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.

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.

The sequence and mom-transactivation function of the C gene of bacteriophage Mu.

The mom gene of bacteriophage Mu encodes a DNA modification function. The gene is regulated on the transcriptional level by Dam-specific methylation and a trans-acting Mu function, and on a post-transcriptional level by the product of gene com. The gene encoding the transactivator has been cloned and mapped. By complementation analysis the activation function (also designated Dad) was shown to be the product of gene C. Transactivation of the mom promoter was shown in the following assay: the mom promoter and N-terminal part of com were fused in frame to lacZ. Cells containing such fusion plasmids were infected with M13 clones expressing C in the presence of IPTG and XGal. Successful transactivation results in the formation of blue plaques. Moreover, we have determined the sequence of gene C and found that it has a coding capacity of 140 amino acids. The promoter for C (pc) is likely to be located at least 0.5 kb upstream from the gene. A transcription terminator is found directly downstream from the C-coding region.

Identification of DNA gyrase A mutations in ciprofloxacin-resistant isolates of Salmonella typhimurium from men and cattle in Germany.

Six multiply resistant isolates of Salmonella typhimurium var. copenhagen with high-level resistance to fluoroquinolones (e.g., MIC of ciprofloxacin: 32 micrograms/ml) were isolated from human patients (n = 3) and from cattle (n = 3). The isolates were examined by complementation tests using a set of broad-host-range plasmids, which carry either the gyrA+ or the gyrB+ genes or a combination of both from Escherichia coli K-12. The results indicated a combination of gyrA and gyrB mutations in all isolates. Subsequent direct sequencing of PCR-generated internal DNA fragments of gyrA revealed an identical double mutation in all six isolates (Ser-83-->Ala and Asp-87-->Asn). In addition, the results of phenotypic (i.e., phagetype, biotype, serotype) and genotypic characterization [i.e., ribotyping and polymerase chain reaction fingerprinting (PCR-fingerprinting)] were identical for all six isolates and were distinguishable from a quinolone-susceptible strain of the same serovar and an unrelated isolate of S. typhimurium. These data indicate the clonal identity of the fluoroquinolone-resistant strains of S. typhimurium isolated from men and cattle in Germany.

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.

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.

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.

High-level fluoroquinolone resistance in a Salmonella typhimurium isolate due to alterations in both gyrA and gyrB genes.

A clinical isolate of Salmonella typhimurium serovar copenhagen (80190) with high-level fluoroquinolone resistance (MIC ciprofloxacin 32 mg/L) was examined for the occurrence of alterations of gyrA and gyrB by a dominance test with the introduction of plasmids carrying either the gyrA or the gyrB gene of Escherichia coli K-12. Either plasmid resulted in enhanced susceptibilities of each of the resulting heterodiploid strains. Introduction of a plasmid carrying both gyrA and gyrB genes into S. typhimurium 80190 restored the wildtype sensitivity. These observations provide evidence that alterations of both gyrA and gyrB are responsible for the high-level fluoroquinolone resistance of this isolate of S. typhimurium.

Genetic evidence for a role of parC mutations in development of high-level fluoroquinolone resistance in Escherichia coli.

Fifteen strains of Escherichia coli with MICs of ciprofloxacin (CIP) between 0.015 and 256 micrograms/ml were examined for the presence of mutations in the quinolone resistance-determining region of the gyrA gene and in an analogous region of the parC gene. No mutation was found in a susceptible isolate (MIC of CIP, 0.015 microgram/ml). Four moderately resistant strains (MIC of CIP 0.06 to 4 micrograms/ml) carried one gyrA mutation affecting serine 83, but in only one strain was an additional parC mutation (Gly-78 to Asp) detected. All ten highly resistant strains examined (MIC of CIP, > 4 micrograms/ml) carried two gyrA mutations affecting residues serine 83 and aspartate 87, and at least one parC mutation. These parC mutations included alterations of serine 80 to arginine or isoleucine and glutamate 84 to glycine or lysine. The parC+ and two mutant alleles (parCI-80 and parCI-80,G-84) were inserted into the mobilizable vector pBP507. Transfer of a plasmid-coded parC+ allele into parC+ strains did not alter the susceptibilities towards ciprofloxacin or nalidixic acid, while a significant increase in susceptibility was detectable for parC mutants. This increase, however, did not restore wild-type susceptibility, whereas transfer of a plasmid-coded gyrA+ allele alone or in combination with parC+ did. These data are in agreement with the view that topoisomerase IV is a secondary, less sensitive target for quinolone action in Escherichia coli and that the development of high-level fluoroquinolone resistance in E. coli requires at least one parC mutation in addition to the gyrA mutation(s).

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.

Characterization of fluoroquinolone-resistant mutants of escherichia coli selected in vitro.

Wild-type mutants highly resistant to fluoroquinolones were selected in vitro from a quinolone-susceptible Escherichia coli isolate by stepwise exposure to increasing concentrations of nalidixic acid and ciprofloxacin (CIP) either in liquid medium or on solid medium. Mutant R17 was selected by serial passage in liquid medium; the MIC of CIP for mutant R17 was 256 micrograms/ml. On solid medium, consecutive mutants MI, MII, MIII, MIVa, and MIVb were selected in four steps. The frequencies of mutations were between 10(-9) and 10(-11), and the MICs of CIP ranged from 0.5 microgram/ml (for mutant MI) to 256 micrograms/ml (for mutant MIVb). From the results of a dominance test with the gyrB+ plasmid (pBP547), no gyrB mutations were detectable. In the first step, mutant MI, a mutation from a Ser to a Leu residue at position 83 (a Ser-83-->Leu mutation), was detected in the quinolone resistance-determining region of the gyrA gene. In addition, the second-step mutation was associated with a reduced uptake of CIP and an altered outer membrane protein profile. The third mutation was identified as an Asp-87-->Gly mutation in the quinolone resistance-determining region of the gyrA gene. Concomitantly, a slight increase in the doubling time was detected. For two different four-step mutants, mutants MIVa and MIVb, the MICs of only some quinolones, including CIP, increased. The accumulation of CIP in the mutants was comparable to that in their parent MIII. The doubling time of mutant MIVa was similar to that of mutant MIII, but differed by a factor of 3 from that of the very slow growing mutant MIVb. In contrast, a clinical isolate of E.coli (isolate 205096) described previously (P. Heisig, H. Schedletzky, and H. Falkenstein-Paul, Antimicrob. Agents Chemother. 37:696-701, 1993) which has the same double mutation in gyrA had a doubling time comparable to that of the wild-type isolate.

[Evaluation of the development of resistance as a factor for the limitation of therapeutic possibilities]. / Beurteilung der Resistenzentwicklung als Faktor für die Einschränkung therapeutischer Möglichkeiten.

From the microbiological point of view a variety of highly active compounds has contributed to improved efficacy of antibacterial chemotherapy during the last few decades. In some cases, however, resistance has increased due to different molecular mechanisms. Resistance to the new generation of broad-spectrum beta-lactams is in the cases of TEM and SHV enzymes based upon the stepwise acquisition of point mutations within the structural gene. Multiresistance to aminoglycosides is caused by a combination of different genes coding for aminoglycoside modifying enzymes on transferable plasmids. Resistance to glycopeptides has recently been detected in enterococci and is due to a new mechanism of resistance. These substances have so far had unlimited activity against methicillin-resistant Staphylococcus aureus and have been widely used for treatment of pseudomembranous colitis. While all the three mechanisms of resistance mentioned above are transferable among different strains, no evidence exists so far for transferable resistance to 4-quinolones. However, for S. aureus and Pseudomonas aeruginosa an increase of resistance has been reported. The underlying mechanisms seem to be unchanged. The detection of global changes in the development of resistance and the discrimination of these changes from local events requires recording of statistically significant data obtained with approved methods and evaluation of the data with standardized international breakpoints. Consequently, the use of new agents should be controlled efficiently.

Use of a broad-host-range gyrA plasmid for genetic characterization of fluoroquinolone-resistant gram-negative bacteria.

The gyrA genotypes of ciprofloxacin-resistant clinical isolates of Escherichia coli (n = 3), Klebsiella pneumoniae (n = 4), Providencia stuartii (n = 2), Pseudomonas aeruginosa (n = 1), and Acinetobacter calcoaceticus (n = 1) were analyzed in a dominance test. This test is based on the dominance of a wild-type gyrA gene (gyrA+) over the quinolone resistance allele (gyrA) in a heterodiploid strain. Plasmid pBP515, developed to carry the gyrA+ gene of E. coli K-12 on a broad-host-range vector derived from pRSF1010, was used to obtain heterodiploid strains. Plasmid pBP515 encodes kanamycin and gentamicin resistance and is transferable via mobilization by a pRP1-derived helper plasmid (pRP1H) to strains of several gram-negative species. After the introduction of pBP515, single-cell MICs (as measured by reduction of the viable cell count) of ciprofloxacin and nalidixic acid decreased by 4- to greater than 8,000-fold for all strains tested, and 8 of the 11 strains regained ciprofloxacin susceptibilities similar to those of the respective wild types. The results indicate that (i) high-level fluoroquinolone resistance in clinical isolates of E. coli, K. pneumoniae, P. aeruginosa, and A. calcoaceticus can result from mutational alteration of the gyrA gene, and (ii) gyrA mutations are involved in high levels of fluoroquinolone resistance in P. stuartii. Additional mutations outside the gyrA locus may contribute to resistance in K. pneumoniae and P. stuartii.

Mechanisms of quinolone resistance.

Two mechanisms of resistance to fluoroquinolones are known: (i) alteration of the molecular target of quinolone action-DNA gyrase, and (ii) reduction of the quinolone accumulation. Mutations altering the N-terminus of the gyrase A subunit, especially those around residues Ser83 and Asp87, significantly reduce the susceptibilities towards all quinolones, while alterations of the gyrase B subunit are rarely found and are of minor importance. Reduced drug accumulation is associated with alterations of the outer membrane protein profile in gram-negative bacteria. Such mutations include the marA locus in Escherichia coli and result in low level resistance towards quinolones and unrelated drugs. Increased activity of naturally existing efflux systems, such as the transmembrane protein NorA of staphylococci, may also lead to reduced accumulation in gram-positive and gram-negative bacteria. Clinical fluoroquinolone resistance is rarely found in intrinsically highly susceptible organisms such as Enterobacteriaceae and involves a combination of at least two mutations. In contrast, species with moderate intrinsic susceptibility such as Campylobacter jejuni, Pseudomonas aeruginosa, and Staphylococcus aureus require only one mutation to become clinically resistant. As a consequence development of resistance during therapy may result from acquisition of already resistant strains in the case of susceptible species, and selection of mutants in the case of less susceptible species.

Antibacterial activity of grepafloxacin.

Grepafloxacin has an extremely broad spectrum of activity. Its activity against Gram-positive bacteria exceeds that of currently available quinolones. Grepafloxacin-resistant mutants seem to occur less frequently than ciprofloxacin- or ofloxacin-resistant mutants, and the increase in MIC against the former mutants is less than that of the latter. This applies only to the relative differences (in dilution steps); the absolute values are similar. Grepafloxacin kills Gram-positive bacteria at concentrations little above the MIC. Its pharmacodynamic profile against pneumococci is promising, favouring use of this drug for respiratory tract infections.

The effect of moxifloxacin on its target topoisomerases from Escherichia coli and Staphylococcus aureus.

The effect of moxifloxacin on its target enzymes was evaluated by three different approaches: (i) the MICs of moxifloxacin and nine other fluoroquinolones were determined for mutants of Escherichia coli (n = 13) and Staphylococcus aureus (n = 5) carrying different combinations of resistance mutations; (ii) the activity of moxifloxacin on isolated targets was determined as IC50 values for wild-type and mutant type II topoisomerases from E. coli; and (iii) the mutation frequencies were determined for two single-step mutants (MI with a Ser83-->Leu mutation in gyrA and WT-4 with a Ser80-->Ile mutation in parC) and their parent strain (WT). Of the quinolones tested, moxifloxacin was the only one showing an equivalent high activity against both targets. This is reflected by a comparable high susceptibility of the test strains of E. coli and S. aureus and by the IC50 values of moxifloxacin which were 50-90% lower than those of ciprofloxacin, norfloxacin and sparfloxacin for the wild-type and single mutant enzymes of gyrase and topoisomerase IV. However, double mutant GyrA was significantly more sensitive to moxifloxacin than to the other fluoroquinolones tested, while wild-type topoisomerase IV was two-fold more refractory. Mutation rates of WT, MI and WT-4 for ciprofloxacin and moxifloxacin were 5 x 10(-8) vs 4 x 10(-10); <6 x 10(-11) vs <6 x 10(-11); and 2 x 10(-6) vs 5 x 10(-7), respectively. These data indicate an equivalent high inhibitory activity of moxifloxacin on DNA gyrase and topoisomerase IV of E. coli.