Effect of polyamines on the inhibition of peptidyltransferase by antibiotics: revisiting the mechanism of chloramphenicol action

Chloramphenicol is thought to interfere competitively with the binding of the aminoacyl-tRNA 3′-terminus to ribosomal A-site. However, noncompetitive or mixed-noncompetitive inhibition, often observed to be dependent on chloramphenicol concentration and ionic conditions, leaves some doubt about the precise mode of action. Here, we examine further the inhibition effect of chloramphenicol, using a model system derived from Escherichia coli in which a peptide bond is formed between puromycin and AcPhe-tRNA bound at the P-site of poly(U)-programmed ribosomes, under ionic conditions (6 mM Mg(2+), 100 mM NH(4)(+), 100 µM spermine) more closely resembling the physiological status. Kinetics reveal that chloramphenicol (I) reacts rapidly with AcPhe-tRNA·poly(U)·70S ribosomal complex (C) to form the encounter complex CI which is then isomerized slowly to a more tight complex, C*I. A similar inhibition pattern is observed, if complex C modified by a photoreactive analogue of spermine, reacts in buffer free of spermine. Spermine, either reversibly interacting with or covalently attached to ribosomes, enhances the peptidyltransferase activity and increases the chloramphenicol potency, without affecting the isomerization step. As indicated by photoaffinity labeling, the peptidyltransferase center at which chloramphenicol binds, is one of the preferred cross-linking sites for polyamines. This fact may explain the effect of spermine on chloramphenicol binding to ribosomes

Polyamines affect diversely the antibiotic potency: insight gained from kinetic studies of the blasticidin S and spiramycin interactions with functional ribosomes

The effects of spermine on peptidyltransferase inhibition by an aminohexosylcytosine nucleoside, blasticidin S, and by a macrolide, spiramycin, were investigated in a model system derived from Escherichia coli, in which a peptide bond is formed between puromycin and AcPhe-tRNA bound at the P-site of poly(U)-programmed ribosomes. Kinetics revealed that blasticidin S, after a transient phase of interference with the A-site, is slowly accommodated near to the P-site so that peptide bond is still formed but with a lower catalytic rate constant. At high concentrations of blasticidin S (>10 x Ki), a second drug molecule binds to a weaker binding site on ribosomes, and this may account for the onset of a subsequent mixed-noncompetitive inhibition phase. Spermine enhances the blasticidin S inhibitory effect by facilitating the drug accommodation to both sites. On the other hand, spiramycin (A) was found competing with puromycin for the A-site of AcPhe-tRNA·poly(U)·70 S ribosomal complex (C) via a two-step mechanism, according to which the fast formation of the encounter complex CA is followed by a slow isomerization to a tighter complex, termed C*A. In contrast to that observed with blasticidin S, spermine reduced spiramycin potency by decreasing the formation and stability of complex C*A. Polyamine effects on drug binding were more pronounced when a mixture of spermine and spermidine was used, instead of spermine alone. Our kinetic results correlate well with cross-linking and crystallographic data and suggest that polyamines bound at the vicinity of the antibiotic binding pockets modulate diversely the interaction of these drugs with ribosomes

Increasing incidence of candidaemia and shifting epidemiology in favor of Candida non-albicans in a 9-year period (2009-2017) in a university Greek hospital.

The aim of the present study was to analyze candidaemia's epidemiology (incidence, species distribution, and susceptibility rates) and antifungal consumption during a 9-year period. All candidaemias recorded at The University General Hospital of Patras, Greece, between 2009 and 2017 were included. Candida isolates were identified using the germ tube test, API 20C AUX System, and/or Vitek-2 YST card. Antifungal susceptibility was determined by the gradient method according to CLSI. During the study period, 505 episodes of candidaemia were observed with an overall incidence of 1.5 episodes per 1000 hospital admissions (1.1 episodes in 2009 to 1.9 in 2017: P 0.038, r 0.694). C. albicans was the leading cause (200 cases; 39.6%), followed by C. parapsilosis (185; 36.6%), C. glabrata (56; 11.1%), C. tropicalis (50; 9.9%), C. krusei (8; 0.2%), C. lusitaniae (5; < 0.1%), and C. guilliermondii (1; < 0.1%). Overall resistance to fluconazole, voriconazole, anidulafungin, caspofungin, and micafungin (according to CLSI) were 11.6%, 4.1%, 2.0%, 6.0%, and 0.8%, respectively. The overall consumption of antifungal drugs was stable, with a significant reduction of fluconazole's use in favor of echinocandins. An increase in the incidence of candidaemia and a predominance of Candida non-albicans due to decreasing use of fluconazole in favor of more potent antifungals, such as echinocandins, are reported in this study

Synthesis and Evaluation of Chloramphenicol Homodimers: Molecular Target, Antimicrobial Activity, and Toxicity against Human Cells

As fight against antibiotic resistance must be strengthened, improving old drugs that have fallen in reduced clinical use because of toxic side effects and/or frequently reported resistance, like chloramphenicol (CAM), is of special interest. Chloramphenicol (CAM), a prototypical wide-spectrum antibiotic has been shown to obstruct protein synthesis via binding to the bacterial ribosome. In this study we sought to identify features intensifying the bacteriostatic action of CAM. Accordingly, we synthesized a series of CAM-dimers with various linker lengths and functionalities and compared their efficiency in inhibiting peptide-bond formation in an Escherichia coli cell-free system. Several CAM-dimers exhibited higher activity, when compared to CAM. The most potent of them, compound 5, containing two CAM bases conjugated via a dicarboxyl aromatic linker of six successive carbon-bonds, was found to simultaneously bind both the ribosomal catalytic center and the exit-tunnel, thus revealing a second, kinetically cryptic binding site for CAM. Compared to CAM, compound 5 exhibited comparable antibacterial activity against MRSA or wild-type strains of Staphylococcus aureus, Enterococcus faecium and E. coli, but intriguingly superior activity against some CAM-resistant E. coli and Pseudomonas aeruginosa strains. Furthermore, it was almost twice as active in inhibiting the growth of T-leukemic cells, without affecting the viability of normal human lymphocytes. The observed effects were rationalized by footprinting tests, crosslinking analysis, and MD-simulations