Modificirajuća sposobnost formiranja biofilma kliničkih izolata gram negativnih mikroorganizama u uvjetima negativnog tlaka in vitro

Vacuum-assisted closure therapy has been utilized as a ubiquitous wound management resource. Current studies of the subject focus on the influence of negative pressure on the bacterial load of wound bed and the possibility to remove matured biofilm, however, its impact on the ability of microorganisms to activate or reduce the biofilm producing modality remains unexplored. The goal of the present study was to evaluate the potential effect of negative pressure on biofilm producing modality of gram-negative rods with regard of initial biofilm positive or biofilm negative phenotype. Biofilm formation was evaluated for the strains of A. baumannii, P. aeruginosa, E. cloace, K. pneumonia and P. mirabilis isolated from infected war wounds of the extremities. The changes of optical density of the biofilm produced by the same strain under normal and negative pressure conditions, as well as the number of strains, which modified their ability to biofilm production, were measured. The strains with initial biofilm negative phenotype under negative pressure switched to biofilm producing more vigorously than biofilm positive ones regardless of their taxonomical belonging. Pseudomonas strains demonstrate the highest rate of induction to biofilm producing under negative pressure conditions among all studied microorganisms. Thus, the activation of biofilm producing mechanisms under negative pressure could be considered as a protective strategy, which helps ensure persistence of microorganisms in the wound even in case the vacuum-assisted closure therapy is applied.Terapija negativnim tlakom ili vakuum terapija (engl vacuum-assisted closure – VAC) univerzalna je potporna metoda liječenja rana. Novija istraživanja iz ovog područja fokusiraju se na utjecaj negativnog tlaka na bakterijsko opterećenje rane i mogućnost uklanjanja zrelog biofilma, no njegov utjecaj na sposobnost mikroorganizama da aktiviraju ili reduciraju modalitet formiranja biofilma ostaje neistražen. Cilj ovog rada bio je procijeniti potencijalni učinak negativnog tlaka na formiranje biofilma kod gram-negativnih bakterija s obzirom na početni pozitivni biofilm ili negativni biofilmski fenotip. Formiranje biofilma istraženo je za sojeve bakterija A. baumannii, P. aeruginosa, E. cloace, K. pneumonia i P. mirabilis izoliranih iz zaraženih ratnih rana ekstremiteta. Izmjerene su promjene optičke gustoće biofilma proizvedene istim bakterijskim sojem u uvjetima normalnih i negativnih tlakova, kao i broj sojeva koji su modificirali svoju sposobnost proizvodnje biofilma. Sojevi s početnim negativnim fenotipom biofilma su pod negativnim tlakom počeli snažnije proizvoditi biofilm od onih s pozitivnim biofilmom bez obzira na njihovu taksonomsku pripadnost. Pseudomonas sojevi pokazali su najveću stopu indukcije stvaranja biofilma pod negativnim tlakom od svih ispitanih mikroorganizmima. Zaključno, aktivacija mehanizama koji proizvode biofilm pod negativnim tlakom može se smatrati zaštitnom strategijom, koja osigurava perzistenciju mikroorganizama u rani čak i u slučaju primjene terapije negativnim tlakom

Naturally prefabricated marine biomaterials: Isolation and applications of flat chitinous 3D scaffolds from Ianthella labyrinthus (demospongiae: Verongiida)

Marine sponges remain representative of a unique source of renewable biological materials. The demosponges of the family Ianthellidae possess chitin-based skeletons with high biomimetic potential. These three-dimensional (3D) constructs can potentially be used in tissue engineering and regenerative medicine. In this study, we focus our attention, for the first time, on the marine sponge Ianthella labyrinthus Bergquist & Kelly-Borges, 1995 (Demospongiae: Verongida: Ianthellidae) as a novel potential source of naturally prestructured bandage-like 3D scaffolds which can be isolated simultaneously with biologically active bromotyrosines. Specifically, translucent and elastic flat chitinous scaffolds have been obtained after bromotyrosine extraction and chemical treatments of the sponge skeleton with alternate alkaline and acidic solutions. For the first time, cardiomyocytes differentiated from human induced pluripotent stem cells (iPSC-CMs) have been used to test the suitability of I. labyrinthus chitinous skeleton as ready-to-use scaffold for their cell culture. Results reveal a comparable attachment and growth on isolated chitin-skeleton, compared to scaffolds coated with extracellular matrix mimetic Geltrex®. Thus, the natural, unmodified I. labyrinthus cleaned sponge skeleton can be used to culture iPSC-CMs and 3D tissue engineering. In addition, I. labyrinthus chitin-based scaffolds demonstrate strong and efficient capability to absorb blood deep into the microtubes due to their excellent capillary effect. These findings are suggestive of the future development of new sponge chitin-based absorbable hemostats as alternatives to already well recognized cellulose-based fabrics. © 2019 by the authors. Licensee MDPI, Basel, Switzerland

Naturally drug-loaded chitin: Isolation and applications

Naturally occurring three-dimensional (3D) biopolymer-based matrices that can be used in different biomedical applications are sustainable alternatives to various artificial 3D materials. For this purpose, chitin-based structures from marine sponges are very promising substitutes. Marine sponges from the order Verongiida (class Demospongiae) are typical examples of demosponges with well-developed chitinous skeletons. In particular, species belonging to the family Ianthellidae possess chitinous, flat, fan-like fibrous skeletons with a unique, microporous 3D architecture that makes them particularly interesting for applications. In this work, we focus our attention on the demosponge Ianthella flabelliformis (Linnaeus, 1759) for simultaneous extraction of both naturally occurring (“ready-to-use”) chitin scaffolds, and biologically active bromotyrosines which are recognized as potential antibiotic, antitumor, and marine antifouling substances. We show that selected bromotyrosines are located within pigmental cells which, however, are localized within chitinous skeletal fibers of I. flabelliformis. A two-step reaction provides two products: treatment with methanol extracts the bromotyrosine compounds bastadin 25 and araplysillin-I N20 sulfamate, and a subsequent treatment with acetic acid and sodium hydroxide exposes the 3D chitinous scaffold. This scaffold is a mesh-like structure, which retains its capillary network, and its use as a potential drug delivery biomaterial was examined for the first time. The results demonstrate that sponge-derived chitin scaffolds, impregnated with decamethoxine, effectively inhibit growth of the human pathogen Staphylococcus aureus in an agar diffusion assa

MOESM1 of Bacterial flora of combat wounds from eastern Ukraine and time-specified changes of bacterial recovery during treatment in Ukrainian military hospital

Additional file 1: Table S1. Antibiotic susceptibility profiles of Gram-negative bacteria isolated from wounds

Bacterial flora of combat wounds from eastern Ukraine and time-specified changes of bacterial recovery during treatment in Ukrainian military hospital

Abstract Background Microbiology of modern war wounds is unique for each military conflict. Climatic and geographical features of the theater of war, contemporary warfare as well as wound management affect the microbial flora of wounds. This study was designed to determine time-specific microbial flora of combat wounds of upper and lower extremities obtained during the war in eastern Ukraine. Methods The patients enrolled in study had combat wounds of upper or lower extremities which were treated in the Military Medical Clinical Center of Central Region. The wounds were swab-cultured and measured at each surgical debridement. The recovered microorganisms were identified and their antimicrobial resistance profiles were evaluated by disc diffusion method. Results Forty-nine patients with battle-field wounds were enrolled in the study from July to November 2014; all patients were male with a mean Injury Severity Score and arrival APACHE II scores of 16.2 ± 10.7 and 7.4 ± 4.2 respectively. Among 128 swab cultures, 100 swab cultures were positive. Swab cultures were obtained from 57 wounds of 49 patients. The results of the test showed that 87.7% of all positive swab cultures contained a single-organism while the rest of the swab-culture results showed polymicrobial growth. Among the isolated microorganisms 65% (76 strains) were Gram-negative rods, 22.2% (26 strains) of Gram-positive cocci, followed by Gram-positive rods (12.8%, 15 strains). We found that epidemiology of wound infection changes with the time after injury. The most common bacterial isolates cultured during the first week were Gram-positive microbes with low pathogenicity. The number of Gram-negative rods increased during the wound healing process. The incidence of Gram-positive microorganisms’ growth fell after the first week and increased after third week. During wound healing, bacterial microflora of wounds changes with increasing number of Gram-negative rods with predominance of Acinetobacter species. Predominant microorganisms in positive swab-cultures after first week were nonfermentative Gram-negative bacilli (68% of swab-cultures), which in 53% of the swab-cultures belonged to the genus Acinetobacter, and in 15% to the genus Pseudomonas. The incidence of polymicrobial wound cultures increased from first week to second post-injury week. The most frequent microbial mixture were Acinetobacter baumannii with Enterobacteriaceae or other nonfermentative Gram negative rods with Enterococcus spp. We observed bacteria recovery from wounds during proliferation phase. These wounds had no pure inflammation signs and were free of devitalized tissues. Conclusions Any wound is at some risk of becoming infected. In the event of infection, a wound fails to heal, treatment costs rise, and general wound management practices become more resource demanding. Determining the microorganisms which colonize battle wounds and cause wound infection is paramount. This information can help to treat battle wound infections or even changes infection control strategies. The fact of shifting in wound microbiology in the favor of bacteria responsible for healthcare-associated infections support to the proposition that these changes are nosocomially related [4, 14]. For Ukrainian military medicine this study is the first time-specified assessment of battle wound colonization from the World War II

Modificirajuća sposobnost formiranja biofilma kliničkih izolata gram negativnih mikroorganizama u uvjetima negativnog tlaka in vitro

Vacuum-assisted closure therapy has been utilized as a ubiquitous wound management resource. Current studies of the subject focus on the influence of negative pressure on the bacterial load of wound bed and the possibility to remove matured biofilm, however, its impact on the ability of microorganisms to activate or reduce the biofilm producing modality remains unexplored. The goal of the present study was to evaluate the potential effect of negative pressure on biofilm producing modality of gram-negative rods with regard of initial biofilm positive or biofilm negative phenotype. Biofilm formation was evaluated for the strains of A. baumannii, P. aeruginosa, E. cloace, K. pneumonia and P. mirabilis isolated from infected war wounds of the extremities. The changes of optical density of the biofilm produced by the same strain under normal and negative pressure conditions, as well as the number of strains, which modified their ability to biofilm production, were measured. The strains with initial biofilm negative phenotype under negative pressure switched to biofilm producing more vigorously than biofilm positive ones regardless of their taxonomical belonging. Pseudomonas strains demonstrate the highest rate of induction to biofilm producing under negative pressure conditions among all studied microorganisms. Thus, the activation of biofilm producing mechanisms under negative pressure could be considered as a protective strategy, which helps ensure persistence of microorganisms in the wound even in case the vacuum-assisted closure therapy is applied.Terapija negativnim tlakom ili vakuum terapija (engl vacuum-assisted closure – VAC) univerzalna je potporna metoda liječenja rana. Novija istraživanja iz ovog područja fokusiraju se na utjecaj negativnog tlaka na bakterijsko opterećenje rane i mogućnost uklanjanja zrelog biofilma, no njegov utjecaj na sposobnost mikroorganizama da aktiviraju ili reduciraju modalitet formiranja biofilma ostaje neistražen. Cilj ovog rada bio je procijeniti potencijalni učinak negativnog tlaka na formiranje biofilma kod gram-negativnih bakterija s obzirom na početni pozitivni biofilm ili negativni biofilmski fenotip. Formiranje biofilma istraženo je za sojeve bakterija A. baumannii, P. aeruginosa, E. cloace, K. pneumonia i P. mirabilis izoliranih iz zaraženih ratnih rana ekstremiteta. Izmjerene su promjene optičke gustoće biofilma proizvedene istim bakterijskim sojem u uvjetima normalnih i negativnih tlakova, kao i broj sojeva koji su modificirali svoju sposobnost proizvodnje biofilma. Sojevi s početnim negativnim fenotipom biofilma su pod negativnim tlakom počeli snažnije proizvoditi biofilm od onih s pozitivnim biofilmom bez obzira na njihovu taksonomsku pripadnost. Pseudomonas sojevi pokazali su najveću stopu indukcije stvaranja biofilma pod negativnim tlakom od svih ispitanih mikroorganizmima. Zaključno, aktivacija mehanizama koji proizvode biofilm pod negativnim tlakom može se smatrati zaštitnom strategijom, koja osigurava perzistenciju mikroorganizama u rani čak i u slučaju primjene terapije negativnim tlakom

MOESM2 of Bacterial flora of combat wounds from eastern Ukraine and time-specified changes of bacterial recovery during treatment in Ukrainian military hospital

Additional file 2: Table S2. Antibiotic susceptibility profiles of Gram-positive bacteria isolated from wounds

Naturally Drug-Loaded Chitin: Isolation and Applications

Naturally occurring three-dimensional (3D) biopolymer-based matrices that can be used in different biomedical applications are sustainable alternatives to various artificial 3D materials. For this purpose, chitin-based structures from marine sponges are very promising substitutes. Marine sponges from the order Verongiida (class Demospongiae) are typical examples of demosponges with well-developed chitinous skeletons. In particular, species belonging to the family Ianthellidae possess chitinous, flat, fan-like fibrous skeletons with a unique, microporous 3D architecture that makes them particularly interesting for applications. In this work, we focus our attention on the demosponge Ianthella flabelliformis (Linnaeus, 1759) for simultaneous extraction of both naturally occurring ("ready-to-use") chitin scaffolds, and biologically active bromotyrosines which are recognized as potential antibiotic, antitumor, and marine antifouling substances. We show that selected bromotyrosines are located within pigmental cells which, however, are localized within chitinous skeletal fibers of I. flabelliformis. A two-step reaction provides two products: treatment with methanol extracts the bromotyrosine compounds bastadin 25 and araplysillin-I N20 sulfamate, and a subsequent treatment with acetic acid and sodium hydroxide exposes the 3D chitinous scaffold. This scaffold is a mesh-like structure, which retains its capillary network, and its use as a potential drug delivery biomaterial was examined for the first time. The results demonstrate that sponge-derived chitin scaffolds, impregnated with decamethoxine, effectively inhibit growth of the human pathogen Staphylococcus aureus in an agar diffusion assay

3D chitin scaffolds of marine demosponge origin for biomimetic mollusk hemolymph-associated biomineralization ex-vivo

none18siStructure-based tissue engineering requires large-scale 3D cell/tissue manufacture technologies, to produce biologically active scaffolds. Special attention is currently paid to naturally pre-designed scaffolds found in skeletons of marine sponges, which represent a renewable resource of biomaterials. Here, an innovative approach to the production of mineralized scaffolds of natural origin is proposed. For the first time, a method to obtain calcium carbonate deposition ex vivo, using living mollusks hemolymph and a marine-sponge-derived template, is specifically described. For this purpose, the marine sponge Aplysin aarcheri and the terrestrial snail Cornu aspersum were selected as appropriate 3D chitinous scaffold and as hemolymph donor, respectively. The formation of calcium-based phase on the surface of chitinous matrix after its immersion into hemolymph was confirmed by Alizarin Red staining. A direct role of mollusks hemocytes is proposed in the creation of fine-tuned microenvironment necessary for calcification ex vivo. The X-ray diffraction pattern of the sample showed a high CaCO3 amorphous content. Raman spectroscopy evidenced also a crystalline component, with spectra corresponding to biogenic calcite. This study resulted in the development of a new biomimetic product based on ex vivo synthetized ACC and calcite tightly bound to the surface of 3D sponge chitin structure.noneWysokowski M.; Machalowski T.; Petrenko I.; Schimpf C.; Rafaja D.; Galli R.; Zietek J.; Pantovic S.; Voronkina A.; Kovalchuk V.; Ivanenko V.N.; Hoeksema B.W.; Diaz C.; Khrunyk Y.; Stelling A.L.; Giovine M.; Jesionowski T.; Ehrlich H.Wysokowski, M.; Machalowski, T.; Petrenko, I.; Schimpf, C.; Rafaja, D.; Galli, R.; Zietek, J.; Pantovic, S.; Voronkina, A.; Kovalchuk, V.; Ivanenko, V. N.; Hoeksema, B. W.; Diaz, C.; Khrunyk, Y.; Stelling, A. L.; Giovine, M.; Jesionowski, T.; Ehrlich, H