominance of two new hot spots for A·T¡G·C transitions that are not well represented in either single mutagen spectrum, and one of which is not found even in the spectrum of a mismatch repair-deficient strain. The strong synergy between 2AP and ZEB could be explained by changes in the dinucleoside triphosphate (dNTP) pools. IMPORTANCEAlthough mutagens have been widely studied, the mutagenic effects of combinations of mutagens have not been fully researched. Here, we show that certain pairwise combinations of base analog mutagens display synergy or suppression. In particular, the combination of 2-aminopurine and zebularine, analogs of adenine and cytidine, respectively, shows a 35-fold increased mutation frequency compared with that of either mutagen alone. Understanding the mechanism of synergy can lead to increased understanding of mutagenic processes. As combinations of base analogs are used in certain chemotherapy regimens, including those involving ZEB and 5AZ, these results indicate that testing the mutagenicity of all drug combinations is prudent. Mutagen-induced mutations have been the subject of intensive investigation for decades (e.g., see references 1 to 4; see reviews in references 5 to 7). However, there are far fewer studies of combinations of mutagens. We are studying possible synergies between mutagens in Escherichia coli and initially examined a set of base analog mutagens ( Fig. 1): the cytidine analogs zebularine (ZEB) and 5-azacytidine (5AZ), the adenine analog 2-aminopurine (2AP), and the uridine/thymidine analog 5-bromodeoxyuridine (5BrdU). ZEB lacks the amino group of cytidine (8) and causes C·G¡T·A changes in the rpoB/rifampin resistance (Rif r ) system in E. coli (9). It is used in chemotherapy as a demethylating agent (8, 10, 11) to reverse the effects of gene-silenced tumor suppressor gene (12-15) and because the hydrated form is a potent inhibitor of cytidine deaminase (16). 5AZ, another cytidine analog that is used as a demethylating agent in chemotherapy (12, 13), possesses a unique mutagenic specificity, stimulating only C·G¡G·C changes (17-19). 5AZ and ZEB have been used in combination in chemotherapy (20). 2-Aminopurine results principally in G·C¡A·T and A·T¡G·C changes (e.g., see references 18 and 21 to 25), as...

We used classical mutagens in Gram-negative Escherichia coli to study synergies with different classes of antibiotics, test models of antibiotic mechanisms of action, and examine the basis of synergy. We used 4-nitroquinoline 1-oxide (4NQO), zebularine (ZEB), 5-azacytidine (5AZ), 2-aminopurine (2AP), and 5-bromodeoxyuridine (5BrdU) as mutagens (with bactericidal potency of 4NQO > ZEB > 5AZ > 2AP > 5BrdU) and vancomycin (VAN), ciprofloxacin (CPR), trimethoprim (TMP), gentamicin (GEN), tetracycline (TET), erythromycin (ERY), and chloramphenicol (CHL) as antibiotics. We detected the strongest synergies with 4NQO, an agent that oxidizes guanines and ultimately results in double-strand breaks when paired with the bactericidal antibiotics VAN, TMP, CPR, and GEN, but no synergies with the bacteriostatic antibiotics TET, ERY, and CHL. Each of the other mutagens displays synergies with the bactericidal antibiotics to various degrees that reflect their potencies, as well as with some of the other mutagens. The results support recent models showing that bactericidal antibiotics kill bacteria principally by ultimately generating more double-strand breaks than can be repaired. We discuss the synergies seen here and elsewhere as representing dose effects of not the proximal target damage but rather the ultimate resulting double-strand breaks. We also used the results of pairwise tests to place the classic mutagens into functional antibacterial categories within a previously defined drug interaction network. N ew strategies are needed to combat the rise of multidrugresistant pathogens (1, 2). One avenue of research takes advantage of the synergy between antibiotics in combination (e.g., reference 3; see also a review in reference 4). Previously, we used the synergy between different antibiotics to potentiate the low concentration of vancomycin that is able to enter Gram-negative cells (5). The outer membrane of these cells normally acts as a barrier to vancomycin and many other drugs (6, 7). A more comprehensive understanding of the basis of synergy between certain pairwise combinations of antibiotics is important for developing this approach more thoroughly. Recently, Kohanski and coworkers (8) proposed that bactericidal antibiotics kill cells in part by generating hydroxyl radicals, causing DNA damage that leads to double-strand breaks (9). Recent works by Dwyer et al. (10) and Belenky et al. (11) strongly support this idea. In this study, we used a new strategy to examine both the mechanism of synergy and the mechanism of action of antibiotics by quantifying interactions between classic mutagens and different classes of commonly used antibiotics. In particular, we focused on mutagens that are strongly bactericidal via known mechanisms, generating doublestrand breaks. The use of synergistic relationships allows us to look at the mechanism of antibiotic killing through a different lens and shows that strongly bactericidal mutagens, particularly 4-nitroquinoline 1-oxide (4NQO), are highly synergistic with bactericidal antibio...

No abstract