An improved assay for the detection of alterations in bacterial DNA supercoiling in vivo.

Due to the increasing prevalence of antibiotic resistance and the yet low output of the genomics-based drug discovery approach novel strategies are urgently needed to detect new antibiotics. One such strategy uses known ubiquitous targets like DNA topoisomerases. However, to detect inhibitors of these enzymes by an in vitro assay time-consuming isolation of enzymes and DNA followed by electrophoretic separation of topoisomers are required. Instead, this study aimed at developing an in vivo assay for the detection of alterations in DNA supercoiling indicative of topoisomerase inhibition by a reporter gene assay. A pair of plasmids was developed which carry the reporter gene luc for firefly luciferase under control of either promoter ptopA (pPHB90) or pgyrA (pPHB91), whose activities are reciprocally affected by alterations of the supercoiling degree. Each plasmid is individually transferred into E. coli cells. The quotient of the luciferase activities determined using cells with either plasmid was taken as relative measure of the global supercoiling degree Qsc (quotient of supercoiling). Using isogenic reference strains with known alterations of the global DNA supercoiling degree due to mutations in either gyrB or topA, the reporter gene system was able to detect both a decrease and an increase of the negative supercoiling degree compared to the isogenic parent strain. Treating cells with known inhibitors of DNA gyrase, like fluoroquinolones, novobiocin as well as simocyclinone D8 from Streptomyces antibioticus which has been identified as an inhibitor of DNA gyrase in vitro, also caused decreases of the Qsc value in vivo. The suitability of this reporter gene system to screen for anti-topoisomerase I and II compounds from various natural sources like plant extracts by sensing alterations of the DNA supercoiling was demonstrated and offers a new application to identify novel compounds active against bacterial topoisomerases I and gyrase.

[Urinary tract infections and antibiotic resistance]. / Harnwegsinfektionen und Antibiotikaresistenz.

Urinary tract infections (UTI) usually are monoinfections caused by the endogenous microflora including gram-negatives, such as Escherichia coli, or gram-positives, like enterococci. This allows for an empiric treatment of uncomplicated UTIs and a short duration of therapy to minimize the probability for the development of resistance. Resistance often is based upon mutations altering the drug target (sulfonamides, trimethoprim, fluoroquinolones, fosfomycin) or acquisition of resistance genes (beta-lactams). The latter can be collocated with other resistance genes on mobile genetic elements mediating multiple drug resistance. Such elements are part of the chromosome of the Escherichia coli clonal group A (cgA). The prevalence of resistance to nitrofurantoin and fosfomycin is below 6%, while that of sulfonamide/trimethoprim varies between 15 and 50% all over Europe. Resistance to fluoroquinolones and amoxycillin approximates 15 and over 30%, respectively. A value of >20% for the empiric treatment of uncomplicated UTIs is associated with a significantly increased duration of therapy suggesting alternative antibiotic regimens.

Inhibitors of bacterial topoisomerases: mechanisms of action and resistance and clinical aspects.

The quinolone class of inhibitors of bacterial type II topoisomerases has gained major clinical importance during the last years due to improvements in both pharmacokinetic and pharmacodynamic properties. These include favorable bioavailability allowing oral administration, good tolerability, high tissue concentrations as well as superior bactericidal activity against a broad spectrum of clinically relevant pathogens, like enterobacteria, Pseudomonas aeruginoso, Staphylococcus aureus, and Streptococcus pneumoniae. In addition, no enzymatic mechanism of drug inactivation exists in bacteria and no indications for transfer of clinically relevant resistance exist. Nevertheless, resistance is being increasingly reported, even for naturally highly susceptible species like Escherichia coli. The underlying mechanisms of resistance include alterations in both bacterial targets, DNA gyrase and topoisomerase IV, often combined with mutations affecting drug accumulation, e.g., by increased drug efflux, reduced drug influx, or both. Investigations aiming at understanding the molecular mechanisms of quinolone action and resistance in more detail should provide a basis for a rational design of more potent derivatives. In addition, a prudent use of these highly valuable "magic bullets" is necessary to preserve their potential for the future.

Clinical significance and spread of fluoroquinolone resistant uropathogens in hospitalised urological patients.

The minimum inhibitory concentrations (MIC) of ciprofloxacin were determined for 441 uropathogens from patients with complicated and/or hospital acquired urinary tract infections (UTI). None of the Enterobacteriaceae was resistant (MIC > or = 4 mg/l), but 21.7% of enterococci, 28.3% of Pseudomonas spp. and 38.5% of staphylococci were. Subtyping of the strains revealed that with staphylococci there was no clonal spread of resistant strains. In the case of enterococci and Pseudomonas spp., however, cross infections played a major role (46% and 40% respectively). In a retrospective analysis of 370 UTI episodes caused by Pseudomonas aeruginosa (74), enterococci (185) or staphylococci (111) there was no difference between sensitive and resistant strains with respect to clinical aspects and rates of elimination by appropriate anti-bacterial therapy. The rates of spontaneous disappearance without antibacterial therapy ranged from 28% in the case of P. aeruginosa up to 63% in the case of coagulase-negative staphylococci. This implies that especially in UTI caused by gram-positive cocci an indication for antibacterial therapy should be weighted thoroughly and fluoroquinolones should only be used in accordance with sensitivity testing.