Renaissance of vancomycin: approaches for breaking antibiotic resistance in multidrug-resistant bacteria.

The emergence of multidrug-resistant bacteria demands innovations in the development of new antibiotics. For decades, the glycopeptide antibiotic vancomycin has been considered as the "last resort" treatment of severe infections caused by Gram-positive bacteria. Since the discovery of the first vancomycin-resistant enterococci strains in the late 1980s, the number of resistances has been steadily rising, with often life-threatening consequences. As an alternative to the generation of completely new substances, novel approaches focus on structural modifications of established antibiotics such as vancomycin to overcome these resistances. Here, we provide an overview of several promising modifications of vancomycin to restore its efficacy against vancomycin-resistant enterococci.

Vancomycin Resistance Is Overcome by Conjugation of Polycationic Peptides.

Multidrug-resistant bacteria represent one of the biggest challenges facing modern medicine. The increasing prevalence of glycopeptide resistance compromises the efficacy of vancomycin, for a long time considered as the last resort for the treatment of resistant bacteria. To reestablish its activity, polycationic peptides were conjugated to vancomycin. By site-specific conjugation, derivatives that bear the peptide moiety at four different sites of the antibiotic were synthesized. The most potent compounds exhibited an approximately 1000-fold increased antimicrobial activity and were able to overcome the most important types of vancomycin resistance. Additional blocking experiments using d-Ala-d-Ala revealed a mode of action beyond inhibition of cell-wall formation. The antimicrobial potential of the lead candidate FU002 for bacterial infection treatments could be demonstrated in an in vivo study. Molecular imaging and biodistribution studies revealed that conjugation engenders superior pharmacokinetics.

Replacement of l-Amino Acids by d-Amino Acids in the Antimicrobial Peptide Ranalexin and Its Consequences for Antimicrobial Activity and Biodistribution.

Infections caused by multidrug-resistant bacteria are a global emerging problem. New antibiotics that rely on innovative modes of action are urgently needed. Ranalexin is a potent antimicrobial peptide (AMP) produced in the skin of the American bullfrog Rana catesbeiana. Despite strong antimicrobial activity against Gram-positive bacteria, ranalexin shows disadvantages such as poor pharmacokinetics. To tackle these problems, a ranalexin derivative consisting exclusively of d-amino acids (named danalexin) was synthesized and compared to the original ranalexin for its antimicrobial potential and its biodistribution properties in a rat model. Danalexin showed improved biodistribution with an extended retention in the organisms of Wistar rats when compared to ranalexin. While ranalexin is rapidly cleared from the body, danalexin is retained primarily in the kidneys. Remarkably, both peptides showed strong antimicrobial activity against Gram-positive bacteria and Gram-negative bacteria of the genus Acinetobacter with minimum inhibitory concentrations (MICs) between 4 and 16 mg/L (1.9-7.6 µM). Moreover, both peptides showed lower antimicrobial activities with MICs ≥32 mg/L (≥15.2 µM) against further Gram-negative bacteria. The preservation of antimicrobial activity proves that the configuration of the amino acids does not affect the anticipated mechanism of action, namely pore formation.

A Polymer/Peptide Complex-Based Sensor Array That Discriminates Bacteria in Urine.

A negatively charged poly(para-phenyleneethynylene) (PPE) forms electrostatic complexes with four positively charged antimicrobial peptides (AMP). The AMPs partially quench the fluorescence of the PPE and discriminate fourteen different bacteria in water and in human urine by pattern-based fluorescence recognition; the AMP-PPE complexes bind differentially to the components of bacterial surfaces. The bacterial species and strains form clusters according to staining properties (Gram-positive and Gram-negative) or genetic similarity (genus, species, and strain). The identification and data treatment is performed by pattern evaluation with linear discriminant analysis (LDA) of the collected fluorescence intensity data.

Impact of Linker Modification and PEGylation of Vancomycin Conjugates on Structure-Activity Relationships and Pharmacokinetics.

As multidrug-resistant bacteria represent a concerning burden, experts insist on the need for a dramatic rethinking on antibiotic use and development in order to avoid a post-antibiotic era. New and rapidly developable strategies for antimicrobial substances, in particular substances highly potent against multidrug-resistant bacteria, are urgently required. Some of the treatment options currently available for multidrug-resistant bacteria are considerably limited by side effects and unfavorable pharmacokinetics. The glycopeptide vancomycin is considered an antibiotic of last resort. Its use is challenged by bacterial strains exhibiting various types of resistance. Therefore, in this study, highly active polycationic peptide-vancomycin conjugates with varying linker characteristics or the addition of PEG moieties were synthesized to optimize pharmacokinetics while retaining or even increasing antimicrobial activity in comparison to vancomycin. The antimicrobial activity of the novel conjugates was determined by microdilution assays on susceptible and vancomycin-resistant bacterial strains. VAN1 and VAN2, the most promising linker-modified derivatives, were further characterized in vivo with molecular imaging and biodistribution studies in rodents, showing that the linker moiety influences both antimicrobial activity and pharmacokinetics. Encouragingly, VAN2 was able to undercut the resistance breakpoint in microdilution assays on vanB and vanC vancomycin-resistant enterococci. Out of all PEGylated derivatives, VAN:PEG1 and VAN:PEG3 were able to overcome vanC resistance. Biodistribution studies of the novel derivatives revealed significant changes in pharmacokinetics when compared with vancomycin. In conclusion, linker modification of vancomycin-polycationic peptide conjugates represents a promising strategy for the modulation of pharmacokinetic behavior while providing potent antimicrobial activity.

What do minerals in the feces of Bearded Vultures reveal about their dietary habits?

The diet of Bearded Vultures Gypaetus barbatus consists mainly of bones, which are completely digested in the gastrointestinal tract, unwanted bone minerals being discarded via the feces. Chemical analyses of feces therefore provide a noninvasive technique for studying the diet of this species. We analysed the inorganic and organic remains in feces collected from Bearded Vulture nests in the Spanish Pyrenees and discussed these results with the diet of individuals determined by video camera observations. Of the food items delivered to the nest, taxonomically 65% were bone fragments of Ovis/Capra spp. (range 56-75%) and anatomically 76% (74-81%) bones from the extremities, indicating a selective preference. At least 15% of the diet was meat based, mainly originating from small prey (e.g. small carnivores, birds). The fecal analyses show that calcium and phosphorus are the most abundant mineral constituents, accounting for 41.3-44.4% of the mineral part of the feces. Among the minor elements identified, the variation in the concentrations of iron, silicon and zinc suggest differences in food selection between territories, although this could be related to varying amounts of accidentally ingested soil particles present in the food. We found variation in the content of uric acid in the feces, ranging between 0.5 and 4.6%. Higher values of uric acid might be due to a more meat or marrow bone-based diet. However, no relationship was found between the amount of calcium and uric acid levels, suggesting that the metabolites of meat digestion (uric acid) and those of bone digestion (calcium) are not negatively correlated as expected. In conclusion, our chemical analyses of feces collected from the nests of Bearded Vultures confirm that their diet consists mainly of bone remains and that these bones are digested completely. However, the direct observations of the prey items delivered to the nest produced more detailed information than the chemical analyses.

Vancomycin-Lipopeptide Conjugates with High Antimicrobial Activity on Vancomycin-Resistant Enterococci.

Multidrug-resistant bacteria represent one of the most important health care problems worldwide. While there are numerous drugs available for standard therapy, there are only a few compounds capable of serving as a last resort for severe infections. Therefore, approaches to control multidrug-resistant bacteria must be implemented. Here, a strategy of reactivating the established glycopeptide antibiotic vancomycin by structural modification with polycationic peptides and subsequent fatty acid conjugation to overcome the resistance of multidrug-resistant bacteria was followed. This study especially focuses on the structure-activity relationship, depending on the modification site and fatty acid chain length. The synthesized conjugates showed high antimicrobial potential on vancomycin-resistant enterococci. We were able to demonstrate that the antimicrobial activity of the vancomycin-lipopeptide conjugates depends on the chain length of the attached fatty acid. All conjugates showed good cytocompatibility in vitro and in vivo. Radiolabeling enabled the in vivo determination of pharmacokinetics in Wistar rats by molecular imaging and biodistribution studies. An improved biodistribution profile in comparison to unmodified vancomycin was observed. While vancomycin is rapidly excreted by the kidneys, the most potent conjugate shows a hepatobiliary excretion profile. In conclusion, these results demonstrate the potential of the structural modification of already established antibiotics to provide highly active compounds for tackling multidrug-resistant bacteria.

Fatty Acid Conjugation Leads to Length-Dependent Antimicrobial Activity of a Synthetic Antibacterial Peptide (Pep19-4LF).

The increasing number of infections caused by multidrug-resistant bacteria requires an intensified search for new antibiotics. Pep19-4LF is a synthetic antimicrobial peptide (GKKYRRFRWKFKGKLFLFG) that was previously designed with the main focus on high antimicrobial activity. The hydrophobic motif, LFLFG, was found to be essential for antimicrobial activity. However, this motif shows several limitations such as aggregation in biological media, low solubility, and small yields in peptide synthesis. In order to obtain more appropriate peptide characteristics, the hydrophobic motif was replaced with fatty acids. For this purpose, a shortened variant of Pep19-4LF (Pep19-short; GKKYRRFRWKFKGK) was synthesized and covalently linked to saturated fatty acids of different chain lengths. The peptide conjugates were tested with respect to their antibacterial activity by microdilution experiments on different bacterial strains. The length of the fatty acid was found to be directly correlated to the antimicrobial activity up to an ideal chain length (undecanoic acid, C11:0). This conjugate showed high antimicrobial activity in absence of toxicity. Time-kill studies revealed a fast and bactericidal mode of action. Furthermore, the first in vivo experiments of the conjugate in rodents demonstrated pharmacokinetics appropriate for application as a drug. These results clearly indicate that the hydrophobic motif of the peptide can be replaced by a single fatty acid of medium length, simplifying the design of this antimicrobial peptide while retaining high antimicrobial activity in the absence of toxicity.

A novel tool against multiresistant bacterial pathogens: lipopeptide modification of the natural antimicrobial peptide ranalexin for enhanced antimicrobial activity and improved pharmacokinetics.

As evolutionarily optimised defence compounds, antimicrobial peptides (AMPs) represent a powerful tool against bacterial infections. Ranalexin, an AMP found in the skin of the American bullfrog (Rana catesbeiana), is primarily active against Gram-positive bacteria, with minimum inhibitory concentrations (MICs) of 8-16 mg/L, but shows weaker activity against Gram-negative bacteria (MICs > 64 mg/L). By substitution of six N-terminal amino acids by saturated fatty acids [decanoic acid (C10:0) to myristic acid (C14:0)], lipopeptide derivatives with enhanced antimicrobial activity were developed. The antimicrobial capacity of the peptides was tested against different bacterial strains, including multiresistant clinical isolates. C13C3lexin, the most potent derivative, showed MICs of 2-8 mg/L against Gram-positive bacteria and 2-16 mg/L against Gram-negative bacteria. In time-kill studies, it was clearly shown that ranalexin and the lipopeptide C13C3lexin function as concentration-dependent, fast-acting substances against different bacteria. Cell viability assays revealed that cytotoxicity towards human cells increases with the chain length of the attached fatty acid (IC50, 12.74-108.10 µg/mL). Furthermore, using positron emission tomography (PET) imaging, pharmacokinetic studies of 68Ga-labelled ranalexin and its derivatives were performed for the first time. Here it was demonstrated that ranalexin is rapidly cleared via the kidneys within 1 h post-injection. In contrast, the lipopeptide showed greatly extended circulation in the bloodstream and a shift from renal to hepatic accumulation characteristics. Therefore, the more favourable pharmacokinetics and enhanced antimicrobial activity clearly demonstrate the potential of the lipopeptide AMPs as novel ammunition against emerging multiresistant bacterial pathogens.