A Multifunctional Subphthalocyanine Nanosphere for Targeting,Labeling, and Killing of Antibiotic‐Resistant Bacteria

Developing a material that can combat antibiotic‐resistant bacteria, a major global health threat, is an urgent requirement. To tackle this challenge, we synthesized a multifunctional subphthalocyanine (SubPc) polymer nanosphere that has the ability to target, label, and photoinactivate antibiotic‐resistant bacteria in a single treatment with more than 99 % efficiency, even with a dose as low as 4.2 J cm⁻² and a loading concentration of 10 nM . The positively charged nanosphere shell composed of covalently linked SubPc units can increase the local concentration of photosensitizers at therapeutic sites. The nanosphere shows superior performance compared to corresponding monomers presumably because of their enhanced water dispersibility, higher efficiency of singlet‐oxygen generation, and phototoxicity. In addition, this material is useful in fluorescence labeling of living cells and shows promise in photoacoustic imaging of bacteria in vivo.     

Enzyme‐Responsive Polymeric Vesicles for Bacterial‐Strain‐Selective Delivery of Antimicrobial Agents

Antimicrobial resistance poses serious public health concerns and antibiotic misuse/abuse further complicates the situation; thus, it remains a considerable challenge to optimize/improve the usage of currently available drugs. We report a general strategy to construct a bacterial strain‐selective delivery system for antibiotics based on responsive polymeric vesicles. In response to enzymes including penicillin G amidase (PGA) and β‐lactamase (Bla), which are closely associated with drug‐resistant bacterial strains, antibiotic‐loaded polymeric vesicles undergo self‐immolative structural rearrangement and morphological transitions, leading to sustained release of antibiotics. Enhanced stability, reduced side effects, and bacterial strain‐selective drug release were achieved. Considering that Bla is the main cause of bacterial resistance to β‐lactam antibiotic drugs, as a further validation, we demonstrate methicillin‐resistant S. aureus (MRSA)‐triggered release of antibiotics from Bla‐degradable polymeric vesicles, in vitro inhibition of MRSA growth, and enhanced wound healing in an in vivo murine model.     

A Multifunctional Subphthalocyanine Nanosphere for Targeting,Labeling, and Killing of Antibiotic‐Resistant Bacteria

Developing a material that can combat antibiotic‐resistant bacteria, a major global health threat, is an urgent requirement. To tackle this challenge, we synthesized a multifunctional subphthalocyanine (SubPc) polymer nanosphere that has the ability to target, label, and photoinactivate antibiotic‐resistant bacteria in a single treatment with more than 99 % efficiency, even with a dose as low as 4.2 J cm^?2 and a loading concentration of 10 nM . The positively charged nanosphere shell composed of covalently linked SubPc units can increase the local concentration of photosensitizers at therapeutic sites. The nanosphere shows superior performance compared to corresponding monomers presumably because of their enhanced water dispersibility, higher efficiency of singlet‐oxygen generation, and phototoxicity. In addition, this material is useful in fluorescence labeling of living cells and shows promise in photoacoustic imaging of bacteria in vivo.     

Capillary electrophoresis for fast detection of heterogeneous population in colistin‐resistant Gram‐negative bacteria

It has been shown that diverse strains of bacteria can be separated according to their characteristic surface properties by means of CE. We employed here this analytical technique to the study of colistin‐resistance in Gram‐negative bacteria, which involves the selection of mutants with modified outer membrane composition resulting in changes of surface cell properties. In the same way as with molecular entities, we performed firstly the validation of an ITP‐based CE method for three common pathogenic Gram‐negative bacteria namely Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Secondly, we compared the electrophoretic profiles of bacterial samples from a colistin‐susceptible clinical isolate of K. pneumoniae and from the corresponding colistin‐resistant derivative. By a simple CE run taking a few minutes, the coexistence of several bacterial subpopulations in the colistin‐resistant derivative was clearly evidenced. This work encourages further research that would allow applications of CE in clinical laboratory for a daily monitoring of bacterial population in cared patients when “last‐chance” colistin treatment is initiated against multidrug‐resistant bacteria.     

Antimicrobial Photodynamic Efficiency of Novel Cationic Porphyrins towards Periodontal Gram‐positive and Gram‐negative Pathogenic Bacteria

The Gram‐negative Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum are major causative agents of aggressive periodontal disease. Due to increase in the number of antibiotic‐resistant bacteria, antimicrobial Photodynamic therapy (aPDT) seems to be a plausible alternative. In this work, photosensitization was performed on Gram‐positive and Gram‐negative bacteria in pure culture using new‐age cationic porphyrins, namely mesoimidazolium‐substituted porphyrin derivative ( ImP ) and pyridinium‐substituted porphyrin derivative ( PyP ). The photophysical properties of both the sensitizers including absorption, fluorescence emission, quantum yields of the triplet excited states and singlet oxygen generation efficiencies were evaluated in the context of aPDT application. The studied porphyrins exhibited high ability to accumulate into bacterial cells with complete penetration into early stage biofilms. As compared with ImP, PyP was found to be more effective for photoinactivation of bacterial strains associated with periodontitis, without any signs of dark toxicity, owing to its high photocytotoxicity.     

Autoinducer Sensing Microarrays by Reporter Bacteria Encapsulated in Hybrid Supramolecular‐Polysaccharide Hydrogels

A generally applicable strategy to obtain mechanically robust hydrogels for the incorporation and containment of functional reporter bacteria for the microarray and microparticle‐based detection and signaling of N‐acyl homoserine lactone autoinducers (3OC₁₂HSL) at relevant concentrations is reported. For reinforcing hydrogels of 1,4‐bi(phenylalanine‐diglycol)‐benzene (PDB), a hybrid hydrogel is formed by the combination of PDB self‐assembly with Ca²⁺ mediated alginate crosslinking. The different assembly mechanisms are shown not to interfere with each other and despite the more than four‐fold increased moduli of the hydrogels, diffusion of autoinducers into the gels remains efficient and Escherichia coli pLuxR‐green fluorescent protein (GFP) reporter bacteria are proliferating. Templating affords reporter bacteria‐loaded hydrogels with controllable shape and size. Upon exposure to 3OC₁₂HSL, the embedded bacteria exhibit an up to 12 ± 3 times increase in fluorescence intensity due to autoinducer‐triggered GFP expression. This approach can serve as a potentially generally applicable strategy to sensitively detect bacteria via their secreted autoinducers.     

Multiplex identification of drug‐resistant Gram‐positive pathogens using stuffer‐free MLPA system

Early detection of pathogens from blood and identification of their drug resistance are essential for sepsis management. However, conventional culture‐based methods require relatively longer time to identify drug‐resistant pathogens, which delays therapeutic decisions. For precise multiplex detection of drug‐resistant Gram‐positive pathogens, we developed a method by using stuffer‐free multiplex ligation‐dependent probe amplification (MLPA) coupled with high‐resolution CE single‐strand conformation polymorphisms (CE‐SSCP) system. We designed three probe sets for genes specific to Gram‐positive species (Staphylococcus aureus: nuc, Enterococcus faecium: sodA, and Streptococcus pneumoniae: lytA) and two sets for genes associated with drug resistance (mecA and vanA) to discriminate major Gram‐positive pathogens with the resistance. A total of 94 different strains (34 reference strains and 60 clinical isolates) were used to validate this method and strain‐specific peaks were successfully observed for all the strains. To improve sensitivity of the method, a target‐specific preamplification step was introduced and, consequently, the sensitivity increased from 10 pg to 100 fg. We also reduced a total assay time to 8 h by optimizing hybridization time without compromising test sensitivity. Taken together, our multiplex detection system can improve detection of drug‐resistant Gram‐positive pathogens from sepsis patients’ blood.     

A Glycosylated Cationic Block Poly(β‐peptide) Reverses Intrinsic Antibiotic Resistance in All ESKAPE Gram‐Negative Bacteria

Carbapenem‐resistant Gram‐negative bacteria (GNB) are heading the list of pathogens for which antibiotics are the most critically needed. Many antibiotics are either unable to penetrate the outer‐membrane or are excluded by efflux mechanisms. Here, we report a cationic block β‐peptide (PAS8‐b‐PDM12) that reverses intrinsic antibiotic resistance in GNB by two distinct mechanisms of action. PAS8‐b‐PDM12 does not only compromise the integrity of the bacterial outer‐membrane, it also deactivates efflux pump systems by dissipating the transmembrane electrochemical potential. As a result, PAS8‐b‐PDM12 sensitizes carbapenem‐ and colistin‐resistant GNB to multiple antibiotics in vitro and in vivo. The β‐peptide allows the perfect alternation of cationic versus hydrophobic side chains, representing a significant improvement over previous antimicrobial α‐peptides sensitizing agents. Together, our results indicate that it is technically possible for a single adjuvant to reverse innate antibiotic resistance in all pathogenic GNB of the ESKAPE group, including those resistant to last resort antibiotics.     

Dual Fluorescent‐ and Isotopic‐Labelled Self‐Assembling Vancomycin for in vivo Imaging of Bacterial Infections

The increase of bacterial resistance demands rapid and accurate diagnosis of bacterial infections. Biosurface‐induced supramolecular assembly for diagnosis and therapy has received little attention in detecting bacterial infections. Herein we present a dual fluorescent‐nuclear probe based on self‐assembly of vancomycin (Van) on Gram‐positive bacteria for imaging bacterial infection. A Van‐ and rhodamine‐modified peptide derivative (Rho‐FF‐Van), as the imaging agent, binds to the terminal peptide of the methicillin‐resistant staphylococcus aureus (MRSA) and self‐assembles to form nanoaggregates on the surface of MRSA . In an in vivo myositis model, Rho‐FF‐Van results in a significant increased fluorescence signal at the MRSA infected site. Radiolabeled with iodine‐125, Rho‐FF‐Van shows strong radioactive signal in the MRSA ‐infected lungs in a murine model. This novel dual fluorescent and nuclear probe promises a new way for in vivo imaging of bacterial infections.     

Long‐subchain hyperbranched poly(aminoethyl acrylate): A potent antimicrobial polymer with low hemolytic toxicity

The emergency of antibiotic‐resistant bacteria and their wide spread has posed a worldwide threat to public health, and traditional antibiotics are gradually overwhelmed by infectious bacteria. Herein, we report an efficient and economical strategy to construct antimicrobial polymers with net cationic component and hyperbranched architecture, which exhibit highly selective toxicity toward bacteria over human cells. To this aim, cationic poly(aminoethyl acrylate) (PAEA) without hydrophobicity is chosen for low hemolysis activity and targeting the negative bacterial membranes, and hyperbranched architecture is introduced to solve the dilemma of low antimicrobial activity. Long‐subchain hyperbranched PAEA (lhb‐PAEA) samples kills >99.99% gram‐negative Escherichia coli and >98% gram‐positive Staphylococcu aureus at the dose of ≤4 μg/mL. Moreover, lhb‐PAEA samples exhibit great biocompatibility, for the hemolysis percentage was ≤35% even at the high dose of 1024 μg/mL. Thus, enhanced antimicrobial activity, reduced hemolytic toxicity, the feasible and low‐cost production are achieved for lhb‐PAEA as antimicrobial agents. © 2016 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016 , 54, 3462–3469     

Combining Topology and Sequence Design for the Discovery of Potent Antimicrobial Peptide Dendrimers against Multidrug‐Resistant Pseudomonas aeruginosa

Multidrug‐resistant opportunistic bacteria, such as Pseudomonas aeruginosa, represent a major public health threat. Antimicrobial peptides (AMPs) and related peptidomimetic systems offer an attractive opportunity to control these pathogens. AMP dendrimers (AMPDs) with high activity against multidrug‐resistant clinical isolates of P. aeruginosa and Acinetobacter baumannii were now identified by a systematic survey of the peptide sequences within the branches of a distinct type of third‐generation peptide dendrimers. Combined topology and peptide sequence design as illustrated here represents a new and general strategy to discover new antimicrobial agents to fight multidrug‐resistant bacterial pathogens.     

Site‐specific reversible immobilization and purification of His‐tagged protein on poly(2‐acetamidoacrylic acid) hydrogel beads

Ni²⁺‐complexed poly(2‐acetamidoacrylic acid) (PAAA) hydrogel beads were developed for the site‐specific reversible immobilization and purification of the histidine‐tagged green fluorescent protein (His‐tagged GFP). PAAA hydrogel beads were prepared by photopolymerization, and significantly improved mechanical properties of PAAA hydrogel beads were observed in comparison with PAAA hydrogel from our previous study. Confocal laser scanning microscopy was used to determine the binding of His‐tagged GFP to the hydrogel beads in three‐dimensional space. Photoluminescence spectroscopy revealed 89% of binding efficiency of His‐tagged GFP to the Ni²⁺‐PAAA hydrogel beads, 51% of yielding recovery. The maximum binding capacity of His‐tagged GFP was estimated to be 0.45 µg/mg of Ni²⁺‐PAAA hydrogel beads. The recombinant His‐tagged GFP from the soluble fraction of E. coli BL21(DE3) cell lysates was purified with Ni²⁺‐PAAA hydrogel beads. The major advantage of the Ni²⁺‐PAAA hydrogel beads system was simple preparation procedures of producing the matrix, because PAAA hydrogel beads had relatively enhanced mechanical strength than soft hydrogels. Copyright © 2012 John Wiley & Sons, Ltd.     

Dual‐Functional Dextran‐PEG Hydrogel as an Antimicrobial Biomedical Material

Microbial infections continually present a major worldwide public healthcare threat, particularly in instances of impaired wound healing and biomedical implant fouling. The development of new materials with the desired antimicrobial property to avoid and treat wound infection is urgently needed in wound care management. This study reports a novel dual‐functional biodegradable dextran‐poly(ethylene glycol) (PEG) hydrogel covalently conjugated with antibacterial Polymyxin B and Vancomycin (Vanco). The hydrogel is designed as a specialized wound dressing that eradicates existing bacteria and inhibits further bacteria growth, while, ameliorating the side effects of antibiotics and accelerating tissue repair and regeneration. The hydrogel exhibits potent antibacterial activities against both gram‐negative bacteria Escherichia coli (E. coli) and gram‐positive bacteria Staphylococcus aureus (S. aureus) with no observable toxicity to mouse fibroblast cell line NIH 3T3. These results demonstrate the immense potential of dextran‐PEG hydrogel as a wound dressing healthcare material in efficiently controlling bacteria growth in complex biological systems.     

Polycyclic Polyprenylated Acylphloroglucinols: An Emerging Class of Non‐Peptide‐Based MRSA‐ and VRE‐Active Antibiotics

In the past 20 years, peptide‐based antibiotics, such as vancomycin, teicoplanin, and daptomycin, have often been considered as second‐line antibiotics. However, in recent years, an increasing number of reports on vancomycin resistance in pathogens appeared, which forces researchers to find novel lead structures for potent new antibiotics. Herein, we report the total synthesis of a defined endo‐type B PPAP library and their antibiotic activity against multiresistant S. aureus and various vancomycin‐resistant Enterococci . Four new compounds that combine high activities and low cytotoxicity were identified, indicating that the PPAP core might become a new non‐peptide‐based lead structure in antibiotic research.     

UV Excited‐State Photoresponse of Biochromophore Negative Ions

Members of the green fluorescent protein (GFP) family may undergo irreversible phototransformation upon irradiation with UV light. This provides clear evidence for the importance of the higher‐energy photophysics of the chromophore, which remains essentially unexplored. By using time‐resolved action and photoelectron spectroscopy together with high‐level electronic structure theory, we directly probe and identify higher electronically excited singlet states of the isolated para‐ and meta‐chromophore anions of GFP. These molecular resonances are found to serve as a doorway for very efficient electron detachment in the gas phase. Inside the protein, this band is found to be resonant with the quasicontinuum of a solvated electron, thus enhancing electron transfer from the GFP to the solvent. This suggests a photophysical pathway for photoconversion of the protein, where GFP resonant photooxidation in solution triggers radical redox reactions inside these proteins.     

3‐aryl‐6‐methoxy‐2‐oxo‐1,2‐dihydroquinoline‐4‐carbonitriles as Solvent and pH Independent Green Fluorescent Dyes

Highly fluorescent and stable 3‐aryl‐6‐methoxy‐2‐oxoquinoline‐4‐carbonitriles 6 (λ_exc = 408 nm and λ_em = 510 nm) were synthesized starting from appropriate arylmalonates 2 . Ring closure reaction with p‐anisidine gave 4‐hydroxyquinolones 3 , which could be bis‐chlorinated to yield quinolines 4 . Regioselective hydrolysis produced reactive 4‐chloroquinolones 5 , which were converted to green fluorescent 4‐cyano quinolones 6 using toluenesulfinates as catalysts.     

Chemical Synthesis of Burkholderia Lipid A Modified with Glycosyl Phosphodiester‐Linked 4‐Amino‐4‐deoxy‐β‐L‐arabinose and Its Immunomodulatory Potential

Modification of the Lipid A phosphates by positively charged appendages is a part of the survival strategy of numerous opportunistic Gram‐negative bacteria. The phosphate groups of the cystic fibrosis adapted Burkholderia Lipid A are abundantly esterified by 4‐amino‐4‐deoxy‐β‐L ‐arabinose (β‐L ‐Ara4N), which imposes resistance to antibiotic treatment and contributes to bacterial virulence. To establish structural features accounting for the unique pro‐inflammatory activity of Burkholderia LPS we have synthesised Lipid A substituted by β‐L ‐Ara4N at the anomeric phosphate and its Ara4N‐free counterpart. The double glycosyl phosphodiester was assembled by triazolyl‐tris‐(pyrrolidinyl)phosphonium‐assisted coupling of the β‐L ‐Ara4N H‐phosphonate to α‐lactol of β(1→6) diglucosamine, pentaacylated with (R)‐(3)‐acyloxyacyl‐ and Alloc‐protected (R)‐(3)‐hydroxyacyl residues. The intermediate 1,1′‐glycosyl‐H‐phosphonate diester was oxidised in anhydrous conditions to provide, after total deprotection, β‐L ‐Ara4N‐substituted Burkholderia Lipid A. The β‐L ‐Ara4N modification significantly enhanced the pro‐inflammatory innate immune signaling of otherwise non‐endotoxic Burkholderia Lipid A.     

An RCM‐Based Total Synthesis of the Antibiotic Disciformycin B

The total synthesis of the potent new antibiotic disciformycin B ( 2 ) is described, which shows significant activity against methicillin‐ and vancomycin‐resistant Staphylococcus aureus (MRSA/VRSA) strains. The synthetic route is based on macrocyclization of a tetraene substrate to the 12‐membered macrolactone core by ring‐closing olefin metathesis (RCM). Although macrocyclization was accompanied by concomitant cyclopentene formation by an alternative RCM pathway, conditions were established to give the macrocycle as the major product. Key steps in the construction of the RCM substrate include a highly efficient Evans syn‐aldol reaction, the asymmetric Brown allylation of angelic aldehyde, and the stereoselective Zn(BH₄)₂‐mediated 1,2‐reduction of an enone. The synthesis was completed by late‐stage dehydrative glycosylation to introduce the d ‐arabinofuranosyl moiety and final chemoselective allylic alcohol oxidation.     

Biotransformation and metabolic profile of caudatin‐2,6‐dideoxy‐3‐O‐methy‐β‐d‐cymaropyranoside with human intestinal microflora by liquid chromatography quadrupole time‐of‐flight mass spectrometry

In our previous studies, caudatin‐2,6‐dideoxy‐3‐O‐methy‐β‐d‐ cymaropyranoside (CDMC) was for the first time isolated from Cynanchum auriculatum Royle ex Wightand and was reported to possess a wide range of biological activities. However, the routes and metabolites of CDMC produced by intestinal bacteria are not well understood. In this study, ultra‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry (UPLC‐Q‐TOF‐MS) technique combined with Metabolynx^TMsoftware was applied to analyze metabolites of CDMC by human intestinal bacteria. The incubated samples collected for 48 h in an anaerobic incubator and extracted with ethyl acetate were analyzed by UPLC‐Q‐TOF‐MS within 12 min. Eight metabolites were identified based on MS and MS/MS data. The results indicated that hydrolysis, hydrogenation, demethylation and hydroxylation were the major metabolic pathways of CDMC in vitro. Seven strains of bacteria including Bacillus sp. 46, Enterococcus sp. 30 and sp. 45, Escherichia sp. 49A, sp. 64, sp. 68 and sp. 75 were further identified using 16S rRNA gene sequencing owing to their relatively strong metabolic capacity toward CDMC. The present study provides important information about metabolic routes of CDMC and the roles of different intestinal bacteria in the metabolism of CDMC. Moreover, those metabolites might influence the biological effect of CDMC in vivo, which affects the clinical effects of this medicinal plant. Copyright © 2015 John Wiley & Sons, Ltd.     

Manipulation of Single‐Stranded DNA by Using an Artificial Site‐Selective DNA Cutter Composed of Cerium(IV)/EDTA and Phosphonate–Oligonucleotide Conjugates

An artificial site‐selective DNA cutter to hydrolyze single‐stranded DNA at a desired site was prepared from Ce^IV/ethylenediamintetraacetic acid (EDTA) and two ethylenediamine‐N,N,N′,N′‐tetrakis(methylenephosphonic acid)–oligonucleotide conjugates. By using this cutter, the sense strand of a blue fluorescent protein (BFP) gene was selectively cut at a predetermined site in the chromophore‐coding region. The upstream fragment obtained by the site‐selective scission was ligated with the downstream fragment of the closely related green fluorescent protein (GFP) gene so that the 5′‐ and 3′‐end portions of the chromophore came from the BFP fragment and the GFP fragment, respectively. The recombinant gene was successfully expressed in E. coli and the chimeric chromophore emitted green fluorescence as expected.