Publications - Publications

The effect of multiple-enzyme treatment on in situ oral biofilm formation in healthy participants

Rikvold, P. D., Møllebjerg, A., Raittio, E. J., et al. — Mon, 01 Dec 2025 11:58:27 +0100

Novel approaches for the prevention of biofilm-mediated oral diseases aim to control dental biofilms rather than eradicating bacteria in the mouth. One such approach is the use of enzymes that specifically target and degrade the dental biofilm matrix and thereby facilitate biofilm removal. Matrix-degrading enzymes have consistently shown promising results in vitro, but data on in situ-grown oral biofilms are limited. This study aimed to investigate the effect of combined treatment with mutanase, beta-glucanase and DNase on in situ biofilm formation and removal, microbial biofilm composition and biofilm pH. Biofilms from healthy participants were grown for 48 or 72 h on lower-jaw splints and enzyme or control-treated during (3x/day, 30 min) or after growth (30 min). Under the tested conditions, enzyme treatment had no significant effect on biofilm formation or removal compared to control, as assessed by optical coherence tomography and confocal microscopy. Likewise, enzymatic treatment did not induce significant changes in the microbial composition of the biofilms that were dominated by Streptococcus, Haemophilus, Neisseria, Veillonella and Fusobacterium species. The biofilm pH response to a sucrose challenge was assessed using confocal microscopy-based pH ratiometry, and the average biofilm pH was not significantly different between the intervention groups. Under the conditions employed in this study, the tested enzymes had no significant impact on in situ grown biofilms. The treatment regimen, the biofilm composition, or the analytical methods employed may explain the difference to previous results. Further studies are warranted to assess the therapeutic potential of multi-enzyme treatment for dental biofilm control.

Coaggregation of oral pathogens by postbiotic lactobacilli

Golletz, P., Jensen, S. D., Collignon, M., et al. — Wed, 01 Jan 2025 11:58:27 +0100

INTRODUCTION: Coaggregation may reduce the abundance of bacteria in physiological fluids, such as saliva, as aggregated bacteria are cleared more easily than planktonic cells. This study aimed to identify Lactobacillus strains that coaggregate with oral pathogens with the perspective of using this approach to improve oral health.

MATERIAL AND METHODS: Coaggregation of 719 postbiotic Lactobacillus strains with target pathogens Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedia was quantified by absorbance. Coaggregation efficacy of selected strains with clinical isolates and in the presence of other salivary bacteria was determined by flow cytometry. Brightfield and confocal microscopy were applied to characterize the size and structure of coaggregates. Pangenome analysis was used to identify genomic regions potentially involved in the coaggregation activity.

RESULTS: Two strains, Lacticaseibacillus rhamnosus 1B06 and Lacticaseibacillus paracasei 8A12, coaggregated efficiently with all three target pathogens and clinical isolates of the same species even in the presence of other salivary bacteria. The coaggregation capability of the selected Lactobacillus strains was unique and could not be reproduced with other genetically similar lactic acid bacteria of the same species.

CONCLUSION: Lactobacillus strains capable of coaggregating oral pathogens were identified as promising candidates for the development of new postbiotic ingredients for oral hygiene products.

Microscale analyses of the effect of arginine on oral biofilms

Assar, S., Kristensen, M. F., Del Rey, Y. C., et al. — Thu, 29 May 2025 11:58:27 +0200

Objectives: Arginine is a caries-preventive agent that modulates the microbial composition of dental biofilms and raises pH via bacterial ammonia production. The effect of arginine on biofilm pH has only been measured in bulk, and it is unknown how treatment affects microscale pH developments inside dental biofilms. Moreover, little is known about arginine's impact on the biofilm matrix. This study investigated the effects of arginine on biofilm pH, matrix architecture and microbial composition in a saliva-derived biofilm model. Methods: 72-h biofilms were grown with or without sucrose, and arginine was either supplemented during growth or provided as pulsed treatment. pH was measured in different locations at the biofilm base using confocal-microscopy-based pH ratiometry. Matrix carbohydrates and extracellular DNA (eDNA) were assessed via fluorescence-lectin-binding-analysis and eDNA mapping. Microbial composition was determined by 16S rRNA gene sequencing. Results: Arginine supplementation during growth considerably raised the biofilm pH, despite the presence of sucrose, and counteracted sucrose-induced shifts in microbial composition. Pulsed arginine treatment, in contrast, had little influence on microbial composition, but still a significant effect on biofilm pH. Importantly, arginine treatment increased the pH variance between different biofilm areas, suggesting that local factors in the biofilm microarchitecture modulate the treatment response. Lastly, arginine supplementation reduced the abundance of matrix carbohydrates, while increasing eDNA levels. Conclusions: Arginine impacts biofilm pH at the microscale and changes the microbial and matrix composition in a saliva-derived biofilm model. Clinical significance: Arginine exerts caries-preventive effects via multiple mechanisms, including modulations of biofilm pH and matrix microarchitecture.

Development and use of microscale biosensors for organic and inorganic ions

Meyer, R.L. — Tue, 01 Jan 2002 11:58:27 +0100

Dental biofilms contain DNase I-resistant Z-DNA and G-quadruplexes but alternative DNase overcomes this resistance

Evans, D. C. S., Kristensen, M. F., Minero, G. A. S., et al. — Mon, 01 Dec 2025 11:58:27 +0100

Extracellular DNA (eDNA) in bacterial biofilms can form non-canonical structures like Z-DNA and G-quadruplex (G4), which enhance biofilm resilience by providing protection against mammalian DNases. However, the conformation of eDNA in dental biofilms remains unexplored. Using fluorescence immunolabeling and confocal microscopy, we examined dental biofilms from healthy and caries-active subjects, revealing B-DNA, G4-, and Z-DNA structures surrounding clusters of bacteria, with some structures directly associated with the bacterial cell surface. We demonstrated that these non-canonical DNA structures were resistant to mammalian DNase I. Using a Streptococcus mutans biofilm model, we visualised fluorescently labelled eDNA during enzyme treatment and identified both an experimental nuclease and a DNase I-chloroquine combination capable of removing eDNA that was resistant to DNase I. These findings suggest that G4 and Z-DNA structures represent novel targets for improved enzyme formulations in controlling dental biofilms and potentially other biofilms containing these secondary DNA structures.

Extracellular DNA and polysaccharide intercellular adhesin protect Staphylococcus epidermidis biofilms from phagocytosis by polymorphonuclear neutrophils

Evans, D. C.S., Mitkin, A. A., Rohde, H., Meyer, R. L. — Fri, 01 Aug 2025 11:58:27 +0200

Staphylococcus epidermidis is the leading cause of implant-associated infections, where it forms biofilms that are highly effective at evading the immune system. Here we investigate to what extent the biofilm extracellular matrix components extracellular DNA (eDNA) and polysaccharide intercellular adhesin (PIA) protect S. epidermidis from phagocytosis by polymorphonuclear neutrophils (PMN). We visualised phagocytosis using time-lapse confocal laser scanning microscopy of PMN interacting with planktonic S. epidermidis and 24 h old biofilms formed by the wildtype strain or mutant strains lacking either eDNA or PIA. We also compared phagocytosis of 24 h vs. 6 h old biofilms. PMN easily moved around and phagocytised S. epidermidis that were adhered to a surface from a planktonic culture. In contrast, PMN quickly became immobilised when interacting with biofilms. Very few PMN were able to phagocytise young (6 h) and mature (24 h) biofilms, suggesting that the accumulation of matrix components quickly provides a protective effect. Biofilms lacking either eDNA or PIA were much less dense, and many more PMN were able to phagocytise bacteria in these biofilms. Our findings suggest that both eDNA and PIA contribute to the ability of S. epidermidis biofilms to resist phagocytosis.

Combination therapy delays antimicrobial resistance after adaptive laboratory evolution of Staphylococcus aureus

Sat, 01 Mar 2025 11:58:27 +0100

Antibiotic resistance, driven by misuse and overuse of antibiotics, is one of the greatest threats against human health. The antimicrobial pressure during prolonged antibiotic treatment of chronic bacterial infections selects for resistance. While antibiotic combinations may reduce resistance emergence, antibiotic-tolerant persister cells can serve as a reservoir for resistance development. Therefore, targeting these cells with anti-persister drugs might provide a novel strategy for resistance prevention. In this study, we conducted 42 days of adaptive laboratory evolution using Staphylococcus aureus exposed to rifampicin, ciprofloxacin, daptomycin, and vancomycin, alone or in combination with the anti-persister drug mitomycin C. We monitored antibiotic susceptibility daily and assessed phenotypic changes in growth and biofilm formation in evolved strains. Whole-genome sequencing revealed mutations linked to antibiotic resistance and phenotypic shifts. Rifampicin resistance developed within a few days, while ciprofloxacin and daptomycin emerged in approximately 3 weeks. Treatments with vancomycin or mitomycin C resulted in minimal changes in susceptibility. While combination therapy delayed resistance, it did not fully prevent it. Notably, the combination of rifampicin with mitomycin C maintained rifampicin susceptibility throughout the long-term evolution experiment. Sub-inhibitory antibiotic treatments selected for both previously characterized and novel mutations, including unprecedented alterations in the nucleotide excision repair system and azoreductase following mitomycin C exposure. The delayed resistance development observed with combination therapy, particularly mitomycin C’s ability to suppress rifampicin resistance, suggests potential therapeutic applications. Future studies should evaluate the clinical efficacy of anti-persister drugs in preventing resistance across different bacterial pathogens and infection models.

Chitosan–saccharide conjugates for eradication of Pseudomonas aeruginosa biofilms

Tue, 27 Feb 2024 11:58:27 +0100

The problem of antibiotic resistance has raised serious concerns globally and hence the development of new materials which can combat these drug-resistant strains has gained a great deal of attention. Herein, we report the use of a biocompatible material, chitosan, as a scaffold to graft saccharides which can specifically target Pseudomonas aeruginosa. We realized this by synthesizing N-functionalized chitosan conjugates by coupling chitosan to fucose and galactose moieties which intercept Pseudomonas aeruginosa lectins and target the bacterial biofilms. A series of six conjugates containing similar proportions of cationic and sugar moieties were synthesized by direct modification of the chitosan backbone using a method that is highly efficient and reproducible. The conjugates showed a bactericidal effect against both Gram positive and Gram negative bacterial strains. An investigation into the antibiofilm activity of the conjugates revealed the optimum combination of the type and positioning of the functionalities that were highly effective in eradicating Pseudomonas aeruginosa biofilms. 2D and 3D imaging of the conjugate-treated biofilms using confocal laser scanning microscopy (CLSM) allowed us to determine that the conjugates not only acted on the surface but also dispersed into deep layers of the biofilm. Interaction between the conjugates and individual bacterial cells in the biofilm was further confirmed by fluorescence labelling of the conjugates and imaging by CLSM.

Polymyxin B complexation enhances the antimicrobial potential of graphene oxide

Pandit, S., Jacquemin, L., Zhang, J., et al. — Sun, 01 Jan 2023 11:58:27 +0100

Introduction: The antibacterial activity of graphene oxide (GO) has been widely explored and tested against various pathogenic bacterial strains. Although antimicrobial activity of GO against planktonic bacterial cells was demonstrated, its bacteriostatic and bactericidal effect alone is not sufficient to damage sedentary and well protected bacterial cells inside biofilms. Thus, to be utilized as an effective antibacterial agent, it is necessary to improve the antibacterial activity of GO either by integration with other nanomaterials or by attachment of antimicrobial agents. In this study, antimicrobial peptide polymyxin B (PMB) was adsorbed onto the surface of pristine GO and GO functionalized with triethylene glycol. Methods: The antibacterial effects of the resulting materials were examined by evaluating minimum inhibitory concentration, minimum bactericidal concentration, time kill assay, live/dead viability staining and scanning electron microscopy. Results and discussion: PMB adsorption significantly enhanced the bacteriostatic and bactericidal activity of GO against both planktonic cells and bacterial cells in biofilms. Furthermore, the coatings of PMB-adsorbed GO applied to catheter tubes strongly mitigated biofilm formation, by preventing bacterial adhesion and killing the bacterial cells that managed to attach. The presented results suggest that antibacterial peptide absorption can significantly enhance the antibacterial activity of GO and the resulting material can be effectively used not only against planktonic bacteria but also against infectious biofilms.

pH-FISH - coupled microscale analysis of microbial identity and acid–base metabolism in complex biofilm samples

Del Rey, Y. C., Kitzinger, K., Lund, M. B., et al. — Sun, 01 Dec 2024 11:58:27 +0100

Background: Correlative structural and chemical imaging of biofilms allows for the combined analysis of microbial identity and metabolism at the microscale. Here, we developed pH-FISH, a method that combines pH ratiometry with fluorescence in situ hybridization (FISH) in structurally intact biofilms for the coupled investigation of microbial acid metabolism and biofilm composition. Careful biofilm handling and modified sample preparation procedures for FISH allowed preservation of the three-dimensional biofilm structure throughout all processing and imaging steps. We then employed pH-FISH to investigate the relationship between local biofilm pH and the distribution of acid-producing (streptococci) and acid-consuming (Veillonella spp.) bacteria in dental biofilms from healthy subjects and caries-active patients. Results: The relative abundance of streptococci correlated with low biofilm pH at the field-of-view level, while the opposite trend was observed for Veillonella spp. These results suggest that clusters of streptococci contribute to the formation of acidic pockets inside dental biofilms, whereas Veillonella spp. may have a protective role against biofilm acidification. Conclusions: pH-FISH combines microscale mapping of biofilm pH in real time with structural imaging of the local microbial architecture, and is a powerful method to explore the interplay between biofilm composition and metabolism in complex biological systems.

Environmental pH and compound structure affect the activity of short-chain carboxylic acids against planktonic growth, biofilm formation, and eradication of the food pathogen Salmonella enterica

Ng, K. S., Bambace, M. F., Andersen, E. B., Meyer, R. L., Schwab, C. — Fri, 01 Nov 2024 11:58:27 +0100

Short-chain carboxylic acids (SCCAs) that are naturally produced by microbial fermentation play an essential role in delaying microbial spoilage. SCCAs are structurally diverse, but only a few of them are routinely used in food biopreservation. This study investigated the effects of environmental pH and intrinsic properties of 21 structurally different SCCAs on the antimicrobial and antibiofilm activity against Salmonella enterica. Inhibition of SCCA toward planktonic and biofilm growth of S. enterica was higher in an acidic environment (pH 4.5) that is common in fermented products, and for SCCA that possessed both a high acid dissociation strength (pKa) (>4.0) and a positive hydrophobicity [octanol/water partition coefficient (log Kow)]. Crotonic and caproic acids were identified as SCCAs with potential as biopreservatives even at near-neutral pH. SCCA with hydrophilic groups such as lactic acid did not inhibit S. enterica at concentrations up to 50 mM, while SCCA with benzene or methyl groups or a double bond prevented S. enterica growth and biofilm formation. Stimulation of biofilm formation was observed for formic, acetic, and propionic acid close to the minimum inhibitory concentration to reduce 50% of cell density (MIC50) of planktonic cells, and for citric and isocitric acid with an MIC50 of ≥50 mM. The presence of low concentrations of formic and propionic acids during biofilm formation conferred protection during eradication possibly due to a pre-adaptation effect, yet two consecutive acid treatments were successful in eradicating biofilms if the first acid treatment was two- to threefold of the MIC50.IMPORTANCEThis study provides a systematic comparison on the antimicrobial and antibiofilm activity of more than 20 structurally different SCCAs against a common food pathogen. We tested the antimicrobial activity at controlled pH and identified the structure-dependent antimicrobial effects of SCCA without the confounding influence of acidification. The combined effect of pKa and log Kow was identified as an important feature that should be considered when deciding for a specific SCCA in the application as antimicrobial. Our results imply that additional phenomena such as the use of SCCA as substrate and cellular pre-adaption effects have to be taken into consideration. We finally present a two-step treatment as an efficient approach to eradicate biofilms, which can be applied for the disinfection of contact surfaces and manufacturing equipment. Results obtained here can serve as guidelines for application of SCCA to avoid the growth of food pathogens and/or to develop biopreserved food systems.

Cooperation between coagulase and von willebrand factor binding protein in Staphylococcus aureus fibrin pseudocapsule formation

Evans, D. C.S., Khamas, A. B., Payne-Dwyer, A., et al. — Sun, 01 Dec 2024 11:58:27 +0100

The major human pathogen Staphylococcus aureus forms biofilms comprising of a fibrin network that increases attachment to surfaces and shields bacteria from the immune system. It secretes two coagulases, Coagulase (Coa) and von Willebrand factor binding protein (vWbp), which hijack the host coagulation cascade and trigger the formation of this fibrin clot. However, it is unclear how Coa and vWbp contribute differently to the localisation and dynamics of clot assembly in growing biofilms. Here, we address this question using high-precision time-resolved confocal microscopy of fluorescent fibrin to establish the spatiotemporal dynamics of fibrin clot formation in functional biofilms. We also use fluorescent fusion proteins to visualise the locations of Coa and vWbp in biofilms using both confocal laser scanning and high resolution highly inclined and laminated optical sheet microscopy. We visualise and quantify the spatiotemporal dynamics of fibrin production during initiation of biofilms in plasma amended with fluorescently labelled fibrinogen. We find that human serum stimulates coagulase production, and that Coa and vWbp loosely associate to the bacterial cell surface. Coa localises to cell surfaces to produce a surface-attached fibrin pseudocapsule but can diffuse from cells to produce matrix-associated fibrin. vWbp produces matrix-associated fibrin in the absence of Coa, and furthermore accelerates pseudocapsule production when Coa is present. Finally, we observe that fibrin production varies across the biofilm. A sub-population of non-dividing cells does not produce any pseudocapsule but remains within the protective extended fibrin network, which could be important for the persistence of S. aureus biofilm infections as antibiotics are more effective against actively growing cells. Our findings indicate a more cooperative role between Coa and vWbp in building fibrin networks than previously thought, and a bet-hedging cell strategy where some cells produce biofilm matrix while others do not, but instead assume a dormant phenotype that could be associated with antibiotic tolerance.

Large-scale screening identifies enzyme combinations that remove in situ grown oral biofilm

Nielsen, S. M., Johnsen, K. K., Hansen, L. B. S., et al. — Sun, 01 Dec 2024 11:58:27 +0100

Bacteria in the oral cavity are responsible for the development of dental diseases such as caries and periodontitis, but it is becoming increasingly clear that the oral microbiome also benefits human health. Many oral care products on the market are antimicrobial, killing a large part of the oral microbiome but without removing the disease-causing biofilm. Instead, non-biocidal matrix-degrading enzymes may be used to selectively remove biofilm without harming the overall microbiome. The challenge of using enzymes to degrade biofilms is to match the narrow specificity of enzymes with the large structural diversity of extracellular polymeric substances that hold the biofilm together. In this study, we therefore perform a large-scale screening of single and multi-enzyme formulations to identify combinations of enzymes that most effectively remove dental biofilm. We tested >400 different treatment modalities using 44 different enzymes in combinations with up to six enzymes in each formulation, on in vitro biofilms inoculated with human saliva. Mutanase was the only enzyme capable of removing biofilm on its own. Multi-enzyme formulations removed up to 69 % of the biofilm volume, and the most effective formulations all contained mutanase. We shortlisted 10 enzyme formulations to investigate their efficacy against biofilms formed on glass slabs on dental splints worn by 9 different test subjects. Three of the ten formulations removed more than 50 % of the biofilm volume. If optimal enzyme concentration and exposure time can be reached in vivo, these enzyme combinations have potential to be used in novel non-biocidal oral care products for dental biofilm control.

Biofilms on RO membranes treating tap water harbour diverse bacteria with opposing salinity optima

Poulsen, J. S., Koren, K., Meyer, R. L., Kjeldsen, K. U. — Sat, 21 Dec 2024 11:58:27 +0100

The escalating scarcity of clean freshwater, a crucial global economic asset, demands advancements in water purification technologies. Reverse osmosis (RO) stands out as an effective method for removing contaminants, yet biofouling of RO membranes poses a significant challenge, lowering the operational and energy efficiency. Through DNA sequencing, we identified a small set of abundant core bacterial species common to the biofilm in the three RO modules treating tap water. The core species are closely related to heterotrophic taxa from soil and groundwater systems and are likely important for the RO membrane biofouling. The community compositions of the RO-biofilms were system-specific. Within a given RO module, the biofilm community composition was uniform across the various sections of the membrane module. We established a method to efficiently extract live cells from the membranes and determined their salinity tolerance. Growth was mainly aerobic and was only inhibited at salinities above 3 to 5 %, i.e. exceeding those that can be reached in the specific RO modules. The species composition of the growing community changed discretely with the salinity level, indicating that salinity fluctuations may have an important role in shaping RO biofilm communities. Our findings highlight a complex interplay between microbial communities, salinity, and growth conditions in RO modules.

Arginine decreases the production of matrix carbohydrates and stimulates eDNA release in saliva-derived dental biofilms

Assar, S., Chokyu Del Rey, Y., Kristensen, M. F., et al. — Mon, 01 Jul 2024 11:58:27 +0200

Arginine modulates the bacterial community composition and raises microscale pH in saliva-derived dental biofilms

Assar, S., Chokyu Del Rey, Y., Kristensen, M. F., et al. — Mon, 01 Jul 2024 11:58:27 +0200

Arginine decreases the production of matrix carbohydrates and stimulates eDNA release in saliva-derived dental biofilms

Assar, S., Chokyu Del Rey, Y., Kristensen, M. F., et al. — Mon, 01 Jul 2024 11:58:27 +0200

Arginine modulates the bacterial community composition and raises microscale pH in saliva-derived dental biofilms

Assar, S., Chokyu Del Rey, Y., Kristensen, M. F., et al. — Mon, 01 Jan 2024 11:58:27 +0100

A high-throughput assay identifies molecules with antimicrobial activity against persister cells

Petersen, M. E., Hansen, L. K., Mitkin, A. A., et al. — Mon, 01 Jul 2024 11:58:27 +0200

Introduction. Persister cells are transiently non-growing antibiotic-tolerant bacteria that cause infection relapse, and there is no effective antibiotic therapy to tackle these infections.Gap statement. High-throughput assays in drug discovery are biased towards detecting drugs that inhibit bacterial growth rather than killing non-growing bacteria. A new and simple assay to discover such drugs is needed.Aim. This study aims to develop a simple and high-throughput assay to identify compounds with antimicrobial activity against persister cells and use it to identify molecular motifs with such activity.Methodology. We quantified Staphylococcus aureus persister cells by enumeration of colony forming units after 24 h ciprofloxacin treatment. We first quantified how the cell concentration, antibiotic concentration, growth phase and presence/absence of nutrients during antibiotic exposure affected the fraction of persister cells in a population. After optimizing these parameters, we screened the antimicrobial activity of compound fragments to identify molecular structures that have activity against persister cells.Results. Exponential- and stationary-phase cultures transferred to nutrient-rich media displayed a bi-phasic time-kill curve and contained 0.001-0.07% persister cells. A short rifampicin treatment resulted in 100% persister cells for 7 h, after which cells resumed activity and became susceptible. Stationary-phase cultures displayed a low but constant death rate but ultimately resulted in similarly low survival rates as the exponential-phase cultures after 24 h ciprofloxacin treatment. The persister phenotype was only maintained in most of the population for 24 h if cells were transferred to a carbon-free minimal medium before exposure to ciprofloxacin. Keeping cells starved enabled the generation of high concentrations of S. aureus cells that tolerate 50× MIC ciprofloxacin, and we used this protocol for rapid screening for biocidal antibiotics. We identified seven compounds from four structural clusters with activity against antibiotic-tolerant S. aureus. Two compounds were moderately cytotoxic, and the rest were highly cytotoxic.Conclusion. Transferring a stationary-phase culture to a carbon-free minimal medium for antimicrobial testing is a simple strategy for high-throughput screening for new antibiotics that kill persister cells. We identified molecule fragments with such activity, but further screening is needed to identify motifs with lower general cytotoxicity.

The efficacy and safety of an enzyme-containing lozenge for dental biofilm control

Thu, 01 Aug 2024 11:58:27 +0200

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.

Bacterial efflux pumps excrete SYTO™ dyes and lead to false-negative staining results

Minero, G. A. S., Larsen, P. B., Hoppe, M. E., Meyer, R. L. — Thu, 01 Feb 2024 11:58:27 +0100

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.

Efficacy of rifampicin combination therapy against MRSA prosthetic vascular graft infections in a rat model

Johansen, M. I., Petersen, M. E., Faddy, E., et al. — Sat, 01 Jun 2024 11:58:27 +0200

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.

Considerations on the use of microsensors to profile dissolved H2 concentrations in microbial electrochemical reactors

Mon, 01 Jan 2024 11:58:27 +0100

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.

Combined pH ratiometry and fluorescence lectin-binding analysis (pH-FLBA) for microscopy-based analyses of biofilm pH and matrix carbohydrates

Del Rey, Y. C., Schramm, A., L Meyer, R., Lund, M. B., Schlafer, S. — Thu, 01 Feb 2024 11:58:27 +0100

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.

Extracellular G-quadruplexes and Z-DNA protect biofilms from DNase I, and G-quadruplexes form a DNAzyme with peroxidase activity

Minero, G. A. S., Møllebjerg, A., Thiesen, C., et al. — Thu, 01 Feb 2024 11:58:27 +0100

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.

The effect of enzymatic treatment with mutanase, beta-glucanase and DNase on a saliva-derived biofilm model

Mon, 01 Jan 2024 11:58:27 +0100

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.

The effect of an enzyme-containing lozenge on dental biofilm accumulation

Sun, 01 Jan 2023 11:58:27 +0100

Multiple-enzyme treatment reduces biofilm formation in a highly acidogenic in vitro model

Rikvold, P., Møllebjerg, A., Meyer, R. L., Schlafer, S. — Sun, 01 Oct 2023 11:58:27 +0200

Background: Biofilm matrix-degrading enzymes are a promising approach to caries control, as they target determinants of biofilm virulence without compromising the microbial balance of the oral cavity. This study aimed to test the effect of treatment with a multiple-enzyme combination on the prevention and removal of a highly acidogenic in vitro-grown biofilm model dominated by Streptococcus spp.
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.

Multiple-enzyme treatment reduces biofilm formation in a highly acidogenic in vitro model

Rikvold, P., Møllebjerg, A., Meyer, R. L., Schlafer, S. — Sat, 01 Jul 2023 11:58:27 +0200

The effect of an enzyme-containing lozenge on dental biofilm accumulation: A clinical pilot study

Sat, 01 Jul 2023 11:58:27 +0200

Dental biofilms consist of microorganisms embedded in an extracellular matrix that comprises a complex mixture of macromolecules, including extracellular DNA and polysaccharides. Matrix-degrading enzymes have been shown to destabilize dental biofilms and thereby contribute to caries control. The aim of this work was to test the effect of a lozenge containing a mixture of glucanase, mutanase and DNAse on biofilm formation, the development of gingivitis and the composition of the oral microbiota.
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.

GFP fusions of Sec-routed extracellular proteins in Staphylococcus aureusreveal surface-associated coagulase in biofilms

Evans, D. C.S., Khamas, A. B., Marcussen, L., et al. — Sat, 01 Jul 2023 11:58:27 +0200

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.

Fibrinolytic and antibiotic treatment of prosthetic vascular graft infections in a novel rat model

Johansen, M. I., Rahbek, S. J., Jensen-Fangel, S., et al. — Sat, 01 Jul 2023 11:58:27 +0200

Objectives
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.

Protein ligand and nanotopography separately drive the phenotype of mouse embryonic stem cells

Ghorbani, S., Christine Füchtbauer, A., Møllebjerg, A., et al. — Sun, 01 Oct 2023 11:58:27 +0200

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.

Antibody-Drug Conjugates to Treat Bacterial Biofilms via Targeting and Extracellular Drug Release

Tvilum, A. S., Johansen, M. I., Glud, L. N., et al. — Tue, 01 Aug 2023 11:58:27 +0200

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.

Polyether Ionophore Antibiotics Target Drug-Resistant Clinical Isolates, Persister Cells, and Biofilms

Wollesen, M., Mikkelsen, K., Tvilum, M. S., et al. — Tue, 01 Aug 2023 11:58:27 +0200

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.

Novel high-throughput screening platform identifies enzymes to tackle biofouling on reverse osmosis membranes

Møllebjerg, A., Zarebska, A., Nielsen, H. B., et al. — Mon, 01 May 2023 11:58:27 +0200

Biofouling is a ubiquitous problem in many industrial systems including reverse osmosis (RO) desalination units where the biofilm impedes the water flow. It is essential to develop novel cleaning methods that are non-damaging and environmentally friendly. The most promising cleaning agents are enzymes, which can selectively cleave the components that stabilize the biofilm. However, biofilm-degrading enzymes are often difficult to identify due to the unknown matrix composition and lack of effective screening methods. To overcome this, a flexible screening platform was developed to evaluate enzyme treatment on fouling removal directly on RO membranes. The developed platform was used to identify enzymes that could degrade biofilms on fouled RO membranes. The membrane biofilms were destabilized by a range of enzyme groups, most effectively by proteases, lipases, DNase, cellulolytic enzymes, and pectin-degrading enzymes. The most effective enzyme formulations could clean the membranes more effectively than conventional chemical cleaning agents, removing 45 % of the biofilm compared with 0 %–19 % for the chemicals (p ≤ 0.001), indicating that enzymes have the potential to replace or complement chemical cleaning. The screening platform thus proved a potent tool for systematically studying biofilms and could be applied to combat biofilms in many other applications.

Biofilm formation and inflammatory potential of Staphylococcus saccharolyticus

Afshar, M., Møllebjerg, A., Minero, G. A., et al. — Tue, 01 Nov 2022 11:58:27 +0100

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.

JMM Profile

Skovdal, S. M., Jørgensen, N. P., Meyer, R. L. — Sat, 01 Oct 2022 11:58:27 +0200

Staphylococcus epidermidis is the most abundant commensal bacterium of human skin. Despite protecting us from foreign invaders, S. epidermidis itself exploits human vulnerability when given the opportunity. Such opportunities arise when patients are immunocompromised or when biomedical implants present an opportunity to colonize the surface and form biofilms. S. epidermidis is one of the most frequently isolated organisms from implanted devices and from bloodstream infections. However, S. epidermidis infections are often recalcitrant to antibiotics because of biofilm-associated antibiotic tolerance. Furthermore, the emergence and spread of nearly pan-resistant strains is a considerable health concern. Symptoms can be subclinical, making diagnosis challenging, and treatment with antibiotics is inefficient. For now, infection prevention remains the best strategy available.

Biofouling Control in Water Filtration Systems

Møllebjerg, A., Meyer, R. L. — Thu, 01 Sep 2022 11:58:27 +0200

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

Erratum for Møllebjerg et al., "The Bacterial Life Cycle in Textiles Is Governed by Fiber Hydrophobicity"

Møllebjerg, A., Palmén, L. G., Gori, K., Meyer, R. L. — Sat, 01 Oct 2022 11:58:27 +0200

The Role of Nanoscale Distribution of Fibronectin in the Adhesion of Staphylococcus aureus Studied by Protein Patterning and DNA-PAINT

Khateb, H., Sørensen, R. S., Cramer, K., et al. — Fri, 01 Jul 2022 11:58:27 +0200

Staphylococcus aureus is a widespread and highly virulent pathogen that can cause superficial and invasive infections. Interactions between S. aureus surface receptors and the extracellular matrix protein fibronectin mediate the bacterial invasion of host cells and is implicated in the colonization of medical implant surfaces. In this study, we investigate the role of distribution of both fibronectin and cellular receptors on the adhesion of S. aureus to interfaces as a model for primary adhesion at tissue interfaces or biomaterials. We present fibronectin in patches of systematically varied size (100-1000 nm) in a background of protein and bacteria rejecting chemistry based on PLL-g-PEG and studied S. aureus adhesion under flow. We developed a single molecule imaging assay for localizing fibronectin binding receptors on the surface of S. aureus via the super-resolution DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) technique. Our results indicate that S. aureus adhesion to fibronectin biointerfaces is regulated by the size of available ligand patterns, with an adhesion threshold of 300 nm and larger. DNA-PAINT was used to visualize fibronectin binding receptor organization in situ at ∼7 nm localization precision and with a surface density of 38-46 μm-2, revealing that the engagement of two or more receptors is required for strong S. aureus adhesion to fibronectin biointerfaces.

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

Khan, N., Aslan, H., Büttner, H., et al. — Fri, 01 Jul 2022 11:58:27 +0200

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

A New Device for In Situ Dental Biofilm Collection Additively Manufactured by Direct Metal Laser Sintering and Vat Photopolymerization

Rikvold, P. T., Johnsen, K. K., Leonhardt, D., et al. — Sun, 01 Oct 2023 11:58:27 +0200

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.

Aptamer-Targeted Drug Delivery for Staphylococcus aureus Biofilm

Ommen, P., Hansen, L., Hansen, B. K., Vu-Quang, H., Kjems, J., Meyer, R. L. — Fri, 29 Apr 2022 11:58:27 +0200

Treatment of Staphylococcus aureus biofilm infections using conventional antibiotic therapy is challenging as only doses that are sublethal to the biofilm can be administered safely to patients. A potential solution to this challenge is targeted drug delivery. In this study, we tailored an aptamer-targeted liposomal drug delivery system for accumulation and delivery of antibiotics locally in S. aureus biofilm. In our search for a suitable targeting ligand, we identified six DNA aptamers that bound to S. aureus cells in biofilms, and we demonstrated that one of these aptamers could facilitate accumulation of liposomes around S. aureus cells inside the biofilm. Aptamer-targeted liposomes encapsulating a combination of vancomycin and rifampicin were able to eradicate S. aureus biofilm upon 24 h of treatment in vitro. Our results point to that aptamer-targeted drug delivery of antibiotics is a potential new strategy for treatment of S. aureus biofilm infections.

Fibrinolytic and Antibiotic Treatment of Prosthetic Vascular Graft Infections in a Novel Rat Model

Johansen, M. I., Rahbek, S. J., Jensen-Fangel, S., et al. — Wed, 09 Jun 2021 11:58:27 +0200

Background
Medical implants are prone to bacterial attachment and development of biofilms. Biofilms cause the bacteria’s recalcitrance to antibiotics, resulting in a chronic infection that can be fatal. Previously, we have shown that the combination of antibiotics with fibrinolytic drugs such as streptokinase and tissue plasminogen activator (tPA) can eradicate methicillin-resistant Staphylococcus aureus (MRSA) biofilms in vitro. In this study, we aim to compare the efficacy of antibiotic therapy with and without tPA to treat MRSA biofilms in a novel rat model of prosthetic vascular graft infections (PVGI)

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

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

Conclusions
This study is the first to treat PVGI in an in vivo model, with clinically relevant doses of a fibrinolytic drug in conjunction with antibiotics. Our model could be used as a basis for future testing of both current antibiotics against PVGI and novel compounds against biofilm producing bacteria. The study drug combinations had limited effect at our samples size, and animal studies on a larger scale are therefore needed to evaluate the potential of this combination against PVGI.

The bacterial life cycle in textiles is governed by fiber hydrophobicity

Møllebjerg, A., Palmén, L. G., Gori, K., Meyer, R. L. — Fri, 01 Oct 2021 11:58:27 +0200

Colonization of textiles and subsequent metabolic degradation of sweat and sebum components by axillary skin bacteria cause the characteristic sweat malodor and discoloring of dirty clothes. Once inside the textile, the bacteria can form biofilms that are hard to remove by conventional washing. When the biofilm persists after washing, the textiles retain the sweat odor. To design biofilm removal and prevention strategies, the bacterial behavior needs to be understood in depth. Here, we aim to study the bacterial behavior in each of the four stages of the bacterial life cycle in textiles: Adhesion, growth, drying, and washing. To accomplish this, we designed a novel in vitro model to mimic physiological sweating in cotton and polyester textiles, in which many of the parameters that influence bacterial behavior could be controlled. Due to the higher hydrophobicity, polyester adhered more bacteria and absorbed more sebum, the bacteria's primary nutrient source. Bacteria were therefore also more active in polyester textiles. However, polyester did not bind water as well as cotton. The increased water content of cotton allowed some species to retain a higher activity after the textile had dried. However, none of the textiles retained enough water upon drying to prevent the bacteria from adhering irreversibly to the textile fibers. This work demonstrates that bacterial colonization of textiles depends partially on the hydrophobic and hygroscopic properties of the textile material, indicating that it might be possible to direct bacterial behavior in a more favorable direction by modifying these surface properties.

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

Najarzadeh, Z., Nielsen, J., Farzadfard, A., et al. — Sun, 01 Aug 2021 11:58:27 +0200

Functional amyloids are highly organized protein/peptide structures that inter alia promote biofilm formation in different bacteria. One such example is provided by a family of 20-45 residue-long peptides called phenol-soluble modulins (PSMs) from Staphylococcus aureus. External components such as eukaryotic host proteins, which alter self-assembly of bacterial amyloids, can affect the biofilm matrix. Here, we studied the effect of the highly prevalent human plasma protein fibrinogen (Fg) on fibrillation of PSMs. Fg inhibits or suppresses fibrillation of most PSMs tested (PSMα1, PSMβ1, and PSMβ2) except for PSMα3, whose already rapid aggregation is accelerated even further by Fg but leads to amorphous β-rich aggregates rather than fibrils. Fg also induces PSMβ2 to form amorphous aggregates and diverts PSMα1 into off-pathway oligomers which consist of both Fg and PSMα1 and cannot seed fibrillation. Peptide arrays showed that Fg bound to the N-terminus of PSMα1, while it bound to the entire length of PSMα3 (except the C terminus) and to the C-termini of PSMβ1 and PSMβ2. The latter peptides are all positively charged, while Fg is negatively charged at physiological pH. The positive charges complement Fg's net negative charge of -7.6 at pH 7.4. Fg's ability to inhibit PSM fibrillation reveals a potential host-defense mechanism to prevent bacterial biofilm growth and infections in the human body.

Management of oral biofilms by nisin delivery in adhesive microdevices

Fri, 01 Oct 2021 11:58:27 +0200

Numerous beneficial microbes thrive in the oral cavity where they form biofilms on dental and mucosal surfaces to get access to nutrients, and to avoid being carried away with the saliva. However, biofilm formation is also a virulence factor as it also protects pathogenic bacteria, providing them with an environment for proliferation causing oral infections. Oral hygiene relies on mechanical removal of biofilms. Some oral care products also contain antimicrobials, but effective eradication of biofilms with antimicrobials requires both a high concentration and long exposure time. In the present communication, we investigate the potential of using miniaturized drug delivery devices, known as microcontainers (MCs), to deliver the antimicrobial peptide, nisin to an oral multi-species biofilm. MCs are loaded with nisin and X-ray micro-computed tomography reveals a full release of nisin through a chitosan lid within 15 min. Chitosan-coated MCs display substantial bioadhesion to the buccal mucosa compared to non-coated MCs (68.6 ± 14.3% vs 33.8 ± 5.2%). Confocal monitoring of multi-species biofilms reveals antibacterial effects of nisin-loaded chitosan-coated MCs with a faster onset (after 3 h) compared to solution-based delivery (after 9 h). Our study shows the potential of using MCs for treatment of multi-species oral biofilms and is encouraging for further design of drug delivery devices to treat oral diseases.

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

Mon, 01 Mar 2021 11:58:27 +0100

Staphylococcus epidermidis is a common cause of implant-associated infections, and this is related to its ability to form biofilms. Strain-to-strain variability in biofilm formation is likely caused by genetic differences. Here, we present a draft genome of S. epidermidis AUH4567, which was isolated from a central venous catheter infection.

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

Aslan, H., Petersen, M. E., De Berardinis, A., et al. — Fri, 01 Jan 2021 11:58:27 +0100

Listeria monocytogenes is a foodborne pathogen which can survive in harsh environmental conditions. It responds to external stimuli through an array of two-component systems (TCS) that sense external cues. Several TCS, including LisRK, have been linked to Listeria’s ability to grow at slightly elevated antibiotic levels. The aim of this study was to determine if the TCS LisRK is also involved in acquiring the high antibiotic tolerance that is characteristic of persister cells. LisRK activates a response that leads to remodeling of the cell envelope, and we therefore hypothesized that activation of LisRK could also increase in the cells’ adhesiveness and initiate the first step in biofilm formation. We used a ΔlisR mutant to study antibiotic tolerance in the presence and absence of LisRK, and a GFP reporter strain to visualize the activation of LisRK in L. monocytogenes LO28 at a single-cell level. LisRK was activated in most cells in stationary phase cultures. Antimicrobial susceptibility tests showed that LisRK was required for the generation of ampicillin tolerance under these conditions. The wildtype strain tolerated exposure to ampicillin at 1,000 × inhibitory levels for 24 h, and the fraction of surviving cells was 20,000-fold higher in the wildtype strain compared to the ΔlisR mutant. The same protection was not offered to other antibiotics (vancomycin, gentamicin, tetracycline), and the mechanism for antibiotic tolerance is thus highly specific. Furthermore, quantification of bacterial attachment rates and attachment force also revealed that the absence of a functional LisRK rendered the cells less adhesive. Hence, LisRK TCS promotes multiple protective mechanisms simultaneously.