Academic literature on the topic 'Intrabacterial concentration'

Antibiotic resistance has become a major issue in the global healthcare system, notably in the case of Gram-negative bacteria. Recent advances in technology with oligonucleotides have an enormous potential for tackling this problem, providing their efficient intrabacterial delivery. The current work aimed to apply this strategy by using a novel nanoformulation consisting of DOTAU, a nucleolipid carrier, in an attempt to simultaneously deliver antibiotic and anti-resistance oligonucleotides. Ceftriaxone, a third-generation cephalosporin, was formulated with DOTAU to form an ion pair, and was then nanoprecipitated. The obtained solid nanocapsules were characterized using FT-IR, XRD, HPLC, TEM and DLS techniques and further functionalized by the anti-resistance ONα sequence. To obtain an optimal anti-resistance activity and encapsulation yield, both the formulation protocol and the concentration of ONα were optimized. As a result, monodispersed negatively charged nanoparticles of CFX–DOTAU-ONα with a molar ratio of 10:24:1 were obtained. The minimum inhibitory concentration of these nanoparticles on the resistant Escherichia coli strain was significantly reduced (by 75%) in comparison with that of non-vectorized ONα. All aforementioned results reveal that our nanoformulation can be considered as an efficient and relevant strategy for oligonucleotide intrabacterial delivery in the fight against antibiotic resistance.

The fluoroquinolones are a series of synthetic antibacterial agents that are used in the treatment of a variety of bacterial infections. These agents inhibit the DNA gyrase, abolishing its activity by interfering with the DNA-rejoining reaction. The inhibition of the resealing leads to the liberation of fragments that are subsequently destroyed by the bacterial exonucleases. All fluoroquinolones accumulate within bacteria very rapidly, so that a steady-state intrabacterial concentration is obtained within a few minutes. Resistance develops slowly and is usually chromosomal and not plasmid mediated. However, development of resistance and transfer between animal and human pathogens has become a fervently argued issue among the microbiologists. Another concern regarding the use of new quinolones in the veterinary field is a possible detrimental effect on the environment. It still seems unlikely that the controlled use of veterinary quinolones will give rise to unfavorable effects on the environment.

Mitocurcumin (a triphenylphosphonium curcumin derivative) was previously reported as a selective antitumoral compound on different cellular lines, as well as a potent bactericidal candidate. In this study, the same compound showed strong antimicrobial efficacy against different strains of methicillin-resistant Staphylococcus aureus (MRSA). The minimum inhibitory concentration was identical for all tested strains (four strains of MRSA and one strain of methicillin-sensitive Staphylococcus aureus), suggesting a new mechanism of action compared with usual antibacterial agents. All tested strains showed a significant sensitivity in the low micromolar range for the curcumin-triphenylphosphonium derivative. This susceptibility was modulated by the menadione/glutathione addition (the addition of glutathione resulted in a significant increase in minimal inhibitory concentration from 1.95 to 3.9 uM, whereas adding menadione resulted in a decrease of 0.49 uM). The fluorescence microscopy showed a better intrabacterial accumulation for the new curcumin-triphenylphosphonium derivative compared with simple curcumin. The MitoTracker staining showed an accumulation of reactive oxygen species (ROS) for a S. pombe superoxide dismutase deleted model. All results suggest a new mechanism of action which is not influenced by the acquired resistance of MRSA. The most plausible mechanism is reactive oxygen species (ROS) overproduction after a massive intracellular accumulation of the curcumin-triphenylphosphonium derivative.

Triaza-coumarin (TA-C) is a Mycobacterium tuberculosis (Mtb) dihydrofolate reductase (DHFR) inhibitor with an IC50 (half maximal inhibitory concentration) of ∼1 µM against the enzyme. Despite this moderate target inhibition, TA-C shows exquisite antimycobacterial activity (MIC50, concentration inhibiting growth by 50% = 10 to 20 nM). Here, we investigated the mechanism underlying this potency disconnect. To confirm that TA-C targets DHFR and investigate its unusual potency pattern, we focused on resistance mechanisms. In Mtb, resistance to DHFR inhibitors is frequently associated with mutations in thymidylate synthase thyA, which sensitizes Mtb to DHFR inhibition, rather than in DHFR itself. We observed thyA mutations, consistent with TA-C interfering with the folate pathway. A second resistance mechanism involved biosynthesis of the redox coenzyme F420. Thus, we hypothesized that TA-C may be metabolized by Mtb F420–dependent oxidoreductases (FDORs). By chemically blocking the putative site of FDOR-mediated reduction in TA-C, we reproduced the F420-dependent resistance phenotype, suggesting that F420H2-dependent reduction is required for TA-C to exert its potent antibacterial activity. Indeed, chemically synthesized TA-C-Acid, the putative product of TA-C reduction, displayed a 100-fold lower IC50 against DHFR. Screening seven recombinant Mtb FDORs revealed that at least two of these enzymes reduce TA-C. This redundancy in activation explains why no mutations in the activating enzymes were identified in the resistance screen. Analysis of the reaction products confirmed that FDORs reduce TA-C at the predicted site, yielding TA-C-Acid. This work demonstrates that intrabacterial metabolism converts TA-C, a moderately active “prodrug,” into a 100-fold-more-potent DHFR inhibitor, thus explaining the disconnect between enzymatic and whole-cell activity.

ABSTRACT The influence of half-life on the postantibiotic effect (PAE) of tobramycin against Pseudomonas aeruginosa andStaphylococcus aureus was investigated during one dosing interval. Tobramycin half-lives of 0.5 to 2.5 h were simulated in an in vitro model, and the PAE was determined by an enzymatic inactivation method at different time points, i.e., when the tobramycin concentrations were 20×, 5×, and 1× the MIC. At the time point during therapy when the tobramycin concentrations had declined to 1× the MIC, at a tobramycin half-life of 0.5 h, the times of the PAEs were approximately 0.7 and 1.7 h for P. aeruginosa andS. aureus, respectively, and the PAE disappeared completely at half-lives corresponding to those found in humans (i.e., 2 to 2.5 h). The PAE itself cannot be fully explained by the presence of free intrabacterial tobramycin or the emergence of resistant subpopulations. The explanation for the disappearance of the PAE during the dosing interval may therefore be explained by the repair of sublethal damage. Since the standard method of determining the PAE in animal models is somewhat different from the method used for measurement of the PAE in vitro, the conditions under which the PAE is measured in vivo were also simulated in the in vitro model. This resulted in PAEs longer than those found by the standard method of obtaining in vitro PAE measurements. We conclude that the PAE of tobramycin, as determined by conventional in vitro methods, has virtually no clinical importance. PAEs determined in vivo may have some clinical relevance, but they are probably primarily caused by sub-MIC effects.

2011/2012
Bac7(1-35) is the smallest fragment of the proline-rich cathelicidin Bac7 that shows the same antibacterial activity as the whole natural peptide of 60 residues. In this work, we remarked that the unique gene whose deletion can confer resistance to E. coli against Bac7(1-35) is sbmA, coding for an inner membrane protein involved in the penetration of this peptide into bacterial cells. Moreover, we provided evidence that SbmA is also involved in the transmembrane transport of a fragment of another proline-rich antimicrobial peptide, arasin1(1-23), isolated from the spider crab. These findings suggest a general role of this membrane protein in the uptake of proline-rich antimicrobial peptides (PR-AMPs) into Gram-negative bacteria. We then measured the intrabacterial concentration reached by Bac7(1-35) in E. coli, and observed that this increases from micromolar in the medium to millimolar within the bacterial cell, suggesting that it may bind to cytosolic structures. For this reason, we looked for possible interactions between Bac7(1-35) and macromolecules involved in viable processes of bacteria. These studies showed that Bac7(1-35) completely inhibits in vitro the transcription/translation process starting from a concentration of 50 μM. Then we demonstrated that inhibition is: i) specific for Bac7(1-35), since it is not exerted by other cathelicidin-derived AMPs not belonging to the Prorich group, and ii) not stereo-specific, since it is exerted at the same level by the all-D isomer of Bac7(1-35). We also demonstrated the ability of Bac7(1-35) to bind DNA in vitro, but we excluded that this binding may represent the primary mechanism of bactericidal action. We also showed that the peptide does not significantly affect in vitro the transcription process, deducing that the inhibition of the transcription/translation targets primarily the translation process. We verified these data in vivo on E. coli cells measuring the incorporation of radioactive precursors of bacterial macromolecules. We observed that the exposure of bacteria to Bac7(1-35) inhibited the incorporation of radioactive leucine, but not of radioactive thymidine and uridine, indicating a specific block at the protein synthesis level and not of DNA and RNA synthesis. In the near future, a clearer definition of the intrabacterial target(s) of Bac7(1-35) would hopefully lead to the experimentation of this molecule or of its derivatives as a new generation antibiotic drug.
Bac7(1-35) è il più breve frammento del peptide Bac7, una catelicidina ricca in prolina di 60 residui, dotato della stessa attività battericida del peptide intero. In questo lavoro di tesi, abbiamo rimarcato che sbmA è l’unico gene la cui delezione conferisce ad E. coli una resistenza a Bac7(1- 35). Tale gene codifica per una proteina della membrana interna coinvolta nell’ingresso del peptide nel citoplasma della cellula batterica. Inoltre, abbiamo dimostrato che SbmA è coinvolta anche nel trasporto transmembrana di un frammento di un altro peptide antimicrobico, l’arasina1(1-23). Tali risultati suggeriscono che questa proteina giochi un ruolo generale nell’internalizzazione di peptidi antimicrobici ricchi in prolina nei batteri Gram-negativi. Abbiamo quindi misurato la concentrazione intrabatterica raggiunta da Bac7(1-35) in E. coli e abbiamo osservato che questa aumenta da valori micromolari nel terreno di coltura a millimolari nel citosol batterico, suggerendo un suo legame a strutture interne della cellula. Per questo abbiamo cercato possibili interazioni tra il peptide e macromolecole coinvolte in processi vitali del batterio. Con questi studi abbiamo appurato che Bac7(1-35) in vitro inibisce completamente il processo di trascrizione/traduzione a partire da una concentrazione di 50 μM. Successivamente abbiamo dimostrato che questa inibizione è una peculiarità di Bac7(1-35), in quanto altri AMP derivati da catelicidine ma non ricchi in prolina non hanno dimostrato un’attività comparabile. Inoltre, questa inibizione non è stereo-specifica, in quanto anche l’isomero D di Bac7(1-35) blocca tale processo esattamente come il suo isomero L. Abbiamo inoltre dimostrato la capacità di Bac7(1-35) di legare in vitro il DNA, ma abbiamo escluso che tale legame rappresenti il meccanismo primario della sua attività battericida. Abbiamo anche dimostrato che il peptide non interferisce in vitro in maniera significativa con il processo di trascrizione, deducendo che l’effetto osservato sul processo di trascrizione/traduzione fosse da attribuirsi prevalentemente all’inibizione della traduzione. Abbiamo verificato tali dati in vivo su cellule di E. coli misurando l’incorporazione di precursori radioattivi delle macromolecole batteriche. Abbiamo osservato che l’esposizione di batteri a Bac7(1-35) bloccava l’incorporazione di leucina radioattiva ma non di timidina ed uridina, indicando un blocco specifico della sintesi proteica ma non di quelle di DNA e RNA. In futuro, una definizione ancora più chiara del target intrabatterico di Bac7(1-35) potrebbe portare alla sperimentazione di tale molecola o di suoi analoghi come farmaci antibiotici di nuova generazione.
XXV Ciclo
1985