Publications by authors named "Tom Coenye"

267 Publications

Organic acids and their salts potentiate the activity of selected antibiotics against biofilms grown in a synthetic cystic fibrosis sputum medium.

Antimicrob Agents Chemother 2021 Nov 22:AAC0187521. Epub 2021 Nov 22.

Laboratory of Pharmaceutical Microbiology, Ghent University, Ghent, Belgium.

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http://dx.doi.org/10.1128/AAC.01875-21DOI Listing
November 2021

is an anti-inflammatory bacterium in the respiratory tract of patients with chronic lung disease.

Eur Respir J 2021 Sep 29. Epub 2021 Sep 29.

Laboratory of Pharmaceutical Microbiology, Ghent University, Gent, Belgium

Chronic airway inflammation is the main driver of pathogenesis in respiratory diseases, such as severe asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), and bronchiectasis. While the role of common pathogens in airway inflammation is widely recognized, the influence of other microbiota members is still poorly understood. Here, we show that , a common resident of the oral cavity that is also often detectable in the lower airways in chronic disease, has an inhibitory effect on pathogen- and LPS-induced pro-inflammatory responses, both (3-D cell culture model) and (mouse model). Furthermore, in a cohort of adults with bronchiectasis, the abundance of spp. was negatively correlated with pro-inflammatory markers (IL-8, IL-1β) and matrix metalloproteinases (MMP-1, MMP-8 and MMP-9) in sputum. Mechanistic studies revealed that inhibits NF-κB pathway activation by reducing the phosphorylation of IκB-α and consequently the expression of NF-κB target genes. These findings indicate that the presence of in the lower airways potentially mitigates inflammation, which could in turn influence severity and progression of chronic respiratory disorders.
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http://dx.doi.org/10.1183/13993003.01293-2021DOI Listing
September 2021

Pillar[5]arene-Based Polycationic Glyco[2]rotaxanes Designed as Antibiofilm Agents.

J Med Chem 2021 Oct 20;64(19):14728-14744. Epub 2021 Sep 20.

Department of Chemistry, Laboratory of Bio-Organic Chemistry - NAmur Research Institute for LIfe Sciences (NARILIS), University of Namur (UNamur), Rue de Bruxelles 61, 5000 Namur, Belgium.

(P.A.) is a human pathogen belonging to the top priorities for the discovery of new therapeutic solutions. Its propensity to generate biofilms strongly complicates the treatments required to cure P.A. infections. Herein, we describe the synthesis of a series of novel rotaxanes composed of a central galactosylated pillar[5]arene, a tetrafucosylated dendron, and a tetraguanidinium subunit. Besides the high affinity of the final glycorotaxanes for the two P.A. lectins LecA and LecB, potent inhibition levels of biofilm growth were evidenced, showing that their three subunits work synergistically. An antibiofilm assay using a double ΔΔ mutant compared to the wild type demonstrated that the antibiofilm activity of the best glycorotaxane is lectin-mediated. Such antibiofilm potency had rarely been reached in the literature. Importantly, none of the final rotaxanes was bactericidal, showing that their antibiofilm activity does not depend on bacteria killing, which is a rare feature for antibiofilm agents.
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http://dx.doi.org/10.1021/acs.jmedchem.1c01241DOI Listing
October 2021

The cystic fibrosis lung microenvironment alters antibiotic activity: causes and effects.

Eur Respir Rev 2021 Sep 15;30(161). Epub 2021 Sep 15.

Laboratory of Pharmaceutical Microbiology, Ghent University, Ghent, Belgium

Chronic airway colonisation by , a hallmark of cystic fibrosis (CF) lung disease, is associated with increased morbidity and mortality and despite aggressive antibiotic treatment, is able to persist in CF airways. antibiotic susceptibility assays are poor predictors of antibiotic efficacy to treat respiratory tract infections in the CF patient population and the selection of the antibiotic(s) is often made on an empirical base. In the current review, we discuss the factors that are responsible for the discrepancies between antibiotic activity and clinical efficacy We describe how the CF lung microenvironment, shaped by host factors (such as iron, mucus, immune mediators and oxygen availability) and the microbiota, influences antibiotic activity and varies widely between patients. A better understanding of the CF microenvironment and population diversity may thus help improve antibiotic susceptibility testing and clinical decision making, in turn increasing the success rate of antibiotic treatment.
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http://dx.doi.org/10.1183/16000617.0055-2021DOI Listing
September 2021

Biosynthesis of Ditropolonyl Sulfide, an Antibacterial Compound Produced by Burkholderia cepacia Complex Strain R-12632.

Appl Environ Microbiol 2021 10 15;87(22):e0116921. Epub 2021 Sep 15.

Laboratory of Microbiology, Department of Biochemistry and Microbiology, Faculty of Sciences, Ghent Universitygrid.5342.0, Ghent, Belgium.

Burkholderia cepacia complex strain R-12632 produces ditropolonyl sulfide, an unusual sulfur-containing tropone, via a yet-unknown biosynthetic pathway. Ditropolonyl sulfide purified from a culture of strain R-12632 inhibits the growth of various Gram-positive and Gram-negative resistant bacteria, with MIC values as low as 16 μg/ml. In the present study, we used a transposon mutagenesis approach combined with metabolite analyses to identify the genetic basis for antibacterial activity of strain R-12632 against Gram-negative bacterial pathogens. Fifteen of the 8304 transposon mutants investigated completely lost antibacterial activity against Klebsiella pneumoniae LMG 2095. In these loss-of-activity mutants, nine genes were interrupted. Four of those genes were involved in assimilatory sulfate reduction, two were involved in phenylacetic acid (PAA) catabolism, and one was involved in glutathione metabolism. Via semipreparative fractionation and metabolite identification, it was confirmed that inactivation of the PAA degradation pathway or glutathione metabolism led to loss of ditropolonyl sulfide production. Based on earlier studies on the biosynthesis of tropolone compounds, the requirement for a functional PAA catabolic pathway for antibacterial activity in strain R-12632 indicated that this pathway likely provides the tropolone backbone for ditropolonyl sulfide. Loss of activity observed in mutants defective in assimilatory sulfate reduction and glutathione biosynthesis suggested that cysteine and glutathione are potential sources of the sulfur atom linking the two tropolone moieties. The demonstrated antibacterial activity of the unusual antibacterial compound ditropolonyl sulfide warrants further studies into its biosynthesis and biological role. bacteria are historically known for their biocontrol properties and have been proposed as a promising and underexplored source of bioactive specialized metabolites. Burkholderia cepacia complex strain R-12632 inhibits various Gram-positive and Gram-negative resistant pathogens and produces numerous specialized metabolites, among which is ditropolonyl sulfide. This unusual antimicrobial has been poorly studied and its biosynthetic pathway remains unknown. In the present study, we performed transposon mutagenesis of strain R-12632 and performed genome and metabolite analyses of loss-of-activity mutants to study the genetic basis for antibacterial activity. Our results indicate that phenylacetic acid catabolism, assimilatory sulfate reduction, and glutathione metabolism are necessary for ditropolonyl sulfide production. These findings contribute to understanding of the biosynthesis and biological role of this unusual antimicrobial.
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http://dx.doi.org/10.1128/AEM.01169-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8552904PMC
October 2021

Antistaphylococcal Activity of the FtsZ Inhibitor C109.

Pathogens 2021 Jul 13;10(7). Epub 2021 Jul 13.

Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, 27100 Pavia, Italy.

infections represent a great concern due to their versatility and involvement in different types of diseases. The shortage of available clinical options, especially to treat multiresistant strains, makes the discovery of new effective compounds essential. Here we describe the activity of the previously described cell division inhibitor C109 against methicillin-sensitive and -resistant strains. Antibiofilm activity was assessed using microtiter plates, confocal microscopy, and in an in vitro biofilm wound model. The ability of C109 to block FtsZ GTPase activity and polymerization was tested in vitro. Altogether, the results show that the FtsZ inhibitor C109 has activity against a wide range of strains and support its use as an antistaphylococcal compound.
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http://dx.doi.org/10.3390/pathogens10070886DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8308607PMC
July 2021

Dynamic Constitutional Frameworks as Antibacterial and Antibiofilm Agents.

Angew Chem Int Ed Engl 2021 10 2;60(41):22505-22512. Epub 2021 Sep 2.

Faculty of Science, University of Namur, Rue de Bruxelles, 61, Namur, Belgium.

Dynamic constitutional frameworks (DCFs) were synthesized and screened for biofilm inhibition or disruption. They are composed of a trialdehyde core reversibly linked to a diamine PEG connector and to a variety of neutral, anionic, or cationic heads, to generate a library of DCFs to generate multivalent dendritic architectures in the presence of Pseudomonas aeruginosa and Staphylococcus aureus. The best DCFs were always polycationic and the nature of the cationic heads significantly impact the antibiofilm activity. The best antibiofilm activity was observed for DCF3B, displaying a polyethyleneimine head. A simple inactive guanidinium functional head strongly inhibited biofilm growth when assayed as a multivalent DCF3C. Using a more advanced in vitro biofilm model of chronic wound infection, DCF3C was found significantly superior than all other DCFs. These results demonstrate the versatility and effectiveness of DCFs as low cost and efficient systems for antibiofilm disruption.
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http://dx.doi.org/10.1002/anie.202109518DOI Listing
October 2021

Interlaboratory study for the evaluation of three microtiter plate-based biofilm quantification methods.

Sci Rep 2021 07 2;11(1):13779. Epub 2021 Jul 2.

Center for Biofilm Engineering, Montana State University, 366 Barnard Hall, Bozeman, MT, 59717, USA.

Microtiter plate methods are commonly used for biofilm assessment. However, results obtained with these methods have often been difficult to reproduce. Hence, it is important to obtain a better understanding of the repeatability and reproducibility of these methods. An interlaboratory study was performed in five different laboratories to evaluate the reproducibility and responsiveness of three methods to quantify Staphylococcus aureus biofilm formation in 96-well microtiter plates: crystal violet, resazurin, and plate counts. An inter-lab protocol was developed for the study. The protocol was separated into three steps: biofilm growth, biofilm challenge, biofilm assessment. For control experiments participants performed the growth and assessment steps only. For treatment experiments, all three steps were performed and the efficacy of sodium hypochlorite (NaOCl) in killing S. aureus biofilms was evaluated. In control experiments, on the log-scale, the reproducibility SD (S) was 0.44 for crystal violet, 0.53 for resazurin, and 0.92 for the plate counts. In the treatment experiments, plate counts had the best responsiveness to different levels of efficacy and also the best reproducibility with respect to responsiveness (Slope/S = 1.02), making it the more reliable method to use in an antimicrobial efficacy test. This study showed that the microtiter plate is a versatile and easy-to-use biofilm reactor, which exhibits good repeatability and reproducibility for different types of assessment methods, as long as a suitable experimental design and statistical analysis is applied.
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http://dx.doi.org/10.1038/s41598-021-93115-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8253847PMC
July 2021

Porphyrins produced by acneic strains activate the inflammasome by inducing K leakage.

iScience 2021 Jun 21;24(6):102575. Epub 2021 May 21.

Laboratory of Pharmaceutical Microbiology, Faculty of Pharmaceutical Sciences, Ghent University, 9000 Ghent, Belgium.

Some subgroups dominate on healthy skin, whereas others are frequently acne associated. Here we provide mechanistic insights into this difference, using an anaerobic keratinocyte-sebocyte- co-culture model. An acneic strain as well as its porphyrins activates NRLP3 inflammasome assembly, whereas this was not observed with a non-acneic strain. Low levels of intracellular K in keratinocytes stimulated with extracted porphyrins or infected with the acneic strain were observed, identifying porphyrin-induced K leakage as trigger for inflammasome activation. Using a panel of strains, we found that porphyrin production and IL-1β release are correlated and are higher in acneic strains. This demonstrates that the latter produce more porphyrins, which interact with the keratinocyte cell membrane, leading to K leakage, NLRP3 inflammasome activation, and IL-1β release and provides an explanation for the observation that some strains are associated with healthy skin, whereas others dominate in acneic skin.
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http://dx.doi.org/10.1016/j.isci.2021.102575DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8188554PMC
June 2021

Model system parameters influence the sodium hypochlorite susceptibility of endodontic biofilms.

Int Endod J 2021 Sep 12;54(9):1557-1570. Epub 2021 Jul 12.

Department of Oral Health Sciences, Section of Endodontology, Ghent University, Ghent, Belgium.

Aim: To evaluate in a laboratory setting the influence of several model system parameters on the sodium hypochlorite (NaOCl) susceptibility of endodontic biofilms. Based on these findings, a relevant in vitro endodontic biofilm model is proposed.

Methodology: In vitro biofilms were cultured, varying the following experimental model parameters: biofilm composition (monospecies Enterococcus faecalis and a multispecies biofilm including E. faecalis, Fusobacterium nucleatum, Prevotella intermedia and Porphyromonas gingivalis), incubation time (24 h or 11 days), incubation atmosphere (aerobically or anaerobically) and biofilm substrate (polystyrene microtiter plate wells, hydroxyapatite or dentine). Biofilms were subjected to treatment with NaOCl (0.025%, 0.1%, 0.5%, 2.5%) for 1 min, control groups included treatment with purified water. Biofilms were harvested and the number of surviving cells was determined by plate counting using general (monospecies biofilms) or selective (multispecies biofilms) media. A two-way ANOVA was used to explore the effect of the model parameters on biofilm eradication. Finally, the most physiologically relevant biofilm model (11-day-old multispecies biofilm grown anaerobically on dentine discs) was characterized by selective media plate counting, NaOCl susceptibility testing, scanning and transmission electron microscopy.

Results: There was no difference in NaOCl eradication between the anaerobically and aerobically grown E. faecalis biofilms. One-day-old biofilms of E. faecalis were more susceptible to most tested NaOCl concentrations than 11-day-old biofilms (p < .05). When grown in a multispecies biofilm, E. faecalis was significantly less susceptible to NaOCl treatment than in a monospecies biofilm (p < .05). E. faecalis in a multispecies biofilm grown in a MTP was more susceptible to NaOCl (0.025% and 0.1%) than when grown on hydroxyapatite or dentine. No difference in biofilm NaOCl susceptibility was seen between hydroxyapatite and dentine. The multispecies biofilm proved to be a reproducible model with high NaOCl resistance, complex structure and organization.

Conclusion: The parameters biofilm age, biofilm composition and substrate had a significant influence on the NaOCl susceptibility of E. faecalis biofilms. Older biofilms, multispecies biofilms and biofilms grown on dentine and hydroxyapatite had reduced NaOCl susceptibility. These findings emphasize the importance of selecting relevant parameters when designing a laboratory biofilm model system for the evaluation of antimicrobial treatments.
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http://dx.doi.org/10.1111/iej.13544DOI Listing
September 2021

The Quorum-Sensing Inhibitor Furanone C-30 Rapidly Loses Its Tobramycin-Potentiating Activity against Pseudomonas aeruginosa Biofilms during Experimental Evolution.

Antimicrob Agents Chemother 2021 06 17;65(7):e0041321. Epub 2021 Jun 17.

Laboratory of Pharmaceutical Microbiology, Ghent University, Ghent, Belgium.

The use of quorum-sensing inhibitors (QSI) has been proposed as an alternative strategy to combat antibiotic resistance. QSI reduce the virulence of a pathogen without killing it and it is claimed that resistance to such compounds is less likely to develop, although there is a lack of experimental data supporting this hypothesis. Additionally, such studies are often carried out in conditions that do not mimic the situation. In the present study, we evaluated whether a combination of the QSI furanone C-30 and the aminoglycoside antibiotic tobramycin would be "evolution-proof" when used to eradicate Pseudomonas aeruginosa biofilms grown in a synthetic cystic fibrosis sputum medium. We found that the biofilm-eradicating activity of the tobramycin/furanone C-30 combination already decreased after 5 treatment cycles. The antimicrobial susceptibility of P. aeruginosa to tobramycin decreased 8-fold after 16 cycles of treatment with the tobramycin/furanone C-30 combination. Furthermore, microcalorimetry revealed changes in the metabolic activity of P. aeruginosa exposed to furanone C-30, tobramycin, and the combination. Whole-genome sequencing analysis of the evolved strains exposed to the combination identified mutations in , , and , genes known to be involved in antibiotic resistance. In P. aeruginosa treated with furanone C-30 alone, a deletion in was also observed. Our data indicate that furanone C-30 is not "evolution-proof" and quickly becomes ineffective as a tobramycin potentiator.
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http://dx.doi.org/10.1128/AAC.00413-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8373219PMC
June 2021

Colloidal silver combating pathogenic Pseudomonas aeruginosa and MRSA in chronic rhinosinusitis.

Colloids Surf B Biointerfaces 2021 Jun 4;202:111675. Epub 2021 Mar 4.

Department of Surgery-Otolaryngology Head and Neck Surgery, Basil Hetzel Institute for Translational Health Research, Central Adelaide Local Health Network, Woodville South, Australia; The University of Adelaide, Adelaide, Australia. Electronic address:

The emergence of antibiotic resistant bacteria requires for the development of new antimicrobial compounds one of which colloidal silver (CS) having strong bactericidal properties and being the most promising inorganic nanoparticles for the treatment of bacterial infectious diseases. However, their production can be slow and cumbersome. Here, we used Corymbia maculata aqueous leaf extract as a reducing agent to synthesize CS in a single 15-minute process. CS was physico-chemically characterized for shape, size, zeta potential and stability. The Minimal Inhibitory Concentration (MIC) and Minimum Biofilm Eradication Concentration (MBEC) of CS against planktonic and biofilm forms of methicillin-resistant Staphylococcus aureus (MRSA, n = 5), Pseudomonas aeruginosa (n = 5), Haemophilus influenzae (n = 5) and Streptococcus pneumoniae (n = 3) chronic rhinosinusitis clinical isolates were investigated using the microdilution method and resazurin assay, respectively. The in vitro cytotoxicity on bronchial epithelial cells (Nuli-1) was analyzed by the crystal violet proliferation assay. The safety and efficacy of CS was evaluated in an in vivo infection model in Caenorhabditis elegans. CS was spherical in shape with a diameter of between 11-16 nm (TEM analysis) in dried form and 40 nm (NanoSight) in colloidal form and was stable at room temperature and 4 °C for one year. Average MIC and MBEC values varied between 11 and 44 ppm for MRSA, H. influenzae and S. pneumoniae and between 0.2 and 3 ppm for P. aeruginosa. CS was not toxic to Nuli-1 cells or C. elegans at concentrations of 44 ppm and reduced the Colony Forming Units counts by 96.9 % and 99.6 % in C. elegans for MRSA and P. aeruginosa, respectively. In conclusion, a novel, green synthesis of stable CS is demonstrated with good safety and efficacy profiles, particularly against P. aeruginosa in planktonic and biofilm forms. These CS have potential applications against clinical infections, including in the context of CRS.
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http://dx.doi.org/10.1016/j.colsurfb.2021.111675DOI Listing
June 2021

Do results obtained with RNA-sequencing require independent verification?

Authors:
Tom Coenye

Biofilm 2021 Dec 13;3:100043. Epub 2021 Jan 13.

Laboratory of Pharmaceutical Microbiology, Ghent University, Ghent, Belgium.

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http://dx.doi.org/10.1016/j.bioflm.2021.100043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7823214PMC
December 2021

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

Mater Sci Eng C Mater Biol Appl 2021 Mar 26;122:111909. Epub 2021 Jan 26.

Department of Biotechnology, Ghent University, 9000 Ghent, Belgium. Electronic address:

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 (CaCO) microparticles in a vaterite-calcite polymorph mixture. CaCO-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 CaCO-mineralized PHB scaffold morphology and resulted in partial recrystallization of vaterite to calcite. In comparison to pristine scaffolds, the loading efficiency of CaCO-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 CaCO-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 CaCO layers and immobilized VCM/ALP are promising materials in bone tissue engineering.
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http://dx.doi.org/10.1016/j.msec.2021.111909DOI Listing
March 2021

Detection of cytosine methylation in by single-molecule real-time sequencing and whole-genome bisulfite sequencing.

Microbiology (Reading) 2021 03 10;167(3). Epub 2021 Feb 10.

Laboratory of Pharmaceutical Microbiology, Ghent University, Ghent, Belgium.

Research on prokaryotic epigenetics, the study of heritable changes in gene expression independent of sequence changes, led to the identification of DNA methylation as a versatile regulator of diverse cellular processes. Methylation of adenine bases is often linked to regulation of gene expression in bacteria, but cytosine methylation is also frequently observed. In this study, we present a complete overview of the cytosine methylome in , an opportunistic respiratory pathogen in cystic fibrosis patients. Single-molecule real-time (SMRT) sequencing was used to map all 4mC-modified cytosines, as analysis of the predicted MTases in the genome revealed the presence of a 4mC-specific phage MTase, M.BceJII, targeting GGC sequences. Methylation motif GCGGCGC was identified, and out of 6850 motifs detected across the genome, 2051 (29.9 %) were methylated at the fifth position. Whole-genome bisulfite sequencing (WGBS) was performed to map 5mC methylation and 1635 5mC-modified cytosines were identified in pG motifs. A comparison of the genomic positions of the modified bases called by each method revealed no overlap, which confirmed the authenticity of the detected 4mC and 5mC methylation by SMRT sequencing and WGBS, respectively. Large inter-strain variation of the 4mC-methylated cytosines was observed when strains J2315 and K56-2 were compared, which suggests that GGC methylation patterns in are strain-specific. It seems likely that 4mC methylation of GGC is not involved in regulation of gene expression but rather is a remnant of bacteriophage invasion, in which methylation of the phage genome was crucial for protection against restriction-modification systems of .
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http://dx.doi.org/10.1099/mic.0.001027DOI Listing
March 2021

Bacteria Produce Multiple Potentially Novel Molecules that Inhibit Carbapenem-Resistant Gram-Negative Bacterial Pathogens.

Antibiotics (Basel) 2021 Feb 2;10(2). Epub 2021 Feb 2.

Laboratory of Microbiology, Department of Biochemistry and Microbiology, Ghent University, 9000 Ghent, Belgium.

Antimicrobial resistance in Gram-negative pathogens represents a global threat to human health. This study determines the antimicrobial potential of a taxonomically and geographically diverse collection of 263 (sensu lato) isolates and applies natural product dereplication strategies to identify potentially novel molecules. Antimicrobial activity is almost exclusively present in sensu stricto bacteria and rarely observed in the novel genera , , , , and . Fourteen isolates show a unique spectrum of antimicrobial activity and inhibited carbapenem-resistant Gram-negative bacterial pathogens. Dereplication of the molecules present in crude spent agar extracts identifies 42 specialized metabolites, 19 of which represented potentially novel molecules. The known identified metabolites include toxoflavin, reumycin, pyrrolnitrin, enacyloxin, bactobolin, cepacidin, ditropolonyl sulfide, and antibiotics BN-227-F and SF 2420B, as well as the siderophores ornibactin, pyochelin, and cepabactin. Following semipreparative fractionation and activity testing, a total of five potentially novel molecules are detected in active fractions. Given the molecular formula and UV spectrum, two of those putative novel molecules are likely related to bactobolins, and another is likely related to enacyloxins. The results from this study confirm and extend the observation that bacteria present exciting opportunities for the discovery of potentially novel bioactive molecules.
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http://dx.doi.org/10.3390/antibiotics10020147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7912996PMC
February 2021

Antibiotic susceptibility of cystic fibrosis lung microbiome members in a multispecies biofilm.

Biofilm 2020 Dec 13;2:100031. Epub 2020 Jun 13.

Laboratory of Pharmaceutical Microbiology, Ghent University, Belgium.

The lungs of cystic fibrosis (CF) patients are often chronically colonized by multiple microbial species that can form biofilms, including the major CF pathogen . Herewith, lower microbial diversity in CF airways is typically associated with worse health outcomes. In an attempt to treat CF lung infections patients are frequently exposed to antibiotics, which may affect microbial diversity. This study aimed at understanding if common antibiotics that target influence microbial diversity. To this end, a microaerophilic multispecies biofilm model of frequently co-isolated members of the CF lung microbiome (, , , , , and ) was exposed to antipseudomonal antibiotics. We found that antibiotics that affected several dominant species (i.e. ceftazidime, tobramycin) resulted in higher species evenness compared to colistin, which is only active against . Furthermore, susceptibility of individual species in the multispecies biofilm following antibiotic treatment was compared to that of the respective single-species biofilms, showing no differences. Adding three anaerobic species (, , and ) to the multispecies biofilm did not influence antibiotic susceptibility. In conclusion, our study demonstrates antibiotic-dependent effects on microbial community diversity of multispecies biofilms comprised of CF microbiome members.
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http://dx.doi.org/10.1016/j.bioflm.2020.100031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7798459PMC
December 2020

Biofilm dispersion: The key to biofilm eradication or opening Pandora's box?

Biofilm 2020 Dec 1;2:100027. Epub 2020 Jun 1.

Laboratory of Pharmaceutical Microbiology, Ghent University, Ghent, Belgium.

Biofilms are extremely difficult to eradicate due to their decreased antibiotic susceptibility. Inducing biofilm dispersion could be a potential strategy to help combat biofilm-related infections. Mechanisms of biofilm dispersion can basically be divided into two groups, i.e. active and passive dispersion. Active dispersion depends on a decrease in the intracellular c-di-GMP levels, leading to the production of enzymes that degrade the biofilm matrix and promote dispersion. In contrast, passive dispersion relies on triggers that directly release cells from the biofilm. In the present review, several active and passive dispersion strategies are discussed. In addition, the disadvantages and possible consequences of using dispersion as a treatment approach for biofilm-related infections are also reviewed.
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http://dx.doi.org/10.1016/j.bioflm.2020.100027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7798462PMC
December 2020

Induction of antibiotic specialized metabolism by co-culturing in a collection of phyllosphere bacteria.

Environ Microbiol 2021 04 12;23(4):2132-2151. Epub 2021 Jan 12.

Laboratory of Microbiology, Department of Biochemistry and Microbiology, Faculty of Sciences, Ghent University, Ghent, Belgium.

A diverse set of bacteria live on the above-ground parts of plants, composing the phyllosphere, and play important roles for plant health. Phyllosphere microbial communities assemble in a predictable manner and diverge from communities colonizing other plant organs or the soil. However, how these communities differ functionally remains obscure. We assembled a collection of 258 bacterial isolates representative of the most abundant taxa of the phyllosphere of Arabidopsis and a shared soil inoculum. We screened the collection for the production of metabolites that inhibit the growth of Gram-positive and Gram-negative bacteria either in isolation or in co-culture. We found that isolates capable of constitutive antibiotic production in monoculture were significantly enriched in the soil fraction. In contrast, the proportion of binary cultures resulting in the production of growth inhibitory compounds differed only marginally between the phyllosphere and soil fractions. This shows that the phyllosphere may be a rich resource for potentially novel molecules with antibiotic activity, but that production or activity is dependent upon induction by external signals or cues. Finally, we describe the isolation of antimicrobial acyloin metabolites from a binary culture of Arabidopsis phyllosphere isolates, which inhibit the growth of clinically relevant Acinetobacter baumannii.
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http://dx.doi.org/10.1111/1462-2920.15382DOI Listing
April 2021

Phylotype I and II Strains Interact Differently With Human Skin Cells.

Front Cell Infect Microbiol 2020 16;10:575164. Epub 2020 Nov 16.

Laboratory of Pharmaceutical Microbiology, Ghent University, Ghent, Belgium.

Acne vulgaris is one of the most common skin disorders and affects the pilosebaceous units. Although the exact pathogenesis of acne is still unknown, (formerly known as ) is considered one of the key contributing factors. In fact, a significant association exists between strains belonging to phylotype I and acne. However, there is still heavy debate on the exact role of in acne and its behavior in the pilosebaceous unit, and more specifically its interactions with the human skin cells. In this study, key elements of the host-pathogen interaction were studied for a collection of strains, belonging to phylotype I and II, including association with HaCaT keratinocytes and SZ95 sebocytes, the effect of on keratinocyte tight junctions in a HaCaT monoculture and in an additional keratinocyte-sebocyte co-culture model, and invasion through the keratinocyte cell layer. Our data showed association of all strains to both skin cell lines, with a significantly higher association of type I strains compared to type II strains. Microscopic imaging and western blot analysis of the tight junction protein ZO-1, together with transepithelial electrical resistance (TEER) measurements revealed an initial induction of keratinocyte tight junctions after 24 h infection but a degradation after 48 h, demonstrating a decline in cell lining integrity during infection. Subsequently, was able to invade after 48 h of infection, although invasion frequency was significantly higher for type II strains compared to type I strains.
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http://dx.doi.org/10.3389/fcimb.2020.575164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7717938PMC
June 2021

Biological activity and antimicrobial property of Cu/a-C:H nanocomposites and nanolayered coatings on titanium substrates.

Mater Sci Eng C Mater Biol Appl 2021 Feb 16;119:111513. Epub 2020 Sep 16.

Research Unit Plasma Technology (RUPT), Department of Applied Physics, Faculty of Engineering & Architecture, Ghent University, Ghent, Belgium.

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.
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http://dx.doi.org/10.1016/j.msec.2020.111513DOI Listing
February 2021

Synergistic Antimicrobial Activity of Supplemented Medical-Grade Honey against Biofilm Formation and Eradication.

Antibiotics (Basel) 2020 Dec 4;9(12). Epub 2020 Dec 4.

Triticum Exploitatie BV, Sleperweg 44, 6222NK Maastricht, The Netherlands.

Biofilms hinder wound healing. Medical-grade honey (MGH) is a promising therapy because of its broad-spectrum antimicrobial activity and the lack of risk for resistance. This study investigated the inhibitory and eradicative activity against multidrug-resistant biofilms by different established MGH-based wound care formulations. Six different natural wound care products (Medihoney, Revamil, Mebo, Melladerm, L-Mesitran Ointment, and L-Mesitran Soft) were tested in vitro. Most of them contain MGH only, whereas some were supplemented. L-Mesitran Soft demonstrated the most potent antimicrobial activity (6.08-log inhibition and 3.18-log eradication). Other formulations ranged between 0.89-log and 4.80-log inhibition and 0.65-log and 1.66-log eradication. Therefore, the contribution of different ingredients of L-Mesitran Soft was investigated in more detail. The activity of the same batch of raw MGH (1.38-log inhibition and 2.35-log eradication), vitamins C and E (0.95-log inhibition and 0.94-log eradication), and all ingredients except MGH (1.69-log inhibition and 0.75-log eradication) clearly support a synergistic activity of components within the L-Mesitran Soft formulation. Several presented clinical cases illustrate its clinical antimicrobial efficacy against biofilms. In conclusion, MGH is a potent treatment for biofilms. L-Mesitran Soft has the strongest antimicrobial activity, which is likely due to the synergistic activity mediated by its supplements.
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http://dx.doi.org/10.3390/antibiotics9120866DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7761815PMC
December 2020

Bacterial Interference With Lactate Dehydrogenase Assay Leads to an Underestimation of Cytotoxicity.

Front Cell Infect Microbiol 2020 15;10:494. Epub 2020 Sep 15.

Laboratory of Pharmaceutical Microbiology, Ghent University, Ghent, Belgium.

Models to study host-pathogen interactions are an important tool for investigating the infectious disease process and evaluating the efficacy of antimicrobial compounds. In these models, the viability of mammalian cells is often determined using the lactate dehydrogenase (LDH) cytotoxicity assay. In the present study we evaluated whether bacteria could interfere with the LDH assay. As a model for host-pathogen interactions, we co-cultured lung epithelial cells with eight bacteria encountered in the lower respiratory tract. We show that LDH activity is affected by , and , and that this depends on the density of the start inoculum and the duration of infection. Two different mechanisms were discovered through which bacteria interfered with LDH activity, i.e., acidification of the cell culture medium (by and ) and protease production (by and ). In addition, we developed and validated a modified protocol to evaluate cytotoxicity using the LDH assay, where bacterial interference with LDH quantification is avoided.
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http://dx.doi.org/10.3389/fcimb.2020.00494DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7523407PMC
May 2021

Does the mode of dispersion determine the properties of dispersed Pseudomonas aeruginosa biofilm cells?

Int J Antimicrob Agents 2020 Dec 9;56(6):106194. Epub 2020 Oct 9.

Laboratory of Pharmaceutical Microbiology, Ghent University, Ghent, Belgium. Electronic address:

Introduction: Actively dispersed Pseudomonas aeruginosa biofilm cells differ from planktonic cells, as they have a lower intracellular cyclic di-guanosine monophosphate (c-di-GMP) concentration and show increased virulence. In addition, the nature of the dispersion trigger has been shown to influence the antibiotic susceptibility of dispersed cells. However, properties of passively-dispersed cells, in which the dispersion trigger directly releases cells from the biofilm, have not been described. The present study determined c-di-GMP concentration, virulence in Galleria mellonella and antibiotic susceptibility of P. aeruginosa cells dispersed from biofilm using various triggers.

Materials And Methods: P. aeruginosa biofilms grown in flow-cells were dispersed actively [exposure to the nitric oxide (NO)-donor sodium nitroprusside (SNP) or to glutamate] or passively [by stopping and restarting the flow or exposure to laser-induced vapor nanobubbles (VNB)], and properties of these dispersed cells were compared to those of spontaneously-dispersed cells.

Results: The passively dispersed P. aeruginosa biofilm cells had significantly lower intracellular c-di-GMP levels than actively-dispersed cells. However, this did not result in differences in virulence in Galleria mellonella, nor in tobramycin and ciprofloxacin susceptibility. Passively-dispersed cells were more susceptible to colistin than actively- and spontaneously-dispersed cells. In cells dispersed by interrupting the flow, increased susceptibility to colistin was immediate, whereas this was delayed for VNB-dispersed cells.

Conclusion: Passively-dispersed P. aeruginosa biofilm cells have a decreased intracellular c-di-GMP concentration and an increased colistin susceptibility compared to actively-dispersed cells. No differences in virulence or susceptibility to tobramycin or colistin were observed.
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http://dx.doi.org/10.1016/j.ijantimicag.2020.106194DOI Listing
December 2020

Complex Taxon K: Where to Split?

Front Microbiol 2020 14;11:1594. Epub 2020 Jul 14.

Laboratory of Microbiology, Department of Biochemistry and Biotechnology, Faculty of Sciences, Ghent University, Ghent, Belgium.

The objective of the present study was to provide an updated classification for complex (Bcc) taxon K isolates. A representative set of 39 taxon K isolates were analyzed through multilocus sequence typing (MLST) and phylogenomic analyses. MLST analysis revealed the presence of at least six clusters of sequence types (STs) within taxon K, two of which contain the type strains of (ST-102) and (ST-101), and four corresponding to the previously defined taxa Other Bcc groups C, G, H and M. This clustering was largely supported by a phylogenomic tree which revealed three main clades. Isolates of and of Other Bcc groups C, G, and H represented a first clade which generally shared average nucleotide identity (ANI) and average digital DNA-DNA hybridization (dDDH) values at or above the 95-96% ANI and 70% dDDH thresholds for species delineation. A second clade consisted of Other Bcc group M bacteria and of four isolates and was supported by average ANI and dDDH values of 97.2 and 76.1% within this clade and average ANI and dDDH values of 94.5 and 57.2% toward the remaining isolates (including the type strain), which represented a third clade. We therefore concluded that isolates known as Other Bcc groups C, G, and H should be classified as , and propose a novel species, sp. nov., to accommodate Other Bcc M and ST-98, ST-103, and ST-119 isolates. Optimized MALDI-TOF MS databases for the identification of clinical isolates may provide correct species-level identification for some of these bacteria but would identify most of them as complex. MLST facilitates species-level identification of many taxon K strains but some may require comparative genomics for accurate species-level assignment. Finally, the inclusion of Other Bcc groups C, G, and H into affects the phenotype of this species minimally and the proposal to classify Other Bcc group M and ST-98, ST-103, and ST-119 strains as a novel species is supported by a distinctive phenotype, i.e., growth at 42°C and lysine decarboxylase activity.
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http://dx.doi.org/10.3389/fmicb.2020.01594DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7372133PMC
July 2020

Low iron-induced small RNA BrrF regulates central metabolism and oxidative stress responses in Burkholderia cenocepacia.

PLoS One 2020 23;15(7):e0236405. Epub 2020 Jul 23.

Laboratory of Pharmaceutical Microbiology, Ghent University, Ghent, Belgium.

Regulatory small RNAs play an essential role in maintaining cell homeostasis in bacteria in response to environmental stresses such as iron starvation. Prokaryotes generally encode a large number of RNA regulators, yet their identification and characterisation is still in its infancy for most bacterial species. Burkholderia cenocepacia is an opportunistic pathogen with high innate antimicrobial resistance, which can cause the often fatal cepacia syndrome in individuals with cystic fibrosis. In this study we characterise a small RNA which is involved in the response to iron starvation, a condition that pathogenic bacteria are likely to encounter in the host. BrrF is a small RNA highly upregulated in Burkholderia cenocepacia under conditions of iron depletion and with a genome context consistent with Fur regulation. Its computationally predicted targets include iron-containing enzymes of the tricarboxylic acid (TCA) cycle such as aconitase and succinate dehydrogenase, as well as iron-containing enzymes responsible for the oxidative stress response, such as superoxide dismutase and catalase. Phenotypic and gene expression analysis of BrrF deletion and overexpression mutants show that the regulation of these genes is BrrF-dependent. Expression of acnA, fumA, sdhA and sdhC was downregulated during iron depletion in the wild type strain, but not in a BrrF deletion mutant. TCA cycle genes not predicted as target for BrrF were not affected in the same manner by iron depletion. Likewise, expression of sodB and katB was dowregulated during iron depletion in the wild type strain, but not in a BrrF deletion mutant. BrrF overexpression reduced aconitase and superoxide dismutase activities and increased sensitivity to hydrogen peroxide. All phenotypes and gene expression changes of the BrrF deletion mutant could be complemented by overexpressing BrrF in trans. Overall, BrrF acts as a regulator of central metabolism and oxidative stress response, possibly as an iron-sparing measure to maintain iron homeostasis under conditions of iron starvation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0236405PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7377471PMC
September 2020

First Report of Candidemia Clonal Outbreak Caused by Emerging Fluconazole-Resistant Candida parapsilosis Isolates Harboring Y132F and/or Y132F+K143R in Turkey.

Antimicrob Agents Chemother 2020 09 21;64(10). Epub 2020 Sep 21.

Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands.

Clonal outbreaks of fluconazole-resistant (FLZR) 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 blood isolates have not been reported in Turkey. In this study, we consecutively collected blood isolates (= 225) from the fifth largest hospital in Turkey (2007 to 2019), assessed their azole susceptibility pattern using CLSI M27-A3/S4, and sequenced for all and , , and for a selected number of 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 , , and 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. 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 blood isolates in Turkey. The , , and mutations exclusively found in FLZR isolates establishes a basis for future studies, which will potentially broaden our knowledge of FLZR mechanisms in MST should be a preferred method for clonal analysis of isolates in outbreak scenarios.
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http://dx.doi.org/10.1128/AAC.01001-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7508617PMC
September 2020

DNA Methylation Epigenetically Regulates Gene Expression in Burkholderia cenocepacia and Controls Biofilm Formation, Cell Aggregation, and Motility.

mSphere 2020 07 15;5(4). Epub 2020 Jul 15.

Laboratory of Pharmaceutical Microbiology, Ghent University, Ghent, Belgium

Respiratory tract infections by the opportunistic pathogen often lead to severe lung damage in cystic fibrosis (CF) patients. New insights in how to tackle these infections might emerge from the field of epigenetics, as DNA methylation is an important regulator of gene expression. The present study focused on two DNA methyltransferases (MTases) in strains J2315 and K56-2 and their role in regulating gene expression. predicted DNA MTase genes BCAL3494 and BCAM0992 were deleted in both strains, and the phenotypes of the resulting deletion mutants were studied: deletion mutant ΔBCAL3494 showed changes in biofilm structure and cell aggregation, while ΔBCAM0992 was less motile. wild-type cultures treated with sinefungin, a known DNA MTase inhibitor, exhibited the same phenotype as DNA MTase deletion mutants. Single-molecule real-time sequencing was used to characterize the methylome of , including methylation at the origin of replication, and motifs CACAG and GTWWAC were identified as targets of BCAL3494 and BCAM0992, respectively. All genes with methylated motifs in their putative promoter region were identified, and qPCR experiments showed an upregulation of several genes, including biofilm- and motility-related genes, in MTase deletion mutants with unmethylated motifs, explaining the observed phenotypes in these mutants. In summary, our data confirm that DNA methylation plays an important role in regulating the expression of genes involved in biofilm formation, cell aggregation, and motility. CF patients diagnosed with infections often experience rapid deterioration of lung function, known as cepacia syndrome. has a large multireplicon genome, and much remains to be learned about regulation of gene expression in this organism. From studies in other (model) organisms, it is known that epigenetic changes through DNA methylation play an important role in this regulation. The identification of genes of which the expression is regulated by DNA methylation and identification of the regulatory systems involved in this methylation are likely to advance the biological understanding of cell adaptation via epigenetic regulation. In time, this might lead to novel approaches to tackle infections in CF patients.
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http://dx.doi.org/10.1128/mSphere.00455-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7364216PMC
July 2020

Complete Genome Sequence of Pseudomonas aeruginosa Strain AA2 (LMG 27630), an Early Isolate Recovered from the Airway of a German Cystic Fibrosis Patient.

Microbiol Resour Announc 2020 Jun 25;9(26). Epub 2020 Jun 25.

Laboratory of Pharmaceutical Microbiology, Ghent University, Ghent, Belgium

is an opportunistic pathogen that is able to cause various infections, including airway infections in cystic fibrosis patients. Here, we present the complete closed and annotated genome sequence of AA2, an isolate obtained early during infection of the respiratory tract of a German cystic fibrosis patient.
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http://dx.doi.org/10.1128/MRA.00526-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7317106PMC
June 2020

Fabrication of Microporous Coatings on Titanium Implants with Improved Mechanical, Antibacterial, and Cell-Interactive Properties.

ACS Appl Mater Interfaces 2020 Jul 25;12(27):30155-30169. Epub 2020 Jun 25.

Research Unit Plasma Technology (RUPT), Department of Applied Physics, Faculty of Engineering and Architecture, Ghent University, Ghent 9000, Belgium.

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 TiO coatings enriched with Ca, P, and Ag on titanium to improve its surface properties and antibacterial 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 TiO, 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 and 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 and a maximal 5-log reduction of 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.
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http://dx.doi.org/10.1021/acsami.0c07234DOI Listing
July 2020
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