Vancomycin-Polyguanidino Dendrimer Conjugates Inhibit Growth of Antibiotic-Resistant Gram-Positive and Gram-Negative Bacteria and Eradicate Biofilm-Associated S. aureus.

The global challenge of antibiotic resistance necessitates the introduction of more effective antibiotics. Here we report a potentially general design strategy, exemplified with vancomycin, that improves and expands antibiotic performance. Vancomycin is one of the most important antibiotics in use today for the treatment of Gram-positive infections. However, it fails to eradicate difficult-to-treat biofilm populations. Vancomycin is also ineffective in killing Gram-negative bacteria due to its inability to breach the outer membrane. Inspired by our seminal studies on cell penetrating guanidinium-rich transporters (e.g., octaarginine), we recently introduced vancomycin conjugates that effectively eradicate Gram-positive biofilm bacteria, persister cells and vancomycin-resistant enterococci (with V-r8, vancomycin-octaarginine), and Gram-negative pathogens (with V-R, vancomycin-arginine). Having shown previously that the spatial array (linear versus dendrimeric) of multiple guanidinium groups affects cell permeation, we report here for the first time vancomycin conjugates with dendrimerically displayed guanidinium groups that exhibit superior efficacy and breadth, presenting the best activity of V-r8 and V-R in single broad-spectrum compounds active against ESKAPE pathogens. Mode-of-action studies reveal cell-surface activity and enhanced vancomycin-like killing. The vancomycin-polyguanidino dendrimer conjugates exhibit no acute mammalian cell toxicity or hemolytic activity. Our study introduces a new class of broad-spectrum vancomycin derivatives and a general strategy to improve or expand antibiotic performance through combined mode-of-action and function-oriented design studies.

Detection of intact vancomycin-arginine as the active antibacterial conjugate in E. coli by whole-cell solid-state NMR.

The introduction of new and improved antibacterial agents based on facile synthetic modifications of existing antibiotics represents a promising strategy to deliver urgently needed antibacterial candidates to treat multi-drug resistant bacterial infections. Using this strategy, vancomycin was transformed into a highly active agent against antibiotic-resistant Gram-negative organisms in vitro and in vivo through the addition of a single arginine to yield vancomycin-arginine (V-R). Here, we report detection of the accumulation of V-R in E. coli by whole-cell solid-state NMR using 15N-labeled V-R. 15N CPMAS NMR revealed that the conjugate remained fully amidated without loss of arginine, demonstrating that intact V-R represents the active antibacterial agent. Furthermore, C{N}REDOR NMR in whole cells with all carbons at natural abundance 13C levels exhibited the sensitivity and selectivity to detect the directly bonded 13C-15N pairs of V-R within E. coli cells. Thus, we also present an effective methodology to directly detect and evaluate active drug agents and their accumulation within bacteria without the need for potentially perturbative cell lysis and analysis protocols.

In Vivo Targeting of Escherichia coli with Vancomycin-Arginine.

The ability of vancomycin-arginine (V-r) to extend the spectrum of activity of glycopeptides to Gram-negative bacteria was investigated. Its MIC towards Escherichia coli, including ß-lactamase expressing Ambler classes A, B, and D, was 8 to 16 µg/ml. Addition of 8 times the MIC of V-r to E. coli was acutely bactericidal and associated with a low frequency of resistance (<2.32 × 10-10). In vivo, V-r markedly reduced E. coli burden by >7 log10 CFU/g in a thigh muscle model. These data warrant further development of V-r in combatting E. coli, including resistant forms.

Multiple patterning of holographic photopolymers for increased refractive index contrast.

We demonstrate that multiple exposures of a two-component holographic photopolymer can quadruple the refractive index contrast of the material beyond the single-exposure saturation limit. Quantitative phase microscopy of isolated structures written by laser direct-write lithography is used to characterize the process. This technique reveals that multiple exposures are made possible by diffusion of the chemical components consumed during writing into the previously exposed regions. The ultimate index contrast is shown to be limited by the solubility of fresh components into the multiply exposed region.

Transport-of-intensity-based phase imaging to quantify the refractive index response of 3D direct-write lithography.

Precise direct-write lithography of 3D waveguides or diffractive structures within the volume of a photosensitive material is hindered by the lack of metrology that can yield predictive models for the micron-scale refractive index profile in response to a range of exposure conditions. We apply the transport of intensity equation in conjunction with confocal reflection microscopy to capture the complete spatial frequency spectrum of isolated 10 µm-scale gradient-refractive index structures written by single-photon direct-write laser lithography. The model material, a high-performance two-component photopolymer, is found to be linear, integrating, and described by a single master dose response function. The sharp saturation of this function is used to demonstrate nearly binary, flat-topped waveguide profiles in response to a Gaussian focus.