Publications - Publications

JMM Profile

Skovdal, S. M., Jørgensen, N. P., Meyer, R. L. — Sat, 01 Oct 2022 23:41:16 +0200

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.

Biofouling Control in Water Filtration Systems

Møllebjerg, A., Meyer, R. L. — Thu, 01 Sep 2022 23:41:16 +0200

Biofouling is one of the biggest impediments to the production of clean and safe water by membrane filtration. As contaminated water is filtered through the membrane, organic matter and microorganisms accumulate on the surface to form a biofilm which obstructs the water flow. A range of strategies have been developed to tackle this issue. Microorganisms and their nutrients can be removed from the feed water by different pretreatment methods to limit colonization and proliferation of microorganisms on the membrane. Furthermore, the membrane surface can be modified to achieve anti-adhesive, biocidal, or quorum quenching properties. These fouling prevention strategies can only delay the development of a biofilm, and the membranes must eventually be cleaned. Physical cleaning methods include air bubbles or ultrasound, but the most commonly used methods use chemical cleaning by applying biocides, acids, caustics, chelators, and surfactants. Chemical cleaning agents come with environmental and economic drawbacks, and biological cleaning solutions are therefore under development. These solutions include treatment with bacteriophages, quorum sensing inhibitors, and matrix-degrading enzymes. Despite extensive research on combatting biofouling, there is still no anti-fouling panacea. However, the additive effect of feedwater pretreatment, anti-adhesive membrane surfaces and the use of gentle biological membrane cleaning methods is bringing the industry closer to long-lasting mitigation of biofouling.

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. — Sat, 01 Oct 2022 23:41:16 +0200

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. — Fri, 01 Jul 2022 23:41:16 +0200

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.

The giant staphylococcal protein Embp facilitates colonization of surfaces through Velcro-like attachment to fibrillated fibronectin

Khan, N., Aslan, H., Büttner, H., et al. — Fri, 01 Jul 2022 23:41:16 +0200

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.

Aptamer-Targeted Drug Delivery for Staphylococcus aureus Biofilm

Ommen, P., Hansen, L., Hansen, B. K., Vu-Quang, H., Kjems, J., Meyer, R. L. — Fri, 01 Apr 2022 23:41:16 +0200

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.

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. — Wed, 09 Jun 2021 23:41:16 +0200

Background
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)

Methods
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.

Results
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.

Conclusions
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.

The bacterial life cycle in textiles is governed by fiber hydrophobicity

Møllebjerg, A., Palmén, L. G., Gori, K., Meyer, R. L. — Fri, 01 Oct 2021 23:41:16 +0200

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.

Human Fibrinogen Inhibits Amyloid Assembly of Most Phenol-Soluble Modulins from Staphylococcus aureus

Najarzadeh, Z., Nielsen, J., Farzadfard, A., et al. — Sun, 01 Aug 2021 23:41:16 +0200

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.

Management of oral biofilms by nisin delivery in adhesive microdevices

Fri, 01 Oct 2021 23:41:16 +0200

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.

Genome sequence of staphylococcus epidermidis AUH4567, a clinical isolate from an infected central venous catheter

Mon, 01 Mar 2021 23:41:16 +0100

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.

Activation of the Two-Component System LisRK Promotes Cell Adhesion and High Ampicillin Tolerance in Listeria monocytogenes

Aslan, H., Petersen, M. E., De Berardinis, A., et al. — Fri, 01 Jan 2021 23:41:16 +0100

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.

Host factors abolish the need for polysaccharides and extracellular matrix-binding protein in Staphylococcus epidermidis biofilm formation

Skovdal, S. M., Hansen, L. K., Ivarsen, D. M., et al. — Mon, 01 Mar 2021 23:41:16 +0100

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.

Phenol-Soluble Modulins Modulate Persister Cell Formation in Staphylococcus aureus

Baldry, M., Bojer, M. S., Najarzadeh, Z., et al. — Sun, 01 Nov 2020 23:41:16 +0100

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.

Polycaprolactone-gelatin nanofibers incorporated with dual antibiotic-loaded carboxyl-modified silica nanoparticles

Tue, 01 Dec 2020 23:41:16 +0100

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.].

Design of a new intraoral splint with 3D-printed inserts for dental biofilm collection

Wed, 01 Jul 2020 23:41:16 +0200

Biofilm models are important tools to study the impact of bacterial metabolism and the effect of therapeutic measures on the caries process. Laboratory models consisting of few bacterial species are useful for high-throughput testing, but in-situ models reflect the complexity of the oral environment much better. The increased complexity typically results in a large variation in the amount of biofilm produced, not only between individuals but also between biofilms collected from the same person. Here, we designed a lower-jaw-splint with 3D-printed silicon inserts that protect the biofilm samples during growth and provide a standardized recession for each individual biofilm sample. We hypothesized that a standardized splint geometry reduces variation between replicate biofilm samples from the same individual and increases the amount of biofilm produced, compared to an in-situ-model without standardized geometry. The splints consisted of a 3D-printed metallic arch and two acrylic vestibular extensions. The extensions contained 3D-printed silicon inserts for biofilm carriers (4x4x1.5 mm). Each individual carrier was surrounded by silicon walls, recessed by 1.5 mm and kept in place by an undercut. 48-h biofilms were collected in 6-tuplicate (2 technical, 3 biological replicates) from five healthy participants, both with standardized splints and with ‘traditional’ splints where the carriers were fixed with sticky wax. The biovolume was determined by confocal microscopy. The coefficient of intra-individual variation was high for both systems, but slightly lower for the standardized splints (69 vs. 80). The average amount of biofilm was slightly lower on the standardized splints (124599 µm3±70284 SD vs. 163616 µm3±135033 SD), with no statistically significant difference (p=0.33, 2-sample t-test). The use of silicon inserts eased the handling of the biofilm samples.

Combination of Rhamnolipid and Chitosan in Nanoparticles Boosts Their Antimicrobial Efficacy

Marangon, C. A., Martins, V. C.A., Ling, M. H., et al. — Wed, 01 Jan 2020 23:41:16 +0100

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.

Delivering antibiotics locally to biofilms by targeted drug delivery and prodrug therapy

Meyer, R. L., Walther, R., Nielsen, S. M., et al. — Tue, 03 Sep 2019 23:41:16 +0200

Distribution of extracellular DNA in Listeria monocytogenes biofilm

Šuláková, M., Pazlarová, J., Meyer, R. L., Demnerová, K. — Tue, 01 Jan 2019 23:41:16 +0100

Development of a label-free LSPR-Apta sensor for Staphylococcus aureus detection

Khateb, H., Klös, G., Meyer, R. L., Sutherland, D. S. — Wed, 01 Apr 2020 23:41:16 +0200

Evaluation of Surface-initiated Polymer brush as Anti-scaling Coating for Plate Heat Exchangers

Friis, J. E., Subbiahdoss, G., Gerved, G., et al. — Fri, 01 Nov 2019 23:41:16 +0100

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.

Antifouling properties of layer by layer DNA coatings

Subbiahdoss, G., Zeng, G., Aslan, H., et al. — Tue, 01 Jan 2019 23:41:16 +0100

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.

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. — Sat, 01 Dec 2018 23:41:16 +0100

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.

Development of an iron oxide nanoparticle drug delivery system against Staphylococcus aureus biofilm infections

Dreier, C., Zelikin, A. N., Kjems, J., Meyer, R. L. — Sat, 01 Sep 2018 23:41:16 +0200

Innate glycosidic activity in metallic implants for localized synthesis of antibacterial drugs

Meer, M. T., Dillion, R., Nielsen, S. M., et al. — Tue, 01 Jan 2019 23:41:16 +0100

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.

Mesoporous silica nanoparticles carrying multiple antibiotics provide enhanced synergistic effect and improved biocompatibility

Gounani, Z., Asadollahi, M. A., Pedersen, J. N., et al. — Fri, 01 Mar 2019 23:41:16 +0100

Treatment of polymicrobial infections requires combination therapy with drugs that have different antimicrobial spectra and possibly work in synergy. However, the different pharmacokinetics and adverse side effects challenge the simultaneous delivery of multiple drugs at the appropriate concentrations to the site of infection. Formulation of multiple drugs in nano-carrier systems may improve therapeutic efficacy by increasing the local concentration and lowering the systemic concentration, leading to fewer side effects. In this study, we loaded
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.

Novel prosthecate bacteria from the candidate phylum Acetothermia

Hao, L., McIlroy, S. J., Kirkegaard, R. H., et al. — Mon, 01 Jan 2018 23:41:16 +0100

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. — Wed, 02 Jan 2019 23:41:16 +0100

Nanozymes, nanoparticles that mimic the natural activity of enzymes, are intriguing academically and are important in the context of the Origin of Life. However, current nanozymes offer mimicry of a narrow range of mammalian enzymes, near‐exclusively performing redox reactions. We present an unexpected discovery of non‐proteinaceous enzymes based on metals, metal oxides, 1D/2D‐materials, and non‐metallic nanomaterials. The specific novelty of these findings lies in the identification of nanozymes with apparent mimicry of diverse mammalian enzymes, including unique pan‐glycosidases. Further novelty lies in the identification of the substrate scope for the lead candidates, specifically in the context of bioconversion of glucuronides, that is, human

Quaternary Ammoniumyl Chitosan Derivatives for Eradication of Staphylococcus aureus Biofilms

Sahariah, P., Masson, M., Meyer, R. L. — Sat, 01 Sep 2018 23:41:16 +0200

Combatting implant-associated biofilms through localized drug synthesis

Walther, R., Nielsen, S. M., Christiansen, R., Meyer, R. L., Zelikin, A. N. — Wed, 10 Oct 2018 23:41:16 +0200

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.

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. — Mon, 01 Jan 2018 23:41:16 +0100

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.

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. — Wed, 01 Aug 2018 23:41:16 +0200

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.

Protein Engineering Reveals Mechanisms of Functional Amyloid Formation in Pseudomonas aeruginosa Biofilms

Bleem, A., Christiansen, G., Madsen, D. J., et al. — Fri, 12 Oct 2018 23:41:16 +0200

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.

Preclinical evaluation of potential infection-imaging probe [⁶⁸Ga]Ga-DOTA-K-A9 in sterile and infectious inflammation

Nielsen, K. M., Jørgensen, N. P., Kyneb, M. H., et al. — Wed, 23 May 2018 23:41:16 +0200

Ag/Fe₃O₄ nanocomposites penetrate and eradicate S.aureus biofilm in an in vitro chronic wound model

Ghaseminezhad, S. M., Shojaosadati, S. A., Meyer, R. L. — Thu, 01 Mar 2018 23:41:16 +0100

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.

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. — Thu, 15 Feb 2018 23:41:16 +0100

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.

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. — Thu, 01 Feb 2018 23:41:16 +0100

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. — Wed, 10 Jan 2018 23:41:16 +0100

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.

Antibacterial isoeugenol coating on stainless steel and polyethylene surfaces prevents biofilm growth

Nielsen, C. K., Subbiahdoss, G., Zeng, G., et al. — Mon, 01 Jan 2018 23:41:16 +0100

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.

Strategies for prevention and treatment of staphylococcal biofilms

Meyer, R. L. — Sat, 01 Apr 2017 23:41:16 +0200

Biofilm formation by bacteria that colonize biomedical implants cause infections that cannot be eradicated by antibiotic therapy. Bacteria in biofilms are tolerant to every antibiotic known today, and this tolerance is partly due to their low metabolic activity, the occurrence of persister cells, and to the protective properties of the biofilm matrix. Strategies for combatting these troublesome infections often fall in to one of two categories: Biomaterial development aimed at preventing biofilm formation, or development of novel drug formulations that more effectively reach their target and kill the bacteria in biofilms.
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.

How can in vitro models best reflect in vivo Staphylococcus biofilms?

Meyer, R. L. — Tue, 15 Aug 2017 23:41:16 +0200

In vitro biofilm models are the basis for most studies of biofilm biology because they enable high-throughput analyses without the expenditure of animals. But how do we ensure that what we learn from in vitro studies is relevant in vivo? Biofilms grown in standard laboratory media do not interact with host factors and are thus profoundly different from in vivo biofilms. We therefore need in vitro models that are as in vivo-like as possible. We investigated how the addition of divalent cations and human plasma to brain heart infusion broth affected biofilm formation by Staphylococcus aureus and Staphylococcus epidermidis. We quantified the biofilm biomass and mechanical properties, imaged the 3D structure, and quantified antibiotic penetration and susceptibility. We also determined how the choice of plasma stabilizer affected biofilm growth and viability. Human plasma stimulated biofilm formation > 100 fold in different S. aureus strains, and increasing the calcium and magnesium concentration to physiological levels further doubled biofilm formation for MRSA. When grown in plasma, S. aureus biofilms consisted of fibrinogen-coated cell clusters interspersed in a matrix of fibrin fibers. This was in stark contrast to the homogenous biofilms formed in the laboratory media. While vancomycin susceptibility was similar for all biofilms, daptomycin more effectively eradicated biofilms grown in plasma. S. epidermidis biofilms were also strongly affected by human plasma. Strains that did not produce polysaccharides could not form biofilms in the absence of plasma, while in the presence of plasma, the structure of polysaccharide-producers and non-producers were indistinguishable. Extracellular polysaccharides may thus be of little consequence to S. epidermidis biofilms in vivo. Access to host factors is critical for the biofilm phenotype of both coagulase-positive and -negative staphylococci, and we urge that biofilms are routinely studied under in vivo-like conditions.

Inhibition of the ATP Synthase Eliminates the Intrinsic Resistance of Staphylococcus aureus towards Polymyxins

Vestergaard, M., Nøhr-Meldgaard, K., Bojer, M. S., et al. — Tue, 05 Sep 2017 23:41:16 +0200

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.

A transposon mutant library of Bacillus cereus ATCC 10987 reveals novel genes required for biofilm formation and implicates motility as an important factor for pellicle-biofilm formation

Sun, 01 Apr 2018 23:41:16 +0200

Bacillus cereus is one of the most common opportunistic pathogens causing foodborne illness, as well as a common source of contamination in the dairy industry. B. cereus can form robust biofilms on food processing surfaces, resulting in food contamination due to shedding of cells and spores. Despite the medical and industrial relevance of this species, the genetic basis of biofilm formation in B. cereus is not well studied. In order to identify genes required for biofilm formation in this bacterium, we created a library of 5000 + transposon mutants of the biofilm-forming strain B. cereusATCC 10987, using an unbiased mariner transposon approach. The mutant library was screened for the ability to form a pellicle biofilm at the air-media interface, as well as a submerged biofilm at the solid-media interface. A total of 91 genes were identified as essential for biofilm formation. These genes encode functions such as chemotaxis, amino acid metabolism and cellular repair mechanisms, and include numerous genes not previously known to be required for biofilm formation. Although the majority of disrupted genes are not directly responsible for motility, further investigations revealed that the vast majority of the biofilm-deficient mutants were also motility impaired. This observation implicates motility as a pivotal factor in the formation of a biofilm by B. cereus. These results expand our knowledge of the fundamental molecular mechanisms of biofilm formation by B. cereus.

A Targeting Agent For Drug Delivery To Staphylococcus Aureus Biofilms

Andersen, P. O., Hansen, L., Vu Quang, H., Kjems, J., Meyer, R. L. — Sun, 01 Jan 2017 23:41:16 +0100

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. — Wed, 15 Nov 2017 23:41:16 +0100

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.

Host Proteins Determine MRSA Biofilm Structure and Integrity

Dreier, C., Nielsen, A., Jørgensen, N. P., Kragstrup, T. W., Meyer, R. L. — Sun, 01 Jan 2017 23:41:16 +0100

Human extracellular matrix (hECM) proteins aids the initial attachment and initiation of an infection, by specific binding to bacterial cell surface proteins. However, the importance of hECM proteins in structure, integrity and antibiotic resilience of a biofilm is unknown. This study aims to determine how specific hECM proteins affect S. aureus USA300 JE2 biofilms.

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.

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. — Sun, 01 Jan 2017 23:41:16 +0100

The bovine milk protein osteopontin (OPN) has been shown to reduce the adhesion of oral bacteria to saliva-coated surfaces, which reduces biofilm formation and may contribute to caries control. We now quantified the effect of OPN (Lacprodan OPN-10) treatment on the adhesion force of Lactobacillus paracasei subsp. paracasei and Actinomyces naeslundii using atomic force microscopy based single cell force spectroscopy (AFM-SCFS). Furthermore, we performed a fluorescent lectin binding analysis (FLBA) to investigate if OPN treatment resulted in the selective blocking of specific cell surfaces glycoconjugates of L. paracasei, A. naeslundii and Streptococcus mitis. For AFM-SCFS, saliva-coated tipless AFM cantilevers were approached on single cells. The cantilevers were then retracted and adhesion was quantified from the recorded force curves. Adhesion on the same cells (n=10) was compared before and after OPN treatment. Adhesion energy was found to be reduced by 94% for L. paracasei and 61% for A. naeslundii (p<0.05). For FLBA, the binding of 75 FITC-labelled lectins to the three bacteria was screened. Lectins BanLec, ConA, VGA and WGA bound well to A. naeslundii, lectins ABA and HPA to L. paracasei, and lectins VGA and WGA to S. mitis. Immobilized bacteria were incubated with these lectins in the presence and absence of OPN. For each combination, 12 confocal images were acquired with fixed microscope settings, and average fluorescence intensities were determined. Experiments were performed in triplicate and analyzed with two-sample t-tests. No statistically significant differences were observed between OPN-treated and OPN-free cells (p>0.05). OPN strongly reduces the adhesion force of oral bacteria to saliva-coated surfaces, but the effect cannot be ascribed to the selective blocking of cell surface glycoconjugates.

Extracellular DNA Contributes to Dental Biofilm Stability

Schlafer, S., Meyer, R. L., Dige, I., Regina, V. R. — Tue, 01 Aug 2017 23:41:16 +0200

Extracellular DNA (eDNA) is a major matrix component of many bacterial biofilms. While the presence of eDNA and its role in biofilm stability have been demonstrated for several laboratory biofilms of oral bacteria, there is no data available on the presence and function of eDNA in in vivo grown dental biofilms. This study aimed to determine whether eDNA was part of the matrix in biofilms grown in situ in the absence of sucrose and whether treatment with DNase dispersed biofilms grown for 2.5, 5, 7.5, 16.5, or 24 h. Three hundred biofilms from 10 study participants were collected and treated with either DNase or heat-inactivated DNase for 1 h. The bacterial biovolume was determined with digital image analysis. Staining with TOTO®-1 allowed visualization of eDNA both on bacterial cell surfaces and, with a cloud-like appearance, in the intercellular space. DNase treatment strongly reduced the amount of biofilm in very early stages of growth (up to 7.5 h), but the treatment effect decreased with increasing biofilm age. This study proves the involvement of eDNA in dental biofilm formation and its importance for biofilm stability in the earliest stages. Further research is required to uncover the interplay of eDNA and other matrix components and to explore the therapeutic potential of DNase treatment for biofilm control.

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. — Wed, 11 Oct 2017 23:41:16 +0200

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.

Quantification of biofilm biomass by staining

Ommen, P., Zobek, N., Meyer, R. L. — Sun, 01 Oct 2017 23:41:16 +0200

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.