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.

EUCAST expert rules in antimicrobial susceptibility testing.

EUCAST expert rules have been developed to assist clinical microbiologists and describe actions to be taken in response to specific antimicrobial susceptibility test results. They include recommendations on reporting, such as inferring susceptibility to other agents from results with one, suppression of results that may be inappropriate, and editing of results from susceptible to intermediate or resistant or from intermediate to resistant on the basis of an inferred resistance mechanism. They are based on current clinical and/or microbiological evidence. EUCAST expert rules also include intrinsic resistance phenotypes and exceptional resistance phenotypes, which have not yet been reported or are very rare. The applicability of EUCAST expert rules depends on the MIC breakpoints used to define the rules. Setting appropriate clinical breakpoints, based on treating patients and not on the detection of resistance mechanisms, may lead to modification of some expert rules in the future.

Identification of variables for aerobic bacterial density at clinically relevant skin sites.

In studies on efficacy testing of topical antimicrobial products, randomisation of test areas and a well-balanced gender ratio are not always standard. Our aim was to generate an evidence-based skin flora map using a systematic review of the literature supplemented by in vivo tests to identify variables that impact on microbial density. Ten out of 83 evaluated studies were reviewed. Microbial density was higher on sebaceous-rich and wet skin sites. In the in vivo study the forehead, upper back, lumbar area, and abdomen of 180 subjects were sampled with a standardised swab method. The highest aerobic microbial density was found on the forehead (mean log10 cfu/cm² = 3.69 ± 1.00), followed by the upper back (3.00 ± 0.90), the abdomen (2.98 ± 0.74), and the lumbar area (2.35 ± 0.70). The difference between all four skin sites was significant (P < 0.001; analysis of variance). On the forehead, we found significantly more micro-organisms on the medial compared to the lateral side (P = 0.002; t-test), on the upper back we found more micro-organisms cranially than caudally (P = 0.006). Males carried significantly more micro-organisms on all sites (P < 0.001). Randomisation of the test areas is essential to obtain representative results in studies on the density of skin flora or the efficacy of skin antiseptics. A well-balanced gender ratio is also strongly recommended for any study population.

[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.

Skin bacteria after chlorhexidine exposure-is there a difference in response to human beta-Defensin-3?

We investigated whether exposure to sub-lethal concentrations of chlorhexidine digluconate (CHG) changed the response of five Staphylococcus spp. to human beta-Defensin-3 (hBD-3). The change in response for each strain was determined in vitro with time-kill experiments in suspension by comparing the mean log(10) reduction caused by hBD-3 at 1.5 and 3 h in exposed and non-exposed bacteria. The identity of staphylococcal species was verified by DNA sequence homology in the gyrA genes in comparison with reference strains. Baseline sub-lethal concentrations allowing visible bacterial growth were between 0.0625 and 0.25 microg/ml. Sub-lethal CHG concentrations increased within 3 days in two isolates. For S. capitis 19/2, CHG-exposed cells were less susceptible to 0.5 microg/ml hBD-3 (log(10) reduction 0.78 versus 2.06 at 1.5 h; p < 0.001; t-test). For S. aureus, however, CHG-exposed cells were more susceptible to 1 microg/ml hBD-3. The observed changes between CHG-exposed and non-exposed cells did not indicate a general trend in response to hBD-3. Overall, we found no consistent evidence that 3 days of exposure to CHG changed the response of five Staphylococcus spp. to hBD-3. The use of CHG for skin antisepsis is, based on our data, unlikely to change the natural defence activity of hBD-3.

Molecular characterisation of linezolid resistance in two vancomycin-resistant (VanB) Enterococcus faecium isolates using Pyrosequencing.

In this paper, we describe the phenotypic and molecular characteristics of two clinically relevant, vancomycin-resistant (VanB), linezolid-resistant Enterococcus faecium isolates. Pyrosequencing showed the G to T single nucleotide polymorphism at bp 2576 in the genes coding for 23S rRNA and was used to quantify the proportion of G to T mutations among six different 23S rRNA genes in E. faecium as a marker for the molecular level of resistance to linezolid. In both isolates, the G to T mutation was found in two of six alleles, and no further mutations in the genes coding for 23S rRNA were found. The dynamic process of linezolid resistance could be demonstrated by the complete reversion of resistant alleles back to only wild type alleles in consecutive isolates of one isolate. Pyrosequencing being used to detect and quantify resistance to linezolid has been proven as a fast and reliable molecular screening method for monitoring linezolid resistance.

Clinically significant borderline resistance of sequential clinical isolates of Klebsiella pneumoniae.

Two sequential clinical isolates of Klebsiella pneumoniae (Kpn) were isolated from bronchoalveolar lavage fluid (Kpn#1) and sputum (Kpn#2) of a patient with pneumonia, complicated by anatomical and immunosuppressive problems due to Wegener's granulomatosis. Despite 4 weeks of systemic treatment with ciprofloxacin (CIP) Kpn#2 was isolated thereafter. A fluoroquinolone-resistant mutant (Kpn#1-SEL) was derived from Kpn#1 in vitro by selecting on agar plates supplemented with ofloxacin. Kpn#1, Kpn#1-SEL and Kpn#2 had an identical pattern in PFGE. CIP MICs were 0.25, 2 and 4 mg/l for Kpn#1, Kpn#2 and Kpn#1-SEL, respectively. Kpn ATCC 10031 (CIP MIC 0.002 mg/l) served as control. We analyzed mechanisms of fluoroquinolone resistance by determining antibiotic susceptibility, organic solvent tolerance, accumulation of fluoroquinolones, dominance testing with wild-type topoisomerase genes (gyrA/B, parC/E), sequencing of the quinolone resistance determining regions of gyrA/B, parC/E and marR and Northern blotting of marR and acrAB genes. Compared with Kpn ATCC 10031, elevated MICs to fluoroquinolones and unrelated antibiotics in Kpn#1 was presumably due to a primary efflux pump other than AcrAB and increased the CIP MIC 125-fold. Although Kpn#1 tested sensitive according to NCCLS breakpoints, the elevated CIP MIC of 0.25 mg/l presumably rendered this isolate clinically resistant and lead to therapeutic failure in this case. Further increase of MIC to fluoroquinolones in vivo and in vitro was distinct. Kpn#1-SEL, selected in vitro, acquired a GyrA target mutation, whereas in Kpn#2 no known resistance mechanism could be detected.

In vitro activities of fluoroquinolones against the spirochete Borrelia burgdorferi.

Little is known to date about the in vitro activity of fluoroquinolones against Borrelia species. Our study aimed at determining the in vitro activities of 15 quinolones against nine isolates of the Borrelia burgdorferi sensu lato complex in addition to one Borrelia valaisiana and one Borrelia bissettii tick isolate. For the determination of MICs, a standardized colorimetric microdilution method was applied. Determination of minimal borreliacidal concentrations providing 100% killing of the final inoculum (MBCs) after 72 h and time-kill experiments were performed by conventional culture in Barbour-Stoenner-Kelly medium in combination with dark-field microscopy. The rank order of potency on a microgram-per-milliliter basis for the substances with in vitro activity against B. burgdorferi was gemifloxacin (MIC at which 90% of the isolates tested are inhibited [MIC(90)], 0.12 microg/ml) > sitafloxacin (MIC(90), 0.5 microg/ml), grepafloxacin (MIC(90), 0.5 microg/ml) > gatifloxacin (MIC(90), 1 microg/ml), sparfloxacin (MIC(90), 1 microg/ml), trovafloxacin (MIC(90), 1 microg/ml) > moxifloxacin (MIC(90), 2 microg/ml), ciprofloxacin (MIC(90), 2 microg/ml) > levofloxacin (MIC(90), 4 microg/ml) > ofloxacin (MIC(90), 8 microg/ml), norfloxacin (MIC(90), 8 microg/ml) > fleroxacin (MIC(90), >16 microg/ml), and pefloxacin (MIC(90), 32 microg/ml) > nalidixic acid (MIC(90), 256 microg/ml). After 72 h of exposure, gemifloxacin was borreliacidal (100% killing) against the isolates investigated at a median MBC of 4 microg/ml. In the other compounds tested, median MBCs were higher (> or =8 microg/ml). Results of electron microscopy and time-kill studies clearly support an in vitro activity of some fluoroquinolones against borreliae. Our study demonstrates for the first time the enhanced in vitro effectiveness of some of the recently introduced 4-quinolones against B. burgdorferi.

Resistance to moxifloxacin in toxigenic Clostridium difficile isolates is associated with mutations in gyrA.

Clostridium difficile is the etiological agent of antibiotic-associated colitis and the most common cause of hospital-acquired infectious diarrhea. Fluoroquinolones such as ciprofloxacin are associated with lower risks of C. difficile-associated diarrhea. In this study, we have analyzed 72 C. difficile isolates obtained from patients with different clinical courses of disease, such as toxic megacolon and relapses; the hospital environment; public places; and horses. They were investigated for their susceptibilities to moxifloxacin (MXF), metronidazole (MEO), and vancomycin (VAN). Mutants highly resistant to fluoroquinolones were selected in vitro by stepwise exposure to increasing concentrations of MXF. The resulting mutants were analyzed for the presence of mutations in the quinolone resistance-determining regions of DNA gyrase (gyrA), the production of toxins A and B, and the epidemiological relationship of these isolates. These factors were also investigated using PCR-based methods. All strains tested were susceptible to MEO and VAN. Twenty-six percent of the clinical isolates (19 of 72) were highly resistant to MXF (MIC > or = 16 microg/ml). Fourteen of these 19 strains contained nucleotide changes resulting in amino acid substitutions at position 83 in the gyrA protein. Resistant strains selected in vitro did not contain mutations at that position. These findings indicate that resistance to MXF in a majority of cases may be due to amino acid substitution in the gyrA gene.

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.

In vitro development of resistance to six quinolones in Streptococcus pneumoniae, Streptococcus pyogenes, and Staphylococcus aureus.

Streptococcus pneumoniae, Streptococcus pyogenes, and Staphylococcus aureus isolates were exposed to subinhibitory MICs of ciprofloxacin, sparfloxacin, gatifloxacin, moxifloxacin, clinafloxacin, and gemifloxacin during a 10-day period. Subculturing led to resistance development, regardless of the initial potencies of the quinolones. None of the quinolones was associated with a significantly slower rate of resistance development.

In vivo increase in resistance to ciprofloxacin in Escherichia coli associated with deletion of the C-terminal part of MarR.

We recovered two isolates (EP1 and EP2) of Escherichia coli from the same patient that had identical pulsed-field gel electrophoresis patterns but required different MICs of ciprofloxacin (CIP): 16 and 256 mg/liter for EP1 and EP2, respectively. Both isolates had mutations in the quinolone resistance-determining regions of GyrA (Ser83Leu and Asp87Tyr) and ParC (Ser80Ile), but not in those regions of GyrB or ParE. Isolate EP2 was also more resistant to chloramphenicol, tetracyclines, cefuroxime, and organic solvents. A deletion of adenine (A) 1821 was found in marR of isolate EP2, which resulted in an 18-amino-acid C-terminal deletion in the MarR protein. The causative relationship between DeltaA1821 and the Mar phenotype was demonstrated both by the replacement of the wild-type marR by marR DeltaA1821 in isolate EP1 and by complementation with the wild-type marR in trans in isolate EP2. In isolate EP2 complemented with wild-type marR, susceptibility to chloramphenicol was restored completely, whereas susceptibility to CIP was restored only incompletely. Northern blotting demonstrated increased expression of marA and acrAB but not of soxS in isolate EP2 compared to EP1. In conclusion, the deletion of A1821 in marR in the clinical isolate EP2 caused an increase in the MICs of CIP and unrelated antibiotics. Presumably, the C-terminal part of MarR is necessary for proper repressor function.

Prevalence of gyrA, gyrB, parC, and parE mutations in clinical isolates of Streptococcus pneumoniae with decreased susceptibilities to different fluoroquinolones and originating from Worldwide Surveillance Studies during the 1997-1998 respiratory season.

From 8,419 worldwide clinical isolates of Streptococcus pneumoniae obtained during 1997-1998, 69 isolates with reduced susceptibility or resistance to fluoroquinolones (FQs) were molecularly characterized. For the isolates in this prevalence study, only parC (Ser-79-->Tyr) and gyrA (Ser-81-->Phe or Tyr) mutations, especially in combination, were found to contribute significantly to resistance. These mutations influenced the FQ MICs to varying degrees, although the rank order of activity remains independent of mutation type, with ciprofloxacin the least active, followed by levofloxacin, gatifloxacin/grepafloxacin/moxifloxacin/sparfloxaci n/trovafloxacin, and clinafloxacin/sitafloxacin. Efflux likely plays a crucial role in reduced susceptibility for new hydrophilic FQs.

Association of alterations in ParC and GyrA proteins with resistance of clinical isolates of Enterococcus faecium to nine different fluoroquinolones.

The parC and gyrA genes of 73 ciprofloxacin-resistant and 6 ciprofloxacin-susceptible Enterococcus faecium clinical isolates were partly sequenced. Alterations in ParC and GyrA, possibly in combination with other resistance mechanisms, severely restricted the in vitro activities of the nine quinolones tested. For all isolates, clinafloxacin and sitafloxacin showed the best activities.