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One of the leading causes of failure for any bone implant is implant-associated infections. The implant-bone interface is in fact the crucial site of infection where both the microorganisms and cells compete to populate the newly introduced implant surface. Most of the work dealing with this issue has focused on the design of implant coatings capable of preventing infection while ignoring cell proliferation or vice versa. The present study is therefore focused on investigating the antibacterial and biological properties of nanocomposite coatings based on an amorphous hydrocarbon (a-C:H) matrix containing silver nanoparticles (AgNPs). a-C:H coatings with varying silver concentrations were generated directly on medical grade titanium substrates using a combination of a gas aggregation source (GAS) and a plasma-enhanced chemical vapor deposition (PE-CVD) process. The obtained results revealed that the surface silver content increased from 1.3 at % to 5.3 at % by increasing the used DC magnetron current in the GAS from 200 to 500 mA. The in vitro antibacterial assays revealed that the nanocomposites with the highest number of silver content exhibited excellent antibacterial activities resulting in a 6-log reduction of Escherichia coli and a 4-log reduction of Staphylococcus aureus after 24 h of incubation. An MTT assay, fluorescence live/dead staining, and SEM microscopy observations of MC3T3 cells seeded on the uncoated and coated Ti substrates also showed that increasing the amount of AgNPs in the nanocomposites had no notable impact on their cytocompatibility, while improved cell proliferation was especially observed for the nanocomposites possessing a low amount of AgNPs. These controllable Ag/a-C:H nanocomposites on Ti substrates, which simultaneously provide an excellent antibacterial performance and good biocompatibility, could thus have promising applications in orthopedics and other biomedical implants.

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.

Clonal outbreaks of fluconazole-resistant (FLZR) Candida parapsilosis isolates have been reported in several countries. Despite its being the second leading cause of candidemia, the azole resistance mechanisms and the clonal expansion of FLZR C. parapsilosis blood isolates have not been reported in Turkey. In this study, we consecutively collected C. parapsilosis blood isolates (n = 225) from the fifth largest hospital in Turkey (2007 to 2019), assessed their azole susceptibility pattern using CLSI M27-A3/S4, and sequenced ERG11 for all and MRR1, TAC1, and UPC2 for a selected number of C. parapsilosis isolates. The typing resolution of two widely used techniques, amplified fragment length polymorphism typing (AFLP) and microsatellite typing (MST), and the biofilm production of FLZR isolates with and without Y132F were compared. Approximately 27% of isolates were FLZR (60/225), among which 90% (54/60) harbored known mutations in Erg11, including Y132F (24/60) and Y132F+K143R (19/60). Several mutations specific to FLZR isolates were found in MRR1, TAC1, and UPC2. AFLP grouped isolates into two clusters, while MST revealed several clusters. The majority of Y132F/Y132F+K143R isolates grouped in clonal clusters, which significantly expanded throughout 2007 to 2019 in neonatal wards. Candida parapsilosis isolates carrying Y132F were associated with significantly higher mortality and less biofilm production than other FLZR isolates. Collectively, we documented the first outbreak of FLZR C. parapsilosis blood isolates in Turkey. The MRR1, TAC1, and UPC2 mutations exclusively found in FLZR isolates establishes a basis for future studies, which will potentially broaden our knowledge of FLZR mechanisms in C. parapsilosis. MST should be a preferred method for clonal analysis of C. parapsilosis isolates in outbreak scenarios. Copyright © 2020 American Society for Microbiology. All Rights Reserved., Shanghai Municipal Health and Family Planning Commission 2018ZX10101003 Science and Technology Commission of Shanghai Municipality, STCSM: 17DZ2272900, 14495800500 National Natural Science Foundation of China, NSFC: 31770161, This work was supported by the Major National R&D Projects of the National Health Department (2018ZX10101003), National Natural Science Foundation of China (31770161), Shanghai Science and Technology Committee (17DZ2272900 and 14495800500), Shanghai Municipal Commission of Health and Family Planning

Infection associated with titanium based implants remains the most serious problem in implant surgery hence it is important to find optimal strategies to prevent infections. In the present study, we investigated the surface properties, antibacterial activity and biocompatibility of nanocomposite coatings based on an amorphous hydrocarbon (a-C:H) film containing copper nanoparticles (CuNPs) deposited on Ti discs via a gas aggregation cluster source. Three different Cu/a-C:H coatings with approximately the same amount of embedded CuNPs with and without barrier a-C:H layer were fabricated. The obtained results revealed that different structures of the produced coatings have significantly different release rates of Cu ions from the coatings into the aqueous media. This subsequently influences the antibacterial efficiency and osteoblast cell viability of the treated coatings. Coatings with the highest number of CuNPs resulted in excellent antibacterial activity exhibiting approximately 4 log reduction of E.coli and S.aureus after 24 h incubation. The cytotoxicity study revealed that after 7 day cell seeding, even the coating with the highest Cu at.% (4 at.%) showed a cell viability of 90%. Consequently, the coating, formed with a properly tailored number of CuNPs and a-C:H barrier thickness offer a strong antibacterial effect without any harm to osteoblast cells.

Bacteria communicate with each other using quorum sensing; i.e. the production and sensing of signalling molecules. Enterococcus faecalis, a Gram-positive bacterium, employs peptides as quorum sensing molecules. These peptides have previously been isolated from culture media by elaborate, time and medium-consuming sample preparation approaches and specific bacteria-based bio-sensors. Here, a method for the detection and quantification of all nine currently reported E. faecalis quorum sensing peptides belonging to the RNPP family in bacterial cell culture medium was developed. The approach developed consists of solid-phase extraction (SPE) sample preparation followed by a UHPLC-triple quadrupole mass spectroscopic method, operated in Multiple Reaction Monitoring (MRM) mode. All nine peptides were quantified with a total analysis time below 90 min per sample and limited cell culture medium volumes of only 1 ml per sample. A method verification, performed in uniplicate, was carried out to obtain an idea of the method performance. The recovery varied between 19.9 and 119.0%, and the limit of detection is in the low nM range. Analytical stability, carry-over and dilution integrity were investigated and were acceptable. This method will be a useful tool in the investigation of the roles of the RNPP-type quorum sensing peptides in microbial processes.

Staphylococcus aureus biofilms are involved in a wide range of infections that are extremely difficult to treat with conventional antibiotic therapy. We aimed to identify potentiators of antibiotics against mature biofilms of S. aureus Mu50, a methicillin-resistant and vancomycin-intermediate-resistant strain. Over 700 off-patent drugs from a repurposing library were screened in combination with vancomycin in a microtitre plate (MTP)-based biofilm model system. This led to the identification of 25 hit compounds, including four phenothiazines among which thioridazine was the most potent. Their activity was evaluated in combination with other antibiotics both against planktonic and biofilm-grown S. aureus cells. The most promising combinations were subsequently tested in an in vitro chronic wound biofilm infection model. Although no synergistic activity was observed against planktonic cells, thioridazine potentiated the activity of tobramycin, linezolid and flucloxacillin against S. aureus biofilm cells. However, this effect was only observed in a general biofilm model and not in a chronic wound model of biofilm infection. Several drug compounds were identified that potentiated the activity of vancomycin against biofilms formed in a MTP-based biofilm model. A selected hit compound lost its potentiating activity in a model that mimics specific aspects of wound biofilms. This study provides a platform for discovering and evaluating potentiators against bacterial biofilms and highlights the necessity of using relevant in vitro biofilm model systems.

The objective of this study was the development of Ag-rich antibacterial coatings on titanium to prevent post-operative infections. A series of Ag-doped TiO2 coatings were synthesized on Ti discs by plasma electrolytic oxidation in an electrolyte containing AgNPs. The incorporation, distribution and chemical composition of the AgNPs on Ti were determined using scanning electron microscopy-energy dispersive spectroscopy. The crystalline structure and wettability of the coating was characterized by X-ray diffraction and water contact angle analysis respectively. Surface roughness and hardness of the coating were examined using surface profilometry and Knoop indentation test respectively, while silver ion release was quantified using inductively coupled plasma-mass spectroscopy. Following PEO, the surface of the Ti substrate was converted to TiO2 composed of anatase and rutile phases. The SEM micrographs showed that the AgNPs were distributed throughout the oxide layer, without changing the morphology of the coating. The coatings also revealed an increased surface roughness, microhardness and improved surface wettability relative to untreated Ti substrates. Furthermore, the incorporation of Ag into the coating did not alter the phase component, roughness, microhardness and wettability. A series of in-vitro antibacterial assays indicated that increasing the number of AgNPs in the electrolyte led to excellent antibacterial activities.

The success of an orthopedic implant therapy depends on successful bone integration and the prevention of microbial infections. In this work, plasma electrolytic oxidation (PEO) was performed to deposit TiO2 coatings enriched with Ca, P, and Ag on titanium to improve its surface properties and antibacterial blasts efficacy while maintaining normal biological functions and thus to enhance the performance of orthopedic implants. After PEO treatment, the surface of Ti was converted to anatase and rutile TiO2, hydroxyapatite, and calcium titanate phases. The presence of these crystalline phases was further increased with an increased Ag content in the coatings. The developed coatings also exhibited a more porous morphology with an improved surface wettability, roughness, microhardness, and frictional coefficient. In vitro antibacterial assays indicated that the Ag-doped coatings can significantly prevent the growth of both Staphylococcus aureus and Escherichia coli by releasing Ag+ ions, and the ability to prevent these bacteria was enhanced by increasing the Ag content in the coatings, resulting in a maximal 6-log reduction of E. coli and a maximal 5-log reduction of S. aureus after 24 h of incubation. Moreover, the in vitro cytocompatibility evaluation of the coatings showed that the osteoblast (MC3T3) cell integration on the PEO-based coatings was greatly improved compared to untreated Ti and no notable impact on their cytocompatibility was observed on increasing the amount of Ag in the coating. In conclusion, the coating with favorable physicochemical and mechanical properties along with controlled silver ion release can offer an excellent antibacterial performance and osteocompatibility and can thus become a prospective coating strategy to face current challenges in orthopedics.

International audience; Twelve new quaternary ammonium sophorolipids with long alkyl chains on the nitrogen atom were synthesized starting from oleic and petroselinic acid based sophorolipids. These novel derivatives were evaluated for their antimicrobial activity against selected Gram-negative and Gram-positive bacteria and their transfection efficacies on three different eukaryotic cell lines in vitro since good activities were demonstrated for previously synthesized derivatives. Self-assembly properties were also evaluated. All compounds proved to possess antimicrobial and transfection properties, and trends could be observed based on the length of the nitrogen substituent and the total length of the sophorolipid tail. Moreover, all long-chain quaternary ammonium sophorolipids form micelles, which proved to be a prerequisite to induce antimicrobial activity and transfection capacity. These results are promising for future healthcare applications of long-chained quaternary ammonium sophorolipids.

Plasma polymerization is gaining popularity as a technique for coating surfaces due to the low cost, ease of operation, and substrate-independent nature. Recently, the plasma polymerization (or deposition) of 2-oxazoline monomers was reported resulting in coatings that have potential applications in regenerative medicine. Despite the structural versatility of 2-oxazolines, only a few monomers have been subjected to plasma polymerization. Within this study, however, we explore the near atmospheric pressure plasma polymerization of a range of 2-oxazoline monomers, focusing on the influence of the aliphatic side-chain length (methyl to butyl) on the plasma polymerization process conditions as well as the properties of the obtained coatings. While side-chain length had only a minor influence on the chemical composition, clear effects on the plasma polymerization conditions were observed, thus gaining valuable insights in the plasma polymerization process as a function of monomer structure. Additionally, cytocompatibility and cell attachment on the coatings obtained by 2-oxazoline plasma polymerization was assessed. The coatings displayed strong cell interactive properties, whereby cytocompatibility increased with increasing aliphatic side-chain length of the monomer, reaching up to 93% cell viability after 1 day of cell culture compared to tissue culture plates. As this is in stark contrast to the antifouling behavior of the parent polymers, we compared the properties and composition of the plasma-polymerized coatings to the parent polymers revealing that a significantly different coating structure was obtained by plasma polymerization.

Antimicrobial peptides (AMPs) are promising templates for the development of novel antibiofilm drugs. Despite the large number of studies on screening and optimization of AMPs, only a few of these evaluated the antibiofilm activity in physiologically relevant model systems. Potent in vitro activity of AMPs often does not translate into in vivo effectiveness due to the interference of the host microenvironment with peptide stability/availability. Hence, mimicking the complex environment found in biofilm-associated infections is essential to predict the clinical potential of novel AMP-based antimicrobials. In the present study, we examined the antibiofilm activity of the semisynthetic peptide lin-SB056-1 and its dendrimeric derivative (lin-SB056-1)(2)-K against Pseudomonas aeruginosa in an in vivo-like three-dimensional (3-D) lung epithelial cell model and an in vitro wound model (consisting of an artificial dermis and blood components at physiological levels). Although moderately active when tested alone, lin-SB056-1 was effective in reducing P aeruginosa biofilm formation in association with 3-D lung epithelial cells in combination with the chelating agent EDTA. The dimeric derivative (Iin-SB056-1)(2)-K demonstrated an enhanced biofilm-inhibitory activity as compared to both lin-SB056-1 and the lin-SB056-1/EDTA combination, reducing the number of biofilm-associated bacteria up to 3-Log units at concentrations causing less than 20% cell death. Biofilm inhibition by (lin-SB056-1)(2)-K was reported both for the reference strain PAO1 and cystic fibrosis lung isolates of P aeruginosa. In addition, using fluorescence microscopy, a significant decrease in biofilm-like structures associated with 3-D cells was observed after peptide exposure. Interestingly, effectiveness of (lin-SB056-1)(2)-K was also demonstrated in the wound model with a reduction of up to 1-Log unit in biofilm formation by P aeruginosa PAO1 and wound isolates. Overall, combination treatment and peptide dendrimerization emerged as promising strategies to improve the efficacy of AMPs, especially under challenging host-mimicking conditions. Furthermore, the results of the present study underlined the importance of evaluating the biological properties of novel AMPs in in vivo-like model systems representative of specific infectious sites in order to make a more realistic prediction of their therapeutic success, and avoid the inclusion of unpromising peptides in animal studies and clinical trials.

New synthetic pathways are proposed for the synthesis of a new set of bolaamphiphilic derivatives starting from microbiologically produced sophorolipids. A total set of 43 new derivatives was synthesized Via reductive amination of a previously synthesized sophorolipid aldehyde with diamines and primary amines. The new derivatives were evaluated for their antimicrobial activity against Gram-negative and Gram-positive bacteria. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were determined for the active compounds. Transfection efficiencies were also evaluated for some of the deprotected derivatives via the assessment of their capacity to transfect three different eukaryotic cell lines in vitro. Finally, the self-assembly properties were evaluated for the deprotected derivatives. Antimicrobial activities were mostly observed for the peracetylated mono- or dicationic bolaamphiphiles and only the deprotected monocationic bolaamphiphile with an octadecyl chain on the nitrogen atom was eligible for the evaluation of its transfection properties. Evaluation of the self-assembly properties indicated that the presence of an octadecyl chain was necessary for micelle formation. Both micelle formation and the net charge of the compounds seem to have an influence on the antimicrobial activity and transfection efficiency. These results are promising for use of bolaamphiphilic sophorolipids in medical and self-assembly applications., Dans ce travail de nouveaux composés amphiphiles cationiques sont préparés à partir de sophoro lipides. Ces composés sont formulés en solution liposomales et évaluées pour leurs propriétés bactéricides et pour la transfection. Des analyses physico-chimiques montrent des assemblages sous forme de micelles

In young cystic fibrosis (CF) patients, Staphylococcus aureus is typically the most prevalent organism, while in adults, Pseudomonas aeruginosa is the major pathogen. More recently, it was observed that also Streptococcus anginosus plays an important role in exacerbations of respiratory symptoms. These species are often coisolated from CF lungs, yet little is known about whether antibiotic killing of one species is influenced by the presence of others. In the present study, we compared the activities of various antibiotics against S. anginosus , S. aureus , and P. aeruginosa when grown in monospecies biofilms with the activity observed in a multispecies biofilm. Our results show that differences in antibiotic activity against species grown in mono- and multispecies biofilms are species and antibiotic dependent. Fewer S. anginosus cells are killed by antibiotics that interfere with cell wall synthesis (amoxicillin plus sulbactam, cefepime, imipenem, meropenem, and vancomycin) in the presence of S. aureus and P. aeruginosa , while for ciprofloxacin, levofloxacin, and tobramycin, no difference was observed. In addition, we observed that the cell-free supernatant of S. aureus , but not that of P. aeruginosa biofilms, also caused this decrease in killing. Overall, S. aureus was more affected by antibiotic treatment in a multispecies biofilm, while for P. aeruginosa , no differences were observed between growth in mono- or multispecies biofilms. The results of the present study suggest that it is important to take the community composition into account when evaluating the effect of antimicrobial treatments against certain species in mixed biofilms.

In vitro models that mimic in vivo host-pathogen interactions are needed to evaluate candidate drugs that inhibit bacterial virulence traits. We established a new approach to study Pseudomonas aeruginosa biofilm susceptibility on biotic surfaces, using a three-dimensional (3-D) lung epithelial cell model. P. aeruginosa formed antibiotic resistant biofilms on 3-D cells without affecting cell viability. The biofilm-inhibitory activity of antibiotics and/or the anti-biofilm peptide DJK-5 were evaluated on 3-D cells compared to a plastic surface, in medium with and without fetal bovine serum (FBS). In both media, aminoglycosides were more efficacious in the 3-D cell model. In serum-free medium, most antibiotics (except polymyxins) showed enhanced efficacy when 3-D cells were present. In medium with FBS, colistin was less efficacious in the 3-D cell model. DJK-5 exerted potent inhibition of P. aeruginosa association with both substrates, only in serum-free medium. DJK-5 showed stronger inhibitory activity against P. aeruginosa associated with plastic compared to 3-D cells. The combined addition of tobramycin and DJK-5 exhibited more potent ability to inhibit P. aeruginosa association with both substrates. In conclusion, lung epithelial cells influence the efficacy of most antimicrobials against P. aeruginosa biofilm formation, which in turn depends on the presence or absence of FBS.

Infections related to fungal biofilms are difficult to treat due to the reduced susceptibility of sessile cells to most antifungal agents. Previous research has shown that 1-10% of sessile Candida cells survive treatment with high doses of miconazole (a fungicidal imidazole). The aim of this study was to identify genes involved in fungal biofilm formation and to unravel the mechanisms of resistance of these biofilms to miconazole. To this end, a screening of a Saccharomyces cerevisiae deletion mutant bank was carried out. Our results revealed that genes involved in peroxisomal transport and the biogenesis of the respiratory chain complex IV play an essential role in biofilm formation. On the other hand, genes involved in transcription and peroxisomal and mitochondrial organization seem to highly influence the susceptibility to miconazole of yeast biofilms. Additionally, our data confirm previous findings on genes involved in biofilm formation and in general stress responses. Our data suggest the involvement of peroxisomes in biofilm formation and miconazole resistance in fungal biofilms.

Propionibacterium acnes is a Gram-positive bacterium that plays an important role in the pathogenesis of acne vulgaris. This organism is capable of biofilm formation and the decreased antimicrobial susceptibility of biofilm-associated cells may hamper efficient treatment. In addition, the prolonged use of systemic antibiotic therapy is likely to lead to the development and spread of antimicrobial resistance. In the present study we investigated whether P. acnes biofilms could be eradicated by plant extracts or their active compounds, and whether other mechanisms besides killing of biofilm cells could be involved. Out of 119 plant extracts investigated, we identified five with potent antibiofilm activity against P. acnes (extracts from Epimedium brevicornum, Malus pumila, Polygonum cuspidatum, Rhodiola crenulata and Dolichos lablab). We subsequently identified icariin, resveratrol and salidroside as active compounds in three of these extracts. Extracts from E. brevicornum and P. cuspidatum, as well as their active compounds (icariin and resveratrol, respectively) showed marked antibiofilm activity when used in subinhibitory concentrations, indicating that killing of microbial cells is not their only mode of action.

We report that certain anthraquinones (AQs) reduce Streptococcus mutans biofilm formation on hydroxyapatite at concentrations below the MIC. Although AQs are known to generate reactive oxygen species, the latter do not underlie the observed effect. Our results suggest that AQs inhibit S. mutans biofilm formation by causing membrane perturbation.