Glycopeptide Antibiotic Vancomycin Research Articles

Identification of pathways to high-level vancomycin resistance in Clostridioides difficile that incur high fitness costs in key pathogenicity traits.

Clostridioides difficile is an important human pathogen, for which there are very limited treatment options, primarily the glycopeptide antibiotic vancomycin. In recent years, vancomycin resistance has emerged as a serious problem in several gram-positive pathogens, but high-level resistance has yet to be reported for C. difficile, although it is not known if this is due to constraints upon resistance evolution in this species. Here, we show that resistance to vancomycin can evolve rapidly under ramping selection but is accompanied by fitness costs and pleiotropic trade-offs, including sporulation defects that would be expected to severely impact transmission. We identified 2 distinct pathways to resistance, both of which are predicted to result in changes to the muropeptide terminal D-Ala-D-Ala that is the primary target of vancomycin. One of these pathways involves a previously uncharacterised D,D-carboxypeptidase, expression of which is controlled by a dedicated two-component signal transduction system. Our findings suggest that while C. difficile is capable of evolving high-level vancomycin resistance, this outcome may be limited clinically due to pleiotropic effects on key pathogenicity traits. Moreover, our data identify potential mutational routes to resistance that should be considered in genomic surveillance.

Genome assembly, comparative genomics, and identification of genes/pathways underlying plant growth-promoting traits of an actinobacterial strain, Amycolatopsis sp. (BCA-696)

The draft genome sequence of an agriculturally important actinobacterial species Amycolatopsis sp. BCA-696 was developed and characterized in this study. Amycolatopsis BCA-696 is known for its biocontrol properties against charcoal rot and also for plant growth-promotion (PGP) in several crop species. The next-generation sequencing (NGS)-based draft genome of Amycolatopsis sp. BCA-696 comprised of ~ 9.05 Mb linear chromosome with 68.75% GC content. In total, 8716 protein-coding sequences and 61 RNA-coding sequences were predicted in the genome. This newly developed genome sequence has been also characterized for biosynthetic gene clusters (BGCs) and biosynthetic pathways. Furthermore, we have also reported that the Amycolatopsis sp. BCA-696 produces the glycopeptide antibiotic vancomycin that inhibits the growth of pathogenic gram-positive bacteria. A comparative analysis of the BCA-696 genome with publicly available closely related genomes of 14 strains of Amycolatopsis has also been conducted. The comparative analysis has identified a total of 4733 core and 466 unique orthologous genes present in the BCA-696 genome The unique genes present in BCA-696 was enriched with antibiotic biosynthesis and resistance functions. Genome assembly of the BCA-696 has also provided genes involved in key pathways related to PGP and biocontrol traits such as siderophores, chitinase, and cellulase production.

Comparison of the adsorption dynamics of leucyl-leucine enantiomers and glycyl-glycine on Chirobiotic R and Chirobiotic V columns

The adsorption dynamics of the LL- and DD-enantiomers of Leu-Leu and Gly-Gly on a Chirobiotic V column packed with a CSP bearing glycopeptide antibiotic vancomycin was studied and compared with the adsorption dynamics of the same dipeptides on a Chirobiotic R column packed with a CSP with bonded antibiotic ristocetin A. The column efficiencies were essentially different. The heights equivalent to a theoretical plate (HETP) on Chirobiotic R were 2-4 times larger than on Chirobiotic V, and the shape of van Deemter plots, strongly convex-upwards on the former column, demonstrated minor (Leu-Leu) or lacking (Gly-Gly) convexity on the latter column. This difference is attributed to differences in the binding kinetics of dipeptides to the antibiotics, fast for vancomycin and slow for ristocetin A. Since the kinetic C term of the van Deemter equation cannot explain such a large discrepancy in HETP for the same analyte on two columns, and since the A and B terms for the Chirobiotic R column were essentially larger than for the Chirobiotic V column, it is suggested that binding kinetics influences eddy and longitudinal diffusion processes associated with those terms. It is also hypothesized that unusually high values of van Deemter B coefficients occasionally found in chiral chromatography are not experimental artifacts but the manifestation of coupling the effects of slow adsorption kinetics and longitudinal diffusion.

Contribution of qPCR to the diagnosis of cervico-vaginal infections at the Hôpital Principal de Dakar, Senegal

To determine the etiology of cervico-vaginal infections by cytobacteriology and the efficacy of qPCR for the diagnosis of sensitive strains such as Streptococcus agalactiae, Borrelia crocidurae, Chlamydia trachomatis, Neisseria gonorrhoeae and Treponema pallidum. This prospective cross-sectional study was performed between January and September 2021 in 346 women who were examined for cervico-vaginal infection at the Hôpital Principal de Dakar (HPD). Cytobacteriological (direct examination, agar culture) and molecular analyses were performed. Vaginal flora imbalances predominated, with a rate of 72.3%. The proportion of type IV vaginal flora was 46.5%. Of the 199 germs isolated, Candida albicans (25.1%), Ureaplasma urealyticum (17.6%), S. agalactiae (7.8%), Gardnerella vaginalis (6.6%) and nonalbicans Candida (5.5%) were the main pathogens responsible for cervico-vaginal infections in patients. Among women tested for mycoplasma, U. urealyticum was identified in 43.3% of patients. Among those tested for C. trachomatis, the proportion of infected women was low (4%). The prevalence of C. albicans was higher in pregnant women (38.3%) than in nonpregnant women (19.2%). S. agalactiae strains showed high resistance to certain beta-lactam antibiotics (pristinamycin 100%, gentamycin 100%, ampicillin 92.5% and cefalotin 85.2%) and to a glycopeptide antibiotic (vancomycin 100%). The Staphylococcus aureus strain had good sensitivity to antibiotics except gentamycin (100%) and kanamycin (100%). The enterobacteria tested were all sensitive to phenicols, carbapenems, cephalosporins and aminoglycosides. However, E. coli showed high resistance to tetracycline. The different methods showed low prevalences of C. trachomatis and N. gonorrhoeae, so comparisons Test RapidChlamydia/qPCR for C. trachomatis and culture/qPCR for N. gonorrhoeae were not possible. For S. agalactiae, on the other hand, qPCR was more advantageous than culture. The χ2 test showed a significant difference (Yates χ2 = 33.77 and p = 1-7) for the diagnosis of S. agalactiae. S. agalactiae qPCR had a sensitivity of 40.7%, a specificity of 94%, and positive and negative predictive values of 36.7% and 94.9% respectively, as well as a kappa = 0.33. The methods applied enabled us to identify the pathogens that cause cervicovaginal infections. The results suggest that qPCR may be an alternative, at least for the diagnosis of S. agalactiae. However, culture remains indispensable for studying antibiotic sensitivity. In order to improve patient care, molecular techniques need to be integrated into the HPD testing toolbox. To broaden the repertoire of pathogens to be diagnosed by qPCR, targeted comparison studies will be needed to increase the probability of encountering infected individuals.

Hematoma-like dynamic hydrogelation through natural glycopeptide molecular recognition for infected bone fracture repair.

Infected bone fractures remain a major clinical challenge for orthopedic surgeons. From a tissue regeneration perspective, biomaterial scaffolds with antibacterial and osteoinductive activities are highly desired, while advanced materials capable of mimicking the pathological microenvironment during the healing process of infected tissues remain an area deserving more research. Hematoma, the gel-like blood coagulum, plays an essential role in bone fracture repair because of its ability to serve as a dynamic and temporary scaffold with cytokines for both pathogen elimination and tissue healing. In light of this, we designed a dynamic hydrogel with hematoma-like antimicrobial or reparative performance for infected bone fracture repair in this study. The proposed dynamic hydrogel network was based on the reversible recognition of a natural glycopeptide antibiotic vancomycin (Van) and its target dipeptide D-Ala-D-Ala (AA), which could serve as a hematoma-like scaffold for obliterating bacteria in the fracture region and promoting bone repair by introducing an endogenous osteogenic peptide (OGP). In vivo experiments demonstrated that the hydrogel could rapidly eradicate bacteria, improve bone regeneration and restore the local inflammatory microenvironment. Together, findings from this study imply that the use of hematoma-like dynamic hydrogel could lead to a biomimetic revolution in surgical strategies against susceptible bone fractures.

Conformationally Restricted Glycopeptide Backbone Inhibits Gas-Phase H/D Scrambling between Glycan and Peptide Moieties.

Protein glycosylation is a common post-translational modification on extracellular proteins. The conformational dynamics of several glycoproteins have been characterized by hydrogen/deuterium exchange mass spectrometry (HDX-MS). However, it is, in most cases, not possible to extract information about glycan conformation and dynamics due to the general difficulty of separating the deuterium content of the glycan from that of the peptide (in particular, for O-linked glycans). Here, we investigate whether the fragmentation of protonated glycopeptides by collision-induced dissociation (CID) can be used to determine the solution-specific deuterium content of the glycan. Central to this concept is that glycopeptides can undergo a facile loss of glycans upon CID, thereby allowing for the determination of their masses. However, an essential prerequisite is that hydrogen and deuterium (H/D) scrambling can be kept in check. Therefore, we have measured the degree of scrambling upon glycosidic bond cleavage in glycopeptides that differ in the conformational flexibility of their backbone and glycosylation pattern. Our results show that complete scrambling precedes the glycosidic bond cleavage in normal glycopeptides derived from a glycoprotein; i.e., all labile hydrogens have undergone positional randomization prior to loss of the glycan. In contrast, the glycosidic bond cleavage occurs without any scrambling in the glycopeptide antibiotic vancomycin, reflecting that the glycan cannot interact with the peptide moiety due to a conformationally restricted backbone as revealed by molecular dynamics simulations. Scrambling is also inhibited, albeit to a lesser degree, in the conformationally restricted glycopeptides ristocetin and its pseudoaglycone, demonstrating that scrambling depends on an intricate interplay between the flexibility and proximity of the glycan and the peptide backbone.

Increased incidence of teicoplanin-non-susceptible Staphylococcus epidermidis strains: a 6-year retrospective study

Glycopeptide antibiotics (vancomycin and teicoplanin) are usually used for the treatment of Staphylococcus epidermidis infections owing to their increased oxacillin resistance. However, S. epidermidis strains with decreased susceptibility to teicoplanin have become increasingly incident in recent years. We aimed to identify the characteristics of teicoplanin-non-susceptible (Teico-NS) S. epidermidis isolated at our hospital and analyze its relationship with teicoplanin usage. We retrospectively evaluated 328 S. epidermidis strains isolated from clinical isolates between January 2016 and December 2021. All strains were susceptible to vancomycin (minimal inhibitory concentration (MIC) ≤ 4 mg/L). The annual incidence for S. epidermidis strains with an elevated teicoplanin MIC of 8 mg/L ranged from 22.2 to 28.9%. In addition, in 2021, the number of S. epidermidis strains with teicoplanin MIC ≥ 16 mg/L rapidly increased (n = 13, 32.5%). Furthermore, teicoplanin use increased annually until 2019; however, in 2020, it decreased abruptly due to the COVID 19 pandemic. Thus, we could not confirm the existence of a clear correlation between teicoplanin usage and increased incidence of S. epidermidis with reduced teicoplanin-susceptibility. We showed the increased incidence of Teico-NS S. epidermidis in recent years. Further studies are needed to identify the mechanisms and risk factors for teicoplanin-resistance in S. epidermidis.

Differential Study of Antimicrobial Activity of Vancomycin and Teicoplanin (Targocid) against Strains of Staphylococcus aureus and Streptococci sp.

The glycopeptide antibiotics vancomycin and teicoplanin (targocid) are widely used in the treatment of infections caused by gram positive bacteria. Vancomyc inhibits both transglycosylation and transpeptidation reaction during peptidoglycan assembly, targocid inhibits peptidoglycan polymerization, resulting in inhibition of bacterial cell wall synthesis and cell death. In this study twenty strains of streptococcus pyogenes and streptococcus pneumoniae and twenty five strains of staphylococcus aureus were collected from different infections. MI Cs and MBCs of vancomycin and targocid were determined against the bacterial strains, the MICs of vancomycin for the streptococcus strains were 4-8 µg /ml compared with 8-16 µg/ml for targocid and MI Cs of targocid for staphylococcus aureus were 4-16 µg/ml MBC for the same strains were 8-32 µg/ml. the conclusion of this study was that targocid was less active than vancomycin against staphylococci but equal or more active against streptococci specifically it presents excellent in vitro activity against streptococcus pneumonia strains.

Degradation of Antibiotic Vancomycin by UV Photolysis and Pulsed Corona Discharge Combined with Extrinsic Oxidants

Antibiotics are the most frequently detected pharmaceuticals in the environment creating conditions for the development of resistant genes in bacteria. Degradation and mineralization of glycopeptide antibiotic vancomycin (VMN) were examined by UV photolysis, pulsed corona discharge (PCD), and their combinations with extrinsic oxidants, hydrogen peroxide (HP), peroxydisulfate (PDS), and peroxymonosulfate (PMS). Both combinations were effective in VMN degradation and faster at pH 11 than in acidic or neutral media. Combined with the UV photolysis, HP showed a higher oxidation rate than other oxidants, whereas PMS and PDS proved to be more efficient in combinations with PCD. In contrast to low-to-moderate mineralization of VMN in the UV/oxidant combinations, PCD and PCD/oxidant combinations appeared to be more effective, reaching up to 90% of TOC removal in acidic/neutral solutions. Application of extrinsic oxidants resulted in an energy efficiency of VMN 90% oxidation improved from 36 to 61 g kW−1 h−1 in HP-assisted photolysis, and from 195 to 250 g kW−1 h−1 in PCD with additions of HP and PDS, thus showing the promising character of the combined treatment.

UV-VUV Photofragmentation Spectroscopy of Isolated Neutral Fragile Macromolecules: A Proof-of-Principle Based on a Deprotonated Vancomycin-Peptide Noncovalent Complex.

The gas phase offers the possibility to analyze organic molecules by ultraviolet-vacuum ultraviolet (UV-VUV) spectroscopy without any solvent effect or limitation in terms of spectral range due to absorption by the solvent. Up to now, the size and chemical composition of neutral molecular systems under study have been limited by the use of vaporization methods based on thermal heating. Soft sources of gas-phase thermolabile molecular systems such as electrospray or matrix-assisted laser desorption ionization are appealing alternatives to heating-based techniques, but they lead to the production of ions. In such cases, UV-VUV action spectroscopy is then the method of choice to study the electronic structure and corresponding photodynamics of these gas-phase molecular ions. However, previous investigations have shown that the UV-VUV action spectrum of a given molecular ion depends on the charge state, which in many cases might be a caveat. Here, by means of synchrotron radiation coupled to mass spectrometry and through the test case of the glycopeptide antibiotic vancomycin noncovalently bound to a deprotonated small peptide, we show that the UV-VUV photofragmentation spectrum of neutral thermally fragile organic molecules can be obtained via charge-tagging action spectroscopy.

A RETROSPECTIVE STUDY: ANALYSIS OF ADVERSE DRUG REACTION IN PEDIATRIC PATIENTS IN A TERTIARY CARE HOSPITAL

Objective: The objective of this study was to analyze various parameters such as admission type, demographics, type of reaction, organ system classification, drugs involved, action is taken, reaction outcome, causality assessment, severity assessment, and the preventability of Adverse drug reactions (ADRs) in pediatric patients. Methods: This retrospective observational study was conducted during the period of September 2017 to June 2020 (34 months) at the ADR monitoring center, Department of Pharmacology, Jawaharlal Nehru Medical College, Ajmer, Rajasthan. All spontaneously reported ADRs were evaluated using various parameters such as type of reaction, causality assessment, preventability, and severity. Results: In the present study, 72 (7.27%) ADRs were reported in relation to 65 pediatric patients. In this study, more ADRs were reported in male (53.85%) as compared to female (46.15%) pediatric patients. The majority of ADRs were considered type B (63.89%), probable (87.5%), moderate (51.39%), and definitely preventable (88.89%) in nature. The majority of ADRs were reported due to antimicrobial classes of drugs, including anthelmintic drug (Albendazole), followed by glycopeptide antibiotic (Vancomycin) and third-generation cephalosporin antibiotics (Ceftriaxone, Cefotaxime, and Cefixime). The organ systems most commonly affected were skin and subcutaneous tissue disorders (47.22%), followed by general disorders and administration site conditions (20.83%) and gastrointestinal disorders (16.67%). Conclusion: The present study 30 different types of suspected ADRs that were reported with multiple frequencies, with included 34 different categories of drugs and combinations of drugs. The majority of patients recovered, with associated ADR, after necessary medical intervention and management. Our purpose is to minimize the incidence rate of ADRs in the pediatric population.

Vancomycin as an Antibacterial Agent Capped with Silver Nanoparticles: An Experimental Potential Analysis.

For the treatment of various infections, a variety of antimicrobial drugs are formulated. Nevertheless, many bacterial infections now exhibit antibiotic resistance due to the widespread utilization antibiotics. Methicillin-resistant among the most dangerous multidrug-resistant bacteria is Staphylococcus aureus (MRSA). Vancomycin became a viable therapy option due to MRSA resistance to methicillin medicines. One of the well-informed antibacterial compounds with wideband antibacterial activity is silver nanoparticles (AgNPs). AgNPs are thus suitable candidates for usage in conjunction alongside vancomycin to increase its antibacterial effect. The goal of the present research work is to boost the antibacterial potency of the glycopeptide antibiotic vancomycin towards Gram-positive (Staphylococcus aureus) but also Gram-negative (Escherichia coli) bacteria. The chemical reduction approach is used to create a colloidal solution of silver nanoparticles utilizing silver nitrate as a precursor in the environment of the ionic surfactant trisodium citrate that serves as covering including reducing reagent. Vancomycin was used to functionalize the synthesized nanoparticles and create the nanodrug complex (Van@AgNPs). The synergistic antibacterial potential of silver nanoparticles coated with vancomycin on both test pathogens was investigated using the agar well diffusion technique. The antibacterial potency for both classes of bacteria has significantly increased, according to the well diffusion test. It has been noted that this improvement is synergistic instead of additive.

Surfaces Containing Sharp Nanostructures Enhance Antibiotic Efficacy.

The ever-increasing rate of medical device implantations is met by a proportionately high burden of implant-associated infections. To mitigate this threat, much research has been directed toward the development of antibacterial surface modifications by various means. One recent approach involves surfaces containing sharp nanostructures capable of killing bacteria upon contact. Herein, we report that the mechanical interaction between Staphylococcus aureus and such surface nanostructures leads to a sensitization of the pathogen to the glycopeptide antibiotic vancomycin. We demonstrate that this is due to cell wall damage and impeded bacterial defenses against reactive oxygen species. The results of this study promise to be impactful in the clinic, as a combination of nanostructured antibacterial surfaces and antibiotics commonly used in hospitals may improve antimicrobial therapy strategies, helping clinicians to prevent and treat implant-associated infections using reduced antibiotic concentrations instead of relying on invasive revision surgeries with often poor outcomes.

Reversible dougong structured receptor–ligand recognition for building dynamic extracellular matrix mimics

Dynamic biomaterials excel at recapitulating the reversible interlocking and remoldable structure of the extracellular matrix (ECM), particularly in manipulating cell behaviors and adapting to tissue morphogenesis. While strategies based on dynamic chemistries have been extensively studied for ECM-mimicking dynamic biomaterials, biocompatible molecular means with biogenicity are still rare. Here, we report a nature-derived strategy for fabrication of dynamic biointerface as well as a three-dimensional (3D) hydrogel structure based on reversible receptor-ligand interaction between the glycopeptide antibiotic vancomycin and dipeptide d-Ala-d-Ala. We demonstrate the reversible regulation of multiple cell types with the dynamic biointerface and successfully implement the dynamic hydrogel as a functional antibacterial 3D scaffold to treat tissue repair. In view of the biogenicity and high applicability, this nature-derived reversible molecular strategy will bring opportunities for malleable biomaterial design with great potential in biomedicine.

Two Novel Semisynthetic Lipoglycopeptides Active against Staphylococcus aureus Biofilms and Cells in Late Stationary Growth Phase.

The increase in antibiotic resistance among Gram-positive bacteria underscores the urgent need to develop new antibiotics. New antibiotics should target actively growing susceptible bacteria that are resistant to clinically accepted antibiotics including bacteria that are not growing or are protected in a biofilm environment. In this paper, we compare the in vitro activities of two new semisynthetic glycopeptide antibiotics, MA79 and ERJ390, with two clinically used glycopeptide antibiotics—vancomycin and teicoplanin. The new antibiotics effectively killed not only exponentially growing cells of Staphylococcus aureus, but also cells in the stationary growth phase and biofilm.

Deciphering Adverse Drug Reactions: In Vitro Priming and Characterization of Vancomycin-Specific T Cells From Healthy Donors Expressing HLA-A*32:01.

Drug rash with eosinophilia with systemic symptoms (DRESS) is a serious adverse event associated with use of the glycopeptide antibiotic vancomycin. Vancomycin-induced drug rash with eosinophilia with systemic symptoms is associated with the expression of human leukocyte antigen (HLA)-A*32:01, suggesting that the drug interacts with this HLA to activate CD8+ T cells. The purpose of this study was to utilize peripheral blood mononuclear cell from healthy donors to: (1) investigate whether expression of HLA-A*32:01 is critical for the priming naïve of T cells with vancomycin and (2) generate T-cell clones (TCC) to determine whether vancomycin exclusively activates CD8+ T cells and to define cellular phenotype, pathways of drug presentation and cross-reactivity. Dendritic cells were cultured with naïve T cells and vancomycin for 2 weeks. On day 14, cells were restimulated with vancomycin and T-cell proliferation was assessed by [3H]-thymidine incorporation. Vancomycin-specific TCC were generated by serial dilution and repetitive mitogen stimulation. Naïve T cells from HLA-A*02:01 positive and negative donors were activated with vancomycin; however the strength of the induced response was significantly stronger in donors expressing HLA-A*32:01. Vancomycin-responsive CD4+ and CD8+ TCC from HLA-A*32:01+ donors expressed high levels of CXCR3 and CCR4, and secreted IFN‐γ, IL-13, and cytolytic molecules. Activation of CD8+ TCC was HLA class I-restricted and dependent on a direct vancomycin HLA binding interaction with no requirement for processing. Several TCC displayed cross-reactivity with teicoplanin and daptomycin. To conclude, this study provides evidence that vancomycin primes naïve T cells from healthy donors expressing HLA-A*32:01 through a direct pharmacological binding interaction. Cross-reactivity of CD8+ TCC with teicoplanin provides an explanation for the teicoplanin reactions observed in vancomycin hypersensitive patients.

Vancomycin-Loaded Microneedle Arrays against Methicillin-Resistant Staphylococcus Aureus Skin Infections.

Skin and soft tissue infections (SSTIs) caused by methicillin‐resistant Staphylococcus aureus (MRSA) are a major healthcare burden, often treated with intravenous injection of the glycopeptide antibiotic vancomycin (VAN). However, low local drug concentration in the skin limits its treatment efficiency, while systemic exposure promotes the development of resistant bacterial strains. Topical administration of VAN on skin is ineffective as its high molecular weight prohibits transdermal penetration. In order to implement a local VAN delivery, microneedle (MN) arrays with a water‐insoluble support layer for the controlled administration of VAN into the skin are developed. The utilization of such a support layer results in water‐insoluble needle shafts surrounded by drug‐loaded water‐soluble tips with high drug encapsulation. The developed MN arrays can penetrate the dermal barriers of both porcine and fresh human skin. Permeation studies on porcine skin reveal that the majority of the delivered VAN is retained within the skin. It is shown that the VAN‐MN array reduces MRSA growth both in vitro and ex vivo on skin. The developed VAN‐MN arrays may be extended to several drugs and may facilitate localized treatment of MRSA‐caused skin infections while minimizing adverse systemic effects.

Sustainable Production of N-methylphenylalanine by Reductive Methylamination of Phenylpyruvate Using Engineered Corynebacterium glutamicum

N-alkylated amino acids occur widely in nature and can also be found in bioactive secondary metabolites such as the glycopeptide antibiotic vancomycin and the immunosuppressant cyclosporine A. To meet the demand for N-alkylated amino acids, they are currently produced chemically; however, these approaches often lack enantiopurity, show low product yields and require toxic reagents. Fermentative routes to N-alkylated amino acids like N-methyl-l-alanine or N-methylantranilate, a precursor of acridone alkaloids, have been established using engineered Corynebacterium glutamicum, which has been used for the industrial production of amino acids for decades. Here, we describe metabolic engineering of C. glutamicum for de novo production of N-methylphenylalanine based on reductive methylamination of phenylpyruvate. Pseudomonas putida Δ-1-piperideine-2-carboxylate reductase DpkA containing the amino acid exchanges P262A and M141L showed comparable catalytic efficiencies with phenylpyruvate and pyruvate, whereas the wild-type enzyme preferred the latter substrate over the former. Deletion of the anthranilate synthase genes trpEG and of the genes encoding branched-chain amino acid aminotransferase IlvE and phenylalanine aminotransferase AroT in a strain engineered to overproduce anthranilate abolished biosynthesis of l-tryptophan and l-phenylalanine to accumulate phenylpyruvate. Upon heterologous expression of DpkAP262A,M141L, N-methylphenylalanine production resulted upon addition of monomethylamine to the medium. In glucose-based minimal medium, an N-methylphenylalanine titer of 0.73 ± 0.05 g L−1, a volumetric productivity of 0.01 g L−1 h−1 and a yield of 0.052 g g−1 glucose were reached. When xylose isomerase gene xylA from Xanthomonas campestris and the endogenous xylulokinase gene xylB were expressed in addition, xylose as sole carbon source supported production of N-methylphenylalanine to a titer of 0.6 ± 0.04 g L−1 with a volumetric productivity of 0.008 g L−1 h−1 and a yield of 0.05 g g−1 xylose. Thus, a fermentative route to sustainable production of N-methylphenylalanine by recombinant C. glutamicum has been established.

Overcoming the Mucosal Barrier: Tetraether Lipid‐Stabilized Liposomal Nanocarriers Decorated with Cell‐Penetrating Peptides Enable Oral Delivery of Vancomycin

AbstractDespite the high medical need for oral peptide delivery, instability in the gastrointestinal tract and low mucosal permeation still impede this preferred route of administration. Herein, a liposomal nanocarrier combining two self‐reliant strategies to overcome these delivery barriers is reported. This approach enables the design of a nanocarrier system with synergistic properties: tetraether lipids derived from archaea are incorporated into liposomes to provide the particles with the stability required to traverse the stomach. When the surface of the resulting inert particles is modified with cell‐penetrating peptides, mucosal permeation can be achieved. The designed nanocarrier is proven effective by the high mucosal uptake of the glycopeptide antibiotic vancomycin in Ussing chamber studies. Efficacy in vivo is demonstrated in naïve rats, where a highly increased oral bioavailability is obtained for vancomycin, a drug known to be minimally absorbed. In contrast, administration of liposomes with single modification (tetraether lipids) leads to a substantially lower bioavailability. Therapeutic efficacy is proven by the antimicrobial activity of vancomycin in a Galleria mellonella and a systemic infection mouse model. The high oral bioavailability in absence of cytotoxic effects demonstrates that this nanocarrier delivery strategy might boost the oral application of macromolecular drugs in general.

Piezoelectric hybrid scaffolds mineralized with calcium carbonate for tissue engineering: Analysis of local enzyme and small-molecule drug delivery, cell response and antibacterial performance

As the next generation of materials for bone reconstruction, we propose a multifunctional bioactive platform based on biodegradable piezoelectric polyhydroxybutyrate (PHB) fibrous scaffolds for tissue engineering with drug delivery capabilities. To use the entire surface area for local drug delivery, the scaffold surface was uniformly biomineralized with biocompatible calcium carbonate (CaCO3) microparticles in a vaterite–calcite polymorph mixture. CaCO3-coated PHB scaffolds demonstrated a similar elastic modulus compared to that of pristine one. However, reduced tensile strength and failure strain of 31% and 67% were observed, respectively. The biomimetic immobilization of enzyme alkaline phosphatase (ALP) and glycopeptide antibiotic vancomycin (VCM) preserved the CaCO3-mineralized PHB scaffold morphology and resulted in partial recrystallization of vaterite to calcite. In comparison to pristine scaffolds, the loading efficiency of CaCO3-mineralized PHB scaffolds was 4.6 and 3.5 times higher for VCM and ALP, respectively. Despite the increased number of cells incubated with ALP-immobilized scaffolds (up to 61% for non-mineralized and up to 36% for mineralized), the CaCO3-mineralized PHB scaffolds showed cell adhesion; those containing both VCM and ALP molecules had the highest cell density. Importantly, no toxicity for pre-osteoblastic cells was detected, even in the VCM-immobilized scaffolds. In contrast with antibiotic-free scaffolds, the VCM-immobilized ones had a pronounced antibacterial effect against gram-positive bacteria Staphylococcus aureus. Thus, piezoelectric hybrid PHB scaffolds modified with CaCO3 layers and immobilized VCM/ALP are promising materials in bone tissue engineering.