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K Drlica

Publications and source records attributed to K Drlica.

At least 19 recordsLinked to original sources
PubMed2001

Mutant prevention concentrations of fluoroquinolones for clinical isolates of Streptococcus pneumoniae.

The mutant prevention concentration (MPC) represents a threshold above which the selective proliferation of resistant mutants is expected to occur only rarely. A provisional MPC (MPC(pr)) was defined and measured for five fluoroquinolones with clinical isolates of Streptococcus pneumoniae. Based on their potential for restricting the selection of resistant mutants, the five fluoroquinolones, in descending order, were found to be moxifloxacin > trovafloxacin > gatifloxacin > grepafloxacin > levofloxacin. For several compounds, 90% of about 90 clinical isolates that lacked a known resistance mutation had a value of MPC(pr) that was close to or below the serum levels that could be attained with a dosing regimen recommended by the manufacturers. Since MPC(pr) overestimates MPC, these data identify moxifloxacin and gatifloxacin as good candidates for determining whether MPC(pr) can be used as a guide for choosing and eventually administering fluoroquinolones to significantly reduce the development of resistance.

Anti-Infective Agents↗
PubMed2000

Selection of antibiotic-resistant bacterial mutants: allelic diversity among fluoroquinolone-resistant mutations.

To obtain a general framework for understanding selection of antibiotic-resistant mutants, allelic diversity was examined with about 600 fluoroquinolone-resistant mutants of mycobacteria. Selection at low fluoroquinolone concentration produced many low-level resistance mutants. Some of these contained mutations that conferred unselected antibiotic resistance; none contained alterations in the quinolone-resistance-determining region of the GyrA protein, the principal drug target. As selection pressure increased, a variety of GyrA variants became prevalent. High concentrations of antibiotic reduced the variety to a few types, and eventually a concentration was reached at which no mutant was recovered. That concentration defined a threshold for preventing the selection of resistance. The pattern of variants selected, which was also strongly influenced by antibiotic structure, readily explained the variants present in clinical isolates. Thus, resistance arises from selection of mutants whose identity depends on drug concentration and structure, both of which can be manipulated to restrict selection.

Alleles↗
PubMed2000

Prokaryotic and eukaryotic chromosomes: what's the difference?

It is widely held that the profound differences in cellular architecture between prokaryotes and eukaryotes, in particular the housing of eukaryotic chromosomes within a nuclear membrane, also extends to the properties of their chromosomes. When chromosomal multiplicity, ploidy, linearity, transcriptional silencing, partitioning, and packaging are considered, no consistent association is found between any of these properties and the presence or absence of a nuclear membrane. Some of the perceived differences can be attributed to cytological limitations imposed by the small size of bacterial nucleoids and the arbitrary choice of representative organisms for comparison. We suggest that the criterion of nucleosome-based packaging of chromosomal DNA may be more useful than the prokaryote/eukaryote dichotomy for inferring the broadest phylogenetic relationships among organisms.

Animals↗
PubMed2000

Mutant prevention concentration as a measure of fluoroquinolone potency against mycobacteria.

Mutant prevention concentration (MPC) has been proposed as a new measure of antibiotic potency by which the ability to restrict selection of resistant mutants is evaluated. To determine whether MPC provides potency information unavailable from the more customary measurement of the MIC, 18 fluoroquinolones were examined for their ability to block the growth of Mycobacterium smegmatis and to select resistant mutants from wild-type populations. Both MPC and MIC were affected by changes in the moiety at the fluoroquinolone C-8 position and in alkyl groups attached to the C-7 piperazinyl ring. When eight resistant mutants, altered in the gyrase A protein, were tested with fluoroquinolones having either a methoxy or a hydrogen at the C-8 position, the MIC for the most resistant mutant correlated better with the MPC than did the MIC for wild-type cells. For C-8-fluorine derivatives, which were generally less active than the C-8-methoxy compounds but which were more active than C-8-hydrogen derivatives, the MICs for both the mutant and the wild type correlated well with the MPCs. Thus, measurement of the MICs for wild-type cells can reflect the ability of a quinolone to restrict the selection of resistance, but often it does not. With the present series of compounds, the most potent contained a C-8-methoxy and a small group attached to the C-7 ring.

Anti-Infective Agents↗
PubMed2000

Selective targeting of topoisomerase IV and DNA gyrase in Staphylococcus aureus: different patterns of quinolone-induced inhibition of DNA synthesis.

The effect of quinolones on the inhibition of DNA synthesis in Staphylococcus aureus was examined by using single resistance mutations in parC or gyrA to distinguish action against gyrase or topoisomerase IV, respectively. Norfloxacin preferentially attacked topoisomerase IV and blocked DNA synthesis slowly, while nalidixic acid targeted gyrase and inhibited replication rapidly. Ciprofloxacin exhibited an intermediate response, consistent with both enzymes being targeted. The absence of RecA had little influence on target choice by this assay, indicating that differences in rebound (repair) DNA synthesis were not responsible for the results. At saturating drug concentrations, norfloxacin and a gyrA mutant were used to show that topoisomerase IV-norfloxacin-cleaved DNA complexes are distributed on the S. aureus chromosome at intervals of about 30 kbp. If cleaved complexes block DNA replication, as indicated by previous work, such close spacing of topoisomerase-quinolone-DNA complexes should block replication rapidly (replication forks are likely to encounter a cleaved complex within a minute). Thus, the slow inhibition of DNA synthesis at growth-inhibitory concentrations suggests that a subset of more distantly distributed complexes is physiologically relevant for drug action and is unlikely to be located immediately in front of the DNA replication fork.

Anti-Infective Agents↗
PubMed2000

Mutant prevention concentration as a measure of antibiotic potency: studies with clinical isolates of Mycobacterium tuberculosis.

The mutant prevention concentration (MPC) of a C-8-methoxy fluoroquinolone exhibited a narrow distribution for 14 genetically diverse clinical isolates of Mycobacterium tuberculosis, indicating that results from single-isolate studies are likely to be representative. When one isolate was challenged with a variety of antituberculosis agents, C-8-methoxy fluoroquinolones were exceptional in having MPCs below the maximum concentration attained in serum by use of commonly recommended doses.

Anti-Infective Agents↗
PubMed2000

Cloning and characterization of secretory tyrosine phosphatases of Mycobacterium tuberculosis.

Two genes with sequence homology to those encoding protein tyrosine phosphatases were cloned from genomic DNA of Mycobacterium tuberculosis H(37)Rv. The calculated molecular masses of these two putative tyrosine phosphatases, designated MPtpA and MPtpB, were 17. 5 and 30 kDa, respectively. MPtpA and MPtpB were expressed as glutathione S-transferase fusion proteins in Escherichia coli. The affinity-purified proteins dephosphorylated the phosphotyrosine residue of myelin basic protein (MBP), but they failed to dephosphorylate serine/threonine residues of MBP. The activity of these phosphatases was inhibited by sodium orthovanadate, a specific inhibitor of tyrosine phosphatases, but not by okadaic acid, an inhibitor of serine/threonine phosphatases. Mutations at the catalytic site motif, cysteine 11 of MPtpA and cysteine 160 of MPtpB, abolished enzyme activity. Southern blot analysis revealed that, while mptpA is present in slow-growing mycobacterial species as well as fast-growing saprophytes, mptpB was restricted to members of the M. tuberculosis complex. These phosphatases were present in both whole-cell lysates and culture filtrates of M. tuberculosis, suggesting that these proteins are secreted into the extracellular medium. Since tyrosine phosphatases are essential for the virulence of several pathogenic bacteria, the restricted distribution of mptpB makes it a good candidate for a virulence gene of M. tuberculosis.

Amino Acid Sequence↗
PubMed2000

The future of fluoroquinolones.

The mutant prevention concentration (MPC) is a new measure of antibiotic potency above which a microbe must attain two concurrent resistance mutations for growth. For some C-8-methoxy fluoroquinolone-pathogen combinations, the value of MPC is below human serum drug concentration achieved with standard doses. Although untested clinically, such a low value of MPC, coupled with high serum concentration, should allow these fluoroquinolones to restrict severely the selection of resistant mutants when used as monotherapy. Compounds that cannot meet the MPC-pharmacokinetic criterion will enrich resistant mutants unless they are a part of combination therapy. Separation of fluoroquinolones into groups suitable for monotherapy or for combination therapy, followed by appropriate adminstration, may help extend the lifespan of the fluoroquinolones.

Anti-Infective Agents↗
PubMed1999

Mechanism of fluoroquinolone action.

When fluoroquinolones bind to gyrase or topoisomerase IV in the presence of DNA, they alter protein conformation. DNA cleavage results with diminished religation, so the enzymes are trapped in ternary complexes with drug and cleaved DNA. Preferential localization of gyrase ahead of replication forks and topoisomerase IV behind them causes fluoroquinolone-mediated complexes with the two enzymes to have different physiological consequences.

Anti-Infective Agents↗
PubMed1999

Cytotoxic hammerhead ribozymes.

Small catalytic RNA molecules of the hammerhead ribozyme type were found to have cytotoxic effects unrelated to their intended activity. An expression library of ribozyme sequence variants was constructed in a recA-deficient strain of Escherichia coli such that individual library members differed in regions designed to form base pairs with human immunodeficiency virus-1 (HIV-1) tat mRNA. The parental ribozyme and many variants exhibited a bacteriostatic effect. One variant studied in detail was also bactericidal. When its expression was induced, ribozyme-dependent inhibition of bacterial growth was not observed in recA+ or recA+ lexA3 (Ind-) cells, suggesting that the recombination function of the RecA protein, not the absence of the SOS response, is sufficient to alleviate the cytotoxic effect. These data document the need for careful testing for toxic effects during intracellular studies of ribozyme action.

Base Sequence↗
PubMed1999

Gatifloxacin activity against quinolone-resistant gyrase: allele-specific enhancement of bacteriostatic and bactericidal activities by the C-8-methoxy group.

Antibacterial activities of gatifloxacin (AM1155), a new C-8-methoxy fluoroquinolone, and two structurally related compounds, AM1121 and ciprofloxacin, were studied with an isogenic set of ten quinolone-resistant, gyrA (gyrase) mutants of Escherichia coli. To compare the effect of each mutation on resistance, the mutant responses were normalized to those of wild-type cells. Alleles exhibiting the most resistance to growth inhibition mapped in alpha-helix 4, which is thought to lie on a GyrA dimer surface that interacts with DNA. The C-8-methoxy group lowered the resistance due to these mutations more than it lowered resistance arising from several gyrA alleles located outside alpha-helix 4. These data are consistent with alpha-helix 4 being a distinct portion of the quinolone-binding site of GyrA. A helix change to proline behaved more like nonhelix alleles, indicating that helix perturbation differs from the other changes at helix residues. Addition of a parC (topoisomerase IV) resistance allele revealed that the C-8-methoxy group also facilitated attack of topoisomerase IV. When lethal effects were measured at a constant multiple of the minimum inhibitory concentration for each fluoroquinolone to normalize for differences in bacteriostatic action, gatifloxacin was more potent than the C-8-H compounds, both in the presence and absence of protein synthesis (an exception was observed when alanine was substituted for aspartic acid at position 82). Collectively, these data show that the C-8-methoxy group contributes to the enhanced activity of gatifloxacin against resistant gyrase and wild-type topoisomerase IV.

Alleles↗
PubMed1999

Fluoroquinolone action against clinical isolates of Mycobacterium tuberculosis: effects of a C-8 methoxyl group on survival in liquid media and in human macrophages.

When the lethal action of a C-8 methoxyl fluoroquinolone against clinical isolates of Mycobacterium tuberculosis in liquid medium was measured, the compound was found to be three to four times more effective (as determined by measuring the 90% lethal dose) than a C-8-H control fluoroquinolone or ciprofloxacin against cells having a wild-type gyrA (gyrase) gene. Against ciprofloxacin-resistant strains, the C-8 methoxyl group enhanced lethality when alanine was replaced by valine at position 90 of the GyrA protein or when aspartic acid 94 was replaced by glycine, histidine, or tyrosine. During infection of a human macrophage model by wild-type Mycobacterium bovis BCG, the C-8 methoxyl group lowered survival 20- to 100-fold compared with the same concentration of a C-8-H fluoroquinolone. The C-8 methoxyl fluoroquinolone was also more effective than ciprofloxacin against a gyrA Asn94 mutant of M. bovis BCG. In an M. tuberculosis-macrophage system the C-8 methoxyl group improved fluoroquinolone action against both quinolone-susceptible and quinolone-resistant clinical isolates. Thus, a C-8 methoxyl group enhances the bactericidal activity of quinolones with N1-cyclopropyl substitutions; these data encourage further refinement of fluoroquinolones as antituberculosis agents.

Anti-Infective Agents↗
PubMed1999

Effect of fluoroquinolone concentration on selection of resistant mutants of Mycobacterium bovis BCG and Staphylococcus aureus.

When Mycobacterium bovis BCG and Staphylococcus aureus were plated on agar containing increasing concentrations of fluoroquinolone, colony numbers exhibited a sharp drop, followed by a plateau and a second sharp drop. The plateau region correlated with the presence of first-step resistant mutants. Mutants were not recovered at concentrations above those required for the second sharp drop, thereby defining a mutant prevention concentration (MPC). A C-8-methoxy group lowered the MPC for an N-1-cyclopropyl fluoroquinolone.

Anti-Infective Agents↗
PubMed1998

Topoisomerase I of Helicobacter pylori: juxtaposition with a flagellin gene (flaB) and functional requirement of a fourth zinc finger motif.

Cloning and nucleotide sequence analysis showed that in Helicobacter pylori the gene encoding topoisomerase I (topA) lies about 170 nucleotides upstream from flaB, a gene encoding one of the two flagellin proteins that is required for virulence. The topA and flaB genes are divergently transcribed. The orientation and spatial relationship between flaB and topA are remarkably conserved among strains of a bacterium in which genomic rearrangements are common. The deduced amino acid sequence of topoisomerase I revealed four zinc finger motifs, one more than has been reported previously for the Escherichia coli homologue. The additional motif, which is near the C-terminus of the protein, appears to be essential for function since mutations in that region are lethal. These data show that TopA proteins can be divided into several classes on the basis of zinc finger motifs and raise the interesting possibility that the H. pylori enzyme has local topological effects focussed on a flagellin gene.

Bacterial Proteins↗
PubMed1998

Degradation of hammerhead ribozymes by human ribonucleases.

Hammerhead ribozymes were used as substrates to examine endoribonucleolytic activities in cell extracts and cultured human cells. Primer-extension analyses showed that ribozymes directed against tumor necrosis factor-alpha mRNA and human immunodeficiency virus type 1 tat mRNA were cleaved at UA and CA dinucleotides by extracts. Preferred cleavage sites were similar to those observed following digestion with RNase A, and cleavage was blocked by RNasin, an inhibitor of pyrimidine-specific ribonucleases. Removal of UA and CA dinucleotides rendered ribozymes more stable when incubated in cell extracts that were not significantly contaminated by extracellular nucleases. Placement of UA dinucleotides adjacent to a ribozyme in mRNA led to excision of the ribozyme from long transcripts during incubation in extracts. UA dinucleotides also made mRNA more labile than a control RNA when expressed from an endogenous plasmid gene in the human myeloid cell line U937. Similarly, UA and CA dinucleotides caused ribozymes to have a shorter half-life when delivered to U937 cells by lipofectin-mediated transformation. Taken together, these data indicate that one or more members of the pyrimidine-specific ribonuclease family is involved in the intracellular degradation of RNA, and they explain the paucity of UA dinucleotides in eukaryotic mRNA. Judicious manipulation of preferred target sequences of pyrimidine-specific ribonucleases may be useful in designing effective hammerhead ribozymes.

Base Sequence↗
PubMed1998

Fluoroquinolone action against mycobacteria: effects of C-8 substituents on growth, survival, and resistance.

Fluoroquinolones trap gyrase on DNA as bacteriostatic complexes from which lethal DNA breaks are released. Substituents at the C-8 position increase activities of N-1-cyclopropyl fluoroquinolones against several bacterial species. In the present study, a C-8-methoxyl group improved bacteriostatic action against gyrA (gyrase-resistant) strains of Mycobacterium tuberculosis and M. bovis BCG. It also enhanced lethal action against gyrase mutants of M. bovis BCG. When cultures of M. smegmatis, M. bovis BCG, and M. tuberculosis were challenged with a C-8-methoxyl fluoroquinolone, no resistant mutant was recovered under conditions in which more than 1, 000 mutants were obtained with a C-8-H control. A C-8-bromo substituent also increased bacteriostatic and lethal activities against a gyrA mutant of M. bovis BCG. When lethal activity was normalized to bacteriostatic activity, the C-8-methoxyl compound was more bactericidal than its C-8-H control, while the C-8-bromo fluoroquinolone was not. The C-8-methoxyl compound was also found to be more effective than the C-8-bromo fluoroquinolone at reducing selection of resistant mutants when each was compared to a C-8-H control over a broad concentration range. These data indicate that a C-8-methoxyl substituent, which facilitates attack of first-step gyrase mutants, may help make fluoroquinolones effective antituberculosis agents.

Anti-Infective Agents↗
PubMed1998

Role of superoxide in catalase-peroxidase-mediated isoniazid action against mycobacteria.

Isoniazid (INH) activation in vitro is associated with reduction of the mycobacterial ferric KatG catalase-peroxidase by hydrazine and reaction with O2 to form an oxyferrous enzyme complex. Since this complex could also form directly via reaction of ferric KatG with superoxide, intracellular activation might be responsive to superoxide concentration. When Mycobacterium smegmatis carrying the M. bovis katG gene was treated with nontoxic levels of plumbagin, a generator of superoxide, the bacteriostatic activity of INH increased unless a plasmid-borne superoxide dismutase gene was also present. Thus, endogenous superoxide probably contributes to intracellular activation of INH.

Antitubercular Agents↗