Mutational Consequences of Ciprofloxacin in Escherichia coli

Abstract

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 ...

The antioxidant drug N-acetylcysteine abolishes SOS-mediated mutagenesis produced by fluoroquinolones in bacteria

“…Consistent with this view, we observed an increase in mutagenesis in the TLS strain when submitted to CIP treatment. This result agrees with recent work in which an increased frequency of ⁻ indels was found upon CIP treatment in a TLS strain (Song et al 2016). Together, these results support the existence of a TLS-independent mutagenic pathway.…”

Ecological effects of stress drive bacterial evolvability under sub-inhibitory antibiotic treatments

“…The following studies contain relevant data, but were excluded from the analysis despite matching our a priori inclusion criteria: Gutierrez et al [5], Schroder et al [36] (data available, but in a different representation or in a summary form); Kohanski et al [4], Nair et al [37], Peng et al [38], Bunnell et al [39], Cairns et al [40], Thi et al [41], Song et al [42], Valencia et al [43], Nagel et al [44] (raw data unavailable or no answer from the author who kept the raw data).…”

Access to high-impact mutations constrains the evolution of antibiotic resistance in soft agar

“…Other antibiotics showed a similar pattern, though with ciprofloxacin increasing the maximal drug concentration from 75x MIC to 125x MIC actually accelerated growth. This may be due to the mutagenic effect of subinhibitory ciprofloxacin concentrations, which could potentially increase the effective mutant supply rate 29 . Of the antibiotics tested, only doxycycline showed a statistically significant difference in growth at 72 hr (10x MIC vs 50x MIC, s = 0.04, two-tailed, unequal variance Student’s t-test).…”