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ABSTRACTStaphylococcus aureussmall-colony variants (SCVs) persist intracellularly, which may contribute to persistence/recurrence of infections and antibiotic failure. We have studied the intracellular fate of menDand hemBmutants (corresponding to menadione- and hemin-dependent SCVs, respectively) of the COL methicillin-resistant S. aureus(MRSA) strain and the antibiotic pharmacodynamic profile against extracellular (broth) and intracellular (human THP-1 monocytes) bacteria. Compared to the parental strain, SCVs showed slower extracellular growth (restored upon medium supplementation with menadione or hemin), reduced phagocytosis, and, for the menDSCV, lower intracellular counts at 24 h postinfection. Against extracellular bacteria, daptomycin, gentamicin, rifampin, moxifloxacin, and oritavancin showed similar profiles of activity against all strains, with a static effect obtained at concentrations close to their MICs and complete eradication as maximal effect. In contrast, vancomycin was not bactericidal against SCVs. Against intracellular bacteria, concentration-effect curves fitted sigmoidal regressions for vancomycin, daptomycin, gentamicin, and rifampin (with maximal effects lower than a 2-log decrease in CFU) but biphasic regressions (with a maximal effect greater than a 3-log decrease in CFU) for moxifloxacin and oritavancin, suggesting a dual mode of action against intracellular bacteria. For all antibiotics, these curves were indistinguishable between the strains investigated, except for the menDmutant, which systematically showed a lower amplitude of the concentration-effect response, with markedly reduced minimal efficacy (due to slower growth) but no change in maximal efficacy. The data therefore show that the maximal efficacies of antibiotics are similar against normal-phenotype and menadione- and hemin-dependent strains despite their different intracellular fates, with oritavancin, and to some extent moxifloxacin, being the most effective.

DD-ligases catalyze the synthesis of the D-Ala-D-Ala and D-Ala-D-Ser dipeptides or the D-Ala-D-Lac depsipeptide in an early step of peptidoglycan synthesis. Their function is essential for bacterial growth and specific to bacteria, making them attractive targets for the development of novel antibiotics. This review examines the biochemical and structural features of these enzymes and presents the main families of inhibitors described so far. Over the last 20 years, 7 structures of DD-ligases have been solved by X-ray crystallography, giving a detailed view of the general topology of the active site and of the residues in the catalytic pocket that play a central role in substrate recognition. This has paved the way to the rational design of inhibitors, which can be classified as (i) analogues of substrates, (ii) analogues of the product of the reaction, (iii) analogues of the transition state, and (iv) original scaffolds discovered by screening or by rational computer-aided design. The three first strategies have led to molecules that are polar by nature and have therefore poor access to their cytosolic target. The fourth one is potentially most promising as it yields more diverse structures. The most active molecules show affinity constants in the M range, but microbiological evaluation remains scarce (typical MIC 1-8 mg/L for the tested compounds). These data strongly suggest targeting DD-ligases is a promising approach for discovery of new antibiotics. Future research should, however, aim at finding more potent inhibitors endowed with the appropriate pharmacokinetic properties that ensure access to their intracellular target.

The potential of 14/15 membered macrolides to cause phospholipidosis has been prospectively assessed, and structure–effects examined, using combined experimental and conformational approaches. Biochemical studies demonstrated drug binding to phosphatidylinositol-containing liposomes and inhibition of the activity of lysosomal phospholipase A1toward phosphatidylcholine included in the bilayer, in close correlation with the number of cationic groups carried by the drugs (erythromycin A ≤ roxithromycin < erythromycylamine ≤ azithromycin). In cultured cells (fibroblasts), phospholipidosis (affecting all major phospholipids except sphingomyelin) was observed after 3 days with the following ranking: erythromycin A ≤ roxithromycin < erythromycylamine < azithromycin (roxithromycin could, however, not be studied in detail due to intrinsic toxicity). The difference between erythromycylamine and azithromycin was accounted for by the lower cellular accumulation of erythromycylamine. In parallel, based on a methodology developed and validated to study drug–membrane interactions, the conformational analyses revealed that erythromycin A, roxithromycin, erythromycylamine, and azithromycin penetrate into the hydrophobic domain of a phosphatidylinositol monolayer through their desosamine and cladinose moieties, whereas their macrocycle is found close to the interface. This position allows the aminogroups carried by the macrocycle of the diaminated macrolides (erythromycylamine and azithromycin) to come into close contact with the negatively charged phosphogroup of phosphatidylinositol, whereas the amine located on the C-3 of the desosamine, common to all four drugs, is located at a greater distance from this phosphogroup. Our study suggests that all macrolides have the potential to cause phospholipidosis but that this effect is modulated by toxicodynamic and toxicokinetic parameters related to the drug structure and mainly to their cationic character.

Computer-aided simulations suggest that the doses and schedules of administration of azithromycin proposed in treatment and prophylaxis of Mycobacterium avium complex (MAC) in AIDS patients will result in drug concentrations in serum and extracellular fluids remaining for sustained periods of time in the 0.03-0.1 mg/L range. We exposed cultured rat embryo fibroblasts to these concentrations (and multiples up to 20 mg/L) for up to 16 days. Electron microscopy showed that after 7 days' incubation in 0.03 mg/L azithromycin, there was conspicuous accumulation of osmiophilic, lamellar structures (myeloid bodies) in lysosomes, suggesting the onset of a phospholipidosis. Assay of total cell phospholipids and cholesterol showed significant increases in cells exposed to > or = 1 to 5 mg/L of azithromycin in association with hyperactivity of the lysosomal enzyme cathepsin B. The data suggest that azithromycin, at extracellular concentrations pertinent to its use for MAC treatment, and perhaps also prophylaxis, causes limited morphological alterations of the lysosomes in cultured cells which are of the same nature as those developing rapidly and extensively at higher concentrations.

&lt;it>Objectives&lt;/it>: Quinolones accumulate in eukaryotic cells and show activity against a large array of intracellular organisms, but systematic studies aimed at examining their pharmacodynamic profile against intracellular bacteria are scarce. The present work aims at comparing intracellular-to-extracellular activities in this context. &lt;it>Methods&lt;/it>: We assessed the activities of ciprofloxacin, levofloxacin, moxifloxacin and garenoxacin against the extracellular (broth) and intracellular (infected J774 macrophages) forms of &lt;it>Listeria monocytogenes&lt;/it> (cytosolic infection) and &lt;it>Staphylococcus aureus&lt;/it> (phagolysosomal infection) using a range of clinically meaningful extracellular concentrations (0.06–4 mg/L). &lt;it>Results&lt;/it>: All four quinolones displayed concentration-dependent bactericidal activity against extracellular and intracellular &lt;it>L. monocytogenes&lt;/it> and &lt;it>S. aureus&lt;/it> for extracellular concentrations in the range 1–4-fold their MIC. Compared at equipotent extracellular concentrations, intracellular activities against &lt;it>L. monocytogenes&lt;/it> were roughly equal to those that were extracellular, but were 50–100 times lower against &lt;it>S. aureus&lt;/it>. Because quinolones accumulate in cells (ciprofloxacin, ∼3 times; levofloxacin, ∼5 times; garenoxacin, ∼10 times, moxifloxacin, ∼13 times), these data show that, intracellularly, quinolones are 5–10 times less potent against &lt;it>L. monocytogenes&lt;/it> (&lt;it>P&lt;/it>=0.065 [ANCOVA]), and at least 100 times less potent (&lt;it>P&lt;/it> &lt; 0.0001) against &lt;it>S. aureus.&lt;/it> Because of their lower MICs and higher accumulation levels, garenoxacin and moxifloxacin were, however, more active than ciprofloxacin and levofloxacin when compared at similar extracellular concentrations. &lt;it>Conclusions&lt;/it>: Quinolone activity is reduced intracellulary. This suggests that either only a fraction of cell-associated quinolones exert an antibacterial effect, or that intracellular activity is defeated by the local environment, or that intracellular bacteria only poorly respond to the action of quinolones.