Activated polyhydroxyalkanoate meshes prevent bacterial adhesion and biofilm development in regenerative medicine applications

Regenerative medicine has become an extremely valuable tool offering an alternative to conventional therapies for the repair and regeneration of tissues. The re-establishment of tissue and organ functions can be carried out by tissue engineering strategies or by using medical devices such as implants. However, with any material being implanted inside the human body, one of the conundrums that remains is the ease with which these materials can get contaminated by bacteria. Bacterial adhesion leads to the formation of mature, alive and complex three-dimensional biofilm structures, further infection of surrounding tissues and consequent development of complicated chronic infections. Hence, novel tissue engineering strategies delivering biofilm-targeted therapies, while at the same time allowing tissue formation are highly relevant. In this study our aim was to develop surface modified polyhydroxyalkanoate-based fiber meshes with enhanced bacterial anti-adhesive and juvenile biofilm disrupting properties for tissue regeneration purposes. Using reactive and amphiphilic star-shaped macromolecules as an additive to a polyhydroxyalkanoate spinning solution, a synthetic antimicrobial peptide, Amhelin, with strong bactericidal and anti-biofilm properties, and Dispersin B, an enzyme promoting the disruption of exopolysaccharides found in the biofilm matrix, were covalently conjugated to the fibers by addition to the solution before the spinning process. Staphylococcus epidermidis is one of the most problematic pathogens responsible for tissue-related infections. The initial antibacterial screening showed that Amhelin proved to be strongly bactericidal at 12 μg/ml and caused >50% reductions of biofilm formation at 6 μg/ml, while Dispersin B was found to disperse >70% of pre-formed biofilms at 3 μg/ml. Regarding the cytotoxicity of the agents toward L929 murine fibroblasts, a CC50 of 140 and 115 μg/ml was measured for Amhelin and Dispersin B, respectively. Optimization of the electrospinning process resulted in aligned fibers. Surface activated fibers with Amhelin and Dispersin B resulted in 83% reduction of adhered bacteria on the surface of the fibers. Additionally, the materials developed were found to be cytocompatible toward L929 murine fibroblasts. The strategy reported in this preliminary study suggests an alternative approach to prevent bacterial adhesion and, in turn biofilm formation, in materials used in regenerative medicine applications such as tissue engineering

In situ Forming Hyperbranched PEG—Thiolated Hyaluronic Acid Hydrogels With Honey-Mimetic Antibacterial Properties

The rapidly increasing resistance of bacteria to currently approved antibiotic drugs makes surgical interventions and the treatment of bacterial infections increasingly difficult. In recent years, complementary strategies to classical antibiotic therapy have, therefore, gained importance. One of these strategies is the use of medicinal honey in the treatment of bacterially colonized wounds. One of the several bactericidal effects of honey is based on the in situ generation of hydrogen peroxide through the activity of the enzyme glucose oxidase. The strategy underlying this work is to mimic this antibacterial redox effect of honey in an injectable, biocompatible, and rapidly forming hydrogel. The hydrogel was obtained by thiol–ene click reaction between hyperbranched polyethylene glycol diacrylate (HB PEGDA), synthesized using reversible addition-fragmentation chain transfer (RAFT) polymerization, and thiolated hyaluronic acid (HA-SH). After mixing 500 µL HB PEGDA (10%, w/w) and 500 µL HA-SH (1%, w/w) solutions, hydrogels formed in ∼60 s (HB PEGDA/HA-SH 10.0–1.0), as assessed by the tube inverting test. The HB PEGDA/HA-SH 10.0–1.0 hydrogel (200 µL) was resistant to in vitro dissolution in water for at least 64 days, absorbing up to 130 wt% of water. Varying glucose oxidase (GO) amounts (0–500 U/L) and constant glucose content (2.5 wt%) were loaded into HB PEGDA and HA-SH solutions, respectively, before hydrogel formation. Then, the release of H2O2 was evaluated through a colorimetric pertitanic acid assay. The GO content of 250 U/L was selected, allowing the formation of 10.8 ± 1.4 mmol H2O2/L hydrogel in 24 h, under static conditions. The cytocompatibility of HB PEGDA/HA-SH 10.0–1.0 hydrogels loaded with different GO activities (≤ 500 U/L) at a constant glucose amount (2.5 wt%) was investigated by in vitro assays at 24 h with L929 and HaCaT cell lines, according to DIN EN ISO 10993-5. The tests showed cytocompatibility for GO enzyme activity up to 250 U/L for both cell lines. The antibacterial activity of HB PEGDA/HA-SH 10.0–1.0 hydrogels loaded with increasing amounts of GO was demonstrated against various gram-positive bacteria (S. aureus and S. epidermidis), antibiotic-resistant gram-positive bacteria (MRSA and MRSE), gram-negative bacteria (P. aeruginosa, E. coli, and A. baumanii), and antibiotic-resistant gram-negative strains (P. aeruginosa and E. coli) using agar diffusion tests. For all gram-positive bacterial strains, increasing efficacy was measured with increasing GO activity. For the two P. aeruginosa strains, efficacy was shown only from an enzyme activity of 125 U/L and for E. coli and A. baumanii, efficacy was shown only from 250 U/L enzyme activity. HB PEGDA/HA-SH 10.0–1.0 hydrogels loaded with ≤250 U/L GO and 2.5 wt% glucose are promising formulations due to their fast-forming properties, cytocompatibility, and ability to produce antibacterial H2O2, warranting future investigations for bacterial infection treatment, such as wound care

Occurrence of genes of putative fibrinogen binding proteins and hemolysins, as well as of their phenotypic correlates in isolates of S. lugdunensis of different origins

<p>Abstract</p> <p>Background</p> <p><it>Staphylococcus lugdunensis </it>is an important human pathogen that causes potentially fatal endocarditis, osteomyelitis and skin and soft tissue infections similar to diseases caused by <it>Staphylococcus aureus</it>. Nevertheless, in contrast to <it>S. aureus</it>, data on pathogenicity factors of <it>S. lugdunensis </it>is scarce. Two adhesins, a fibrinogen and a von Willebrand factor binding protein, and a <it>S. lugdunensis </it>synergistic hemolysin (SLUSH) have been previously described. Moreover, the newly sequenced genome of <it>S. lugdunensis </it>revealed genes of other putative fibrinogen binding adhesins and hemolysins. The aim of this study was to gain more insight into the occurrence of genes likely coding for fibrinogen binding adhesins and hemolysins using clinical strains of <it>S. lugdunensis</it>.</p> <p>Findings</p> <p>Most of the putative adhesin genes and hemolysin genes investigated in this study were highly prevalent, except for the SLUSH gene cluster. In contrast to previous reports, binding to fibrinogen was detected in 29.3% of the <it>S. lugdunensis </it>strains. In most strains, hemolysis on blood agar plates was weak after 24 h and distinct after 48 h of incubation. The fibrinogen binding and hemolysis phenotypes were also independent of the type of clinical specimen, from which the isolates were obtained.</p> <p>Conclusion</p> <p>In this study we described a pyrrolidonyl arylamidase negative <it>S. lugdunensis </it>isolate. Our data indicate that a matrix-assisted laser desorption ionisation time-of-flight MS-based identification of <it>S. lugdunensis </it>or species-specific PCR's should be performed in favour of pyrrolidonyl arylamidase testing. In contrast to the high occurrence of putative fibrinogen binding protein genes, 29.3% of the <it>S. lugdunensis </it>strains bound to fibrinogen. Putative hemolysin genes were also prevalent in most of the <it>S. lugdunensis </it>strains, irrespective of their hemolysis activity on Columbia blood agar plates. Similar to a previous report, hemolysis after 48 h of incubation is also indicative for <it>S. lugdunensis</it>. The SLUSH gene cluster was detected in an estimated 50% of the strains, indicating that this locus is different or non-prevalent in many strains.</p

SHOCK WAVES IN THE TREATMENT OF STRESS FRACTURES

In soccer players, lower extremity stress fractures are common injuries and are the result of repetitive use damage that exceeds the intrinsic ability of the bone to repair itself. They may be treated conservatively but this may cause long-term complications, such as delayed union, muscle atrophy and chronic pain. Stress fractures that fail to respond to this management require surgical treatment, which is also not without risks and complications. Extracorporeal shock wave therapy (ESWT) has been used successfully on fracture complications, such as delayed union and nonunion. As such, we want to examine ESWT in the management of stress fractures. In this article, we present a retrospective study of 10 athletes affected by chronic stress fractures of the fifth metatarsus and tibia that received three to four sessions of low-middle energy ESWT. At the follow-up (8 wk on average), the clinical and radiography results were excellent and enabled all players to gradually return to sports activities. These reports show that ESWT is a noninvasive and effective treatment for resistant stress fractures in soccer players

Evaluation of six commercial products for colistin susceptibility testing in Enterobacterales

Objectives: The recommended technique for colistin susceptibility testing by both EUCAST and CLSI is broth microdilution (BMD). However, many routine laboratories still use other methods such as gradient strips or semi-automated systems. The objective of this study was to compare six of the most widespread commercial products for colistin susceptibility testing in Europe with in-house prepared BMD. Methods: A collection of 325 carbapenemase-producing Enterobacterales was tested for colistin susceptibility with three semi-automated systems (Vitek 2, BD Phoenix, MicroScan WalkAway), one gradient-strip test (Etest (R)) and two commercial BMD products (MICRONAUT-S, TREK Sensititre). BMD, in-house prepared according to ISO standard 20776-1, served as reference. Results: The MICRONAUT-S BMD performed best with only one false-resistant (major error, ME) and four false-susceptible (very major error, VME) results while the TREK BMD performed poorer with 16 ME and seven VME. The semi-automated systems Vitek 2 and Phoenix performed poorly with 31 and 26 VME, respectively. The WalkAway semi-automated system showed 16 and 13 false results, depending on the inoculation method. The Etest (R) showed six ME and 10 VME. Conclusions: This study shows that colistin susceptibility testing remains a challenging task for laboratories. It emphasizes the need for selecting the most reliable test method to advocate proper treatment and shows that critical evaluation and precautious usage of colistin susceptibility testing results is constantly required. (C) 2019 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved