Publications - Publications en-us PURE Extension (Web Department) 30 <![CDATA[The effect of an enzyme-containing lozenge on dental biofilm accumulation]]> Schlafer, S., K. Johnsen, K., Jørgensen, M., Kjærbølling, I., Schramm, A., Meyer, R. L. Research Sun, 01 Jan 2023 00:49:38 +0100 71a4711b-1dad-4a89-b828-23d65742b765 <![CDATA[Multiple-enzyme treatment reduces biofilm formation in a highly acidogenic in vitro model]]> Rikvold, P., Møllebjerg, A., Meyer, R. L., Schlafer, S. Materials and methods: Biofilms were grown in 96-well plates under aerobic conditions at 37°C for 24 hours using pooled saliva as inoculum and brain-heart infusion broth supplied with 5% sucrose and sterile saliva as growth medium. The effect of treatment with a combination of mutanase, glucanase and DNase on biofilm prevention and removal was investigated. Treatment was performed either during (prevention) or after biofilm growth (removal) and the remaining biofilm was quantified by crystal violet staining. Additionally, the treatment effect on biofilm matrix compounds was analyzed using confocal microscopy. Extracellular DNA was stained with TOTO-1, microbial cells with SYTO41 and matrix polysaccharides were visualized by including fluorescently labeled dextran in the growth medium. Treatment effects were analyzed using unpaired t-tests (p<0.05 was considered statistically significant).
Results: Enzyme treatment reduced mean biofilm formation with 94%±1% SD (p=0.0001) and 69%±2% SD (p<0.0001) when performed during and after growth, respectively. Extracellular polysaccharides were on average reduced by 28%±6% SD compared to control treatment (p=0.02), but the mean ratio between polysaccharides and microbial cells remained unchanged (0.70±0.37 SD vs. 0.77±0.22 SD; p=0.74). No significant reduction in the amount of eDNA (p=0.74) was observed.
Conclusion: Multiple-enzyme treatment is a promising non-biocidal approach to biofilm control.
Research Sun, 01 Oct 2023 00:49:38 +0200 c34c0044-7b4c-4db3-936c-91169af99e84
<![CDATA[Multiple-enzyme treatment reduces biofilm formation in a highly acidogenic in vitro model]]> Rikvold, P., Møllebjerg, A., Meyer, R. L., Schlafer, S. Research Sat, 01 Jul 2023 00:49:38 +0200 a4554b30-214a-4f5c-90aa-c3059d549165 <![CDATA[The effect of an enzyme-containing lozenge on dental biofilm accumulation: A clinical pilot study]]> Schlafer, S., K. Johnsen, K., Jørgensen, M., Kjærbølling, I., Schramm, A., Meyer, R. L. 24 healthy volunteers were enrolled in a randomized, double-blind placebo-controlled study with two parallel arms. After an initial professional tooth cleaning, participants refrained from oral hygiene and used three enzyme-containing or placebo lozenges/d for 7 d. After 1 and 7 d, the Turesky-modification of the Quigley-and-Hein Plaque Index (TM-QHPI) was assessed in the first and second quadrant, respectively and compared to TM-QHPI after a one-day baseline period without oral hygiene (two-sample t-tests). At baseline and after 7 d of intervention, the gingival index (GI) was recorded and compared (two-sample t-test), and the species richness was analysed in plaque and saliva samples by 16S rRNA sequencing (linear mixed model). The protocol was approved by the local Ethics Commitee (1-10-72-260-20).
Enzyme treatment resulted in a reduced mean TM-QHPI compared to control treatment after 1 d (Δ=-0.61±1.00 SD vs. 0.03±0.59 SD; p=0.07; Δ=-0.82±0.74 SD vs. -0.09±0.45 SD; p=0.01 without outliers) but not 7 d (Δ=1.03±1.42 SD vs. 1.61±1.36 SD; p=0.39), and in a reduced increase in average species richness (17±9 SE vs. 35±7 SE; p=0.04) from baseline to day 7. Mean GI did not increase over the intervention period for both treatment groups (Δ=-0.06±0.63 SD vs. 0.02±0.44 SD; p=0.39). Lozenges containing matrix-degrading enzymes may be a promising adjunct to oral hygiene.]]>
Research Sat, 01 Jul 2023 00:49:38 +0200 26b01e07-d567-4765-9adb-2bcbc78ce933
<![CDATA[GFP fusions of Sec-routed extracellular proteins in Staphylococcus aureusreveal surface-associated coagulase in biofilms]]> Evans, D. C.S., Khamas, A. B., Marcussen, L., et al. Staphylococcus aureus is a major human pathogen that utilises many surface-associated and secreted proteins to form biofilms and cause disease. However, our understanding of these processes is limited by chal-lenges of using fluorescent protein reporters in their native environment, be-cause they must be exported and fold correctly to become fluorescent. Here, we demonstrate the feasibility of using the monomeric superfolder GFP (msfGFP) exported from S. aureus. By fusing msfGFP to signal peptides for the Secretory (Sec) and Twin Arginine Translocation (Tat) pathways, the two ma-jor secretion pathways in S. aureus, we quantified msfGFP fluorescence in bacterial cultures and cell-free supernatant from the cultures. When fused to a Tat signal peptide, we detected msfGFP fluorescence inside but not outside bacterial cells, indicating a failure to export msfGFP. However, when fused to a Sec signal peptide, msfGFP fluorescence was present outside cells, indicat-ing successful export of the msfGFP in the unfolded state, followed by extra-cellular folding and maturation to the photoactive state. We applied this strategy to study coagulase (Coa), a secreted protein and a major contributor to the formation of a fibrin network in S. aureus biofilms that protects bacte-ria from the host immune system and increases attachment to host surfaces. We confirmed that a genomically integrated C-terminal fusion of Coa to msfGFP does not impair the activity of Coa or its localisation within the bio-film matrix. Our findings demonstrate that msfGFP is a good candidate fluo-rescent reporter to consider when studying proteins secreted by the Sec pathway in S. aureus.

Research Sat, 01 Jul 2023 00:49:38 +0200 efdfe5a1-3c1f-4b17-8ada-7ff8ad44e166
<![CDATA[Fibrinolytic and antibiotic treatment of prosthetic vascular graft infections in a novel rat model]]> Johansen, M. I., Rahbek, S. J., Jensen-Fangel, S., et al. We developed a rat model of prosthetic vascular graft infection to assess, whether the fibrinolytic tissue plasminogen activator (tPA) could increase the efficacy of antibiotic therapy.

Materials and methods
Rats were implanted a polyethylene graft in the common carotid artery, pre-inoculated with approx. 6 log10 colony forming units (CFU) of methicillin resistant Staphylococcus aureus. Ten days after surgery, rats were randomized to either: 0.9% NaCl (n = 8), vancomycin (n = 8), vancomycin + tPA (n = 8), vancomycin + rifampicin (n = 18) or vancomycin + rifampicin + tPA (n = 18). Treatment duration was seven days. Approximately 36 hours after the end of treatment, the rats were euthanized, and grafts and organs were harvested for CFU enumeration.

All animals in the control group had significantly higher CFU at the time of euthanization compared to bacterial load found on the grafts prior to inoculation (6.45 vs. 4.36 mean log10 CFU/mL, p = 0.0011), and both the procedure and infection were well tolerated.

Vancomycin and rifampicin treatment were superior to monotherapy with vancomycin, as it lead to a marked decrease in median bacterial load on the grafts (3.50 vs. 6.56 log10 CFU/mL, p = 0.0016). The addition of tPA to vancomycin and rifampicin combination treatment did not show a further decrease in bacterial load (4.078 vs. 3.50 log10 CFU/mL, p = 0.26). The cure rate was 16% in the vancomycin + rifampicin group vs. 37.5% cure rate in the vancomycin + rifampicin + tPA group. Whilst interesting, this trend was not significant at our sample size (p = 0.24).

We developed the first functional model of an arterial prosthetic vascular graft infection in rats. Antibiotic combination therapy with vancomycin and rifampicin was superior to vancomycin monotherapy, and the addition of tPA did not significantly reduce bacterial load, nor significantly increase cure rate.]]>
Research Sat, 01 Jul 2023 00:49:38 +0200 846ae713-5cb9-440d-b500-5937f21f6aac
<![CDATA[Protein ligand and nanotopography separately drive the phenotype of mouse embryonic stem cells]]> Ghorbani, S., Christine Füchtbauer, A., Møllebjerg, A., et al. Biochemical and biomechanical signals regulate stem cell function in the niche environments in vivo. Current in vitro culture of mouse embryonic stem cells (mESC) uses laminin (LN-511) to provide mimetic biochemical signaling (LN-521 for human systems) to maintain stemness. Alternative approaches propose topographical cues to provide biomechanical cues, however combined biochemical and topographic cues may better mimic the in vivo environment, but are largely unexplored for in vitro stem cell expansion. In this study, we directly compare in vitro signals from LN-511 and/or topographic cues to maintain stemness, using systematically-varied submicron pillar patterns or flat surfaces with or without preadsorbed LN-511. The adhesion of cells, colony formation, expression of the pluripotency marker,octamer-binding transcription factor 4 (Oct4), and transcriptome profiling were characterized. We observed that either biochemical or topographic signals could maintain stemness of mESCs in feeder-free conditions, indicated by high-level Oct4 and gene profiling by RNAseq. The combination of LN-511 with nanotopography reduced colony growth, while maintaining stemness markers, shifted the cellular phenotype indicating that the integration of biochemical and topographic signals is antagonistic. Overall, significantly faster (up to 2.5 times) colony growth was observed at nanotopographies without LN-511, suggesting for improved ESC expansion.

Research Sun, 01 Oct 2023 00:49:38 +0200 362a42dd-df10-4b40-8216-3a7699d88025
<![CDATA[Antibody-Drug Conjugates to Treat Bacterial Biofilms via Targeting and Extracellular Drug Release]]> Tvilum, A. S., Johansen, M. I., Glud, L. N., et al. The treatment of implant-associated bacterial infections and biofilms is an urgent medical need and a grand challenge because biofilms protect bacteria from the immune system and harbor antibiotic-tolerant persister cells. This need is addressed herein through an engineering of antibody-drug conjugates (ADCs) that contain an anti-neoplastic drug mitomycin C, which is also a potent antimicrobial against biofilms. The ADCs designed herein release the conjugated drug without cell entry, via a novel mechanism of drug release which likely involves an interaction of ADC with the thiols on the bacterial cell surface. ADCs targeted toward bacteria are superior by the afforded antimicrobial effects compared to the non-specific counterpart, in suspension and within biofilms, in vitro, and in an implant-associated murine osteomyelitis model in vivo. The results are important in developing ADC for a new area of application with a significant translational potential, and in addressing an urgent medical need of designing a treatment of bacterial biofilms.

Research Tue, 01 Aug 2023 00:49:38 +0200 f4effb5f-6ebb-4824-bab0-b9a925f62858
<![CDATA[Polyether Ionophore Antibiotics Target Drug-Resistant Clinical Isolates, Persister Cells, and Biofilms]]> Wollesen, M., Mikkelsen, K., Tvilum, M. S., et al. Polyether ionophores are complex natural products known to transport various cations across biological membranes. While several members of this family are used in agriculture (e.g., as anti-coccidiostats) and have potent antibacterial activity, they are not currently being pursued as antibiotics for human use. Polyether ionophores are typically grouped as having similar functions, despite the fact that they significantly differ in structure; for this reason, how their structure and activity are related remains unclear. To determine whether certain members of the family constitute particularly interesting springboards for in-depth investigations and future synthetic optimization, we conducted a systematic comparative study of eight different polyether ionophores for their potential as antibiotics. This includes clinical isolates from bloodstream infections and studies of the compounds' effects on bacterial biofilms and persister cells. We uncover distinct differences within the compound class and identify the compounds lasalocid, calcimycin, and nanchangmycin as having particularly interesting activity profiles for further development. IMPORTANCE Polyether ionophores are complex natural products used in agriculture as anti-coccidiostats in poultry and as growth promoters in cattle, although their precise mechanism is not understood. They are widely regarded as antimicrobials against Gram-positive bacteria and protozoa, but fear of toxicity has so far prevented their use in humans. We show that ionophores generally have very different effects on Staphylococcus aureus, both in standard assays and in more complex systems such as bacterial biofilms and persister cell populations. This will allow us to focus on the most interesting compounds for future in-depth investigations and synthetic optimizations.

Research Tue, 01 Aug 2023 00:49:38 +0200 8241a10e-5778-4631-abdd-509176b0f370
<![CDATA[Novel high-throughput screening platform identifies enzymes to tackle biofouling on reverse osmosis membranes]]> Møllebjerg, A., Zarebska, A., Nielsen, H. B., et al. Research Mon, 01 May 2023 00:49:38 +0200 26230021-488e-45a3-9f57-5b1ca6953c99 <![CDATA[Biofilm formation and inflammatory potential of <i>Staphylococcus saccharolyticus</i>]]> Afshar, M., Møllebjerg, A., Minero, G. A., et al. Staphylococcus saccharolyticus, a coagulase-negative staphylococcal species, has some unusual characteristics for human-associated staphylococci, such as slow growth and its preference for anoxic culture conditions. This species is a relatively abundant member of the human skin microbiota, but its microbiological properties, as well as the pathogenic potential, have scarcely been investigated so far, despite being occasionally isolated from different types of infections including orthopedic implant-associated infections. Here, we investigated the growth and biofilm properties of clinical isolates of S. saccharolyticus and determined host cell responses. Growth assessments in anoxic and oxic conditions revealed strain-dependent outcomes, as some strains can also grow aerobically. All tested strains of S. saccharolyticus were able to form biofilm in a microtiter plate assay. Strain-dependent differences were determined by optical coherence tomography, revealing that medium supplementation with glucose and sodium chloride enhanced biofilm formation. Visualization of the biofilm by confocal laser scanning microscopy revealed the role of extracellular DNA in the biofilm structure. In addition to attached biofilms, S. saccharolyticus also formed bacterial aggregates at an early stage of growth. Transcriptome analysis of biofilm-grown versus planktonic cells revealed a set of upregulated genes in biofilm-embedded cells, including factors involved in adhesion, colonization, and competition such as epidermin, type I toxin-antitoxin system, and phenol-soluble modulins (beta and epsilon). To investigate consequences for the host after encountering S. saccharolyticus, cytokine profiling and host cell viability were assessed by infection experiments with differentiated THP-1 cells. The microorganism strongly triggered the secretion of the tested pro-inflammatory cyto- and chemokines IL-6, IL-8, and TNF-alpha, determined at 24 h post-infection. S. saccharolyticus was less cytotoxic than Staphylococcus aureus. Taken together, the results indicate that S. saccharolyticus has substantial pathogenic potential. Thus, it can be a potential cause of orthopedic implant-associated infections and other types of deep-seated infections.

Research Tue, 01 Nov 2022 00:49:38 +0100 7dfcdc91-10b3-4428-bac4-02f6a3e12c89
<![CDATA[JMM Profile]]> Skovdal, S. M., Jørgensen, N. P., Meyer, R. L. Staphylococcus epidermidis is the most abundant commensal bacterium of human skin. Despite protecting us from foreign invaders, S. epidermidis itself exploits human vulnerability when given the opportunity. Such opportunities arise when patients are immunocompromised or when biomedical implants present an opportunity to colonize the surface and form biofilms. S. epidermidis is one of the most frequently isolated organisms from implanted devices and from bloodstream infections. However, S. epidermidis infections are often recalcitrant to antibiotics because of biofilm-associated antibiotic tolerance. Furthermore, the emergence and spread of nearly pan-resistant strains is a considerable health concern. Symptoms can be subclinical, making diagnosis challenging, and treatment with antibiotics is inefficient. For now, infection prevention remains the best strategy available.

Research Sat, 01 Oct 2022 00:49:38 +0200 1f396264-a26a-4384-8132-f49b679245ea
<![CDATA[Biofouling Control in Water Filtration Systems]]> Møllebjerg, A., Meyer, R. L. Research Thu, 01 Sep 2022 00:49:38 +0200 468bc437-8e0e-4902-a34c-545392da571a <![CDATA[Erratum for Møllebjerg et al., "The Bacterial Life Cycle in Textiles Is Governed by Fiber Hydrophobicity"]]> Møllebjerg, A., Palmén, L. G., Gori, K., Meyer, R. L. Research Sat, 01 Oct 2022 00:49:38 +0200 42418e5b-06e7-4086-bc05-ca60601d6452 <![CDATA[The Role of Nanoscale Distribution of Fibronectin in the Adhesion of Staphylococcus aureus Studied by Protein Patterning and DNA-PAINT]]> Khateb, H., Sørensen, R. S., Cramer, K., et al. Staphylococcus aureus is a widespread and highly virulent pathogen that can cause superficial and invasive infections. Interactions between S. aureus surface receptors and the extracellular matrix protein fibronectin mediate the bacterial invasion of host cells and is implicated in the colonization of medical implant surfaces. In this study, we investigate the role of distribution of both fibronectin and cellular receptors on the adhesion of S. aureus to interfaces as a model for primary adhesion at tissue interfaces or biomaterials. We present fibronectin in patches of systematically varied size (100-1000 nm) in a background of protein and bacteria rejecting chemistry based on PLL-g-PEG and studied S. aureus adhesion under flow. We developed a single molecule imaging assay for localizing fibronectin binding receptors on the surface of S. aureus via the super-resolution DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) technique. Our results indicate that S. aureus adhesion to fibronectin biointerfaces is regulated by the size of available ligand patterns, with an adhesion threshold of 300 nm and larger. DNA-PAINT was used to visualize fibronectin binding receptor organization in situ at ∼7 nm localization precision and with a surface density of 38-46 μm-2, revealing that the engagement of two or more receptors is required for strong S. aureus adhesion to fibronectin biointerfaces.

Research Fri, 01 Jul 2022 00:49:38 +0200 eed6fe74-a3be-4778-becf-6dc23a6b5c8b
<![CDATA[The giant staphylococcal protein Embp facilitates colonization of surfaces through Velcro-like attachment to fibrillated fibronectin]]> Khan, N., Aslan, H., Büttner, H., et al. Staphylococcus epidermidis causes some of the most hard-to-treat clinical infections by forming biofilms: Multicellular communities of bacteria encased in a protective matrix, supporting immune evasion and tolerance against antibiotics. Biofilms occur most commonly on medical implants, and a key event in implant colonization is the robust adherence to the surface, facilitated by interactions between bacterial surface proteins and host matrix components. S. epidermidis is equipped with a giant adhesive protein, Embp, which facilitates bacterial interactions with surface-deposited, but not soluble fibronectin. The structural basis behind this selective binding process has remained obscure. Using a suite of single-cell and single-molecule analysis techniques, we show that S. epidermidis is capable of such distinction because Embp binds specifically to fibrillated fibronectin on surfaces, while ignoring globular fibronectin in solution. S. epidermidis adherence is critically dependent on multi-valent interactions involving 50 fibronectin-binding repeats of Embp. This unusual, Velcro-like interaction proved critical for colonization of surfaces under high flow, making this newly identified attachment mechanism particularly relevant for colonization of intravascular devices, such as prosthetic heart valves or vascular grafts. Other biofilm-forming pathogens, such as Staphylococcus aureus, express homologs of Embp and likely deploy the same mechanism for surface colonization. Our results may open for a novel direction in efforts to combat devastating, biofilm-associated infections, as the development of implant materials that steer the conformation of adsorbed proteins is a much more manageable task than avoiding protein adsorption altogether.

Research Fri, 01 Jul 2022 00:49:38 +0200 fb012b0b-fe1f-4a9c-b0c0-a898c4ffd6f9
<![CDATA[A New Device for In Situ Dental Biofilm Collection Additively Manufactured by Direct Metal Laser Sintering and Vat Photopolymerization]]> Rikvold, P. T., Johnsen, K. K., Leonhardt, D., et al. Dental biofilms are complex medical biofilms that cause caries, the most prevalent disease of humankind. They are typically collected using handcrafted intraoral devices with mounted carriers for biofilm growth. As the geometry of handcrafted devices is not standardized, the shear forces acting on the biofilms and the access to salivary nutrients differ between carriers. The resulting variability in biofilm growth renders the comparison of different treatment modalities difficult. The aim of the present work was to design and validate an additively manufactured intraoral device with a dental bar produced by direct metal laser sintering and vat photopolymerized inserts with standardized geometry for the mounting of biofilm carriers. Additive manufacturing reduced the production time and cost, guaranteed an accurate fit of the devices and facilitated the handling of carriers without disturbing the biofilm. Biofilm growth was robust, with increasing thickness over time and moderate inter- and intraindividual variation (coefficients of variance 0.48-0.87). The biofilms showed the typical architecture and composition of dental biofilms, as evidenced by confocal microscopy and 16S rRNA gene sequencing. Deeper inserts offering increased protection from shear tended to increase the biofilm thickness, whereas prolonged exposure to sucrose during growth increased the biofilm volume but not the thickness. Ratiometric pH imaging revealed considerable pH variation between participants and also inside single biofilms. Intraoral devices for biofilm collection constitute a new application for medical additive manufacturing and offer the best possible basis for studying the influence of different treatment modalities on biofilm growth, composition, and virulence. The Clinical Trial Registration number is: 1-10-72-193-20.

Research Sun, 01 Oct 2023 00:49:38 +0200 5e85749a-17c0-4dda-b2df-18570ca5a7cb
<![CDATA[Aptamer-Targeted Drug Delivery for <i>Staphylococcus aureus </i>Biofilm]]> Ommen, P., Hansen, L., Hansen, B. K., Vu-Quang, H., Kjems, J., Meyer, R. L. Treatment of Staphylococcus aureus biofilm infections using conventional antibiotic therapy is challenging as only doses that are sublethal to the biofilm can be administered safely to patients. A potential solution to this challenge is targeted drug delivery. In this study, we tailored an aptamer-targeted liposomal drug delivery system for accumulation and delivery of antibiotics locally in S. aureus biofilm. In our search for a suitable targeting ligand, we identified six DNA aptamers that bound to S. aureus cells in biofilms, and we demonstrated that one of these aptamers could facilitate accumulation of liposomes around S. aureus cells inside the biofilm. Aptamer-targeted liposomes encapsulating a combination of vancomycin and rifampicin were able to eradicate S. aureus biofilm upon 24 h of treatment in vitro. Our results point to that aptamer-targeted drug delivery of antibiotics is a potential new strategy for treatment of S. aureus biofilm infections.

Research Fri, 01 Apr 2022 00:49:38 +0200 592b8007-49cb-4a7d-bfe4-cd21bbe751d5
<![CDATA[Fibrinolytic and Antibiotic Treatment of Prosthetic Vascular Graft Infections in a Novel Rat Model]]> Johansen, M. I., Rahbek, S. J., Jensen-Fangel, S., et al. Medical implants are prone to bacterial attachment and development of biofilms. Biofilms cause the bacteria’s recalcitrance to antibiotics, resulting in a chronic infection that can be fatal. Previously, we have shown that the combination of antibiotics with fibrinolytic drugs such as streptokinase and tissue plasminogen activator (tPA) can eradicate methicillin-resistant Staphylococcus aureus (MRSA) biofilms in vitro. In this study, we aim to compare the efficacy of antibiotic therapy with and without tPA to treat MRSA biofilms in a novel rat model of prosthetic vascular graft infections (PVGI)

Rats received a pre-inoculated vascular graft implanted in a. carotis communis. Implants were inoculated with approx. 4.4 CFU/graft of MRSA USA300 FPR3757. 10 days following surgery rats were randomized to either 1) vancomycin (50 mg/kg); 2) vancomycin + rifampicin (25 mg/kg); 3) vancomycin + tPA (0.9 mg/kg); 4) vancomycin + rifampicin + tPA or; 5) 0.9% NaCl, as a seven-day treatment. Hereafter, the rats were euthanized, and implants and organs were harvested for CFU enumeration.

Vancomycin and rifampicin treatment was superior compared to monotherapy with vancomycin, with a decrease in bacterial load on the prosthetics (2.89 ± 0.77, p= 0.0012, mean ± SD). Addition of tPA to antibiotic combination therapy did not show a further decrease in bacterial load (0.32±0.50, p= 0.526, mean ± SD), however, we saw a 16% cure rate in the vancomycin + rifampicin vs. 40% cure rate in the vancomycin + rifampicin + tPA group. Whilst interesting, this trend was at our sample size not significant (p=0.24, Fisher’s exact test.

This study is the first to treat PVGI in an in vivo model, with clinically relevant doses of a fibrinolytic drug in conjunction with antibiotics. Our model could be used as a basis for future testing of both current antibiotics against PVGI and novel compounds against biofilm producing bacteria. The study drug combinations had limited effect at our samples size, and animal studies on a larger scale are therefore needed to evaluate the potential of this combination against PVGI.
Communication Wed, 09 Jun 2021 00:49:38 +0200 9f47fe18-228e-4c52-98d8-adab50540619
<![CDATA[The bacterial life cycle in textiles is governed by fiber hydrophobicity]]> Møllebjerg, A., Palmén, L. G., Gori, K., Meyer, R. L. Colonization of textiles and subsequent metabolic degradation of sweat and sebum components by axillary skin bacteria cause the characteristic sweat malodor and discoloring of dirty clothes. Once inside the textile, the bacteria can form biofilms that are hard to remove by conventional washing. When the biofilm persists after washing, the textiles retain the sweat odor. To design biofilm removal and prevention strategies, the bacterial behavior needs to be understood in depth. Here, we aim to study the bacterial behavior in each of the four stages of the bacterial life cycle in textiles: Adhesion, growth, drying, and washing. To accomplish this, we designed a novel in vitro model to mimic physiological sweating in cotton and polyester textiles, in which many of the parameters that influence bacterial behavior could be controlled. Due to the higher hydrophobicity, polyester adhered more bacteria and absorbed more sebum, the bacteria's primary nutrient source. Bacteria were therefore also more active in polyester textiles. However, polyester did not bind water as well as cotton. The increased water content of cotton allowed some species to retain a higher activity after the textile had dried. However, none of the textiles retained enough water upon drying to prevent the bacteria from adhering irreversibly to the textile fibers. This work demonstrates that bacterial colonization of textiles depends partially on the hydrophobic and hygroscopic properties of the textile material, indicating that it might be possible to direct bacterial behavior in a more favorable direction by modifying these surface properties.

Research Fri, 01 Oct 2021 00:49:38 +0200 52139771-0566-4cd9-b709-81d27c18bcf1
<![CDATA[Human Fibrinogen Inhibits Amyloid Assembly of Most Phenol-Soluble Modulins from Staphylococcus aureus]]> Najarzadeh, Z., Nielsen, J., Farzadfard, A., et al. Functional amyloids are highly organized protein/peptide structures that inter alia promote biofilm formation in different bacteria. One such example is provided by a family of 20-45 residue-long peptides called phenol-soluble modulins (PSMs) from Staphylococcus aureus. External components such as eukaryotic host proteins, which alter self-assembly of bacterial amyloids, can affect the biofilm matrix. Here, we studied the effect of the highly prevalent human plasma protein fibrinogen (Fg) on fibrillation of PSMs. Fg inhibits or suppresses fibrillation of most PSMs tested (PSMα1, PSMβ1, and PSMβ2) except for PSMα3, whose already rapid aggregation is accelerated even further by Fg but leads to amorphous β-rich aggregates rather than fibrils. Fg also induces PSMβ2 to form amorphous aggregates and diverts PSMα1 into off-pathway oligomers which consist of both Fg and PSMα1 and cannot seed fibrillation. Peptide arrays showed that Fg bound to the N-terminus of PSMα1, while it bound to the entire length of PSMα3 (except the C terminus) and to the C-termini of PSMβ1 and PSMβ2. The latter peptides are all positively charged, while Fg is negatively charged at physiological pH. The positive charges complement Fg's net negative charge of -7.6 at pH 7.4. Fg's ability to inhibit PSM fibrillation reveals a potential host-defense mechanism to prevent bacterial biofilm growth and infections in the human body.

Research Sun, 01 Aug 2021 00:49:38 +0200 05e1ac47-4311-4def-956c-58ba8d3b3f28
<![CDATA[Management of oral biofilms by nisin delivery in adhesive microdevices]]> Birk, S. E., Mosgaard, M. D., Kjeldsen, R. B., Boisen, A., Meyer, R. L., Nielsen, L. H. Numerous beneficial microbes thrive in the oral cavity where they form biofilms on dental and mucosal surfaces to get access to nutrients, and to avoid being carried away with the saliva. However, biofilm formation is also a virulence factor as it also protects pathogenic bacteria, providing them with an environment for proliferation causing oral infections. Oral hygiene relies on mechanical removal of biofilms. Some oral care products also contain antimicrobials, but effective eradication of biofilms with antimicrobials requires both a high concentration and long exposure time. In the present communication, we investigate the potential of using miniaturized drug delivery devices, known as microcontainers (MCs), to deliver the antimicrobial peptide, nisin to an oral multi-species biofilm. MCs are loaded with nisin and X-ray micro-computed tomography reveals a full release of nisin through a chitosan lid within 15 min. Chitosan-coated MCs display substantial bioadhesion to the buccal mucosa compared to non-coated MCs (68.6 ± 14.3% vs 33.8 ± 5.2%). Confocal monitoring of multi-species biofilms reveals antibacterial effects of nisin-loaded chitosan-coated MCs with a faster onset (after 3 h) compared to solution-based delivery (after 9 h). Our study shows the potential of using MCs for treatment of multi-species oral biofilms and is encouraging for further design of drug delivery devices to treat oral diseases.

Research Fri, 01 Oct 2021 00:49:38 +0200 c6ff27ff-396a-4ac4-abde-f71e0d70880f
<![CDATA[Genome sequence of staphylococcus epidermidis AUH4567, a clinical isolate from an infected central venous catheter]]> Meyer, R. L., Skovdal, S. M., Marshall, I. P.G., Schreiber, L., Nørskov-Lauritsen, N., Jørgensen, N. P. Staphylococcus epidermidis is a common cause of implant-associated infections, and this is related to its ability to form biofilms. Strain-to-strain variability in biofilm formation is likely caused by genetic differences. Here, we present a draft genome of S. epidermidis AUH4567, which was isolated from a central venous catheter infection.

Research Mon, 01 Mar 2021 00:49:38 +0100 ae825aed-81dc-40c9-81f9-f9aec62cc8a1
<![CDATA[Activation of the Two-Component System LisRK Promotes Cell Adhesion and High Ampicillin Tolerance in Listeria<i> monocytogenes</i>]]> Aslan, H., Petersen, M. E., De Berardinis, A., et al. Listeria monocytogenes is a foodborne pathogen which can survive in harsh environmental conditions. It responds to external stimuli through an array of two-component systems (TCS) that sense external cues. Several TCS, including LisRK, have been linked to Listeria’s ability to grow at slightly elevated antibiotic levels. The aim of this study was to determine if the TCS LisRK is also involved in acquiring the high antibiotic tolerance that is characteristic of persister cells. LisRK activates a response that leads to remodeling of the cell envelope, and we therefore hypothesized that activation of LisRK could also increase in the cells’ adhesiveness and initiate the first step in biofilm formation. We used a ΔlisR mutant to study antibiotic tolerance in the presence and absence of LisRK, and a GFP reporter strain to visualize the activation of LisRK in L. monocytogenes LO28 at a single-cell level. LisRK was activated in most cells in stationary phase cultures. Antimicrobial susceptibility tests showed that LisRK was required for the generation of ampicillin tolerance under these conditions. The wildtype strain tolerated exposure to ampicillin at 1,000 × inhibitory levels for 24 h, and the fraction of surviving cells was 20,000-fold higher in the wildtype strain compared to the ΔlisR mutant. The same protection was not offered to other antibiotics (vancomycin, gentamicin, tetracycline), and the mechanism for antibiotic tolerance is thus highly specific. Furthermore, quantification of bacterial attachment rates and attachment force also revealed that the absence of a functional LisRK rendered the cells less adhesive. Hence, LisRK TCS promotes multiple protective mechanisms simultaneously.

Research Fri, 01 Jan 2021 00:49:38 +0100 9d688fce-9187-4795-aaf0-472f2a2c53be
<![CDATA[Host factors abolish the need for polysaccharides and extracellular matrix-binding protein in <i>Staphylococcus epidermidis </i>biofilm formation]]> Skovdal, S. M., Hansen, L. K., Ivarsen, D. M., et al. Introduction. Staphylococcus epidermidis is predominant in implant-associated infections due to its capability to form biofilms. It can deploy several strategies for biofilm development using either polysaccharide intercellular adhesin (PIA), extracellular DNA (eDNA) and/or proteins, such as the extracellular matrix-binding protein (Embp). Hypothesis/Gap Statement. We hypothesize that the dichotomic regulation of S. epidermidis adhesins is linked to whether it is inside a host or not, and that in vitro biofilm investigations in laboratory media may not reflect actual biofilms in vivo. Aim. We address the importance of PIA and Embp in biofilm grown in 'humanized' media to understand if these components play different roles in biofilm formation under conditions where bacteria can incorporate host proteins in the biofilm matrix. Methodology. S. epidermidis 1585 WT (deficient in icaADBC), and derivative strains that either lack embp, express embp from an inducible promotor, or express icaADBC from a plasmid, were cultivated in standard laboratory media, or in media with human plasma or serum. The amount, structure, elasticity and antimicrobial penetration of biofilms was quantified to describe structural differences caused by the different matrix components and growth conditions. Finally, we quantified the initiation of biofilms as suspended aggregates in response to host factors to determine how quickly the cells aggregate in response to the host environment and reach a size that protects them from phagocytosis. Results. S. epidermidis 1585 required polysaccharides to form biofilm in laboratory media. However, these observations were not representative of the biofilm phenotype in the presence of human plasma. If human plasma were present, polysaccharides and Embp were redundant for biofilm formation. Biofilms formed in human plasma were loosely attached and existed mostly as suspended aggregates. Aggregation occurred after 2 h of exposing cells to plasma or serum. Despite stark differences in the amount and composition of biofilms formed by polysaccharide-producing and Embp-producing strains in different media, there were no differences in vancomycin penetration or susceptibility. Conclusion. We suggest that the assumed importance of polysaccharides for biofilm formation is an artefact from studying biofilms in laboratory media void of human matrix components. The cell-cell aggregation of S. epidermidis can be activated by host factors without relying on either of the major adhesins, PIA and Embp, indicating a need to revisit the basic question of how S. epidermidis deploys self-produced and host-derived matrix components to form antibiotic-tolerant biofilms in vivo.

Research Mon, 01 Mar 2021 00:49:38 +0100 bea19448-194f-4abd-86fe-cd3f63f28c23
<![CDATA[Phenol-Soluble Modulins Modulate Persister Cell Formation in <i>Staphylococcus aureus</i>]]> Baldry, M., Bojer, M. S., Najarzadeh, Z., et al. Staphylococcus aureus is a human pathogen that can cause chronic and recurrent infections and is recalcitrant to antibiotic chemotherapy. This trait is partly attributed to its ability to form persister cells, which are subpopulations of cells that are tolerant to lethal concentrations of antibiotics. Recently, we showed that the phenol-soluble modulins (PSMs) expressed by S. aureus reduce persister cell formation. PSMs are a versatile group of toxins that, in addition to toxicity, form amyloid-like fibrils thought to support biofilm structures. Here, we examined individual or combined synthetic PSMα peptides and their equivalent amyloid-like fibrils on ciprofloxacin-selected S. aureus persister cells. We found that PSMα2 and the mixture of all four alpha peptides consistently were able to reduce persister frequency in all growth phases, and this activity was specifically linked to the presence of the soluble peptide as no effect was seen with fibrillated peptides. Persister reduction was particularly striking in a mutant that, due to mutations in the Krebs cycle, has enhanced ability to form persisters with PSMα4 and the combination of peptides being most effective. In biofilms, only the combination of peptides displayed persister reducing activity. Collectively, we report the individual contributions of PSMα peptides to persister cell reduction and that the combination of peptides generally was most effective. Strikingly, the fibrillated peptides lost activity and thus, if formed in bacterial cultures, they will be inactive against persister cells. Further studies will be needed to address the biological role of phenol-soluble modulins in reducing persister cells.

Research Sun, 01 Nov 2020 00:49:38 +0100 8fd35108-006e-488c-94e0-f751f050ac74
<![CDATA[Polycaprolactone-gelatin nanofibers incorporated with dual antibiotic-loaded carboxyl-modified silica nanoparticles]]> Gounani, Z., Pourianejad, S., Asadollahi, M. A., Meyer, R. L., Rosenholm, J. M., Arpanaei, A. Abstract: In this study, we used electrospun polycaprolactone (PCL) or a mixture of PCL and gelatin (Gel) in a mixed acidic solvent to develop antimicrobial electrospun nanofibers. Carboxyl-modified mesoporous silica nanoparticles (CMSNs) or CMSNs loaded with antibiotic drugs polymyxin B and vancomycin (CMSNs/ABs) were mixed with the electrospinning solution in concentrations of 1%, 2.5% and 5%. The nanofibers diameter measured between 122 and138 nm. Higher concentrations of gelatin or CMSNs increased hydrophilicity and degradability of the nanofibers. CMSNs enhanced nanofibers mechanical strength. PCL/Gel nanofibers incorporated with CMSNs/ABs (2.5% and 5%) showed high antibacterial efficiency against Pseudomonas aeruginosa and Staphylococcus aureus. Also bacterial cell adhesion decreased when 2.5% and 5% of CMSNs/ABs were incorporated in PCL/Gel mats. MTT and hemolysis assays indicated excellent biocompatibility of all types of electrospun nanofibers. This study confirms that a proper mixture of PCL, gelatin and CMSNs loaded with two antibiotics could offer antimicrobial activities with high biocompatibility and biodegradability properties. Graphic abstract: [Figure not available: see fulltext.].

Research Tue, 01 Dec 2020 00:49:38 +0100 e2d5d3b2-8b21-40b6-819d-d5ee4eebe604
<![CDATA[Design of a new intraoral splint with 3D-printed inserts for dental biofilm collection]]> Schlafer, S., Møllebjerg, A., Kambourakis Johnsen, K., Nielsen, S. M., Meyer, R. L. Research Wed, 01 Jul 2020 00:49:38 +0200 50eb4975-6769-4c09-a17b-8f03e1ac118b <![CDATA[Combination of Rhamnolipid and Chitosan in Nanoparticles Boosts Their Antimicrobial Efficacy]]> Marangon, C. A., Martins, V. C.A., Ling, M. H., et al. Nanomaterials have emerged as antimicrobial agents due to their unique physical and chemical properties. The development of nanoparticles (NPs) composed of natural biopolymers and biosurfactants have sparked interest, as they can be obtained without the use of complex chemical synthesis and toxic materials. In this study, we develop antimicrobial nanoparticles combining the biopolymer chitosan with the biosurfactant rhamnolipid. Addition of rhamnolipid reduced the size and polydispersity index of chitosan nanoparticles showing a more positive surface charge with improved stability, suggesting that chitosan-free amino groups are predominantly present on the surface of nanoparticles. Antimicrobial activity of chitosan/rhamnolipid nanoparticles (C/RL-NPs) against Staphylococcus strains surpassed that of either single rhamnolipid or chitosan, both in planktonic bacteria and biofilms. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of C/RL-NPs were determined considering the concentration of each individual molecule in NPs. MIC values of 14/19 μg mL-1 and MBC of 29/37 μg mL-1 were observed for S. aureus DSM 1104 and MIC and MBC of 29/37 and 58/75 μg mL-1 were observed against S. aureus ATCC 29213, respectively. For S. epidermidis, MIC and MBC of 7/9 and 14/19 μg mL-1 were noticed. Chitosan and chitosan nanoparticles eliminate the bacteria present in the upper parts of biofilms, while C/RL-NPs were more effective, eradicating most sessile bacteria and reducing the number of viable cells below the detection limit, when NPs concentration of 58/75 μg mL-1 was applied for both S. aureus DSM 1104 and S. epidermidis biofilms. The improved antibacterial efficacy of C/RL-NPs was linked to the increased local delivery of chitosan and rhamnolipid at the cell surface and, consequently, to their targets in Gram-positive bacteria. The combination of chitosan and rhamnolipid offers a promising strategy to the design of novel nanoparticles with low cytotoxicity, which can be exploited in pharmaceutical and food industries.

Research Wed, 01 Jan 2020 00:49:38 +0100 d3692f0e-fc42-4165-8dc0-d5a5b090ca0b
<![CDATA[Delivering antibiotics locally to biofilms by targeted drug delivery and prodrug therapy]]> Meyer, R. L., Walther, R., Nielsen, S. M., et al. Research Tue, 03 Sep 2019 00:49:39 +0200 d710cec8-95de-4ba2-8aaa-8df38625c30a <![CDATA[Distribution of extracellular DNA in <i>Listeria monocytogenes</i> biofilm]]> Šuláková, M., Pazlarová, J., Meyer, R. L., Demnerová, K. Extracellular DNA (eDNA) is an abundant matrix component that protects biofilm from environmental stress, facilitate horizontal gene transfer, and serve as a source of nutrients. eDNA is also found in Listeria monocytogenes biofilm, but it is unknown to which extent its importance as a matrix component varies in terms of phylogenetic relatedness. This study aims to determine if these variations exist. Biofilm forming capacity of ten L. monocytogenes strains of different phylogenetic lineages and serotypes was examined using crystal violet assay at 37°C and 22°C. eDNA content was evaluated fluorometrically at 37°C and at 22°C, then the 3D structure of biofilm was studied by confocal laser scanning microscopy (CLSM). Biofilm forming capacity differed significantly between the culturing conditions and was higher at 37°C than at ambient temperature. eDNA signal distribution was found to be influenced by strain and lineage. CLSM images revealed information about spatial distribution in the biofilm. The information about the eDNA spatial organisation in the biofilm contributes to the understanding of the role of eDNA in a biofilm formation.

Research Tue, 01 Jan 2019 00:49:39 +0100 51b7eaac-17a5-48d9-8f35-30aa6811d7f1
<![CDATA[Development of a label-free LSPR-Apta sensor for Staphylococcus aureus detection]]> Khateb, H., Klös, G., Meyer, R. L., Sutherland, D. S. The risk of foodborne diseases has increased over the last years. We have developed a simple, portable, and label-free optical sensor via aptamer recognition of Staphylococcus aureus at nanostructured plasmonic elements. The developed aptamers conjugated to a localized surface plasmon resonance (LSPR) sensing device were applied in both pure culture and artificially contaminated milk samples enabling a limit of detection of 10 3 CFU/mL for S. aureus in milk. There was no need for a pre-enrichment step, and the total analysis time decreased from 30 min to 120 s. Finite-difference time-domain was used to simulate the experimentally measured optical responses for a range of different sensor designs (100 and 200 nm disks), addressing the role of the near field and intrinsic refractive index sensitivity. A comparison of the aptamer to antibody-based recognition approaches showed that the thickness of the sensing layer was critical with a significantly larger response for the thinner aptamer layer. Comparison of differently sized metal nanostructures showed a significantly higher sensitivity for 200 nm diameter compared to 100 nm diameter disk structures resulting from both increases in bulk refractive index sensitivity and the extent to which the local field extends out from the metal surface. These findings confirmed that the developed gold nanodisk-based LSPR sensing chips could facilitate sensitive detection of S. aureus in food samples.

Research Wed, 01 Apr 2020 00:49:39 +0200 741b1dfc-bb24-4d9e-9096-9945cc9ec8c6
<![CDATA[Evaluation of Surface-initiated Polymer brush as Anti-scaling Coating for Plate Heat Exchangers]]> Friis, J. E., Subbiahdoss, G., Gerved, G., et al. Inorganic fouling is one of the challenging problems in heat exchanger applications. One approach to mitigate fouling is to employ surface coatings. In this study, we evaluated the feasibility of surface-initiated polymerization (SIP) as thin coating technology to mitigate CaCO3 formation for heat transfer applications. The extent of formation of CaCO3 on different types of poly(oligoethyleneglycol) methacrylate brushes (POEGMA) was investigated under stagnant and flow heat-exchanging conditions. Polymer brushes of high graft density reduced the surface coverage of CaCO3more effectively than the low graft density brushes. By contrast, the thickness of the brush did not correlate with the surface coverage of CaCO3. The comparison of stagnant and flow experiments revealed that the antiscaling property of- POEGMA brushes was due to low adhesion CaCO3 deposits though the brushes themselves do not prevent the nucleation of CaCO3. b. Finally, the SIP process was successfully scaled-up to coat commercial heat exchanger plates with thickness and homogeneity comparable to lab-scale surfaces. Under industrial testing, the POEGMA brushes extended the performance by 50 h before the commencement of complete blockage.

Research Fri, 01 Nov 2019 00:49:39 +0100 e9621489-1a41-4f98-9440-ce085d10f4fb
<![CDATA[Antifouling properties of layer by layer DNA coatings]]> Subbiahdoss, G., Zeng, G., Aslan, H., et al. Fouling is a major concern for solid/liquid interfaces of materials used in different applications. One approach of fouling control is the use of hydrophilic polymer coatings made from poly-anions and poly-cations using the layer-by-layer (LBL) method. The authors hypothesized that the poly-anionic properties and the poly-phosphate backbone of DNA would provide anti-biofouling and anti-scaling properties. To this end, poly(ethyleneimine)/DNA LBL coatings against microbial and inorganic fouling were developed, characterized and evaluated. DNA LBL coatings reduced inorganic fouling from tap water by 90% when incubated statically or under flow conditions mimicking surfaces in heat exchangers. The coatings also impaired biofilm formation by 93% on stainless steel from tap water, and resulted in a 97% lower adhesion force and reduced initial attachment of the human pathogens Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa on glass. This study demonstrates a proof of concept that LBL coatings with poly-anions harboring phosphate groups can address fouling in several applications.

Research Tue, 01 Jan 2019 00:49:39 +0100 3d7ce3b3-8157-436b-b83d-6f2ce004de97
<![CDATA[Cell wall associated protein TasA provides an initial binding component to extracellular polysaccharides in dual-species biofilm]]> Duanis-Assaf, D., Duanis-Assaf, T., Zeng, G., et al. Many bacteria in biofilm surround themselves by an extracellular matrix composed mainly of extracellular polysaccharide (EP), proteins such as amyloid-like fibers (ALF) and nucleic acids. While the importance of EP in attachment and acceleration of biofilm by a number of different bacterial species is well established, the contribution of ALF to attachment in multispecies biofilm remains unknown. The study presented here aimed to investigate the role of TasA, a precursor for ALF, in cell-cell interactions in dual-species biofilms of Bacillus subtilis and Streptococcus mutans. Expression of major B. subtilis matrix operons was significantly up-regulated in the presence of S. mutans during different stages of biofilm formation, suggesting that the two species interacted and modulated gene expression in each other. Wild-type B. subtilis expressing TasA adhered strongly to S. mutans biofilm, while a TasA-deficient mutant was less adhesive and consequently less abundant in the dual-species biofilm. Dextran, a biofilm polysaccharide, induced aggregation of B. subtilis and stimulated adhesion to S. mutans biofilms. This effect was only observed in the wild-type strain, suggesting that interactions between TasA and dextran-associated EP plays an important role in inter-species interactions during initial stages of multispecies biofilm development.

Research Sat, 01 Dec 2018 00:49:39 +0100 11bdd111-083a-4202-8131-15f4b224c32e
<![CDATA[Development of an iron oxide nanoparticle drug delivery system against Staphylococcus aureus biofilm infections]]> Dreier, C., Zelikin, A. N., Kjems, J., Meyer, R. L. Research Sat, 01 Sep 2018 00:49:39 +0200 04cd94a7-c25e-4ccc-884c-010100109eab <![CDATA[Innate glycosidic activity in metallic implants for localized synthesis of antibacterial drugs]]> Meer, M. T., Dillion, R., Nielsen, S. M., et al. Iron-containing metallic implants are shown herein to mediate hydrolysis of glycosidic linkages. Using glucuronide prodrugs for broad-spectrum fluoroquinolone antibacterial agents, we capitalize on this behaviour and perform localized synthesis of antimicrobials which affords a significant zone of inhibition of bacterial growth around the metallic material.

Research Tue, 01 Jan 2019 00:49:39 +0100 df8941a9-f777-455f-8db5-915e0fc48b98
<![CDATA[Mesoporous silica nanoparticles carrying multiple antibiotics provide enhanced synergistic effect and improved biocompatibility]]> Gounani, Z., Asadollahi, M. A., Pedersen, J. N., et al. polymyxin B and vancomycin on bare and carboxyl-modified mesoporous silica nanoparticles (B-MSNs and CMSNs, respectively) to achieve simulataneous local delivery of antibiotics against Gram-positive and –negative bacteria. Polymyxin B adsorbed preferentially to nanoparticles compared to vancomycin. The total antibiotic loading was 563 μg and 453 μg per mg B-MSNs or C-MSNs, respectively. Both B-MSNs and C-MSNs loaded with antibiotics were effective against Gram-negative and Gram-positive bacteria. The antibiotics had synergistic interactions against Gram-negative bacteria, and the antimicrobial efficacy was higher for antibiotic-loaded CMSNs compared to free antibiotics at the same concentration even though the cytotoxicity was lower. Our study
shows that formulations of existing antibiotics in nanocarrier systems can improve their therapeutic efficiency, indicating that combination therapy with drug-loaded silica nanoparticles may provide a better treatment outcome for infections that require high concentrations of multiple drugs.]]>
Research Fri, 01 Mar 2019 00:49:39 +0100 6e6892ff-db00-4098-8f48-d38c880bbff6
<![CDATA[Novel prosthecate bacteria from the candidate phylum Acetothermia]]> Hao, L., McIlroy, S. J., Kirkegaard, R. H., et al. Members of the candidate phylum Acetothermia are globally distributed and detected in various habitats. However, little is known about their physiology and ecological importance. In this study, an operational taxonomic unit belonging to Acetothermia was detected at high abundance in four full-scale anaerobic digesters by 16S rRNA gene amplicon sequencing. The first closed genome from this phylum was obtained by differential coverage binning of metagenomes and scaffolding with long nanopore reads. Genome annotation and metabolic reconstruction suggested an anaerobic chemoheterotrophic lifestyle in which the bacterium obtains energy and carbon via fermentation of peptides, amino acids, and simple sugars to acetate, formate, and hydrogen. The morphology was unusual and composed of a central rod-shaped cell with bipolar prosthecae as revealed by fluorescence in situ hybridization combined with confocal laser scanning microscopy, Raman microspectroscopy, and atomic force microscopy. We hypothesize that these prosthecae allow for increased nutrient uptake by greatly expanding the cell surface area, providing a competitive advantage under nutrient-limited conditions.

Research Mon, 01 Jan 2018 00:49:39 +0100 f7d4ca69-e3da-45af-bd2e-bdb8314eab63
<![CDATA[Identification and Directed Development of Non‐Organic Catalysts with Apparent Pan‐Enzymatic Mimicry into Nanozymes for Efficient Prodrug Conversion]]> Walther, R., Winther, A., Fruergaard, A. S., et al. Research Wed, 02 Jan 2019 00:49:39 +0100 212299d1-9b09-42bd-8c9b-aa2f9fc85ae3 <![CDATA[Quaternary Ammoniumyl Chitosan Derivatives for Eradication of Staphylococcus aureus Biofilms]]> Sahariah, P., Masson, M., Meyer, R. L. Bacterial biofilms tolerate extreme levels of antibiotics. Treatment of biofilm infections therefore requires the development of new or modified antimicrobials that can penetrate biofilms and are effective against dormant persistent cells. One such new approach uses the biodegradable biopolymer chitosan and its derivatives as antimicrobials. In this study, we performed synthetic modification of chitosan to selectively introduce different cationic and hydrophobic moieties at varying ratios on chitosan. This improved its aqueous solubility and antimicrobial activity toward bacterial biofilms. Initial evaluation of the chitosan derivatives showed increased activity toward planktonic Staphylococcus aureus. The effect of the quaternary ammoniumyl chitosan derivatives against Staphylococcus aureus biofilms was more variable. The most effective derivatives contained hydrophobic groups, and their efficacy against biofilms depended on the ratio and length of the alkyl chains. Three-dimensional imaging of biofilms confirmed the accessibility and antimicrobial effect of chitosan derivatives with alkyl chains in the full depth of the biofilms.

Research Sat, 01 Sep 2018 00:49:39 +0200 a98c7b75-c031-4e8e-a9f8-3875f4e94200
<![CDATA[Combatting implant-associated biofilms through localized drug synthesis]]> Walther, R., Nielsen, S. M., Christiansen, R., Meyer, R. L., Zelikin, A. N. Bacterial contamination of implantable biomaterials is a significant socioeconomic and healthcare burden. Indeed, bacterial colonization of implants after surgery has a high rate of incidence whereas concurrent prophylaxis using systemic antibiotics has limited clinical success. In this work, we develop enzyme-prodrug therapy (EPT) to prevent and to treat bacteria at interfaces. Towards the overall goal, novel prodrugs for fluoroquinolone antibiotics were developed on a privileged glucuronide scaffold. Whereas carbamoyl prodrugs were not stable and not suitable for EPT, glucuronides containing self-immolative linker between glucuronic acid masking group and the antibiotic were stable in solution and readily underwent bioconversion in the presence of β-glucuronidase. Surface coatings for model biomaterials were engineered using sequential polymer deposition technique. Resulting coatings afforded fast prodrug conversion and mediated antibacterial measures against planktonic species as evidenced by pronounced zone of bacterial growth inhibition around the biomaterial surface. These biomaterials coupled with the glucuronide prodrugs also effectively combatted bacteria within established biofilms and also successfully prevented bacterial colonization of the surface. To our knowledge, this is the first report of EPT engineered to the surface of biomaterials to mediate antibacterial measures.

Research Wed, 10 Oct 2018 00:49:39 +0200 3e01125b-d714-40c3-b1b1-84e5032d08df
<![CDATA[Effect of DNase treatment on adhesion and early biofilm formation of Enterococcus faecalis]]> Schlafer, S., Garcia, J., Meyer, R. L., Vaeth, M., Neuhaus, K. W. Objective: Extracellular DNA (eDNA) has been shown to be important for biofilm stability of the endodontic pathogen Enterococcus faecalis. In this study, we hypothesized that treatment with DNase prevents adhesion and disperses young E. faecalis biofilms in 96-well plates and root canals of extracted teeth.

Methods: E. faecalis eDNA in 96-well plates was visualized with TOTO-1 (R). The effect of DNase treatment was assessed in 96-well plates and in extracted single-rooted premolars (n=37) using a two-phase crossover design. E. faecalis was treated with DNase (50 Kunitz/mL) or heat-inactivated DNase for 1 h during adhesion or after 24 h of biofilm formation. In 96-well plates, adhering cells were quantified using confocal microscopy and digital image analysis. In root canals, the number of adhering cells was determined in dentine samples based on colony forming unit counts. Data from the 96-well plate were analyzed using one-tailed t-tests, and data from extracted teeth were analyzed using mixed-effect Poisson regressions.

Results: eDNA was present in wells colonized by E. faecalis after 1 h of adhesion and 24 h of biofilm formation; it was removed by DNase treatment, as evidenced by TOTO (R)-1 staining. DNase treatment reduced the area covered by cells in 96-well plates after 1 h (p

Conclusion: DNase treatment does not disperse endodontic E. faecalis biofilms. The sole use of DNase as an anti-biofilm agent in root canal treatments is not recommendable.

Research Mon, 01 Jan 2018 00:49:39 +0100 6648f0ab-4c0b-4a2b-b9f7-60aa9eb6f83f
<![CDATA[Ultra-dense polymer brush coating reduces Staphylococcus epidermidis biofilms on medical implants and improves antibiotic treatment outcome]]> Skovdal, S. M., Jørgensen, N. P., Petersen, E., et al. Staphylococcal biofilm formation is a severe complication of medical implants, leading to high antibiotic tolerance and treatment failure. Ultra-dense poly(ethylene glycol) (udPEG) coating resists adsorption of proteins, polysaccharides and extracellular DNA. It is therefore uniquely resistant to attachment by Staphylococcus epidermidis, which remains loosely adhered to the surface. Our aim was to determine if S. epidermidis remains susceptible to antibiotics when adhering to udPEG, and if udPEG coatings can improve the treatment outcome for implant-associated infections. We tested the in vitro efficacy of vancomycin treatment on recently adhered S. epidermidis AUH4567 on udPEG, conventional PEG or titanium surfaces using live/dead staining and microscopy. udPEG was then applied to titanium implants and inserted subcutaneously in mice and inoculated with S. epidermidis to induce infection. Mice were given antibiotic prophylaxis or a short antibiotic treatment. One group was given immunosuppressive therapy. After five days, implants and surrounding tissue were harvested for CFU enumeration. Only few S. epidermidis cells adhered to udPEG compared to conventional PEG and uncoated titanium, and a much lower fraction of cells on udPEG survived antibiotic treatment in vitro. In vivo, the bacterial load on implants in mice receiving vancomycin treatment was significantly lower on udPEG-coated compared to uncoated implants, also in neutropenic mice. Our results suggest that the improved outcome results from the coating's anti-adhesive properties that leads to less biofilm and increased efficacy of antibiotic treatment. Thus, the combination of udPEG with antibiotics is a promising strategy to prevent acute implant-associated infections that arise due to perioperative contaminations.

STATEMENT OF SIGNIFICANCE: Infections of medical implants is an ever-present danger. Here, bacteria develop biofilms that cannot be eradicated with antibiotics. By using an ultra-dense polymer-brush coating (udPEG), bacterial attachment and the subsequent biofilm formation can be reduced, resulting in increased antibiotic susceptibility of bacteria surrounding the implant. udPEG combined with antibiotics proved to significantly reduce bacteria on implants inserted into mice, in our animal model. As the coating is not antibacterial per se, it does not induce antimicrobial resistance and its effect is independent of the bacterial species. Our results are encouraging for the prospect of preventing and treating implant-associated infections that arise due to perioperative contaminations.

Research Wed, 01 Aug 2018 00:49:39 +0200 573b29b9-ab72-47c0-82f0-325f2f4cb390
<![CDATA[Protein Engineering Reveals Mechanisms of Functional Amyloid Formation in <i>Pseudomonas aeruginosa</i> Biofilms]]> Bleem, A., Christiansen, G., Madsen, D. J., et al. Amyloids are typically associated with neurodegenerative diseases, but recent research demonstrates that several bacteria utilize functional amyloid fibrils to fortify the biofilm extracellular matrix and thereby resist antibiotic treatments. In Pseudomonas aeruginosa, these fibrils are composed predominantly of FapC, a protein with high sequence conservation among the genera. Previous studies established FapC as the major amyloid subunit, but its mechanism of fibril formation in P. aeruginosa remained largely unexplored. Here, we examine the FapC sequence in greater detail through a combination of bioinformatics and protein engineering, and we identify specific motifs that are implicated in amyloid formation. Sequence regions of high evolutionary conservation tend to coincide with regions of high amyloid propensity, and mutation of amyloidogenic motifs to a designed, non-amyloidogenic motif suppresses fibril formation in a pH-dependent manner. We establish the particular significance of the third repeat motif in promoting fibril formation and also demonstrate emergence of soluble oligomer species early in the aggregation pathway. The insights reported here expand our understanding of the mechanism of amyloid polymerization in P. aeruginosa, laying the foundation for development of new amyloid inhibitors to combat recalcitrant biofilm infections.

Research Fri, 12 Oct 2018 00:49:39 +0200 fab62c4f-cce6-481d-bd23-1c938f99dbe1
<![CDATA[Preclinical evaluation of potential infection-imaging probe [<sup>68</sup>Ga]Ga-DOTA-K-A9 in sterile and infectious inflammation]]> Nielsen, K. M., Jørgensen, N. P., Kyneb, M. H., et al. The development of bacteria-specific infection radiotracers is of considerable interest to improve diagnostic accuracy and enabling therapy monitoring. The aim of this study was to determine if the previously reported radiolabelled 1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid (DOTA) conjugated peptide [ 68Ga]Ga-DOTA-K-A9 could detect a staphylococcal infection in vivo and distinguish it from aseptic inflammation. An optimized [ 68Ga]Ga-DOTA-K-A9 synthesis omitting the use of acetone was developed, yielding 93 ± 0.9% radiochemical purity. The in vivo infection binding specificity of [ 68Ga]Ga-DOTA-K-A9 was evaluated by micro positron emission tomography/magnetic resonance imaging of 15 mice with either subcutaneous Staphylococcus aureus infection or turpentine-induced inflammation and compared with 2-deoxy-2-[ 18F]fluoro-D-glucose ([ 18F]FDG). The scans showed that [ 68Ga]Ga-DOTA-K-A9 accumulated in all the infected mice at injected doses ≥3.6 MBq. However, the tracer was not found to be selective towards infection, since the [ 68Ga]Ga-DOTA-K-A9 also accumulated in mice with inflammation. In a concurrent in vitro binding evaluation performed with a 5-carboxytetramethylrhodamine (TAMRA) fluorescence analogue of the peptide, TAMRA-K-A9, the microscopy results suggested that TAMRA-K-A9 bound to an intracellular epitope and therefore preferentially targeted dead bacteria. Thus, the [ 68Ga]Ga-DOTA-K-A9 uptake observed in vivo is presumably a combination of local hyperemia, vascular leakiness and/or binding to an epitope present in dead bacteria.

Research Wed, 23 May 2018 00:49:39 +0200 b54d1ae6-a607-47be-9ee5-38b1e38f8767
<![CDATA[Ag/Fe<sub>3</sub>O<sub>4</sub> nanocomposites penetrate and eradicate <i>S.aureus</i> biofilm in an <i>in vitro</i> chronic wound model]]> Ghaseminezhad, S. M., Shojaosadati, S. A., Meyer, R. L. Bacterial biofilms are a common cause of the persistence of chronic wounds, and continue to be an unsolved problem in infection microbiology due to their tolerance to antibiotics. Silver nanoparticles (Ag-NPs) have attracted attention as an alternative to antibiotics for treatment of wound infections, but their use is challenged by limited tissue penetration and high cytotoxicity. The aim of this study was to show that combination of Ag nanoparticles with Fe3O4 to produce Ag/Fe3O4 nanocomposites (NCs) can overcome these problems, as they penetrate and eradicate biofilms when applying a magnetic field. Ag/Fe3O4-NCs were synthesized using starch as a stabilizer and linker between Ag and Fe3O4 NPs, resulting in agglomerations of 20 nm Ag-NPs and 5 nm Fe3O4 NPs. The antibacterial activity was evaluated against an in vitro chronic wound biofilm model, and cytotoxicity was evaluated on human fibroblasts. Increasing the amount of starch during synthesis led to formation of NCs with increased antibacterial activity. In comparison to Ag-NPs, the NCs showed lower Ag+ release, less ROS production, were less cytotoxic, but nevertheless, their antimicrobial efficacy was higher. Furthermore, their efficiency against biofilm could be enhanced by applying a magnetic field, which ensured penetration of the entire biofilm. In conclusion, Ag/Fe3O4-NCs display important advantages over Ag-NPs as a potential avenue for development of novel therapeutic strategies for treatment of chronic wound infections. (C) 2017 Elsevier B.V. All rights reserved.

Research Thu, 01 Mar 2018 00:49:39 +0100 2279d8c4-ad9f-4e42-858c-e72d7f7f8dd5
<![CDATA[Loading of polymyxin B onto anionic mesoporous silica nanoparticles retains antibacterial activity and enhances biocompatibility]]> Gounani, Z., Asadollahi, M. A., Meyer, R. L., Arpanaei, A. Polymyxin B is a polycationic antibiotic used as the last line treatment against antibiotic-resistant Gram negative bacteria. However, application of polymyxin B is limited because of its toxicity effects. Herein, we used bare and surface modified mesoporous silica nanoparticles (MSNs) with an average diameter of 72.29 +/- 8.17 nm as adsorbent for polymyxin B to improve its therapeutic properties. The polymyxin B adsorption onto MSN surfaces was explained as a function of pH, type of buffer and surface charge of nanoparticles, according to the zeta-potential of silica nanoparticles and adsorption kinetics results. The highest value of the adsorption capacity (about 401 +/- 15.38 mg polymyxin B/g silica nanoparticles) was obtained for the bare nanoparticles in Tris buffer, pH 9. Release profiles of polymyxin B showed a sustained release pattern, fitting Power law and Hill models. The antibiotic molecules-loaded nanoparticles showed enhanced antibacterial activity compared to free antibiotic against different Gram negative bacteria. Biocompatibility evaluation results revealed that loading of polymyxin B onto MSNs can decrease the cytotoxicity effects of the drug by reducing ROS generation. Our results suggest that formulation of drugs by adsorption onto MSNs may offer a way forward to overcome the adverse effects of some antibiotics such as polymyxin B without compromising their antimicrobial properties.

Research Thu, 15 Feb 2018 00:49:39 +0100 ffd5eb32-d954-4dc3-9c2e-8745b849272d
<![CDATA[Prospective role of indigenous Exiguobacterium profundum PT2 in arsenic biotransformation and biosorption by planktonic cultures and biofilms]]> Rehman, S. ., Andreasen, R., li, Y., et al. Aims: The aim of this study was to analyse arsenic (As) transformation and biosorption by indigenous As-resistant bacteria both in planktonic and biofilm modes of growth. Methods and Results: As-resistant bacteria were isolated from industrial waste water and strain PT2, and identified as Exiguobacterium profundum through 16S rRNA gene sequencing was selected for further study. As transformation and biosorption by E. profundumPT2 was determined by HPLC-ICP-MS analysis. Planktonic cultures reduced 3·73 mmol l −1 As 5+ into As 3+ from artificial waste water effluent after 48-h incubation. In case of biosorption, planktonic cultures and biofilms exhibited 25·2 and 29·4 mg g −1 biomass biosorption, respectively. As biosorption kinetics followed Freundlich isotherm and pseudo second-order model. Biofilm formation peaked after 3 days of incubation, and in the presence of As stress, biofilm formation was significantly affected in contrast to control (P < 0·05). Homogeneous nature of mature biofilms with an increased demand of nutrients was revealed by minimum roughness and maximum surface to biovolume ratio measured through CLSM analysis. Conclusion: Indigenous As-resistant E. profundumPT2 was found capable of As transformation and biosorption both in the form of planktonic cultures and biofilms. Significance and Impact of the Study: Indigenous biofilm forming E. profundum PT2 revealing As biosorption and biotransformation potential is presented an eco-friendly and cost-effective source for As remediation that can be implemented for waste water treatment.

Research Thu, 01 Feb 2018 00:49:39 +0100 f3e8a5ca-b363-4965-bc00-243698cf616b
<![CDATA[Pan-genome analysis of the genus Finegoldia identifies two distinct clades, strain-specific heterogeneity, and putative virulence factors]]> Brüggemann, H., Jensen, A., Nazipi, S., et al. Finegoldia magna, a Gram-positive anaerobic coccus, is an opportunistic pathogen, associated with medical device-related infections. F. magna is the only described species of the genus Finegoldia. We report the analysis of 17 genomes of Finegoldia isolates. Phylogenomic analyses showed that the Finegoldia population can be divided into two distinct clades, with an average nucleotide identity of 90.7%. One clade contains strains of F. magna, whereas the other clade includes more heterogeneous strains, hereafter tentatively named "Finegoldia nericia". The latter species appears to be more abundant in the human microbiome. Surface structure differences between strains of F. magna and "F. nericia" were detected by microscopy. Strain-specific heterogeneity is high and previously identified host-interacting factors are present only in subsets of "F. nericia" and F. magna strains. However, all genomes encode multiple host factor-binding proteins such as albumin-, collagen-, and immunoglobulin-binding proteins, and two to four copies of CAMP (Christie-Atkins-Munch-Petersen) factors; in accordance, most strains show a positive CAMP reaction for co-hemolysis. Our work sheds new light of the genus Finegoldia and its ability to bind host components. Future research should explore if the genomic differences identified here affect the potential of different Finegoldia species and strains to cause opportunistic infections.

Research Wed, 10 Jan 2018 00:49:39 +0100 f889ea41-584a-4d09-80e4-064354a70ac6