Influence of Conversion of Penicillin G into a Basic Derivative on Its Accumulation and Subcellular Localization in Cultured Macrophages

Renard, Christine; Vanderhaeghe, Hubert; Claes, Paul; Zenebergh, A.; Tulkens, Paul M.

doi:10.1128/aac.31.9.1450

Cited by 31 publications

(44 citation statements)

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“…Eighteen to 25% of delafloxacin was associated with the nuclear-unbroken cells fraction, 10 to 17% with the organelle fraction, and 62 to 73% with the soluble fraction. Marker enzymes showed the expected distribution (38), attesting to the quality of the preparations. In particular, 85% of lactate dehydrogenase (LDH) activity was detected in the soluble fraction, suggesting that both fluoroquinolones were mostly localized in the cytosol and that only a minimal proportion was associated with organelles.…”

Section: Resultsmentioning

confidence: 99%

“…The resulting cytoplasmic extract was further fractionated into a "granule/membrane" fraction (containing the bulk of the cell organelles and membranes; MLP fraction) and a final supernatant fraction (S fraction) by high-speed centrifugation (145,000 ϫ g) for 30 min (Ti 50 rotor; Beckman Instruments, Inc., Fullerton, CA). Antibiotic concentrations and activities of marker enzymes of the main organelles (cytochrome c oxidase for mitochondria, N-acetyl-beta-glucosaminidase for lysosomes, and lactate dehydrogenase for cytosol) were measured in each fraction (38,41).…”

Section: Methodsmentioning

confidence: 99%

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Contrasting Effects of Acidic pH on the Extracellular and Intracellular Activities of the Anti-Gram-Positive Fluoroquinolones Moxifloxacin and Delafloxacin againstStaphylococcus aureus

Lemaire

Tulkens

Bambeke

2011

Antimicrob Agents Chemother

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169

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In contrast to currently marketed fluoroquinolones, which are zwitterionic, delafloxacin is an investigational fluoroquinolone with an anionic character that is highly active against Gram-positive bacteria. We have examined the effect of acidic pH on its accumulation in Staphylococcus aureus and in human THP-1 cells, in parallel with its activity against extracellular and intracellular S. aureus. Moxifloxacin was used as a comparator. Delafloxacin showed MICs 3 to 5 log 2 dilutions lower than those of moxifloxacin for a collection of 35 strains with relevant resistance mechanisms and also proved to be 10-fold more potent against intracellular S. aureus ATCC 25923. In medium at pH 5.5, this difference was further enhanced, with the MIC decreasing by 5 log 2 dilutions. In infected cells incubated in acidic medium, the relative potency was 10-fold higher than that at neutral pH and the maximal relative efficacy reached a bactericidal effect at 24 h. These results can be explained by a 10-fold increase in delafloxacin accumulation in both bacteria and cells at acidic pH, making delafloxacin one of the most efficient drugs tested in this model. Opposite effects were seen for moxifloxacin with respect to both activity and accumulation. As reported for zwitterionic fluoroquinolones, delafloxacin was found associated with the soluble fraction in homogenates of eukaryotic cells. Taken together, these properties may confer to delafloxacin an advantage for the eradication of S. aureus in acidic environments, including intracellular infections.Fluoroquinolones are one of the first families of fully synthetic antibiotics with large clinical use, and they represent a wide range of molecules, which permits the establishment of in-depth structure-activity relationships (16,17). Over the last 20 years, most fluoroquinolone research efforts have been focused on new molecules with improved activity toward Grampositive cocci because of the increasing spread of multiresistant staphylococci and streptococci. The 8-methoxy fluoroquinolone moxifloxacin is the first example of this new generation to reach the commercial market. It combines many desirable microbiological and pharmacokinetic properties (rapid bactericidal activity, a spectrum covering pertinent pathogens, excellent oral bioavailability, a large tissue distribution, and a propensity to accumulate within eukaryotic cells [see reference 45 for a review]). Moxifloxacin shows extensive activity toward bacteria thriving in the cytosol (Listeria monocytogenes [14]), phagosomes (Legionella pneumophila, Mycobacterium avium, and Mycobacterium tuberculosis [5,9,46]), and phagolysosomes (Staphylococcus aureus [6]), suggesting that it easily diffuses between different subcellular compartments. Yet moxifloxacin activity is partially defeated by the acidic pH prevailing in vacuolar subcellular compartments (6).Delafloxacin [RX-3341; 1-(6-amino-3,5-difluoropyridin-2-yl)-8-chloro-6-fluoro-7-(3-hydroxyazetidin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid] is an investigational fluoro...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Contrasting Effects of Acidic pH on the Extracellular and Intracellular Activities of the Anti-Gram-Positive Fluoroquinolones Moxifloxacin and Delafloxacin againstStaphylococcus aureus

Lemaire

Tulkens

Bambeke

2011

Antimicrob Agents Chemother

Self Cite

169

140

View full text Add to dashboard Cite

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“…There was concern about the efficacy of ampicillin on intracellular bacteria (21,29). However, ampicillin and chloramphenicol were similar in efficacy with respect to C. burnetii growth inhibition in BHK cells.…”

Section: Discussionmentioning

confidence: 99%

Transformation of Coxiella burnetii to ampicillin resistance

1996

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. In the present study, an ars replicon was used to transform C. burnetii to ampicillin resistance. Plasmid pSKO(؉)1000 contained the C. burnetii ars sequence cloned into a ColE1-type replicon encoding ␤-lactamase. pSKO(؉)1000 was introduced into C. burnetii by electroporation. Ampicillin-resistant cells were selected, and survivors were examined for the transformed genotype by Southern hybridization. Transformants stably maintained the pSKO(؉)1000 bla DNA sequence in the chromosome as a result of homologous recombination. The recombination event resulted in the duplication of the 5.8-kb ars sequence in the C. burnetii chromosome. The bla gene was also located in an episome. However, an ampicillin resistance plasmid lacking the C. burnetii ars sequence did not stably transform C. burnetii. A biological assay analyzing ␤-lactamase activity of C. burnetii transformants during acid activation in vitro provided evidence for expression of the bla (␤-lactamase) gene.Coxiella burnetii is the etiological agent of human Q fever (9). It is an obligate intracellular bacterium replicating within the acidic phagolysosomes of eukaryotic cells (1,4,15,24). Although C. burnetii becomes metabolically active and synthesizes protein and DNA during in vitro acid activation assays, axenic growth has not been observed (7,15,43). This obligate intracellular parasitism poses difficulties in studying virulence factors associated with this organism. Classical genetic studies involve the mutation of genes to eliminate a virulent phenotype and then complementation using DNA from wild-type organisms to restore virulence; this approach ascertains the contribution of specific genes to virulence. However, these types of studies have been unavailable for C. burnetii, since genetic transformation of this organism has not yet been demonstrated.Information on enzymes produced by C. burnetii and on gene structure within the chromosome and the endogenous plasmid QpHI has been obtained by the cloning and expression of C. burnetii genes in Escherichia coli (19). The use of E. coli as a host for C. burnetii genes was also implemented for the cloning of the C. burnetii chromosomal origin of DNA replication. These origin search techniques resulted in the isolation of a 5.8-kb C. burnetii chromosomal DNA fragment which initiates plasmid DNA replication within E. coli (36). The minimal ars sequence required for plasmid replication in an E. coli polA strain is 403 bp (6, 36); it demonstrates limited similarity to origins of other bacteria (Fig. 1). The ars sequence contains two consensus sequences for the binding of DnaA, an AϩT-rich region, and a potential binding site for integration host factor and for factor of inversion stimulation; these sequences are characteristic of bacterial chromosomal origins (3,12,28). The open reading frames flanking one side of the C. burnetii ars sequence demonstrate similarity to genes located in bacterial origin regions; these open reading frames include rnpA and rpmH genes and genes encoding 9K and 60K proteins (Fig. 1). ...

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“…All studies have so far reported a lack of accumulation (ie, an apparent intracellular concentration lower than the extracellular one at equilibrium) for all b-lactams whether in phagocytic [9][10][11][12][13][14][15] or nonphagocytic cells and tissues in general [16]. It has often been concluded that b-lactams are unable to penetrate cells, which is probably incorrect because most of the representatives of this class of drug do diffuse reasonably well through biologic membranes.…”

Section: B-lactamsmentioning

confidence: 99%

“…Because the cell cytosol is more acidic than the extracellular milieu, single acid b-lactams are prevented from accumulating in cells even if they can pass across membranes. Masking the free carboxyl group of a single acid b-lactam, such as penicillin G, by a basic moiety is actually all that is needed to allow substantial accumulation of the corresponding derivative [13]. The situation may be more complex for zwitterionic b-lactams, such as ampicillin, or most of the third-and fourth-generation cephalosporins, but none of them has ever been shown to accumulate in cells.…”

Section: B-lactamsmentioning

confidence: 99%