Enzyme oxidation of plant galactomannans yielding biomaterials with novel properties and applications, including as delivery systems.

New biomaterials from renewable sources and the development of "functionalized biopolymers" are fields of growing industrial interest. Plant polysaccharides represent a valid alternative to traditional synthetic polymers, which are obtained from monomers of fossil, non-renewable origin. Several polysaccharides, either in their natural or chemically/biochemically modified forms, are currently employed in the biomedical, food and feed, and industrial fields, including packaging. Sustainable biochemical reactions, such as enzyme modifications of polysaccharides, open further possibilities for new product and process innovation. In the present review, we summarize the recent progress on enzyme oxidation of galactomannans (GM) from few leguminous plants (performed either with galactose oxidase or laccase) and we focus on the versatile and easily accessible laccase/TEMPO oxidative reaction. The latter causes a steep viscosity increase of GM water solutions and a transition of the gels from a viscous to an elastic form, due to formation of emiacetalic bonds and thus of internal cross-linking of the polymers. Following lyophilization of these hydrogels, stable aerogels can be obtained, which were shown to have good potential as delivery systems (DS) of actives. The active molecules tested and herewith described are polymyxin B, an antibiotic; nisin, an antimicrobial peptide; the enzymes lysozyme, protease and lipase; the mixture of the industrial microbiocides 5-chloro-2-methyl-4-isothiazolin-3-one (CIT) and 2-methyl-4-isothiazolin-3-one (MIT). The advantages of such aerogel systems and the possibilities they open for future developments, including as DS, are described.

Aerogel from chemo-enzymatically oxidized fenugreek gum: an innovative delivery system of isothiazolinones biocides.

Aerogels of chemo-enzymatically oxidized, lyophilized fenugreek gum (EOLFG) were evaluated as "delivery system" (DS) of the microbiocide mix 5-chloro-2-methyl-4-isothiazolin-3-one (CIT) and 2-methyl-4-isothiazolin-3-one (MIT). These biocides have a broad activity spectrum and a low MIC (minimal inhibitory concentration) in the ppm range. They are approved under the EU Biocidal Product Directive for various applications, including cosmetics, are classified as skin sensitizers, and are under increasing scrutiny to limit or eliminate their use.We demonstrate that CIT/MIT can be uptaken into EOLFG aerogel corks by immersion into aqueous solutions of microbiocides, followed by blotting and re-lyophilization to generate "loaded" EOLFG aerogels. Incubation of "loaded" corks in water brings about a slow and almost complete release of the absorbed actives having the same MIC of free biocides against selected bacterial pathogens.This new biomaterial could represent an innovative DS of microbiocides and other actives for a variety of industrial, food, cosmetic, and biomedical applications.

"Aerogels of enzymatically oxidized galactomannans from leguminous plants: Versatile delivery systems of antimicrobial peptides and enzymes".

We describe aerogels obtained by laccase/TEMPO-oxidation and lyophilization of galactomannans (GM) from fenugreek, sesbania and guar. Enzymatic oxidation of GM in aqueous solution caused viscosity increase up to fifteen-fold, generating structured, elastic, stable hydrogels, presumably due to establishment of hemiacetalic bonds between newly formed carbonyl groups and free hydroxyl groups. Upon lyophilization, water-insoluble aerogels were obtained, whose mechanical properties and porosity were investigated. Active principles were absorbed into the aerogels from aqueous solutions and, following rinsing, blotting, re-lyophilization, were released in an appropriate medium. The release of the antibiotic polymyxin B was tested against six different Gram-negative bacterial strains, of the antimicrobial peptide nisin against two Gram-positive and of the muraminidase lysozyme against one anaerobic strain. Protease and lipase release in solution from "enzyme loaded" aerogels was monitored by the increase in enzymatic activity. These biomaterials could represent new versatile, biocompatible "delivery systems" of actives for biomedical and industrial applications.

An aerogel obtained from chemo-enzymatically oxidized fenugreek galactomannans as a versatile delivery system.

We describe a new aerogel obtained from laccase-oxidized galactomannans of the leguminous plant fenugreek (Trigonella foenum-graecum) and suggest its potential practical use. Laccase/TEMPO oxidation of fenugreek in aqueous solution caused a viscosity increase of over 15-fold. A structured, elastic, stable hydrogel was generated, due to formation of carbonyl groups from primary OH of galactose side units and subsequent establishment of hemiacetalic bonds with available free hydroxyl groups. Upon lyophilization of this hydrogel, a water-insoluble aerogel was obtained (EOLFG), capable of uptaking aqueous or organic solvents over 20 times its own weight. The material was characterized by scanning electron microscopy, FT-IR, elemental analysis and (13)C CP-MAS NMR spectroscopy and its mechanical properties were investigated. To test the EOLFG as a delivery system, the anti-microbial enzyme lysozyme was used as model active principle. Lysozyme was added before or after formation of the aerogel, entrapped or absorbed in the gel, retained and released in active form, as proven by its hydrolytic glycosidase activity on lyophilized Micrococcus lysodeikticus cells wall peptidoglycans. This new biomaterial, composed of a chemo-enzymatically modified plant polysaccharide, might represent a versatile, biocompatible "delivery system" of active principles in food and non-food products.

Carbapenem-Resistant Klebsiella pneumoniae: Results of a Laboratory Surveillance Program in an Italian General Hospital (August 2014-January 2015) : Surveillance of Carbapenem-resistant Klebsiella pneumoniae.

In this study we report the analysis of 131 Klebsiella pneumoniae (K. pneumoniae) clinical isolates from patients hospitalized in various wards, of Perugia General Hospital, from August 2014 to January 2015. Forty two isolates (32.1 %), were resistant to at least one carbapenem antibiotic and, among these isolates, 14 (33.3 %) exhibited resistance to colistin. All isolates were carbapenemases producers and 41 (97.6 %) harboured the bla KPC gene. Carbapenem-resistant K. pneumoniae isolates (CRKPs) were, also, typed for the genotypic diversity and the results revealed the circulation of two major clusters.This surveillance study evidences the spread of CRKP isolates in Perugia General Hospital and confirms that carbapenem-resistant K. pneumoniae isolates have reached epidemic dissemination in Italy. In addition the percentage of resistance to colistin resulted to be less than that observed in other hospital laboratories across Italy. In conclusion the circulation of these isolates should be monitored and appropriate policy of surveillance must be used, in a target manner, in order to reduce the spread of carbapenem-resistant isolates.

Enzymatic and chemical oxidation of polygalactomannans from the seeds of a few species of leguminous plants and characterization of the oxidized products.

Plant polysaccharides are used in a growing number of applications, in their native or in chemically and/or biochemically modified forms. In the present work, we compare TEMPO-mediated oxidation with laccase of polygalactomannans (PGM) from different species of plant leguminous to chemical oxidation with NaClO/NaBr/TEMPO. We have investigated the gums from: locust bean (Ceratonia siliqua), tara (Caesalpinia spinosa), guar (Cyamopsis tetragonolobus), sesbania (Sesbania bispinosa) and fenugreek (Trigonella foenum-graecum). Upon laccase/TEMPO oxidation, PGM viscosity and concentration of reducing groups increased up to five-fold and structured, elastic, stable gels were formed, which could be degraded by hydrolysis with ß-mannanase. Conversely, chemical oxidation with NaClO/NaBr/TEMPO caused a rapid, intermediate transition of the gum solutions to compact gels, that immediately reverted to liquid, with a lower viscosity than at the start and an increased concentration of reducing groups, similar to the reaction with laccase. We interpret the above as due to, in the case of laccase, oxidation of primary hydroxyl groups to aldehydes, able to form stable hemiacetalic bonds with free hydroxyl groups. While upon chemical oxidation, primary OH's are only transiently oxidized to aldehydes, followed by rapid oxidation of all carbonyl groups to carboxylates. In either cases, TEMPO appeared to cause a limited splitting of glycosidic bonds of PGM. Native and oxidized PGM were further characterized by 1D and 2D NMR spectroscopy and by rheology.

Typing of nosocomial outbreaks of Acinetobacter baumannii by use of matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been evaluated for the identification of multidrug-resistant Acinetobacter baumannii nosocomial outbreaks in comparison with the repetitive sequence-based PCR DiversiLab system. The results suggest that MALDI-TOF MS can be used for real-time detection of Acinetobacter outbreaks before results from DNA-based systems are available.