Publications - Publications https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Bcontroller%5D=Publications&cHash=39d6d332fdfef510f4d682d7ec0120a7 en-us PURE Extension typo3support@science.au.dk (Web Department) 30 <![CDATA[The efficacy and safety of an enzyme-containing lozenge for dental biofilm control]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=45008183-2787-42df-9cec-8bb95eabd939&tx_pure_pure5%5BshowType%5D=pub&cHash=3c059b6d6d073776a2872995e603c073 Schlafer, S., Johnsen, K. K., Kjærbølling, I., Schramm, A., Meyer, R. L., Jørgensen, M. R. Objectives: To evaluate the effect of daily use of a multiple-enzyme lozenge on de novo plaque formation, on gingivitis development, and on the oral microbiome composition. Methods: This trial with two parallel arms included 24 healthy adults allocated to the Active (n = 12) or Placebo (n = 12) group. Subjects consumed one lozenge three times daily for seven days, and no oral hygiene procedures were allowed. Differences in de novo plaque accumulation between a baseline period, and one and seven days of intervention were assessed by the Turesky-modification of the Quigley-and-Hein-Plaque-Index (TM-QHPI). The development of gingivitis after seven days of intervention was assessed by the Gingival Index (GI). Plaque and saliva samples were collected at baseline and after seven days of intervention, and evaluated by 16S rRNA gene sequencing. Results: All subjects completed the study, and no adverse events were reported. After one day, the average TM-QHPI was significantly lower in the Active than in the Placebo group, as compared to baseline (p = 0.012). After 7 days, average TM-QHPI values did not differ significantly between groups (p = 0.37). GI values did not increase during the intervention period, with no difference between groups (p = 0.62). Bacterial richness increased in both plaque and saliva samples over a seven-day oral hygiene-free period, with a statistically significant difference for the saliva samples (p = 0.0495) between groups. Conclusions: A multiple-enzymes lozenge decreased the build-up of de novo plaque after one day and slowed down the process of species increment in saliva. The lozenge may be an adjunct to regular mechanical plaque removal. Clinical significance: Dental plaque is the main cause of caries, gingivitis, and periodontitis. The search for therapeutic adjuncts to mechanical plaque removal that have no harmful effects on the oral microbiome is important. Treatment with multiple plaque-matrix degrading enzymes is a promising non-biocidal approach to plaque control.

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Research Thu, 01 Aug 2024 17:39:52 +0200 45008183-2787-42df-9cec-8bb95eabd939
<![CDATA[Bacterial efflux pumps excrete SYTO™ dyes and lead to false-negative staining results]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=cc11dbe8-bb4e-4091-aeaa-901716bb0113&tx_pure_pure5%5BshowType%5D=pub&cHash=815e3086a649e705a623901aaa962e50 Minero, G. A. S., Larsen, P. B., Hoppe, M. E., Meyer, R. L. Multidrug efflux pumps excrete a range of small molecules from bacterial cells. In this study, we show that bacterial efflux pumps have affinity for a range of SYTO™ dyes that are commonly used to label bacteria. Efflux pump activity will there lead to false negative results from bacterial staining and SYTO™ dyes should be used with caution on live samples.

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Research Thu, 01 Feb 2024 17:39:52 +0100 cc11dbe8-bb4e-4091-aeaa-901716bb0113
<![CDATA[Efficacy of rifampicin combination therapy against MRSA prosthetic vascular graft infections in a rat model]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=c609ada9-3af6-4ee0-b9d7-ac6b002aeacf&tx_pure_pure5%5BshowType%5D=pub&cHash=3e5eab33fc02c150cadcf845a6e3daac Johansen, M. I., Petersen, M. E., Faddy, E., et al. Staphylococcus aureus is a major cause of prosthetic vascular graft or endograft infections (VGEIs) and the optimal choice of antibiotics is unclear. We investigated various antibiotic choices as either monotherapy or combination therapy with rifampicin against MRSA in vitro and in vivo. Fosfomycin, daptomycin and vancomycin alone or in combination with rifampicin was used against MRSA USA300 FPR3757. Each antibiotic was tested for synergism or antagonism with rifampicin in vitro, and all antibiotic regimens were tested against actively growing bacteria in media and non-growing bacteria in buffer, both as planktonic cells and in biofilms. A rat model of VGEI was used to quantify the therapeutic efficacy of antibiotics in vivo by measuring bacterial load on grafts and in spleen, liver and kidneys. In vitro, rifampicin combinations did not reveal any synergism or antagonism in relation to growth inhibition. However, quantification of bactericidal activity revealed a strong antagonistic effect, both on biofilms and planktonic cells. This effect was only observed when treating active bacteria, as all antibiotics had little or no effect on inactive cells. Only daptomycin showed some biocidal activity against inactive cells. In vivo evaluation of therapy against VGEI contrasted the in vitro results. Rifampicin significantly increased the efficacy of both daptomycin and vancomycin. The combination of daptomycin and rifampicin was by far the most effective, curing 8 of 13 infected animals. Our study demonstrates that daptomycin in combination with rifampicin shows promising potential against VGEI caused by MRSA. Furthermore, we show how in vitro evaluation of antibiotic combinations in laboratory media does not predict their therapeutic effect against VGEI in vivo, presumably due to a difference in the metabolic state of the bacteria.

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Research Sat, 01 Jun 2024 17:39:52 +0200 c609ada9-3af6-4ee0-b9d7-ac6b002aeacf
<![CDATA[Considerations on the use of microsensors to profile dissolved H2 concentrations in microbial electrochemical reactors]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=d09456fc-ecc7-4d34-87a3-308c6bc08670&tx_pure_pure5%5BshowType%5D=pub&cHash=748b169a0d8f9c903841fa8d755155b7 Sandfeld, T. ., Grøn, L. V., Munoz Duarte, L. D., Meyer, R. L., Koren, K., Philips, J. Measuring the distribution and dynamics of H 2 in microbial electrochemical reactors is valuable to gain insights into the processes behind novel bioelectrochemical technologies, such as microbial electrosynthesis. Here, a microsensor method to measure and profile dissolved H 2 concentrations in standard H-cell reactors is described. Graphite cathodes were oriented horizontally to enable the use of a motorized microprofiling system and a stereomicroscope was used to place the H 2 microsensor precisely on the cathode surface. Profiling was performed towards the gas-liquid interface, while preserving the electric connections and flushing the headspace (to maintain anoxic conditions) and under strict temperature control (to overcome the temperature sensitivity of the microsensors). This method was tested by profiling six reactors, with and without inoculation of the acetogen Sporomusa ovata, at three different time points. H 2 accumulated over time in the abiotic controls, while S. ovata maintained low H 2 concentrations throughout the liquid phase (< 4 μM) during the whole experimental period. These results demonstrate that this setup generated insightful H 2 profiles. However, various limitations of this microsensor method were identified, as headspace flushing lowered the dissolved H 2 concentrations over time. Moreover, microsensors can likely not accurately measure H 2 in the immediate vicinity of the solid cathode, because the solids cathode surface obstructs H 2 diffusion into the microsensor. Finally, the reactors had to be discarded after microsensor profiling. Interested users should bear these considerations in mind when applying microsensors to characterize microbial electrochemical reactors.

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Research Mon, 01 Jan 2024 17:39:52 +0100 d09456fc-ecc7-4d34-87a3-308c6bc08670
<![CDATA[Combined pH ratiometry and fluorescence lectin-binding analysis (pH-FLBA) for microscopy-based analyses of biofilm pH and matrix carbohydrates]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=75956075-f9ad-4309-96f4-5b9e72d714eb&tx_pure_pure5%5BshowType%5D=pub&cHash=e8f4aae98923abffe1dde5a2109ae829 Del Rey, Y. C., Schramm, A., L Meyer, R., Lund, M. B., Schlafer, S. Bacterial biofilms have a complex and heterogeneous three-dimensional architecture that is characterized by chemically and structurally distinct microenvironments. Confocal microscopy-based pH ratiometry and fluorescence lectin-binding analysis (FLBA) are well-established methods to characterize pH developments and the carbohydrate matrix architecture of biofilms at the microscale. Here, we developed a combined analysis, pH-FLBA, to concomitantly map biofilm pH and the distribution of matrix carbohydrates in bacterial biofilms while preserving the biofilm microarchitecture. As a proof of principle, the relationship between pH and the presence of galactose- and fucose-containing matrix components was investigated in dental biofilms grown with and without sucrose. The pH response to a sucrose challenge was monitored in different areas at the biofilm base using the ratiometric pH-sensitive dye C-SNARF-4. Thereafter, the fucose- and galactose-specific fluorescently labeled lectins Aleuria aurantia lectin (AAL) and Morus nigra agglutinin G (MNA-G) were used to visualize carbohydrate matrix components in the same biofilm areas and their immediate surroundings. Sucrose during growth significantly decreased biofilm pH ( P < 0.05) and increased the amounts of both MNA-G- and AAL-targeted matrix carbohydrates ( P < 0.05). Moreover, it modulated the biofilm composition towards a less diverse community dominated by streptococci, as determined by 16S rRNA gene sequencing. Altogether, these results suggest that the production of galactose- and fucose-containing matrix carbohydrates is related to streptococcal metabolism and, thereby, pH profiles in dental biofilms. In conclusion, pH-FLBA using lectins with different carbohydrate specificities is a useful method to investigate the association between biofilm pH and the complex carbohydrate architecture of bacterial biofilms.IMPORTANCEBiofilm pH is a key regulating factor in several biological and biochemical processes in environmental, industrial, and medical biofilms. At the microscale, microbial biofilms are characterized by steep pH gradients and an extracellular matrix rich in carbohydrate components with diffusion-modifying properties that contribute to bacterial acid-base metabolism. Here, we propose a combined analysis of pH ratiometry and fluorescence lectin-binding analysis, pH-FLBA, to concomitantly investigate the matrix architecture and pH developments in microbial biofilms, using complex saliva-derived biofilms as an example. Spatiotemporal changes in biofilm pH are monitored non-invasively over time by pH ratiometry, while FLBA with lectins of different carbohydrate specificities allows mapping the distribution of multiple relevant matrix components in the same biofilm areas. As the biofilm structure is preserved, pH-FLBA can be used to investigate the in situ relationship between the biofilm matrix architecture and biofilm pH in complex multispecies biofilms.

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Research Thu, 01 Feb 2024 17:39:52 +0100 75956075-f9ad-4309-96f4-5b9e72d714eb
<![CDATA[Extracellular G-quadruplexes and Z-DNA protect biofilms from DNase I, and G-quadruplexes form a DNAzyme with peroxidase activity]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=2e05dc3a-ae94-4aec-920a-9ee888ae65c0&tx_pure_pure5%5BshowType%5D=pub&cHash=e6baa19df6cf43df95c186042605594c Minero, G. A. S., Møllebjerg, A., Thiesen, C., et al. Many bacteria form biofilms to protect themselves from predators or stressful environmental conditions. In the biofilm, bacteria are embedded in a protective extracellular matrix composed of polysaccharides, proteins and extracellular DNA (eDNA). eDNA most often is released from lysed bacteria or host mammalian cells, and it is the only matrix component most biofilms appear to have in common. However, little is known about the form DNA takes in the extracellular space, and how different non-canonical DNA structures such as Z-DNA or G-quadruplexes might contribute to its function in the biofilm. The aim of this study was to determine if non-canonical DNA structures form in eDNA-rich staphylococcal biofilms, and if these structures protect the biofilm from degradation by nucleases. We grew Staphylococcus epidermidis biofilms in laboratory media supplemented with hemin and NaCl to stabilize secondary DNA structures and visualized their location by immunolabelling and fluorescence microscopy. We furthermore visualized the macroscopic biofilm structure by optical coherence tomography. We developed assays to quantify degradation of Z-DNA and G-quadruplex DNA oligos by different nucleases, and subsequently investigated how these enzymes affected eDNA in the biofilms. Z-DNA and G-quadruplex DNA were abundant in the biofilm matrix, and were often present in a web-like structures. In vitro, the structures did not form in the absence of NaCl or mechanical shaking during biofilm growth, or in bacterial strains deficient in eDNA or exopolysaccharide production. We thus infer that eDNA and polysaccharides interact, leading to non-canonical DNA structures under mechanical stress when stabilized by salt. We also confirmed that G-quadruplex DNA and Z-DNA was present in biofilms from infected implants in a murine implant-associated osteomyelitis model. Mammalian DNase I lacked activity against Z-DNA and G-quadruplex DNA, while Micrococcal nuclease could degrade G-quadruplex DNA and S1 Aspergillus nuclease could degrade Z-DNA. Micrococcal nuclease, which originates from Staphylococcus aureus, may thus be key for dispersal of biofilm in staphylococci. In addition to its structural role, we show for the first time that the eDNA in biofilms forms a DNAzyme with peroxidase-like activity in the presence of hemin. While peroxidases are part of host defenses against pathogens, we now show that biofilms can possess intrinsic peroxidase activity in the extracellular matrix.

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Research Thu, 01 Feb 2024 17:39:52 +0100 2e05dc3a-ae94-4aec-920a-9ee888ae65c0
<![CDATA[The effect of enzymatic treatment with mutanase, beta-glucanase and DNase on a saliva-derived biofilm model]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=b647402f-95e8-4e2c-93fb-8f700d66dfbf&tx_pure_pure5%5BshowType%5D=pub&cHash=aefee96b9f3b0c6deb3edf3a618a40b9 Rikvold, P. D., Hansen Skov, L. B., Louise Meyer, R., Rose Jorgensen, M., Tiwari, M. K., Schlafer, S. Introduction: The dental biofilm matrix is an important determinant of virulence for caries development and comprises a variety of extracellular polymeric substances that contribute to biofilm stability. Enzymes that break down matrix components may be a promising approach to caries control, and in light of the compositional complexity of the dental biofilm matrix, treatment with multiple enzymes may enhance the reduction of biofilm formation compared to single enzyme therapy. The present study investigated the effect of the three matrix-degrading enzymes mutanase, beta-glucanase, and DNase, applied separately or in combinations, on biofilm prevention and removal in a saliva-derived in vitro-grown model. Methods: Biofilms were treated during growth to assess biofilm prevention or after 24 h of growth to assess biofilm removal by the enzymes. Biofilms were quantified by crystal violet staining and impedance-based real-time cell analysis, and the biofilm structure was visualized by confocal microscopy and staining of extracellular DNA (eDNA) and polysaccharides. Results: The in vitro model was dominated by Streptococcus spp., as determined by 16S rRNA gene amplicon sequencing. All tested enzymes and combinations had a significant effect on biofilm prevention, with reductions of >90% for mutanase and all combinations including mutanase. Combined application of DNase and beta-glucanase resulted in an additive effect (81.0% ± 1.3% SD vs. 36.9% ± 21.9% SD and 48.2% ± 14.9% SD). For biofilm removal, significant reductions of up to 73.2% ± 5.5% SD were achieved for combinations including mutanase, whereas treatment with DNase had no effect. Glucans, but not eDNA decreased in abundance upon treatment with all three enzymes. Conclusion: Multi-enzyme treatment is a promising approach to dental biofilm control that needs to be validated in more diverse biofilms.

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Research Mon, 01 Jan 2024 17:39:52 +0100 b647402f-95e8-4e2c-93fb-8f700d66dfbf
<![CDATA[The effect of an enzyme-containing lozenge on dental biofilm accumulation]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=71a4711b-1dad-4a89-b828-23d65742b765&tx_pure_pure5%5BshowType%5D=pub&cHash=084ff41a61194c15ec6c7281ccf3be8f Schlafer, S., K. Johnsen, K., Jørgensen, M., Kjærbølling, I., Schramm, A., Meyer, R. L. Research Sun, 01 Jan 2023 17:39:52 +0100 71a4711b-1dad-4a89-b828-23d65742b765 <![CDATA[Multiple-enzyme treatment reduces biofilm formation in a highly acidogenic in vitro model]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=c34c0044-7b4c-4db3-936c-91169af99e84&tx_pure_pure5%5BshowType%5D=pub&cHash=0f39fa3cd49b9b769732b40d65d28bc8 Rikvold, P., Møllebjerg, A., Meyer, R. L., Schlafer, S. Materials and methods: Biofilms were grown in 96-well plates under aerobic conditions at 37°C for 24 hours using pooled saliva as inoculum and brain-heart infusion broth supplied with 5% sucrose and sterile saliva as growth medium. The effect of treatment with a combination of mutanase, glucanase and DNase on biofilm prevention and removal was investigated. Treatment was performed either during (prevention) or after biofilm growth (removal) and the remaining biofilm was quantified by crystal violet staining. Additionally, the treatment effect on biofilm matrix compounds was analyzed using confocal microscopy. Extracellular DNA was stained with TOTO-1, microbial cells with SYTO41 and matrix polysaccharides were visualized by including fluorescently labeled dextran in the growth medium. Treatment effects were analyzed using unpaired t-tests (p<0.05 was considered statistically significant).
Results: Enzyme treatment reduced mean biofilm formation with 94%±1% SD (p=0.0001) and 69%±2% SD (p<0.0001) when performed during and after growth, respectively. Extracellular polysaccharides were on average reduced by 28%±6% SD compared to control treatment (p=0.02), but the mean ratio between polysaccharides and microbial cells remained unchanged (0.70±0.37 SD vs. 0.77±0.22 SD; p=0.74). No significant reduction in the amount of eDNA (p=0.74) was observed.
Conclusion: Multiple-enzyme treatment is a promising non-biocidal approach to biofilm control.
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Research Sun, 01 Oct 2023 17:39:52 +0200 c34c0044-7b4c-4db3-936c-91169af99e84
<![CDATA[Multiple-enzyme treatment reduces biofilm formation in a highly acidogenic in vitro model]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=a4554b30-214a-4f5c-90aa-c3059d549165&tx_pure_pure5%5BshowType%5D=pub&cHash=611f0336ec5d837bd0669923ea1c1ec8 Rikvold, P., Møllebjerg, A., Meyer, R. L., Schlafer, S. Research Sat, 01 Jul 2023 17:39:52 +0200 a4554b30-214a-4f5c-90aa-c3059d549165 <![CDATA[The effect of an enzyme-containing lozenge on dental biofilm accumulation: A clinical pilot study]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=26b01e07-d567-4765-9adb-2bcbc78ce933&tx_pure_pure5%5BshowType%5D=pub&cHash=582173e01fb99ac6aa70827ac69f1764 Schlafer, S., K. Johnsen, K., Jørgensen, M., Kjærbølling, I., Schramm, A., Meyer, R. L. 24 healthy volunteers were enrolled in a randomized, double-blind placebo-controlled study with two parallel arms. After an initial professional tooth cleaning, participants refrained from oral hygiene and used three enzyme-containing or placebo lozenges/d for 7 d. After 1 and 7 d, the Turesky-modification of the Quigley-and-Hein Plaque Index (TM-QHPI) was assessed in the first and second quadrant, respectively and compared to TM-QHPI after a one-day baseline period without oral hygiene (two-sample t-tests). At baseline and after 7 d of intervention, the gingival index (GI) was recorded and compared (two-sample t-test), and the species richness was analysed in plaque and saliva samples by 16S rRNA sequencing (linear mixed model). The protocol was approved by the local Ethics Commitee (1-10-72-260-20).
Enzyme treatment resulted in a reduced mean TM-QHPI compared to control treatment after 1 d (Δ=-0.61±1.00 SD vs. 0.03±0.59 SD; p=0.07; Δ=-0.82±0.74 SD vs. -0.09±0.45 SD; p=0.01 without outliers) but not 7 d (Δ=1.03±1.42 SD vs. 1.61±1.36 SD; p=0.39), and in a reduced increase in average species richness (17±9 SE vs. 35±7 SE; p=0.04) from baseline to day 7. Mean GI did not increase over the intervention period for both treatment groups (Δ=-0.06±0.63 SD vs. 0.02±0.44 SD; p=0.39). Lozenges containing matrix-degrading enzymes may be a promising adjunct to oral hygiene.]]>
Research Sat, 01 Jul 2023 17:39:52 +0200 26b01e07-d567-4765-9adb-2bcbc78ce933
<![CDATA[GFP fusions of Sec-routed extracellular proteins in Staphylococcus aureusreveal surface-associated coagulase in biofilms]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=efdfe5a1-3c1f-4b17-8ada-7ff8ad44e166&tx_pure_pure5%5BshowType%5D=pub&cHash=9e1fb9c78d5c3073028316508662a3e6 Evans, D. C.S., Khamas, A. B., Marcussen, L., et al. Staphylococcus aureus is a major human pathogen that utilises many surface-associated and secreted proteins to form biofilms and cause disease. However, our understanding of these processes is limited by chal-lenges of using fluorescent protein reporters in their native environment, be-cause they must be exported and fold correctly to become fluorescent. Here, we demonstrate the feasibility of using the monomeric superfolder GFP (msfGFP) exported from S. aureus. By fusing msfGFP to signal peptides for the Secretory (Sec) and Twin Arginine Translocation (Tat) pathways, the two ma-jor secretion pathways in S. aureus, we quantified msfGFP fluorescence in bacterial cultures and cell-free supernatant from the cultures. When fused to a Tat signal peptide, we detected msfGFP fluorescence inside but not outside bacterial cells, indicating a failure to export msfGFP. However, when fused to a Sec signal peptide, msfGFP fluorescence was present outside cells, indicat-ing successful export of the msfGFP in the unfolded state, followed by extra-cellular folding and maturation to the photoactive state. We applied this strategy to study coagulase (Coa), a secreted protein and a major contributor to the formation of a fibrin network in S. aureus biofilms that protects bacte-ria from the host immune system and increases attachment to host surfaces. We confirmed that a genomically integrated C-terminal fusion of Coa to msfGFP does not impair the activity of Coa or its localisation within the bio-film matrix. Our findings demonstrate that msfGFP is a good candidate fluo-rescent reporter to consider when studying proteins secreted by the Sec pathway in S. aureus.

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Research Sat, 01 Jul 2023 17:39:52 +0200 efdfe5a1-3c1f-4b17-8ada-7ff8ad44e166
<![CDATA[Fibrinolytic and antibiotic treatment of prosthetic vascular graft infections in a novel rat model]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=846ae713-5cb9-440d-b500-5937f21f6aac&tx_pure_pure5%5BshowType%5D=pub&cHash=e682b1b8ff4ff422ee5a9b7eab05cdf0 Johansen, M. I., Rahbek, S. J., Jensen-Fangel, S., et al. We developed a rat model of prosthetic vascular graft infection to assess, whether the fibrinolytic tissue plasminogen activator (tPA) could increase the efficacy of antibiotic therapy.

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

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

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

Conclusion
We developed the first functional model of an arterial prosthetic vascular graft infection in rats. Antibiotic combination therapy with vancomycin and rifampicin was superior to vancomycin monotherapy, and the addition of tPA did not significantly reduce bacterial load, nor significantly increase cure rate.]]>
Research Sat, 01 Jul 2023 17:39:52 +0200 846ae713-5cb9-440d-b500-5937f21f6aac
<![CDATA[Protein ligand and nanotopography separately drive the phenotype of mouse embryonic stem cells]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=362a42dd-df10-4b40-8216-3a7699d88025&tx_pure_pure5%5BshowType%5D=pub&cHash=c4f12a40bc7cbd361d93570217d7c831 Ghorbani, S., Christine Füchtbauer, A., Møllebjerg, A., et al. Biochemical and biomechanical signals regulate stem cell function in the niche environments in vivo. Current in vitro culture of mouse embryonic stem cells (mESC) uses laminin (LN-511) to provide mimetic biochemical signaling (LN-521 for human systems) to maintain stemness. Alternative approaches propose topographical cues to provide biomechanical cues, however combined biochemical and topographic cues may better mimic the in vivo environment, but are largely unexplored for in vitro stem cell expansion. In this study, we directly compare in vitro signals from LN-511 and/or topographic cues to maintain stemness, using systematically-varied submicron pillar patterns or flat surfaces with or without preadsorbed LN-511. The adhesion of cells, colony formation, expression of the pluripotency marker,octamer-binding transcription factor 4 (Oct4), and transcriptome profiling were characterized. We observed that either biochemical or topographic signals could maintain stemness of mESCs in feeder-free conditions, indicated by high-level Oct4 and gene profiling by RNAseq. The combination of LN-511 with nanotopography reduced colony growth, while maintaining stemness markers, shifted the cellular phenotype indicating that the integration of biochemical and topographic signals is antagonistic. Overall, significantly faster (up to 2.5 times) colony growth was observed at nanotopographies without LN-511, suggesting for improved ESC expansion.

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Research Sun, 01 Oct 2023 17:39:52 +0200 362a42dd-df10-4b40-8216-3a7699d88025
<![CDATA[Antibody-Drug Conjugates to Treat Bacterial Biofilms via Targeting and Extracellular Drug Release]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=f4effb5f-6ebb-4824-bab0-b9a925f62858&tx_pure_pure5%5BshowType%5D=pub&cHash=95d4000a0f0e2f39315b3e4e7ecbd124 Tvilum, A. S., Johansen, M. I., Glud, L. N., et al. The treatment of implant-associated bacterial infections and biofilms is an urgent medical need and a grand challenge because biofilms protect bacteria from the immune system and harbor antibiotic-tolerant persister cells. This need is addressed herein through an engineering of antibody-drug conjugates (ADCs) that contain an anti-neoplastic drug mitomycin C, which is also a potent antimicrobial against biofilms. The ADCs designed herein release the conjugated drug without cell entry, via a novel mechanism of drug release which likely involves an interaction of ADC with the thiols on the bacterial cell surface. ADCs targeted toward bacteria are superior by the afforded antimicrobial effects compared to the non-specific counterpart, in suspension and within biofilms, in vitro, and in an implant-associated murine osteomyelitis model in vivo. The results are important in developing ADC for a new area of application with a significant translational potential, and in addressing an urgent medical need of designing a treatment of bacterial biofilms.

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Research Tue, 01 Aug 2023 17:39:52 +0200 f4effb5f-6ebb-4824-bab0-b9a925f62858
<![CDATA[Polyether Ionophore Antibiotics Target Drug-Resistant Clinical Isolates, Persister Cells, and Biofilms]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=8241a10e-5778-4631-abdd-509176b0f370&tx_pure_pure5%5BshowType%5D=pub&cHash=e3a335381b6c1b2cb868b4e1d82fb836 Wollesen, M., Mikkelsen, K., Tvilum, M. S., et al. Polyether ionophores are complex natural products known to transport various cations across biological membranes. While several members of this family are used in agriculture (e.g., as anti-coccidiostats) and have potent antibacterial activity, they are not currently being pursued as antibiotics for human use. Polyether ionophores are typically grouped as having similar functions, despite the fact that they significantly differ in structure; for this reason, how their structure and activity are related remains unclear. To determine whether certain members of the family constitute particularly interesting springboards for in-depth investigations and future synthetic optimization, we conducted a systematic comparative study of eight different polyether ionophores for their potential as antibiotics. This includes clinical isolates from bloodstream infections and studies of the compounds' effects on bacterial biofilms and persister cells. We uncover distinct differences within the compound class and identify the compounds lasalocid, calcimycin, and nanchangmycin as having particularly interesting activity profiles for further development. IMPORTANCE Polyether ionophores are complex natural products used in agriculture as anti-coccidiostats in poultry and as growth promoters in cattle, although their precise mechanism is not understood. They are widely regarded as antimicrobials against Gram-positive bacteria and protozoa, but fear of toxicity has so far prevented their use in humans. We show that ionophores generally have very different effects on Staphylococcus aureus, both in standard assays and in more complex systems such as bacterial biofilms and persister cell populations. This will allow us to focus on the most interesting compounds for future in-depth investigations and synthetic optimizations.

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Research Tue, 01 Aug 2023 17:39:52 +0200 8241a10e-5778-4631-abdd-509176b0f370
<![CDATA[Novel high-throughput screening platform identifies enzymes to tackle biofouling on reverse osmosis membranes]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=26230021-488e-45a3-9f57-5b1ca6953c99&tx_pure_pure5%5BshowType%5D=pub&cHash=b5921c6c7b8683814d054cf1d815f2ac Møllebjerg, A., Zarebska, A., Nielsen, H. B., et al. Research Mon, 01 May 2023 17:39:52 +0200 26230021-488e-45a3-9f57-5b1ca6953c99 <![CDATA[Biofilm formation and inflammatory potential of <i>Staphylococcus saccharolyticus</i>]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=7dfcdc91-10b3-4428-bac4-02f6a3e12c89&tx_pure_pure5%5BshowType%5D=pub&cHash=67c042bdeacc2f234fa1561a82908663 Afshar, M., Møllebjerg, A., Minero, G. A., et al. Staphylococcus saccharolyticus, a coagulase-negative staphylococcal species, has some unusual characteristics for human-associated staphylococci, such as slow growth and its preference for anoxic culture conditions. This species is a relatively abundant member of the human skin microbiota, but its microbiological properties, as well as the pathogenic potential, have scarcely been investigated so far, despite being occasionally isolated from different types of infections including orthopedic implant-associated infections. Here, we investigated the growth and biofilm properties of clinical isolates of S. saccharolyticus and determined host cell responses. Growth assessments in anoxic and oxic conditions revealed strain-dependent outcomes, as some strains can also grow aerobically. All tested strains of S. saccharolyticus were able to form biofilm in a microtiter plate assay. Strain-dependent differences were determined by optical coherence tomography, revealing that medium supplementation with glucose and sodium chloride enhanced biofilm formation. Visualization of the biofilm by confocal laser scanning microscopy revealed the role of extracellular DNA in the biofilm structure. In addition to attached biofilms, S. saccharolyticus also formed bacterial aggregates at an early stage of growth. Transcriptome analysis of biofilm-grown versus planktonic cells revealed a set of upregulated genes in biofilm-embedded cells, including factors involved in adhesion, colonization, and competition such as epidermin, type I toxin-antitoxin system, and phenol-soluble modulins (beta and epsilon). To investigate consequences for the host after encountering S. saccharolyticus, cytokine profiling and host cell viability were assessed by infection experiments with differentiated THP-1 cells. The microorganism strongly triggered the secretion of the tested pro-inflammatory cyto- and chemokines IL-6, IL-8, and TNF-alpha, determined at 24 h post-infection. S. saccharolyticus was less cytotoxic than Staphylococcus aureus. Taken together, the results indicate that S. saccharolyticus has substantial pathogenic potential. Thus, it can be a potential cause of orthopedic implant-associated infections and other types of deep-seated infections.

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Research Tue, 01 Nov 2022 17:39:52 +0100 7dfcdc91-10b3-4428-bac4-02f6a3e12c89
<![CDATA[JMM Profile]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=1f396264-a26a-4384-8132-f49b679245ea&tx_pure_pure5%5BshowType%5D=pub&cHash=60ab4380d4bedb0d8c4af7ca16a59ba7 Skovdal, S. M., Jørgensen, N. P., Meyer, R. L. Staphylococcus epidermidis is the most abundant commensal bacterium of human skin. Despite protecting us from foreign invaders, S. epidermidis itself exploits human vulnerability when given the opportunity. Such opportunities arise when patients are immunocompromised or when biomedical implants present an opportunity to colonize the surface and form biofilms. S. epidermidis is one of the most frequently isolated organisms from implanted devices and from bloodstream infections. However, S. epidermidis infections are often recalcitrant to antibiotics because of biofilm-associated antibiotic tolerance. Furthermore, the emergence and spread of nearly pan-resistant strains is a considerable health concern. Symptoms can be subclinical, making diagnosis challenging, and treatment with antibiotics is inefficient. For now, infection prevention remains the best strategy available.

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Research Sat, 01 Oct 2022 17:39:52 +0200 1f396264-a26a-4384-8132-f49b679245ea
<![CDATA[Biofouling Control in Water Filtration Systems]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=468bc437-8e0e-4902-a34c-545392da571a&tx_pure_pure5%5BshowType%5D=pub&cHash=37be214917979c2c5017c6b5c8db05c6 Møllebjerg, A., Meyer, R. L. Research Thu, 01 Sep 2022 17:39:52 +0200 468bc437-8e0e-4902-a34c-545392da571a <![CDATA[Erratum for Møllebjerg et al., "The Bacterial Life Cycle in Textiles Is Governed by Fiber Hydrophobicity"]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=42418e5b-06e7-4086-bc05-ca60601d6452&tx_pure_pure5%5BshowType%5D=pub&cHash=8041d8ff177ee11ec39473e7b03f5ee7 Møllebjerg, A., Palmén, L. G., Gori, K., Meyer, R. L. Research Sat, 01 Oct 2022 17:39:52 +0200 42418e5b-06e7-4086-bc05-ca60601d6452 <![CDATA[The Role of Nanoscale Distribution of Fibronectin in the Adhesion of Staphylococcus aureus Studied by Protein Patterning and DNA-PAINT]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=eed6fe74-a3be-4778-becf-6dc23a6b5c8b&tx_pure_pure5%5BshowType%5D=pub&cHash=8d1c44b93f5033878d63978d14f4e2c8 Khateb, H., Sørensen, R. S., Cramer, K., et al. Staphylococcus aureus is a widespread and highly virulent pathogen that can cause superficial and invasive infections. Interactions between S. aureus surface receptors and the extracellular matrix protein fibronectin mediate the bacterial invasion of host cells and is implicated in the colonization of medical implant surfaces. In this study, we investigate the role of distribution of both fibronectin and cellular receptors on the adhesion of S. aureus to interfaces as a model for primary adhesion at tissue interfaces or biomaterials. We present fibronectin in patches of systematically varied size (100-1000 nm) in a background of protein and bacteria rejecting chemistry based on PLL-g-PEG and studied S. aureus adhesion under flow. We developed a single molecule imaging assay for localizing fibronectin binding receptors on the surface of S. aureus via the super-resolution DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) technique. Our results indicate that S. aureus adhesion to fibronectin biointerfaces is regulated by the size of available ligand patterns, with an adhesion threshold of 300 nm and larger. DNA-PAINT was used to visualize fibronectin binding receptor organization in situ at ∼7 nm localization precision and with a surface density of 38-46 μm-2, revealing that the engagement of two or more receptors is required for strong S. aureus adhesion to fibronectin biointerfaces.

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Research Fri, 01 Jul 2022 17:39:52 +0200 eed6fe74-a3be-4778-becf-6dc23a6b5c8b
<![CDATA[The giant staphylococcal protein Embp facilitates colonization of surfaces through Velcro-like attachment to fibrillated fibronectin]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=fb012b0b-fe1f-4a9c-b0c0-a898c4ffd6f9&tx_pure_pure5%5BshowType%5D=pub&cHash=10f4b2cb349ab29dd630c290e29ec65e Khan, N., Aslan, H., Büttner, H., et al. Staphylococcus epidermidis causes some of the most hard-to-treat clinical infections by forming biofilms: Multicellular communities of bacteria encased in a protective matrix, supporting immune evasion and tolerance against antibiotics. Biofilms occur most commonly on medical implants, and a key event in implant colonization is the robust adherence to the surface, facilitated by interactions between bacterial surface proteins and host matrix components. S. epidermidis is equipped with a giant adhesive protein, Embp, which facilitates bacterial interactions with surface-deposited, but not soluble fibronectin. The structural basis behind this selective binding process has remained obscure. Using a suite of single-cell and single-molecule analysis techniques, we show that S. epidermidis is capable of such distinction because Embp binds specifically to fibrillated fibronectin on surfaces, while ignoring globular fibronectin in solution. S. epidermidis adherence is critically dependent on multi-valent interactions involving 50 fibronectin-binding repeats of Embp. This unusual, Velcro-like interaction proved critical for colonization of surfaces under high flow, making this newly identified attachment mechanism particularly relevant for colonization of intravascular devices, such as prosthetic heart valves or vascular grafts. Other biofilm-forming pathogens, such as Staphylococcus aureus, express homologs of Embp and likely deploy the same mechanism for surface colonization. Our results may open for a novel direction in efforts to combat devastating, biofilm-associated infections, as the development of implant materials that steer the conformation of adsorbed proteins is a much more manageable task than avoiding protein adsorption altogether.

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Research Fri, 01 Jul 2022 17:39:52 +0200 fb012b0b-fe1f-4a9c-b0c0-a898c4ffd6f9
<![CDATA[A New Device for In Situ Dental Biofilm Collection Additively Manufactured by Direct Metal Laser Sintering and Vat Photopolymerization]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=5e85749a-17c0-4dda-b2df-18570ca5a7cb&tx_pure_pure5%5BshowType%5D=pub&cHash=585d902c49e9476bc2e92f2c609b48d5 Rikvold, P. T., Johnsen, K. K., Leonhardt, D., et al. Dental biofilms are complex medical biofilms that cause caries, the most prevalent disease of humankind. They are typically collected using handcrafted intraoral devices with mounted carriers for biofilm growth. As the geometry of handcrafted devices is not standardized, the shear forces acting on the biofilms and the access to salivary nutrients differ between carriers. The resulting variability in biofilm growth renders the comparison of different treatment modalities difficult. The aim of the present work was to design and validate an additively manufactured intraoral device with a dental bar produced by direct metal laser sintering and vat photopolymerized inserts with standardized geometry for the mounting of biofilm carriers. Additive manufacturing reduced the production time and cost, guaranteed an accurate fit of the devices and facilitated the handling of carriers without disturbing the biofilm. Biofilm growth was robust, with increasing thickness over time and moderate inter- and intraindividual variation (coefficients of variance 0.48-0.87). The biofilms showed the typical architecture and composition of dental biofilms, as evidenced by confocal microscopy and 16S rRNA gene sequencing. Deeper inserts offering increased protection from shear tended to increase the biofilm thickness, whereas prolonged exposure to sucrose during growth increased the biofilm volume but not the thickness. Ratiometric pH imaging revealed considerable pH variation between participants and also inside single biofilms. Intraoral devices for biofilm collection constitute a new application for medical additive manufacturing and offer the best possible basis for studying the influence of different treatment modalities on biofilm growth, composition, and virulence. The Clinical Trial Registration number is: 1-10-72-193-20.

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Research Sun, 01 Oct 2023 17:39:52 +0200 5e85749a-17c0-4dda-b2df-18570ca5a7cb
<![CDATA[Aptamer-Targeted Drug Delivery for <i>Staphylococcus aureus </i>Biofilm]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=592b8007-49cb-4a7d-bfe4-cd21bbe751d5&tx_pure_pure5%5BshowType%5D=pub&cHash=10519948618226b3ef093c2a5f130111 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.

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Research Fri, 01 Apr 2022 17:39:52 +0200 592b8007-49cb-4a7d-bfe4-cd21bbe751d5
<![CDATA[Fibrinolytic and Antibiotic Treatment of Prosthetic Vascular Graft Infections in a Novel Rat Model]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=9f47fe18-228e-4c52-98d8-adab50540619&tx_pure_pure5%5BshowType%5D=pub&cHash=fa04b6a7bf616d9d6c89900d5da7b793 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)

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.
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Communication Wed, 09 Jun 2021 17:39:52 +0200 9f47fe18-228e-4c52-98d8-adab50540619
<![CDATA[The bacterial life cycle in textiles is governed by fiber hydrophobicity]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=52139771-0566-4cd9-b709-81d27c18bcf1&tx_pure_pure5%5BshowType%5D=pub&cHash=69e78af1c5518ee24c02c145a9aaa7a3 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.

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Research Fri, 01 Oct 2021 17:39:52 +0200 52139771-0566-4cd9-b709-81d27c18bcf1
<![CDATA[Human Fibrinogen Inhibits Amyloid Assembly of Most Phenol-Soluble Modulins from Staphylococcus aureus]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=05e1ac47-4311-4def-956c-58ba8d3b3f28&tx_pure_pure5%5BshowType%5D=pub&cHash=bcfc11aaa613e9d67d94260e672fb9c2 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.

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Research Sun, 01 Aug 2021 17:39:52 +0200 05e1ac47-4311-4def-956c-58ba8d3b3f28
<![CDATA[Management of oral biofilms by nisin delivery in adhesive microdevices]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=c6ff27ff-396a-4ac4-abde-f71e0d70880f&tx_pure_pure5%5BshowType%5D=pub&cHash=652c6a63eaf783626851bbac81208a64 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.

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Research Fri, 01 Oct 2021 17:39:52 +0200 c6ff27ff-396a-4ac4-abde-f71e0d70880f
<![CDATA[Genome sequence of staphylococcus epidermidis AUH4567, a clinical isolate from an infected central venous catheter]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=ae825aed-81dc-40c9-81f9-f9aec62cc8a1&tx_pure_pure5%5BshowType%5D=pub&cHash=3aab3af3f447b299f32017f1aae635ed 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.

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Research Mon, 01 Mar 2021 17:39:52 +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>]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=9d688fce-9187-4795-aaf0-472f2a2c53be&tx_pure_pure5%5BshowType%5D=pub&cHash=093f5b03e3170ab72e05961407024b99 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.

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Research Fri, 01 Jan 2021 17:39:52 +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]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=bea19448-194f-4abd-86fe-cd3f63f28c23&tx_pure_pure5%5BshowType%5D=pub&cHash=6e31b02da3ae7539c12858b8ae3ddf36 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.

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Research Mon, 01 Mar 2021 17:39:52 +0100 bea19448-194f-4abd-86fe-cd3f63f28c23
<![CDATA[Phenol-Soluble Modulins Modulate Persister Cell Formation in <i>Staphylococcus aureus</i>]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=8fd35108-006e-488c-94e0-f751f050ac74&tx_pure_pure5%5BshowType%5D=pub&cHash=5078a29d95775ae6682b9125c9785b8f 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.

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Research Sun, 01 Nov 2020 17:39:52 +0100 8fd35108-006e-488c-94e0-f751f050ac74
<![CDATA[Polycaprolactone-gelatin nanofibers incorporated with dual antibiotic-loaded carboxyl-modified silica nanoparticles]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=e2d5d3b2-8b21-40b6-819d-d5ee4eebe604&tx_pure_pure5%5BshowType%5D=pub&cHash=aebf4992fc62fb0eb0c5035912e499da 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.].

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Research Tue, 01 Dec 2020 17:39:52 +0100 e2d5d3b2-8b21-40b6-819d-d5ee4eebe604
<![CDATA[Design of a new intraoral splint with 3D-printed inserts for dental biofilm collection]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=50eb4975-6769-4c09-a17b-8f03e1ac118b&tx_pure_pure5%5BshowType%5D=pub&cHash=43a2f54772d21acb3b12be1ae0b4c1bf Schlafer, S., Møllebjerg, A., Kambourakis Johnsen, K., Nielsen, S. M., Meyer, R. L. Research Wed, 01 Jul 2020 17:39:52 +0200 50eb4975-6769-4c09-a17b-8f03e1ac118b <![CDATA[Combination of Rhamnolipid and Chitosan in Nanoparticles Boosts Their Antimicrobial Efficacy]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=d3692f0e-fc42-4165-8dc0-d5a5b090ca0b&tx_pure_pure5%5BshowType%5D=pub&cHash=b82e2ba2fad16ee12ca281bca1a5ec08 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.

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Research Wed, 01 Jan 2020 17:39:52 +0100 d3692f0e-fc42-4165-8dc0-d5a5b090ca0b
<![CDATA[Delivering antibiotics locally to biofilms by targeted drug delivery and prodrug therapy]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=d710cec8-95de-4ba2-8aaa-8df38625c30a&tx_pure_pure5%5BshowType%5D=pub&cHash=4abd93852e52324316720267a55768f9 Meyer, R. L., Walther, R., Nielsen, S. M., et al. Research Tue, 03 Sep 2019 17:39:52 +0200 d710cec8-95de-4ba2-8aaa-8df38625c30a <![CDATA[Distribution of extracellular DNA in <i>Listeria monocytogenes</i> biofilm]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=51b7eaac-17a5-48d9-8f35-30aa6811d7f1&tx_pure_pure5%5BshowType%5D=pub&cHash=cdc16305e4c9df76a2f2aa479b4ae2cf Šuláková, M., Pazlarová, J., Meyer, R. L., Demnerová, K. Extracellular DNA (eDNA) is an abundant matrix component that protects biofilm from environmental stress, facilitate horizontal gene transfer, and serve as a source of nutrients. eDNA is also found in Listeria monocytogenes biofilm, but it is unknown to which extent its importance as a matrix component varies in terms of phylogenetic relatedness. This study aims to determine if these variations exist. Biofilm forming capacity of ten L. monocytogenes strains of different phylogenetic lineages and serotypes was examined using crystal violet assay at 37°C and 22°C. eDNA content was evaluated fluorometrically at 37°C and at 22°C, then the 3D structure of biofilm was studied by confocal laser scanning microscopy (CLSM). Biofilm forming capacity differed significantly between the culturing conditions and was higher at 37°C than at ambient temperature. eDNA signal distribution was found to be influenced by strain and lineage. CLSM images revealed information about spatial distribution in the biofilm. The information about the eDNA spatial organisation in the biofilm contributes to the understanding of the role of eDNA in a biofilm formation.

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Research Tue, 01 Jan 2019 17:39:52 +0100 51b7eaac-17a5-48d9-8f35-30aa6811d7f1
<![CDATA[Development of a label-free LSPR-Apta sensor for Staphylococcus aureus detection]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=741b1dfc-bb24-4d9e-9096-9945cc9ec8c6&tx_pure_pure5%5BshowType%5D=pub&cHash=f8e306cfa0fe877b170667c755ce054c Khateb, H., Klös, G., Meyer, R. L., Sutherland, D. S. The risk of foodborne diseases has increased over the last years. We have developed a simple, portable, and label-free optical sensor via aptamer recognition of Staphylococcus aureus at nanostructured plasmonic elements. The developed aptamers conjugated to a localized surface plasmon resonance (LSPR) sensing device were applied in both pure culture and artificially contaminated milk samples enabling a limit of detection of 10 3 CFU/mL for S. aureus in milk. There was no need for a pre-enrichment step, and the total analysis time decreased from 30 min to 120 s. Finite-difference time-domain was used to simulate the experimentally measured optical responses for a range of different sensor designs (100 and 200 nm disks), addressing the role of the near field and intrinsic refractive index sensitivity. A comparison of the aptamer to antibody-based recognition approaches showed that the thickness of the sensing layer was critical with a significantly larger response for the thinner aptamer layer. Comparison of differently sized metal nanostructures showed a significantly higher sensitivity for 200 nm diameter compared to 100 nm diameter disk structures resulting from both increases in bulk refractive index sensitivity and the extent to which the local field extends out from the metal surface. These findings confirmed that the developed gold nanodisk-based LSPR sensing chips could facilitate sensitive detection of S. aureus in food samples.

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Research Wed, 01 Apr 2020 17:39:52 +0200 741b1dfc-bb24-4d9e-9096-9945cc9ec8c6
<![CDATA[Evaluation of Surface-initiated Polymer brush as Anti-scaling Coating for Plate Heat Exchangers]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=e9621489-1a41-4f98-9440-ce085d10f4fb&tx_pure_pure5%5BshowType%5D=pub&cHash=7ebe4dd4d2d72d84bd9d4631c8aeeab7 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.

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Research Fri, 01 Nov 2019 17:39:52 +0100 e9621489-1a41-4f98-9440-ce085d10f4fb
<![CDATA[Antifouling properties of layer by layer DNA coatings]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=3d7ce3b3-8157-436b-b83d-6f2ce004de97&tx_pure_pure5%5BshowType%5D=pub&cHash=96f51a02c9ef2a2d89cf7a8ed0e414b7 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.

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Research Tue, 01 Jan 2019 17:39:52 +0100 3d7ce3b3-8157-436b-b83d-6f2ce004de97
<![CDATA[Cell wall associated protein TasA provides an initial binding component to extracellular polysaccharides in dual-species biofilm]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=11bdd111-083a-4202-8131-15f4b224c32e&tx_pure_pure5%5BshowType%5D=pub&cHash=11630162a87d42f4b577affc15d0aa58 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.

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Research Sat, 01 Dec 2018 17:39:52 +0100 11bdd111-083a-4202-8131-15f4b224c32e
<![CDATA[Development of an iron oxide nanoparticle drug delivery system against Staphylococcus aureus biofilm infections]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=04cd94a7-c25e-4ccc-884c-010100109eab&tx_pure_pure5%5BshowType%5D=pub&cHash=816a38bca231a3809c538b0eed204a25 Dreier, C., Zelikin, A. N., Kjems, J., Meyer, R. L. Research Sat, 01 Sep 2018 17:39:52 +0200 04cd94a7-c25e-4ccc-884c-010100109eab <![CDATA[Innate glycosidic activity in metallic implants for localized synthesis of antibacterial drugs]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=df8941a9-f777-455f-8db5-915e0fc48b98&tx_pure_pure5%5BshowType%5D=pub&cHash=a56391cd26eb349c149ac21d4436a869 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.

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Research Tue, 01 Jan 2019 17:39:52 +0100 df8941a9-f777-455f-8db5-915e0fc48b98
<![CDATA[Mesoporous silica nanoparticles carrying multiple antibiotics provide enhanced synergistic effect and improved biocompatibility]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=6e6892ff-db00-4098-8f48-d38c880bbff6&tx_pure_pure5%5BshowType%5D=pub&cHash=a9a42f2fcf1a198caedc2d576bb57c8c 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 17:39:52 +0100 6e6892ff-db00-4098-8f48-d38c880bbff6
<![CDATA[Novel prosthecate bacteria from the candidate phylum Acetothermia]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=f7d4ca69-e3da-45af-bd2e-bdb8314eab63&tx_pure_pure5%5BshowType%5D=pub&cHash=9f09ecf2bb471bafbae0f66510cf8683 Hao, L., McIlroy, S. J., Kirkegaard, R. H., et al. Members of the candidate phylum Acetothermia are globally distributed and detected in various habitats. However, little is known about their physiology and ecological importance. In this study, an operational taxonomic unit belonging to Acetothermia was detected at high abundance in four full-scale anaerobic digesters by 16S rRNA gene amplicon sequencing. The first closed genome from this phylum was obtained by differential coverage binning of metagenomes and scaffolding with long nanopore reads. Genome annotation and metabolic reconstruction suggested an anaerobic chemoheterotrophic lifestyle in which the bacterium obtains energy and carbon via fermentation of peptides, amino acids, and simple sugars to acetate, formate, and hydrogen. The morphology was unusual and composed of a central rod-shaped cell with bipolar prosthecae as revealed by fluorescence in situ hybridization combined with confocal laser scanning microscopy, Raman microspectroscopy, and atomic force microscopy. We hypothesize that these prosthecae allow for increased nutrient uptake by greatly expanding the cell surface area, providing a competitive advantage under nutrient-limited conditions.

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Research Mon, 01 Jan 2018 17:39:52 +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]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=212299d1-9b09-42bd-8c9b-aa2f9fc85ae3&tx_pure_pure5%5BshowType%5D=pub&cHash=73e4ff4eea509c342172882773d9deaa Walther, R., Winther, A., Fruergaard, A. S., et al. 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 metabolites and privileged prodrugs in the field of enzyme-prodrug therapies. Lastly, nanozymes are employed for conversion of glucuronide prodrugs into marketed anti-inflammatory and antibacterial agents, as well as “nanozyme prodrug therapy” to mediate antibacterial measures.

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Research Wed, 02 Jan 2019 17:39:52 +0100 212299d1-9b09-42bd-8c9b-aa2f9fc85ae3
<![CDATA[Quaternary Ammoniumyl Chitosan Derivatives for Eradication of Staphylococcus aureus Biofilms]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=a98c7b75-c031-4e8e-a9f8-3875f4e94200&tx_pure_pure5%5BshowType%5D=pub&cHash=c01c07adf64179145f73d6ab3fd8abce Sahariah, P., Masson, M., Meyer, R. L. Bacterial biofilms tolerate extreme levels of antibiotics. Treatment of biofilm infections therefore requires the development of new or modified antimicrobials that can penetrate biofilms and are effective against dormant persistent cells. One such new approach uses the biodegradable biopolymer chitosan and its derivatives as antimicrobials. In this study, we performed synthetic modification of chitosan to selectively introduce different cationic and hydrophobic moieties at varying ratios on chitosan. This improved its aqueous solubility and antimicrobial activity toward bacterial biofilms. Initial evaluation of the chitosan derivatives showed increased activity toward planktonic Staphylococcus aureus. The effect of the quaternary ammoniumyl chitosan derivatives against Staphylococcus aureus biofilms was more variable. The most effective derivatives contained hydrophobic groups, and their efficacy against biofilms depended on the ratio and length of the alkyl chains. Three-dimensional imaging of biofilms confirmed the accessibility and antimicrobial effect of chitosan derivatives with alkyl chains in the full depth of the biofilms.

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Research Sat, 01 Sep 2018 17:39:52 +0200 a98c7b75-c031-4e8e-a9f8-3875f4e94200
<![CDATA[Combatting implant-associated biofilms through localized drug synthesis]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=3e01125b-d714-40c3-b1b1-84e5032d08df&tx_pure_pure5%5BshowType%5D=pub&cHash=ee19dde39f2649d63dddb2315cafef3e 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.

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Research Wed, 10 Oct 2018 17:39:52 +0200 3e01125b-d714-40c3-b1b1-84e5032d08df
<![CDATA[Effect of DNase treatment on adhesion and early biofilm formation of Enterococcus faecalis]]> https://inano.au.dk/about/research-groups/biofilm-group/publications?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=6648f0ab-4c0b-4a2b-b9f7-60aa9eb6f83f&tx_pure_pure5%5BshowType%5D=pub&cHash=773636a8958c5c25dcb14582727adff7 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®. 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®-1 staining. DNase treatment reduced the area covered by cells in 96-well plates after 1 h (P<0.05), but not after 24 h (P=0.96). No significant differences in the number of adhering cells were observed in extracted teeth after 1 (P=0.14) and 24 h (P=0.98). 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.

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Research Mon, 01 Jan 2018 17:39:52 +0100 6648f0ab-4c0b-4a2b-b9f7-60aa9eb6f83f