Publications - Publications en-us PURE Extension (Web Department) 30 <![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 21:55:59 +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 21:55:59 +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 21:55:59 +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 21:55:59 +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 21:55:59 +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 21:55:59 +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 21:55:59 +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 21:55:59 +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 21:55:59 +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 21:55:59 +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 21:55:59 +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 21:55:59 +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 21:55:59 +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. Research Tue, 01 Jan 2019 21:55:59 +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. Research Wed, 01 Apr 2020 21:55:59 +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 21:55:59 +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 21:55:59 +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 21:55:59 +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 21:55:59 +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 21:55:59 +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 21:55:59 +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. Research Mon, 01 Jan 2018 21:55:59 +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 21:55:59 +0100 212299d1-9b09-42bd-8c9b-aa2f9fc85ae3 <![CDATA[Quaternary Ammoniumyl Chitosan Derivatives for Eradication of Staphylococcus aureus Biofilms]]> Sahariah, P., Masson, M., Meyer, R. L. Research Sat, 01 Sep 2018 21:55:59 +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 21:55:59 +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 21:55:59 +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 21:55:59 +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 21:55:59 +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. Research Wed, 23 May 2018 21:55:59 +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 21:55:59 +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 21:55:59 +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. Research Thu, 01 Feb 2018 21:55:59 +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 21:55:59 +0100 f889ea41-584a-4d09-80e4-064354a70ac6
<![CDATA[Antibacterial isoeugenol coating on stainless steel and polyethylene surfaces prevents biofilm growth]]> Nielsen, C. K., Subbiahdoss, G., Zeng, G., et al. Aims: Pathogenic bacteria can spread between individuals or between food items via the surfaces they share. Limiting the survival of pathogens on surfaces, therefore, presents an opportunity to limit at least one route of how pathogens spread. In this study, we propose that a simple coating with the essential oil isoeugenol can be used to circumvent the problem of bacterial transfer via surfaces.

Methods and Results: Two commonly used materials, stainless steel and polyethylene, were coated by physical adsorption, and the coatings were characterized by Raman spectroscopy, atomic force microscopy and water contact angle measurements. We quantified and visualized the colonization of coated and uncoated surfaces by three bacteria: Staphylococcus aureus, Listeria monocytogenes and Pseudomonas fluorescens. No viable cells were detected on surfaces coated with isoeugenol.

Conclusions: The isoeugenol coating prepared with simple adsorption proved effective in preventing biofilm formation on stainless steel and polyethylene surfaces. The result was caused by the antibacterial effect of isoeugenol, as the coating did not diminish the adhesive properties of the surface.

Significance and Impact of the Study: Our study demonstrates that a simple isoeugenol coating can prevent biofilm formation of S. aureus, L. monocytogenes and P. fluorescens on two commonly used surfaces.

Research Mon, 01 Jan 2018 21:55:59 +0100 f11f98f2-cdbe-4c61-9007-b4f1bec3217a
<![CDATA[Strategies for prevention and treatment of staphylococcal biofilms]]> Meyer, R. L. Innovative biomaterials may at best delay biofilm formation and an important question in this context is to understand how the material can contribute to more successful antibiotic treatment by not providing the cues that trigger the onset of antibiotic tolerance in the attached bacteria. This is an aspect we are currently studying in the development of anti-adhesive polymer coatings.
While novel biomaterials may lower the risk of acute infections shortly after surgery, it is unlikely that such infections can be prevented indefinitely. It is therefore also important to work towards developing treatments that more effectively tackle biofilm infections. We have explored how the combination of antibiotic therapy with matrix-targeting enzymes can enhance the efficacy of antibiotics. The matrix composition is highly variable among different bacterial species, and this strategy will not produce a one-fits-all solution. However, for Staphylococcus aureus biofilms, we have found a promising lead in targeting the matrix with fibrinolytic drugs, and we are now moving on to test our treatment strategy in vivo.
Research Sat, 01 Apr 2017 21:55:59 +0200 89acb321-67a1-4945-be9a-c2f5921cabfa
<![CDATA[How can in vitro models best reflect in vivo Staphylococcus biofilms?]]> Meyer, R. L. Research Tue, 15 Aug 2017 21:55:59 +0200 fc7ec0a7-397f-4f08-b260-5a8bd387904f <![CDATA[Inhibition of the ATP Synthase Eliminates the Intrinsic Resistance of Staphylococcus aureus towards Polymyxins]]> Vestergaard, M., Nøhr-Meldgaard, K., Bojer, M. S., et al. Staphylococcus aureus is intrinsically resistant to polymyxins (polymyxin B and colistin), an important class of cationic antimicrobial peptides used in treatment of Gram-negative bacterial infections. To understand the mechanisms underlying intrinsic polymyxin resistance in S. aureus, we screened the Nebraska Transposon Mutant Library established in S. aureus strain JE2 for increased susceptibility to polymyxin B. Nineteen mutants displayed at least 2-fold reductions in MIC, while the greatest reductions (8-fold) were observed for mutants with inactivation of either graS, graR, vraF, or vraG or the subunits of the ATP synthase (atpA, atpB, atpG, or atpH), which during respiration is the main source of energy. Inactivation of atpA also conferred hypersusceptibility to colistin and the aminoglycoside gentamicin, whereas susceptibilities to nisin, gallidermin, bacitracin, vancomycin, ciprofloxacin, linezolid, daptomycin, and oxacillin were unchanged. ATP synthase activity is known to be inhibited by oligomycin A, and the presence of this compound increased polymyxin B-mediated killing of S. aureus Our results demonstrate that the ATP synthase contributes to intrinsic resistance of S. aureus towards polymyxins and that inhibition of the ATP synthase sensitizes S. aureus to this group of compounds. These findings show that by modulation of bacterial metabolism, new classes of antibiotics may show efficacy against pathogens towards which they were previously considered inapplicable. In light of the need for new treatment options for infections with serious pathogens like S. aureus, this approach may pave the way for novel applications of existing antibiotics.IMPORTANCE Bacterial pathogens that cause disease in humans remain a serious threat to public health, and antibiotics are still our primary weapon in treating bacterial diseases. The ability to eradicate bacterial infections is critically challenged by development of resistance to all clinically available antibiotics. Polymyxins constitute an important class of antibiotics for treatment of infections caused by Gram-negative pathogens, whereas Gram-positive bacteria remain largely insusceptible towards class of antibiotics. Here we performed a whole-genome screen among nonessential genes for polymyxin intrinsic resistance determinants in Staphylococcus aureus We found that the ATP synthase is important for polymyxin susceptibility and that inhibition of the ATP synthase sensitizes S. aureus towards polymyxins. Our study provides novel insights into the mechanisms that limit polymyxin activity against S. aureus and provides valuable targets for inhibitors to potentially enable the use of polymyxins against S. aureus and other Gram-positive pathogens.

Research Tue, 05 Sep 2017 21:55:59 +0200 5ac7ccbf-6cf5-4ceb-9d5e-59e454361206
<![CDATA[A transposon mutant library of<i> Bacillus cereus</i> ATCC 10987 reveals novel genes required for biofilm formation and implicates motility as an important factor for pellicle-biofilm formation]]> Okshevsky, M. U., Louw, M. G., Otero Lamela, E., Nilsson, M., Tolker-Nielsen, T., Meyer, R. L. Research Sun, 01 Apr 2018 21:55:59 +0200 5493a809-0722-4a07-b777-2239920f476a <![CDATA[A Targeting Agent For Drug Delivery To Staphylococcus Aureus Biofilms]]> Andersen, P. O., Hansen, L., Vu Quang, H., Kjems, J., Meyer, R. L. Research Sun, 01 Jan 2017 21:55:59 +0100 589e6e65-c901-428c-87be-cccf5dbeefe7 <![CDATA[The Immunomodulatory Drug Glatiramer Acetate is Also an Effective Antimicrobial Agent that Kills Gram-negative Bacteria]]> Christiansen, S. H., Murphy, R. A., Juul-Madsen, K., et al. Classic drug development strategies have failed to meet the urgent clinical needs in treating infections with Gram-negative bacteria. Repurposing drugs can lead to timely availability of new antibiotics, accelerated by existing safety profiles. Glatiramer acetate (GA) is a widely used and safe formulation for treatment of multiple sclerosis. It contains a large diversity of essentially isomeric polypeptides with the cationic and amphiphilic character of many antimicrobial peptides (AMP). Here, we report that GA is antibacterial, targeting Gram-negative organisms with higher activity towards Pseudomonas aeruginosa than the naturally-occurring AMP LL-37 in human plasma. As judged from flow cytometric assays, bacterial killing by GA occurred within minutes. Laboratory strains of Escherichia coli and P. aeruginosa were killed by a process of condensing intracellular contents. Efficient killing by GA was also demonstrated in Acinetobacter baumannii clinical isolates and approximately 50% of clinical isolates of P. aeruginosa from chronic airway infection in CF patients. By contrast, the Gram-positive Staphylococcus aureus cells appeared to be protected from GA by an increased formation of nm-scale particulates. Our data identify GA as an attractive drug repurposing candidate to treat infections with Gram-negative bacteria.

Research Wed, 15 Nov 2017 21:55:59 +0100 a0d3e505-53a5-4a41-8160-0904cffb8608
<![CDATA[Host Proteins Determine MRSA Biofilm Structure and Integrity]]> Dreier, C., Nielsen, A., Jørgensen, N. P., Kragstrup, T. W., Meyer, R. L.
Biofilms were grown in the presence of synovial fluid from rheumatoid arteritis patients to mimic in vivo conditions, where bacteria incorporate hECM proteins into the biofilm matrix. Difference in biofilm structure, with and without addition of hECM to growth media, was visualized by confocal laser scanning microscopy. Two enzymatic degradation experiments were used to study biofilm matrix composition and importance of hECM proteins: enzymatic removal of specific hECM proteins from growth media, before biofilm formation, and enzymatic treatment of 24-hour-old biofilms.

hECM addition changed the overall biofilm structure, with larger dispersion of cells within the biofilm matrix. Fibrin, elastin, and collagen were important in forming and maintaining the biofilm structure. Their absence, from growth media, reduced biofilm formation 5-fold, indicating that they are important for biofilm initiation. Their enzymatic degradation, in an established biofilm, caused dispersal, showing that these proteins are critical for structural integrity. A combination of antibiotics with hECM degrading enzymes did not improve the treatment outcome.

We conclude that while hECM proteins are an integral part of the biofilm matrix, we find no evidence that these matrix components are directly responsible for the biofilm’s unique antibiotic resilience. The hECM proteins are however highly important in determining biofilm structure and initiation. When utilizing in vitro biofilm models, we therefore recommend addition of hECM proteins to standard growth media, in order to mimic biofilm properties and structure seen in vivo.]]>
Research Sun, 01 Jan 2017 21:55:59 +0100 b1fe3020-f630-4b52-8acf-e776ae3dcf04
<![CDATA[Osteopontin Reduces the Adhesion Force of Dental Bacteria Without Blocking Bacterial Cell Surface Glycoconjugates]]> Kristensen, M. F., Zeng, G., Neu, T. R., Meyer, R. L., Schlafer, S. Research Sun, 01 Jan 2017 21:55:59 +0100 9fe1c7fe-3947-48d6-911b-654ffb781066 <![CDATA[Extracellular DNA Contributes to Dental Biofilm Stability]]> Schlafer, S., Meyer, R. L., Dige, I., Regina, V. R. Research Tue, 01 Aug 2017 21:55:59 +0200 11544a52-7824-4bb7-a481-10fc3460fc9d <![CDATA[Osteopontin adsorption to Gram-positive cells reduces adhesion forces and attachment to surfaces under flow]]> Kristensen, M. F., Zeng, G., Neu, T. R., Meyer, R. L., Baelum, V., Schlafer, S. The bovine milk protein osteopontin (OPN) may be an efficient means to prevent bacterial adhesion to dental tissues and control biofilm formation. This study sought to determine to what extent OPN impacts adhesion forces and surface attachment of different bacterial strains involved in dental caries or medical device-related infections. It further investigated if OPN's effect on adhesion is caused by blocking the accessibility of glycoconjugates on bacterial surfaces. Bacterial adhesion was determined in a shear-controlled flow cell system in the presence of different concentrations of OPN, and interaction forces of single bacteria were quantified using single-cell force spectroscopy before and after OPN exposure. Moreover, the study investigated OPN's effect on the accessibility of cell surface glycoconjugates through fluorescence lectin-binding analysis. OPN strongly affected bacterial adhesion in a dose-dependent manner for all investigated species (Actinomyces naeslundii, Actinomyces viscosus, Lactobacillus paracasei subsp. paracasei, Staphylococcus epidermidis, Streptococcus mitis, and Streptococcus oralis). Likewise, adhesion forces decreased after OPN treatment. No effect of OPN on the lectin-accessibility to glycoconjugates was found. OPN reduces the adhesion and adhesion force/energy of a variety of bacteria and has a potential therapeutic use for biofilm control. OPN acts upon bacterial adhesion without blocking cell surface glycoconjugates.

Research Wed, 11 Oct 2017 21:55:59 +0200 6ccca2ba-c465-4bd7-8347-f5f5363a8385
<![CDATA[Quantification of biofilm biomass by staining]]> Ommen, P., Zobek, N., Meyer, R. L. Crystal violet staining is commonly used for quantification of biofilm formation, although it is highly toxic. Here we test safranin as a non-toxic replacement. Safranin staining provided similar results as crystal violet, but with higher reproducibility. We therefore recommend safranin staining for biofilm biomass quantification.

Research Sun, 01 Oct 2017 21:55:59 +0200 0b3764bd-dcf7-4e7c-a295-e1483cad40fc
<![CDATA[Differences in Gene Expression Profiles between Early and Late Isolates in Monospecies Achromobacter Biofilm]]> Nielsen, S. M., Meyer, R. L., Nørskov-Lauritsen, N. Bacteria of genus Achromobacter are emerging pathogens in cystic fibrosis (CF) capable of biofilm formation and development of antimicrobial resistance. Evolutionary adaptions in the transition from primary to chronic infection were assessed by transcriptomic analysis of successive isolates of Achromobacter xylosoxidans from a single CF patient. Several efflux pump systems targeting antimicrobial agents were upregulated during the course of the disease, whereas all genes related to motility were downregulated. Genes annotated to subsystems of sulfur metabolism, protein metabolism and potassium metabolism exhibited the strongest upregulation. K+ channel genes were hyperexpressed, and a putative sulfite oxidase was more than 1500 times upregulated. The transcriptome patterns indicated a pivotal role of sulfur metabolism and electrical signalling in Achromobacter biofilms during late stage CF lung disease.

Research Fri, 19 May 2017 21:55:59 +0200 092ddc74-c3c6-4966-865a-5a7135293ff4
<![CDATA[Hyperbaric Oxygen Therapy is Ineffective as an Adjuvant to Daptomycin with Rifampicin Treatment in a Murine Model of Staphylococcus aureus in Implant-Associated Osteomyelitis]]> Jørgensen, N. P., Hansen, K., Andreasen, C. M., et al. Implant-associated infections caused by bacterial biofilms are difficult to treat. Surgical intervention is often necessary to cure the patient, as the antibiotic recalcitrance of biofilms renders them untreatable with conventional antibiotics. Intermittent hyperbaric oxygen treatment (HBOT) has been proposed as an adjuvant to conventional antibiotic treatment and it has been speculated that combining HBOT with antibiotics could improve treatment outcomes for biofilm infections. In this study we addressed whether HBOT could improve treatment outcomes of daptomycin and rifampicin combination therapy. The effect of HBOT on the treatment outcomes of daptomycin and rifampicin against implant-associated osteomyelitis was quantified in a murine model. In total, 80 mice were randomized into two groups receiving antibiotics, either alone or in combination with daily intermittent HBOT (304 kPa for 60 min) following injection of antibiotics. Treatment was initiated 11 days after animals were infected with Staphylococcus aureus and treatment duration was 14 days. We found that HBOT did not improve the cure rate and did not reduce the bacterial load on the implant surface or in the surrounding tissue. Cure rates of daptomycin + rifampicin were 40% in infected tibias and 75% for implants while cure rates for HBOT-daptomycin + rifampicin were 50% and 85%, respectively, which were not significantly higher (Fisher's exact test). While it is encouraging that the combination of daptomycin and rifampicin is very effective, our study demonstrates that this efficacy cannot be improved by adjuvant HBOT.

Research Tue, 25 Apr 2017 21:55:59 +0200 f3164d33-e478-43d4-b374-812cd1d9ef1e
<![CDATA[Confocal microscopy imaging of the biofilm matrix]]> Schlafer, S., Meyer, R. L. Research Sat, 01 Jul 2017 21:55:59 +0200 cfce6ca2-c52b-467d-a962-804af78d6dfe <![CDATA[Achromobacter Species Isolated from Cystic Fibrosis Patients Reveal Distinctly Different Biofilm Morphotypes]]> Nielsen, S. M., Nørskov-Lauritsen, N., Bjarnsholt, T., Meyer, R. L. Achromobacter species have attracted attention as emerging pathogens in cystic fibrosis. The clinical significance of Achromobacter infection is not yet fully elucidated; however, their intrinsic resistance to antimicrobials and ability to form biofilms renders them capable of establishing long-term chronic infections. Still, many aspects of Achromobacter biofilm formation remain uncharacterized. In this study, we characterized biofilm formation in clinical isolates of Achromobacter and investigated the effect of challenging the biofilm with antimicrobials and/or enzymes targeting the extracellular matrix. In vitro biofilm growth and subsequent visualization by confocal microscopy revealed distinctly different biofilm morphotypes: a surface-attached biofilm morphotype of small aggregates and an unattached biofilm morphotype of large suspended aggregates. Aggregates consistent with our in vitro findings were visualized in sputum samples from cystic fibrosis patients using an Achromobacter specific peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) probe, confirming the presence of Achromobacter biofilms in the CF lung. High antibiotic tolerance was associated with the biofilm phenotype, and biocidal antibiotic concentrations were up to 1000 fold higher than for planktonic cultures. Treatment with DNase or subtilisin partially dispersed the biofilm and reduced the tolerance to specific antimicrobials, paving the way for further research into using dispersal mechanisms to improve treatment strategies.

Research Wed, 14 Sep 2016 21:55:59 +0200 cc04246e-282c-4a4d-a1f2-495d33f48366
<![CDATA[Critical review on biofilm methods]]> Azeredo, J., Azevedo, N. F., Briandet, R., et al. Biofilms are widespread in nature and constitute an important strategy implemented by microorganisms to survive in sometimes harsh environmental conditions. They can be beneficial or have a negative impact particularly when formed in industrial settings or on medical devices. As such, research into the formation and elimination of biofilms is important for many disciplines. Several new methodologies have been recently developed for, or adapted to, biofilm studies that have contributed to deeper knowledge on biofilm physiology, structure and composition. In this review, traditional and cutting-edge methods to study biofilm biomass, viability, structure, composition and physiology are addressed. Moreover, as there is a lack of consensus among the diversity of techniques used to grow and study biofilms. This review intends to remedy this, by giving a critical perspective, highlighting the advantages and limitations of several methods. Accordingly, this review aims at helping scientists in finding the most appropriate and up-to-date methods to study their biofilms.

Research Sun, 01 Jan 2017 21:55:59 +0100 17700b93-2a6f-487f-8f61-5b37185785c9