RpoS role in antibiotic resistance, tolerance and persistence in E. coli natural isolates.

BACKGROUND: The intrinsic concentration of RpoS, the second most abundant sigma factor, varies widely across the E. coli species. Bacterial isolates that express high levels of RpoS display high resistance to environmental stresses, such as temperature, pH and osmolarity shifts, but are less nutritional competent, making them less capable of utilising alternative nutrient sources. The role of RpoS in antibiotic resistance and persistence in standard laboratory domesticated strains has been examined in several studies, most demonstrating a positive role for RpoS. RESULTS: Using disk diffusion assays we examined bacterial resistance to 15 different antibiotics, including ß -lactams (penicillins, monobactams, carbapenems and cephalosporins), aminoglycosides, quinolones and anti-folates, in a representative collection of 328 E. coli natural isolates displaying a continuum of different levels of RpoS. There was an overall trend that isolates with higher levels of RpoS were slightly more resistant to these antibiotics. In addition, the effect of RpoS on bacterial tolerance and persistence to 3 different antibiotics - ampicillin, ciprofloxacin and kanamycin was evaluated through time-kill curves. Again, there was a small beneficial effect of RpoS on tolerance and persistence to these antibiotics, but this difference was not statistically significant. Finally, a K-12 strain expressing high levels of RpoS was compared with its isogenic RpoS-null counterpart, and no significant effect of RpoS was found. CONCLUSION: Based on a representative collection of the species E. coli, RpoS was found to have a very small impact on antibiotic resistance, tolerance, or persistence.

A Broad Continuum of E. coli Traits in Nature Associated with the Trade-off Between Self-preservation and Nutritional Competence.

A trade-off between reproduction and survival is a characteristic of many organisms. In bacteria, growth is constrained when cellular resources are channelled towards environmental stress protection. At the core of this trade-off in Escherichia coli is RpoS, a sigma factor that diverts transcriptional resources towards general stress resistance. The constancy of RpoS levels in natural isolates is unknown. A uniform RpoS content in E. coli would impart a narrow range of resistance properties to the species, whereas a diverse set of RpoS levels in nature should result in a diverse range of stress susceptibilities. We explore the diversity of trade-off settings and phenotypes by measuring the level of RpoS protein in strains of E. coli cohabiting in a natural environment. Strains from a stream polluted with domestic waste were investigated in monthly samples. Analyses included E. coli phylogroup classification, RpoS protein level, RpoS-dependent stress phenotypes and the sequencing of rpoS mutations. The most striking finding was the continuum of RpoS levels, with a 100-fold range of RpoS amounts consistently found in individuals in the stream. Approximately 1.8% of the sampled strains carried null or non-synonymous mutations in rpoS. The natural isolates also exhibited a broad (>100-fold) range of stress resistance responses. Our results are consistent with the view that a multiplicity of survival-multiplication trade-off settings is a feature of the species E. coli. The phenotypic diversity resulting from the trade-off permits bet-hedging and the adaptation of E. coli strains to a very broad range of environments.

N-acetylcysteine blocks SOS induction and mutagenesis produced by fluoroquinolones in Escherichia coli.

BACKGROUND: Fluoroquinolones such as ciprofloxacin induce the mutagenic SOS response and increase the levels of intracellular reactive oxygen species (ROS). Both the SOS response and ROS increase bacterial mutagenesis, fuelling the emergence of resistant mutants during antibiotic treatment. Recently, there has been growing interest in developing new drugs able to diminish the mutagenic effect of antibiotics by modulating ROS production and the SOS response. OBJECTIVES: To test whether physiological concentrations of N-acetylcysteine, a clinically safe antioxidant drug currently used in human therapy, is able to reduce ROS production, SOS induction and mutagenesis in ciprofloxacin-treated bacteria without affecting antibiotic activity. METHODS: The Escherichia coli strain IBDS1 and its isogenic mutant deprived of SOS mutagenesis (TLS-) were treated with different concentrations of ciprofloxacin, N-acetylcysteine or both drugs in combination. Relevant parameters such as MICs, growth rates, ROS production, SOS induction, filamentation and antibiotic-induced mutation rates were evaluated. RESULTS: Treatment with N-acetylcysteine reduced intracellular ROS levels (by ∼40%), as well as SOS induction (by up to 75%) and bacterial filamentation caused by subinhibitory concentrations of ciprofloxacin, without affecting ciprofloxacin antibacterial activity. Remarkably, N-acetylcysteine completely abolished SOS-mediated mutagenesis. CONCLUSIONS: Collectively, our data strongly support the notion that ROS are a key factor in antibiotic-induced SOS mutagenesis and open the possibility of using N-acetylcysteine in combination with antibiotic therapy to hinder the development of antibiotic resistance.

Ciprofloxacin-Mediated Mutagenesis Is Suppressed by Subinhibitory Concentrations of Amikacin in Pseudomonas aeruginosa.

Resistance to antibiotics is a global health problem. Activation of the SOS response, and the subsequent elevation in mutagenesis, contributes to the appearance of resistance mutations. Among currently used drugs, quinolones are the most potent inducers of the SOS response. In the present study, we show that amikacin inhibits ciprofloxacin-mediated SOS induction and mutagenesis in Pseudomonas aeruginosa.

N-acetylcysteine selectively antagonizes the activity of imipenem in Pseudomonas aeruginosa by an OprD-mediated mechanism.

The modulating effect of N-acetylcysteine (NAC) on the activity of different antibiotics has been studied in Pseudomonas aeruginosa. Our results demonstrate that, in contrast to previous reports, only the activity of imipenem is clearly affected by NAC. MIC and checkerboard determinations indicate that the NAC-based modulation of imipenem activity is dependent mainly on OprD. SDS-PAGE of outer membrane proteins (OMPs) after NAC treatments demonstrates that NAC does not modify the expression of OprD, suggesting that NAC competitively inhibits the uptake of imipenem through OprD. Similar effects on imipenem activity were obtained with P. aeruginosa clinical isolates. Our results indicate that imipenem-susceptible P. aeruginosa strains become resistant upon simultaneous treatment with NAC and imipenem. Moreover, the generality of the observed effects of NAC on antibiotic activity was assessed with two additional bacterial species, Escherichia coli and Acinetobacter baumannii. Caution should be taken during treatments, as the activity of imipenem may be modified by physiologically attainable concentrations of NAC, particularly during intravenous and nebulized regimes.