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  • 1.
    Alshammari, Hatem
    et al.
    Malmö University, Faculty of Odontology (OD).
    Neilands, Jessica
    Malmö University, Faculty of Odontology (OD). Malmö University, Biofilms Research Center for Biointerfaces.
    Svensäter, Gunnel
    Malmö University, Faculty of Odontology (OD). Malmö University, Biofilms Research Center for Biointerfaces.
    Stavropoulos, Andreas
    Malmö University, Faculty of Odontology (OD). University of Geneva, Switzerland.
    Antimicrobial Potential of Strontium Hydroxide on Bacteria Associated with Peri-Implantitis2021In: Antibiotics, ISSN 0066-4774, E-ISSN 2079-6382, Vol. 10, no 2, article id 150Article in journal (Refereed)
    Abstract [en]

    Background: Peri-implantitis due to infection of dental implants is a common complication that may cause significant patient morbidity. In this study, we investigated the antimicrobial potential of Sr(OH)2 against different bacteria associated with peri-implantitis. Methods: The antimicrobial potential of five concentrations of Sr(OH)2 (100, 10, 1, 0.1, and 0.01 mM) was assessed with agar diffusion test, minimal inhibitory concentration (MIC), and biofilm viability assays against six bacteria commonly associated with biomaterial infections: Streptococcus mitis, Staphylococcus epidermidis, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Escherichia coli, and Fusobacterium nucleatum. Results: Zones of inhibition were only observed for, 0.01, 0.1, and 1 mM of Sr(OH)2 tested against P. gingivalis, in the agar diffusion test. Growth inhibition in planktonic cultures was achieved at 10 mM for all species tested (p < 0.001). In biofilm viability assay, 10 and 100 mM Sr(OH)2 showed potent bactericidal affect against S. mitis, S. epidermidis, A. actinomycetemcomitans, E. coli, and P. gingivalis. Conclusions: The findings of this study indicate that Sr(OH)2 has antimicrobial properties against bacteria associated with peri-implantitis. 

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  • 2. Basic, Amina
    et al.
    Blomqvist, Madeleine
    Malmö högskola, Faculty of Odontology (OD). Malmö högskola, Biofilms Research Center for Biointerfaces.
    Dahlen, Gunnar
    Svensäter, Gunnel
    Malmö högskola, Faculty of Odontology (OD). Malmö högskola, Biofilms Research Center for Biointerfaces.
    The proteins of Fusobacterium spp. involved in hydrogen sulfide production from L-cysteine2017In: BMC Microbiology, E-ISSN 1471-2180, Vol. 17, no 61Article in journal (Refereed)
    Abstract [en]

    Background: Hydrogen sulfide (H2S) is a toxic foul-smelling gas produced by subgingival biofilms in patients with periodontal disease and is suggested to be part of the pathogenesis of the disease. We studied the H2S-producing protein expression of bacterial strains associated with periodontal disease. Further, we examined the effect of a cysteine-rich growth environment on the synthesis of intracellular enzymes in F. nucleatum polymorphum ATCC 10953. The proteins were subjected to one-dimensional (1DE) and two-dimensional (2DE) gel electrophoresis An in-gel activity assay was used to detect the H2S-producing enzymes; Sulfide from H2S, produced by the enzymes in the gel, reacted with bismuth forming bismuth sulfide, illustrated as brown bands (1D) or spots (2D) in the gel. The discovered proteins were identified with liquid chromatography - tandem mass spectrometry (LC-MS/MS). Results: Cysteine synthase and proteins involved in the production of the coenzyme pyridoxal 5'phosphate (that catalyzes the production of H2S) were frequently found among the discovered enzymes. Interestingly, a higher expression of H2S-producing enzymes was detected from bacteria incubated without cysteine prior to the experiment. Conclusions: Numerous enzymes, identified as cysteine synthase, were involved in the production of H2S from cysteine and the expression varied among Fusobacterium spp. and strains. No enzymes were detected with the in-gel activity assay among the other periodontitis-associated bacteria tested. The expression of the H2S-producing enzymes was dependent on environmental conditions such as cysteine concentration and pH but less dependent on the presence of serum and hemin.

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  • 3. Charyeva, Olga
    et al.
    Neilands, Jessica
    Malmö högskola, Faculty of Odontology (OD). Malmö högskola, Biofilms Research Center for Biointerfaces.
    Svensäter, Gunnel
    Malmö högskola, Faculty of Odontology (OD). Malmö högskola, Biofilms Research Center for Biointerfaces.
    Wennerberg, Ann
    Malmö högskola, Faculty of Odontology (OD). Malmö högskola, Biofilms Research Center for Biointerfaces.
    Bacterial Biofilm Formation on Resorbing Magnesium Implants2015In: Open Journal of Medical Microbiology, ISSN 2165-3372, Vol. 5, no 1Article in journal (Refereed)
    Abstract [en]

    Background: Implant-associated infections are a result of bacterial adhesion to an implant surface and subsequent biofilm formation at the implantation site. This study compares different magnesium materials based on their ability to resist bacterial adhesion as well as further biofilm formation. Material and Methods: The surfaces of four magnesium-based materials (Mg2Ag, Mg10Gd, WE43 and 99.99% pure Mg) were characterized using atomic force microscope. In addition, the samples were tested for their ability to resist biofilm formation. Planktonic bacteria of either S. epidermidis or E. faecalis were allowed to adhere to the magnesium surfaces for two hour followed by rinsing and, for S. epidermidis, further incubation of 24, 72 and 168 h was carried out. Results: E. faecalis had a significantly stronger adhesion to all magnesium surfaces compared to S. epidermidis (p = 0.001). Biofilm growth of S. epidermidis was different on various magnesium materials: the amount of bacteria increased up to 72 h but interestingly a significant decrease was seen at 168 h on Mg2Ag and WE43 surfaces. For pure Mg and Mg10Gd the biofilm formation reached plateau at 72 h. Surface characteristics of resorbable magnesium materials were changing over time, and the surface was generally less rough at 168 h compared to earlier time points. No correlation was found between the surface topology and the amount of adherent bacteria. Conclusion: In early stages of biofilm adhesion, no differences between magnesium materials were observed. However, after 72 h Mg2Ag and WE43 had the best ability to suppress S. epidermidis’ biofilm formation. Also, bacterial adhesion to magnesium materials was not dependent on samples’ surface topology.

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  • 4.
    Chavez de Paz, Luis E.
    et al.
    Malmö högskola, Faculty of Odontology (OD).
    Davies, Julia R
    Malmö högskola, Faculty of Odontology (OD).
    Bergenholtz, Gunnar
    Svensäter, Gunnel
    Malmö högskola, Faculty of Odontology (OD).
    Strains of Enterococcus faecalis differ in their ability to coexist in biofilms with other root canal bacteria2015In: International Endodontic Journal, ISSN 0143-2885, E-ISSN 1365-2591, Vol. 48, no 10, p. 916-925Article in journal (Refereed)
    Abstract [en]

    AIM To investigate the relationship between protease production and the ability of Enterococcus faecalis strains to coexist in biofilms with other bacteria commonly recovered from infected root canals. METHODOLOGY: Biofilms with bacteria in mono-, dual- and four-species communities were developed in flow chambers. The organisms used were Lactobacillus salivarius, Streptococcus gordonii and Actinomyces naeslundii and E. faecalis strains, GUL1 and OG1RF. Biovolume and species distribution were examined using 16S rRNA fluorescence in situ hybridization in combination with confocal microscopy and image analysis. The full proteome of the E. faecalis strains was studied using two-dimensional gel electrophoresis. Spots of interest were identified using tandem mass spectroscopy and quantified using Delta 2D software. RESULTS: All bacteria formed biofilms and an anova analysis revealed that the biofilm biomass increased significantly (P ≤ 0.01) between 6 and 24 h. L. salivarius, S. gordonii and A. naeslundii formed mutualistic biofilm communities, and this pattern was unchanged when E. faecalis GUL1 was included in the consortium. However, with OG1RF, L. salivarius and S. gordonii were outcompeted in a 24-h biofilm. Proteomic analysis revealed that OG1RF secreted higher levels of proteases, GelE (P = 0.02) and SprE (P = 0.002) and a previously unidentified serine protease (P = 0.05), than GUL1. CONCLUSIONS: Different strains of E. faecalis can interact synergistically or antagonistically with a consortium of root canal bacteria. A possible mechanism underlying this, as well as potential differences in virulence, is production of different levels of proteases, which can cause detachment of neighbouring bacteria and tissue damage.

  • 5. Chávez de Paz, Luis Eduardo
    et al.
    Bergenholtz, Gunnar
    Dahlén, Gunnar
    Svensäter, Gunnel
    Malmö högskola, Faculty of Odontology (OD).
    Response to alkaline stress by root canal bacteria in biofilms2007In: International Endodontic Journal, ISSN 0143-2885, E-ISSN 1365-2591, Vol. 40, no 5, p. 344-355Article in journal (Refereed)
    Abstract [en]

    To determine whether bacteria isolated from infected root canals survive alkaline shifts better in biofilms than in planktonic cultures. METHODOLOGY: Clinical isolates of Enterococcus faecalis, Lactobacillus paracasei, Olsenella uli, Streptococcus anginosus, S. gordonii, S. oralis and Fusobacterium nucleatum in biofilm and planktonic cultures were stressed at pH 10.5 for 4 h, and cell viability determined using the fluorescent staining LIVE/DEAD BacLight bacterial viability kit. In addition, proteins released into extracellular culture fluids were identified by Western blotting. RESULTS: Enterococcus faecalis, L. paracasei, O. uli and S. gordonii survived in high numbers in both planktonic cultures and in biofilms after alkaline challenge. S. anginosus, S. oralis and F. nucleatum showed increased viability in biofilms compared with planktonic cultures. Alkaline exposure caused all planktonic cultures to aggregate into clusters and resulted in a greater extrusion of cellular proteins compared with cells in biofilms. Increased levels of DnaK, HPr and fructose-1,6-bisphosphate aldolase were observed in culture fluids, especially amongst streptococci. CONCLUSIONS: In general, bacteria isolated from infected roots canals resisted alkaline stress better in biofilms than in planktonic cultures, however, planktonic cells appeared to use aggregation and the extracellular transport of specific proteins as survival mechanisms.

  • 6. Chávez de Paz, Luis Eduardo
    et al.
    Bergenholtz, Gunnar
    Svensäter, Gunnel
    Malmö högskola, Faculty of Odontology (OD).
    The effects of antimicrobials on endodontic biofilm bacteria2010In: Journal of Endodontics, ISSN 0099-2399, E-ISSN 1878-3554, Vol. 36, no 1, p. 70-77Article in journal (Refereed)
    Abstract [en]

    Introduction In the present study, confocal microscopy, a miniflow cell system, and image analysis were combined to test in situ the effect of antimicrobials and alkali on biofilms of Enterococcus faecalis, Lactobacillus paracasei, Streptococcus anginosus, and Streptococcus gordonii isolated from root canals with persistent infections. Methods Biofilms formed for 24 hours were exposed for 5 minutes to alkali (pH = 12), chlorhexidine digluconate (2.5%), EDTA (50 mmol/L), and sodium hypochlorite (1%). The biofilms were then characterized by using fluorescent markers targeting cell membrane integrity (LIVE/DEAD) and metabolic activity (5-cyano-2,3-ditolyl tetrazolium chloride and fluorescein diacetate). In addition, the biofilm architecture and the extent to which coating of the substrate surface with collagen influenced the resistance pattern to the chemicals were also analyzed. Results NaOCl (1%) affected the membrane integrity of all organisms and removed most biofilm cells. Exposure to EDTA (50 mmol/L) affected the membrane integrity in all organisms but failed to remove more than a few cells in biofilms of E. faecalis, L. paracasei, and S. anginosus. Chlorhexidine (2.5%) had a mild effect on the membrane integrity of E. faecalis and removed only 50% of its biofilm cells The effects were substratum-dependent, and most organisms displayed increased resistance to the antimicrobials on collagen-coated surfaces. Conclusions The biofilm system developed here was sensitive and differences in cell membrane integrity and removal of biofilm cells after exposure to antimicrobials commonly used in endodontics was discernible.

  • 7. Chávez de Paz, Luis Eduardo
    et al.
    Hamilton, Ian R
    Svensäter, Gunnel
    Malmö högskola, Faculty of Odontology (OD).
    Oral bacteria in biofilms exhibit slow reactivation from nutrient deprivation2008In: Microbiology, ISSN 1350-0872, E-ISSN 1465-2080, Vol. 154, p. 1927-1938Article in journal (Refereed)
    Abstract [en]

    The ability of oral bacteria to enter a non-growing state is believed to be an important mechanism for survival in the starved micro-environments of the oral cavity. In this study, we examined the reactivation of nutrient-deprived cells of two oral bacteria in biofilms, Streptococcus anginosus and Lactobacillus salivarius. Non-growing cells were generated by incubation in 10 mM potassium phosphate buffer for 24 h and the results were compared to those of planktonic cultures. When both types of cells were shifted from a rich, peptone-yeast extract-glucose (PYG) medium to buffer for 24 h, dehydrogenase and esterase activity measured by the fluorescent dyes 5-cyano-2,3-ditolyl-tetrazolium chloride (CTC) and fluorescein diacetate (FDA), respectively, was absent in both species. However, the membranes of the vast majority of nutrient-deprived cells remained intact as assessed by LIVE/DEAD staining. Metabolic reactivation of the nutrient-deprived biofilm cells was not observed for at least 48 h following addition of fresh PYG medium, whereas the non-growing planktonic cultures of the same two strains were in rapid growth in less than 2 h. At 72 h, the S. anginosus biofilm cells had recovered 78 % of the dehydrogenase activity and 61 % of the esterase activity and the biomass mm(-2) had increased by 30-35 %. With L. salivarius at 72 h, the biofilms had recovered 56 % and 75 % of dehydrogenase and esterase activity, respectively. Reactivation of both species in biofilms was enhanced by removal of glucose from PYG, and S. anginosus cells were particularly responsive to yeast extract (YE) medium. The data suggest that the low reactivity of non-growing biofilm cells to the introduction of fresh nutrients may be a survival strategy employed by micro-organisms in the oral cavity.

  • 8.
    Davies, J
    et al.
    Malmö högskola, Faculty of Odontology (OD).
    Svensäter, G
    Malmö högskola, Faculty of Odontology (OD).
    Adhesion of streptococci to oral mucosa. 9th European Oral Microbiology Workshop.2008Conference paper (Other (popular science, discussion, etc.))
  • 9.
    Davies, Julia
    et al.
    Malmö högskola, Faculty of Odontology (OD).
    Herzberg, Mark C
    Svensäter, Gunnel
    Malmö högskola, Faculty of Odontology (OD).
    Adhesion capacity of Streptococcus gordonii– effects of an srtA mutation (Malmö)2008Conference paper (Other academic)
  • 10.
    Davies, Julia R
    et al.
    Malmö högskola, Faculty of Odontology (OD).
    Herzberg, Mark C
    Svensäter, Gunnel
    Malmö högskola, Faculty of Odontology (OD).
    Adhesion capacity of Streptococcus gordonii– effects of an srtA mutation (Toronto)2008Conference paper (Other academic)
  • 11.
    Davies, Julia R
    et al.
    Malmö University, Faculty of Odontology (OD). Malmö University, Biofilms Research Center for Biointerfaces.
    Kad, Trupti
    Malmö University, Faculty of Odontology (OD). Malmö University, Biofilms Research Center for Biointerfaces.
    Neilands, Jessica
    Malmö University, Faculty of Odontology (OD). Malmö University, Biofilms Research Center for Biointerfaces.
    Kinnby, B
    Malmö University, Faculty of Odontology (OD). Malmö University, Biofilms Research Center for Biointerfaces.
    Prgomet, Zdenka
    Malmö University, Faculty of Odontology (OD). Malmö University, Biofilms Research Center for Biointerfaces.
    Bengtsson, Torbjörn
    School of Medical Sciences, Örebro University, Örebro, Sweden..
    Khalaf, Hazem
    School of Medical Sciences, Örebro University, Örebro, Sweden..
    Svensäter, Gunnel
    Malmö University, Faculty of Odontology (OD). Malmö University, Biofilms Research Center for Biointerfaces.
    Polymicrobial synergy stimulates Porphyromonas gingivalis survival and gingipain expression in a multi-species subgingival community.2021In: BMC Oral Health, ISSN 1472-6831, E-ISSN 1472-6831, Vol. 21, no 1, article id 639Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Dysbiosis in subgingival microbial communities, resulting from increased inflammatory transudate from the gingival tissues, is an important factor in initiation and development of periodontitis. Dysbiotic communities are characterized by increased numbers of bacteria that exploit the serum-like transudate for nutrients, giving rise to a proteolytic community phenotype. Here we investigate the contribution of interactions between members of a sub-gingival community to survival and development of virulence in a serum environment-modelling that in the subgingival pocket.

    METHODS: Growth and proteolytic activity of three Porphyromonas gingivalis strains in nutrient broth or a serum environment were assessed using A600 and a fluorescent protease substrate, respectively. Adherence of P. gingivalis strains to serum-coated surfaces was studied with confocal microscopy and 2D-gel electrophoresis of bacterial supernatants used to investigate extracellular proteins. A model multi-species sub-gingival community containing Fusobacterium nucleatum, Streptococcus constellatus, Parvimonas micra with wild type or isogenic mutants of P. gingivalis was then created and growth and proteolytic activity in serum assessed as above. Community composition over time was monitored using culture techniques and qPCR.

    RESULTS: The P. gingivalis strains showed different growth rates in nutrient broth related to the level of proteolytic activity (largely gingipains) in the cultures. Despite being able to adhere to serum-coated surfaces, none of the strains was able to grow alone in a serum environment. Together in the subgingival consortium however, all the included species were able to grow in the serum environment and the community adopted a proteolytic phenotype. Inclusion of P. gingivalis strains lacking gingipains in the consortium revealed that community growth was facilitated by Rgp gingipain from P. gingivalis.

    CONCLUSIONS: In the multi-species consortium, growth was facilitated by the wild-type and Rgp-expressing strains of P. gingivalis, suggesting that Rgp is involved in delivery of nutrients to the whole community through degradation of complex protein substrates in serum. Whereas they are constitutively expressed by P. gingivalis in nutrient broth, gingipain expression in the model periodontal pocket environment (serum) appeared to be orchestrated through signaling to P. gingivalis from other members of the community, a phenomenon which then promoted growth of the whole community.

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  • 12.
    Davies, Julia
    et al.
    Malmö högskola, Faculty of Odontology (OD).
    Svensäter, Gunnel
    Malmö högskola, Faculty of Odontology (OD).
    Herzberg, Mark C
    Identification of novel LPXTG-linked surface proteins from Streptococcus gordonii2009In: Microbiology, ISSN 1350-0872, E-ISSN 1465-2080, Vol. 155, p. 1977-1988Article in journal (Refereed)
    Abstract [en]

    Surface adhesion plays an essential part in the survival of the commensal organism Streptococcus gordonii in the oral cavity as well as during opportunistic infections such as endocarditis. At least two types of cell surface protein involved in adhesion are found on the surface of Gram-positive bacteria: those anchored via an LPXTG motif by the enzyme sortase A (SrtA) and those associated with the cell surface by, as yet, unknown mechanisms. In srtA(-) mutants, LPXTG-containing proteins have been shown to be released rather than cross-linked to the cell wall. We have therefore used 2D gel electrophoresis of released proteins from an srtA(-) mutant as well as the wild-type strain, followed by peptide identification by MS, to identify a set of novel proteins predicted to be present on the surface of S. gordonii DL1. This includes two large LPXTG-linked proteins (SGO_0707 and SGO_1487), which both contain tandemly repeated sequences similar to those present in known fibrillar adhesins. A 5'-nucleotidase and a protein with a putative collagen-binding domain, both containing LPXTG motifs, were also identified. Anchorless proteins with known chaperone, stress response and elongation factor functions, apparently responsible for bacterial binding to keratinocytes and saliva-coated surfaces in the absence of the LPXTG-linked adhesins, were also associated with the cell surface. These data reveal a range of proteins to be present on the S. gordonii DL1 cell surface, the expression of which plays an important role in adhesion to epithelia and which represent likely candidates for novel virulence factors in S. gordonii.

  • 13.
    Dorkhan, Marjan
    et al.
    Malmö högskola, Faculty of Odontology (OD).
    Chávez de Paz, Luis E.
    Malmö högskola, Faculty of Odontology (OD).
    Skepö, Marie
    Svensäter, Gunnel
    Malmö högskola, Faculty of Odontology (OD).
    Davies, Julia
    Malmö högskola, Faculty of Odontology (OD).
    Effects of saliva or serum coating on adherence of Streptococcus oralis strains to titanium2012In: Microbiology, ISSN 1350-0872, E-ISSN 1465-2080, Vol. 158, no 2, p. 390-397Article in journal (Refereed)
    Abstract [en]

    The use of dental implants to treat tooth loss has increased rapidly over recent years. 'Smooth' implants showing high long-term success rates have successively been replaced by implants with rougher surfaces, designed to stimulate rapid osseointegration and promote tissue healing. If exposed in the oral cavity, rougher surfaces may promote bacterial adhesion leading to formation of microbial biofilms which can induce peri-implant inflammation. Streptococcus oralis is an early colonizer of oral surfaces and has been recovered from titanium surfaces in vivo. The purpose of this study was to examine the adherence of clinical strains of S. oralis to titanium with smooth or moderately rough surface topography and to determine the effect of a saliva- or serum-derived coating on this process. Adherence was studied using a flow-cell system with confocal laser scanning microscopy, while putative adhesins were analysed using proteomics of bacterial cell wall proteins. This showed that adherence to moderately rough was greater than to smooth surfaces. Serum did not promote binding of any studied S. oralis strains to titanium whereas a saliva-coating increased adherence in two of three strains tested. The high level of adherence to the moderately rough surfaces was maintained even in the presence of a saliva coating. The S. oralis strains that bound to saliva expressed an LPXTG-linked protein which was not present in the non-adherent strain. Thus strains of S. oralis differ in their capacity to bind to saliva-coated titanium and we propose that this is due to differential expression of a novel adhesin.

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  • 14.
    Dorkhan, Marjan
    et al.
    Malmö högskola, Faculty of Odontology (OD).
    Hall, Jan
    Uvdal, Per
    Sandell, Anders
    Svensäter, Gunnel
    Malmö högskola, Faculty of Odontology (OD).
    Davies, Julia
    Malmö högskola, Faculty of Odontology (OD).
    Crystalline anatase-rich titanium can reduce adherence of oral streptococci2014In: Biofouling (Print), ISSN 0892-7014, E-ISSN 1029-2454, Vol. 30, no 6, p. 751-759Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Titanium implants in the oral cavity are covered with a saliva-derived pellicle to which early colonizing microorganisms such as Streptococcus oralis can bind. The protein profiles of salivary pellicles on titanium have not been well characterized and the proteins of importance for binding are thus unknown. Biofilm bacteria exhibit different phenotypes from their planktonic counterparts and contact with salivary proteins may be one factor contributing to the induction of changes in physiology. We have characterized salivary pellicles from titanium surfaces and investigated how contact with uncoated and saliva-coated titanium surfaces affects metabolic activity in adherent cells of S. oralis. METHODS: Salivary pellicles on smooth titanium surfaces were desorbed and these, as well as purified human saliva, were subjected to two-dimensional gel electrophoresis and mass spectroscopy. A parallel plate flow-cell model was used to study binding of a fresh isolate of S. oralis to uncoated and saliva-coated titanium surfaces. Metabolic activity was assessed using the BacLight CTC Vitality Kit and confocal scanning laser microscopy. Experiments were carried out in triplicate and the results analyzed using Student's t-test or ANOVA. RESULTS: Secretory IgA, α-amylase and cystatins were identified as dominant proteins in the salivary pellicles. Selective adsorption of proteins was demonstrated by the enrichment of prolactin-inducible protein and absence of zinc-α₂-glycoprotein relative to saliva. Adherence of S. oralis to titanium led to an up-regulation of metabolic activity in the population after 2 hours. In the presence of a salivary pellicle, this effect was enhanced and sustained over the following 22 hour period. CONCLUSIONS: We have shown that adherence to smooth titanium surfaces under flow causes an up-regulation of metabolic activity in the early oral colonizer S. oralis, most likely as part of an adaptation to the biofilm mode of life. The effect was enhanced by a salivary pellicle containing sIgA, α-amylase, cystatins and prolactin-inducible protein which was, for the first time, identified as an abundant component of salivary pellicles on titanium. Further studies are needed to clarify the mechanisms underlying the effect of surface contact on metabolic activity as well as to identify the salivary proteins responsible for enhancing the effect.

  • 15.
    Dorkhan, Marjan
    et al.
    Malmö högskola, Faculty of Odontology (OD).
    Svensäter, Gunnel
    Malmö högskola, Faculty of Odontology (OD).
    Davies, Julia
    Malmö högskola, Faculty of Odontology (OD).
    Salivary pellicles on titanium and their effect on metabolic activity in Streptococcus oralis2013In: BMC Oral Health, ISSN 1472-6831, E-ISSN 1472-6831, Vol. 13, article id 32Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Titanium implants in the oral cavity are covered with a saliva-derived pellicle to which early colonizing microorganisms such as Streptococcus oralis can bind. The protein profiles of salivary pellicles on titanium have not been well characterized and the proteins of importance for binding are thus unknown. Biofilm bacteria exhibit different phenotypes from their planktonic counterparts and contact with salivary proteins may be one factor contributing to the induction of changes in physiology. We have characterized salivary pellicles from titanium surfaces and investigated how contact with uncoated and saliva-coated titanium surfaces affects metabolic activity in adherent cells of S. oralis. METHODS: Salivary pellicles on smooth titanium surfaces were desorbed and these, as well as purified human saliva, were subjected to two-dimensional gel electrophoresis and mass spectroscopy. A parallel plate flow-cell model was used to study binding of a fresh isolate of S. oralis to uncoated and saliva-coated titanium surfaces. Metabolic activity was assessed using the BacLight CTC Vitality Kit and confocal scanning laser microscopy. Experiments were carried out in triplicate and the results analyzed using Student's t-test or ANOVA. RESULTS: Secretory IgA, α-amylase and cystatins were identified as dominant proteins in the salivary pellicles. Selective adsorption of proteins was demonstrated by the enrichment of prolactin-inducible protein and absence of zinc-α₂-glycoprotein relative to saliva. Adherence of S. oralis to titanium led to an up-regulation of metabolic activity in the population after 2 hours. In the presence of a salivary pellicle, this effect was enhanced and sustained over the following 22 hour period. CONCLUSIONS: We have shown that adherence to smooth titanium surfaces under flow causes an up-regulation of metabolic activity in the early oral colonizer S. oralis, most likely as part of an adaptation to the biofilm mode of life. The effect was enhanced by a salivary pellicle containing sIgA, α-amylase, cystatins and prolactin-inducible protein which was, for the first time, identified as an abundant component of salivary pellicles on titanium. Further studies are needed to clarify the mechanisms underlying the effect of surface contact on metabolic activity as well as to identify the salivary proteins responsible for enhancing the effect.

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  • 16.
    Dorkhan, Marjan
    et al.
    Malmö högskola, Faculty of Odontology (OD).
    Yucel-Lindberg, Tulay
    Hall, Jan
    Svensäter, Gunnel
    Malmö högskola, Faculty of Odontology (OD).
    Davies, Julia
    Malmö högskola, Faculty of Odontology (OD).
    Adherence of human oral keratinocytes and gingival fibroblasts to nano-structured titanium surfaces2014In: BMC Oral Health, ISSN 1472-6831, E-ISSN 1472-6831, Vol. 14, no 75, article id 74Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: A key element for long-term success of dental implants is integration of the implant surface with the surrounding host tissues. Modification of titanium implant surfaces can enhance osteoblast activity but their effects on soft-tissue cells are unclear. Adherence of human keratinocytes and gingival fibroblasts to control commercially pure titanium (CpTi) and two surfaces prepared by anodic oxidation was therefore investigated. Since implant abutments are exposed to a bacteria-rich environment in vivo, the effect of oral bacteria on keratinocyte adhesion was also evaluated. METHODS: The surfaces were characterized using scanning electron microscopy (SEM). The number of adhered cells and binding strength, as well as vitality of fibroblasts and keratinocytes were evaluated using confocal scanning laser microscopy after staining with Live/Dead Baclight. To evaluate the effect of bacteria on adherence and vitality, keratinocytes were co-cultured with a four-species streptococcal consortium. RESULTS: SEM analysis showed the two anodically oxidized surfaces to be nano-structured with differing degrees of pore-density. Over 24 hours, both fibroblasts and keratinocytes adhered well to the nano-structured surfaces, although to a somewhat lesser degree than to CpTi (range 42-89% of the levels on CpTi). The strength of keratinocyte adhesion was greater than that of the fibroblasts but no differences in adhesion strength could be observed between the two nano-structured surfaces and the CpTi. The consortium of commensal streptococci markedly reduced keratinocyte adherence on all the surfaces as well as compromising membrane integrity of the adhered cells. CONCLUSION: Both the vitality and level of adherence of soft-tissue cells to the nano-structured surfaces was similar to that on CpTi. Co-culture with streptococci reduced the number of keratinocytes on all the surfaces to approximately the same level and caused cell damage, suggesting that commensal bacteria could affect adherence of soft-tissue cells to abutment surfaces in vivo.

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  • 17. Eriksen, Harald M
    et al.
    Dimitrov, Vladimir
    Rohlin, Madeleine
    Malmö högskola, Faculty of Odontology (OD).
    Petersson, Kerstin
    Malmö högskola, Faculty of Odontology (OD).
    Svensäter, Gunnel
    Malmö högskola, Faculty of Odontology (OD).
    The oral ecosystem: implications for education2006In: European journal of dental education, ISSN 1396-5883, E-ISSN 1600-0579, Vol. 10, no 4, p. 192-196Article in journal (Refereed)
    Abstract [en]

    We propose a model that is applicable to oral health education. The model describes the oral cavity in a complexity-based ecological context. This concept includes the premise that factors from different organisational levels (biological, individual, community, society) interact in a complex way with the potential to 'stress' the ecosystem and thereby provoke changes. This mode of action complies with the understanding of the oral cavity as a complex adaptive system. An ecological model is actively used in the undergraduate problem-based curriculum at the Faculty of Odontology, Malmo University, Sweden and has recently been applied as a conceptual basis for the new dental curriculum being established at the University of Tromso in Northern Norway. The purpose is to encourage and promote an ecological, health-oriented view and to stimulate reflections on premises for oral health and diseases in an integrated context.

  • 18. Fröjd, Victoria
    et al.
    Chávez de Paz, Luis Eduardo
    Andersson, Martin
    Wennerberg, Ann
    Malmö högskola, Faculty of Odontology (OD).
    Davies, Julia
    Malmö högskola, Faculty of Odontology (OD).
    Svensäter, Gunnel
    Malmö högskola, Faculty of Odontology (OD).
    In situ analysis of multispecies biofilm formation on customized titanium surfaces2011In: Molecular Oral Microbiology, ISSN 2041-1006, E-ISSN 2041-1014, Vol. 26, no 4, p. 241-252Article in journal (Refereed)
    Abstract [en]

    Many studies to identify surfaces that enhance the incorporation of dental implants into bone and soft-tissue have been undertaken previously. However, to succeed in the clinical situation, an implant surface must not support development of microbial biofilms with a pathogenic potential. As a first step in investigating this, we used two-species and three-species biofilm models with 16S ribosomal RNA fluorescence in situ hybridization and confocal laser scanning microscopy to examine the effect of surface characteristics on biofilm formation by species that can colonize titanium implants in vivo: Streptococcus sanguinis, Actinomyces naeslundii and Lactobacillus salivarius. Surfaces blasted with Al(2) O(3) (S(a) = 1.0-2.0 μm) showed a seven-fold higher bacterial adhesion after 2 h than turned surfaces (S(a) = 0.18 μm) whereas porous surfaces, generated by anodic oxidation (S(a) = 0.4 μm), showed four-fold greater adhesion than turned surfaces. Hence, increased roughness promoted adhesion, most likely through protection of bacteria from shear forces. Chemical modification of the blasted and oxidized surfaces by incorporation of Ca(2+) ions reduced adhesion compared with the corresponding non-modified surfaces. After 14 h, biofilm growth occurred in the three-species model but not in the two-species consortium (containing S. sanguinis and A. naeslundii only). The biofilm biovolume on all surfaces was similar, suggesting that the influence of surface characteristics on adhesion was compensated for by biofilm development.

  • 19. Fröjd, Victoria
    et al.
    Linderbäck, Paula
    Wennerberg, Ann
    Malmö högskola, Faculty of Odontology (OD).
    Chávez de Paz, Luis
    Svensäter, Gunnel
    Malmö högskola, Faculty of Odontology (OD).
    Davies, Julia R
    Malmö högskola, Faculty of Odontology (OD).
    Effect of nanoporous TiO2 coating and anodized Ca2+ modification of titanium surfaces on early microbial biofilm formation2011In: BMC Oral Health, ISSN 1472-6831, E-ISSN 1472-6831, Vol. 11, no 8, article id 8Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: The soft tissue around dental implants forms a barrier between the oral environment and the peri-implant bone and a crucial factor for long-term success of therapy is development of a good abutment/soft-tissue seal. Sol-gel derived nanoporous TiO2 coatings have been shown to enhance soft-tissue attachment but their effect on adhesion and biofilm formation by oral bacteria is unknown. METHODS: We have investigated how the properties of surfaces that may be used on abutments: turned titanium, sol-gel nanoporous TiO2 coated surfaces and anodized Ca2+ modified surfaces, affect biofilm formation by two early colonizers of the oral cavity: Streptococcus sanguinis and Actinomyces naeslundii. The bacteria were detected using 16S rRNA fluorescence in situ hybridization together with confocal laser scanning microscopy. RESULTS: Interferometry and atomic force microscopy revealed all the surfaces to be smooth (Sa≤0.22 μm). Incubation with a consortium of S. sanguinis and A. naeslundii showed no differences in adhesion between the surfaces over 2 hours. After 14 hours, the level of biofilm growth was low and again, no differences between the surfaces were seen. The presence of saliva increased the biofilm biovolume of S. sanguinis and A. naeslundii ten-fold compared to when saliva was absent and this was due to increased adhesion rather than biofilm growth. CONCLUSIONS: Nano-topographical modification of smooth titanium surfaces had no effect on adhesion or early biofilm formation by S. sanguinis and A. naeslundii as compared to turned surfaces or those treated with anodic oxidation in the presence of Ca2+. The presence of saliva led to a significantly greater biofilm biovolume but no significant differences were seen between the test surfaces. These data thus suggest that modification with sol-gel derived nanoporous TiO2, which has been shown to improve osseointegration and soft-tissue healing in vivo, does not cause greater biofilm formation by the two oral commensal species tested than the other surfaces.

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  • 20.
    Havsed, Kristian
    et al.
    Malmö University, Faculty of Odontology (OD). Department of Pediatric Dentistry, Institute for Postgraduate Dental Education, Jönköping, Sweden; Centre for Oral Health, School of Health and Welfare, Jönköping University, Jönköping, Sweden.
    Hänsel Petersson, Gunnel
    Malmö University, Faculty of Odontology (OD).
    Isberg, Per-Erik
    Department of Statistics, Lund University, Lund, Sweden.
    Pigg, Maria
    Malmö University, Faculty of Odontology (OD). Malmö University, Foresight.
    Svensäter, Gunnel
    Malmö University, Faculty of Odontology (OD).
    Rohlin, Madeleine
    Malmö University, Faculty of Odontology (OD).
    Multivariable prediction models of caries increment: a systematic review and critical appraisal.2023In: Systematic Reviews, E-ISSN 2046-4053, Vol. 12, no 1, article id 202Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Multivariable prediction models are used in oral health care to identify individuals with an increased likelihood of caries increment. The outcomes of the models should help to manage individualized interventions and to determine the periodicity of service. The objective was to review and critically appraise studies of multivariable prediction models of caries increment.

    METHODS: Longitudinal studies that developed or validated prediction models of caries and expressed caries increment as a function of at least three predictors were included. PubMed, Cochrane Library, and Web of Science supplemented with reference lists of included studies were searched. Two reviewers independently extracted data using CHARMS (Critical Appraisal and Data Extraction for Systematic Reviews of Prediction Modelling Studies) and assessed risk of bias and concern regarding applicability using PROBAST (Prediction model Risk Of Bias ASessment Tool). Predictors were analysed and model performance was recalculated as estimated positive (LR +) and negative likelihood ratios (LR -) based on sensitivity and specificity presented in the studies included.

    RESULTS: Among the 765 reports identified, 21 studies providing 66 prediction models fulfilled the inclusion criteria. Over 150 candidate predictors were considered, and 31 predictors remained in studies of final developmental models: caries experience, mutans streptococci in saliva, fluoride supplements, and visible dental plaque being the most common predictors. Predictive performances varied, providing LR + and LR - ranges of 0.78-10.3 and 0.0-1.1, respectively. Only four models of coronal caries and one root caries model scored LR + values of at least 5. All studies were assessed as having high risk of bias, generally due to insufficient number of outcomes in relation to candidate predictors and considerable uncertainty regarding predictor thresholds and measurements. Concern regarding applicability was low overall.

    CONCLUSIONS: The review calls attention to several methodological deficiencies and the significant heterogeneity observed across the studies ruled out meta-analyses. Flawed or distorted study estimates lead to uncertainty about the prediction, which limits the models' usefulness in clinical decision-making. The modest performance of most models implies that alternative predictors should be considered, such as bacteria with acid tolerant properties.

    TRIAL REGISTRATION: PROSPERO CRD#152,467 April 28, 2020.

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  • 21.
    Havsed, Kristian
    et al.
    Malmö University, Faculty of Odontology (OD). Department of Pediatric Dentistry, Institute for Postgraduate Dental Education, Jönköping, Sweden; Centre for Oral Health, School of Health and Welfare, Jönköping University, Jönköping, Sweden.
    Stensson, Malin
    Centre for Oral Health, School of Health and Welfare, Jönköping University, Jönköping, Sweden; Folktandvården Skåne, The Swedish Dental Service of Skåne, Lund, Sweden.
    Jansson, Henrik
    Malmö University, Faculty of Odontology (OD). Centre for Oral Health, School of Health and Welfare, Jönköping University, Jönköping, Sweden; Folktandvården Skåne, The Swedish Dental Service of Skåne, Lund, Sweden.
    Carda-Diéguez, Miguel
    Department of Health & Genomics, Foundation for the Promotion of Health and Biomedical Research (FISABIO) Foundation, Valencia, Spain.
    Pedersen, Anders
    Swedish NMR Centre, The University of Gothenburg, Gothenburg, Sweden.
    Neilands, Jessica
    Malmö University, Faculty of Odontology (OD). Malmö University, Biofilms Research Center for Biointerfaces.
    Svensäter, Gunnel
    Malmö University, Faculty of Odontology (OD). Malmö University, Biofilms Research Center for Biointerfaces.
    Mira, Alex
    Centre for Oral Health, School of Health and Welfare, Jönköping University, Jönköping, Sweden; Department of Health & Genomics, Foundation for the Promotion of Health and Biomedical Research (FISABIO) Foundation, Valencia, Spain.
    Bacterial Composition and Metabolomics of Dental Plaque From Adolescents2021In: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 11, article id 716493Article in journal (Refereed)
    Abstract [en]

    Supragingival dental plaque samples were collected from 40 Swedish adolescents, including 20 with caries lesions (CAR) and 20 caries-free (CF). Fresh plaque samples were subjected to an ex vivo acid tolerance (AT) test where the proportion of bacteria resistant to an acid shock was evaluated through confocal microscopy and live/dead staining, and the metabolites produced were quantified by 1H Nuclear Magnetic Resonance (1H NMR). In addition, DNA was extracted and the 16S rRNA gene was sequenced by Illumina sequencing, in order to characterize bacterial composition in the same samples. There were no significant differences in AT scores between CAR and CF individuals. However, 7 out of the 10 individuals with highest AT scores belonged to the CAR group. Regarding bacterial composition, Abiotrophia, Prevotella and Veillonella were found at significantly higher levels in CAR individuals (p=0.0085, 0.026 and 0.04 respectively) and Rothia and Corynebacterium at significantly higher levels in CF individuals (p=0.026 and 0.003). The caries pathogen Streptococcus mutans was found at low frequencies and was absent in 60% of CAR individuals. Random-forest predictive models indicate that at least 4 bacterial species or 9 genera are needed to distinguish CAR from CF adolescents. The metabolomic profile obtained by NMR showed a significant clustering of organic acids with specific bacteria in CAR and/or high AT individuals, being Scardovia wiggsiae the species with strongest associations. A significant clustering of ethanol and isopropanol with health-associated bacteria such as Rothia or Corynebacterium was also found. Accordingly, several relationships involving these compounds like the Ethanol : Lactate or Succinate : Lactate ratios were significantly associated to acid tolerance and could be of predictive value for caries risk. We therefore propose that future caries risk studies would benefit from considering not only the use of multiple organisms as potential microbial biomarkers, but also their functional adaptation and metabolic output.

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  • 22.
    Hix Janssens, Thomas
    et al.
    Malmö University, Faculty of Health and Society (HS), Department of Biomedical Science (BMV).
    Shinde, Sudhirkumar
    Malmö University, Faculty of Health and Society (HS), Department of Biomedical Science (BMV). Malmö University, Biofilms Research Center for Biointerfaces.
    Abouhany, Rahma
    Malmö University, Faculty of Health and Society (HS), Department of Biomedical Science (BMV). Malmö University, Biofilms Research Center for Biointerfaces.
    Davies, Julia R
    Malmö University, Faculty of Odontology (OD). Malmö University, Biofilms Research Center for Biointerfaces.
    Neilands, Jessica
    Malmö University, Faculty of Odontology (OD). Malmö University, Biofilms Research Center for Biointerfaces.
    Svensäter, Gunnel
    Malmö University, Faculty of Odontology (OD). Malmö University, Biofilms Research Center for Biointerfaces.
    Sellergren, Börje
    Malmö University, Faculty of Health and Society (HS), Department of Biomedical Science (BMV). Malmö University, Biofilms Research Center for Biointerfaces.
    Microcontact-Imprinted Optical Sensors for Virulence Factors of Periodontal Disease2023In: ACS Omega, E-ISSN 2470-1343, Vol. 8, no 17, p. 15259-15265Article in journal (Refereed)
    Abstract [en]

    Periodontitis (gum disease) is a common biofilm-mediated oral condition, with around 7% of the adult population suffering from severe disease with risk for tooth loss. Moreover, periodontitis virulence markers have been found in atherosclerotic plaque and brain tissue, suggesting a link to cardiovascular and Alzheimer’s diseases. The lack of accurate, fast, and sensitive clinical methods to identify patients at risk leads, on the one hand, to patients being undiagnosed until the onset of severe disease and, on the other hand, to overtreatment of individuals with mild disease, diverting resources from those patients most in need. The periodontitis-associated bacterium, Porphyromonas gingivalis, secrete gingipains which are highly active proteases recognized as key virulence factors during disease progression. This makes them interesting candidates as predictive biomarkers, but currently, there are no methods in clinical use for monitoring them. Quantifying the levels or proteolytic activity of gingipains in the periodontal pocket surrounding the teeth could enable early-stage disease diagnosis. Here, we report on a monitoring approach based on high-affinity microcontact imprinted polymer-based receptors for the Arg and Lys specific gingipains Rgp and Kgp and their combination with surface plasmon resonance (SPR)-based biosensor technology for quantifying gingipain levels in biofluids and patient samples. Therefore, Rgp and Kgp were immobilized on glass coverslips followed by microcontact imprinting of poly-acrylamide based films anchored to gold sensor chips. The monomers selected were N-isopropyl acrylamide (NIPAM), N-hydroxyethyl acrylamide (HEAA) and N-methacryloyl-4-aminobenzamidine hydrochloride (BAM), with N,N′-methylene bis(acrylamide) (BIS) as the crosslinker. This resulted in imprinted surfaces exhibiting selectivity towards their templates high affinity and selectivity for the templated proteins with dissociation constants (Kd) of 159 and 299 nM for the Rgp- and Kgp-imprinted, surfaces respectively. The former surface displayed even higher affinity (Kd = 71 nM) when tested in dilute cell culture supernatants. Calculated limits of detection for the sensors were 110 and 90 nM corresponding to levels below clinically relevant concentrations.

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  • 23.
    Kindblom, Christian
    et al.
    Malmö högskola, Faculty of Odontology (OD).
    Davies, Julia R
    Malmö högskola, Faculty of Odontology (OD).
    Herzberg, Mark C
    Svensäter, Gunnel
    Malmö högskola, Faculty of Odontology (OD).
    Wickström, Claes
    Malmö högskola, Faculty of Odontology (OD).
    Salivary proteins promote proteolytic activity in Streptococcus mitis biovar 2 and Streptococcus mutans2012In: Molecular Oral Microbiology, ISSN 2041-1006, E-ISSN 2041-1014, Vol. 27, no 5, p. 362-372Article in journal (Refereed)
    Abstract [en]

    A major function of the salivary pellicle on oral surfaces is to promote colonization of the commensal microbiota by providing binding sites for adherence. Streptococcus mitis is an early colonizer of the oral cavity whereas Streptococcus mutans represents a later colonizer. To survive and grow, oral bacteria produce enzymes, proteases and glycosidases, which allow them to exploit salivary proteins as a nutrient source. In this study, adherence and proteolytic activity of S. mitis biovar 2 and S. mutans were investigated in a flow-cell model in the presence of different populations of surface-associated salivary proteins. Streptococcus mitis biovar 2 adhered well to surfaces coated with both a MUC5B-enriched fraction and a pool of low-density proteins containing MUC7, amylase, cystatin, gp340, immunoglobulin A, lactoferrin, lysozyme and statherin, whereas adherence of S. mutans to these proteins was poor. In environments of MUC5B or the low-density proteins, both S. mitis biovar 2 and S. mutans showed high levels of proteolytic activity. For S. mitis in the MUC5B environment, most of this activity may be attributable to contact with the molecules in the fluid phase although activity was also enhanced by adherence to surface-associated MUC5B. These data suggest that although they differ in their capacity to adhere to surface-associated salivary proteins, in the natural environment exploitation of saliva as a nutrient source can contribute to survival and colonization of the oral cavity by both S. mitis biovar 2 and S. mutans.

  • 24.
    Kinnby, Bertil
    et al.
    Malmö högskola, Faculty of Odontology (OD).
    Booth, Nuala A
    Svensäter, Gunnel
    Malmö högskola, Faculty of Odontology (OD).
    Plasminogen binding by oral streptococci from dental plaque and inflammatory lesions2008In: Microbiology, ISSN 1350-0872, E-ISSN 1465-2080, Vol. 154, no 3, p. 924-931Article in journal (Other (popular science, discussion, etc.))
    Abstract [en]

    Plasminogen binding by bacteria is a virulence factor important for the entry and dissemination of bacteria in the body. A wide variety of bacteria bind plasminogen, including both organisms causing disease and components of the normal oral flora. The purpose of this study was to examine the characteristics of plasminogen binding by six clinical isolates of oral streptococci from both dental plaque and inflammatory lesions. All the strains bound plasminogen with approximately the same affinity, and binding was specific and lysine-dependent as evidenced by its inhibition by epsilon-aminocaproic acid. All of the test strains were capable of activating bound plasminogen to plasmin without the addition of a plasminogen activator, and subsequent analysis revealed the presence of streptokinase in all strains. However, the streptococci exhibited fibrinolytic activity only in the presence of plasminogen and this could be inhibited by the addition of epsilon-aminocaproic acid. SDS-PAGE and 2D gel electrophoresis coupled with plasminogen ligand blotting showed that only a subset of the total proteins (2-15) were involved in the binding of plasminogen. Partial identification of the binding proteins revealed that four glycolytic enzymes, enolase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate mutase, were predominant in binding plasminogen. The binding of plasminogen by bacteria from pus did not differ from that of the strains from supragingival plaque. The findings illustrate how apparently innocuous commensal bacteria are capable of utilizing a mechanism that is generally regarded as being of importance to pathogenicity and suggest an additional role of plasminogen binding.

  • 25.
    Lager, Anders
    et al.
    Malmö högskola, Faculty of Odontology (OD).
    Ericson, Dan
    Malmö högskola, Faculty of Odontology (OD).
    Svensäter, Gunnel
    Malmö högskola, Faculty of Odontology (OD).
    Microbiota in dentine caries cultivable on pH-selective agars2008Conference paper (Refereed)
    Abstract [en]

    Objective: To investigate the acid tolerant microflora at different levels in established dentine caries lesions using solid pH-selective media, as acid stress might be a major selective determinant in dentine caries ecosystems. Methods: Primary dentine caries lesions (vital teeth, no symptoms) in five patients were sampled aseptically with a rose-bur at three levels: superficially, in the centre and the bottom of the lesion, when it was considered clinically caries free using visual and tactile criteria. Samples were incubated on neutral (blood agar) and pH-selective (Todd-Hewitt agar buffered to pH 4.0, 4.5, 5.0, 5.5) agar. Numbers of colony-forming units (cfu) were determined and colonies were characterized morphologically and with enzymatic- and sugar fermentation tests. Results: The total numbers of bacteria recovered from the pH-neutral agars did not decrease significantly with lesion depth (median blood agar: 6.3×103 superficially; 2.2×103 bottom) whereas cfu recovery from low pH agars decreased with increasing agar acidity. The composition of the aciduric microflora varied both between subjects and between sample sites within the lesions. Gram-positive cocci were most abundant, but with lower pH and deeper sampling sites, the numbers of lactobacilli and other Gram-positive rods increased. Mutans streptococci were found at all sampling depths. S. anginosus, S. constellatus, S. crista, S. gordonii, S. intermedius and S. sanguis were found less frequently. Conclusions: The study clearly indicates that many different microorganisms can be recovered on pH 5.5 agars and thus survive in low pH environments. pH 5.5 is quite sufficient to moderately demineralize dentine, and aciduric microorganisms should thus have the potential to contribute to the dentine caries process. Approved by the ethical committee at Lund University. Funded by Faculty grants.

  • 26.
    Lager, Anders
    et al.
    Malmö högskola, Faculty of Odontology (OD).
    Ericson, Dan
    Malmö högskola, Faculty of Odontology (OD).
    Thornqvist, E
    Svensäter, G
    Malmö högskola, Faculty of Odontology (OD).
    Lindberg, Birgitta
    Acid tolerant streptococci in dentine caries using pH-selective agars2006Conference paper (Other academic)
    Abstract [en]

    Objectives: To investigate the acid tolerant microflora in dentine caries, with special respect to oral streptococci cultivable on pH-selective agar media, as acid stress might be a major selective force in dentine caries. Methods: Five patients with primary dentine caries lesions (vital teeth, no symptoms) participated in the study. Caries lesions were excavated aseptically under rubber-dam using rose-burs. Dentine sampling was performed using sterile rose-burs at 3 levels: superficially, in the centre and in the cavity floor. Samples were incubated on neutral (blood agar) and pH-selective agars (Todd-Hewitt agar with citrate-phosphate buffer; pH 4.5, 5.0 and 5.5). Numbers of colony-forming units (CFUs) were determined, characterized morphologically, isolated to blood agar and then identified (Gram-reaction, cell- and colony morphology). Each colony type was frozen in skim milk (158 isolates). Isolates described as streptococci were revived, re-incubated (65 isolates) and further characterized by enzymatic- and sugar fermentation tests. Results: The same bacterial counts were recovered from superficial, center and cavity floor, respectively. Each dentine sample exhibited a unique microflora. There was no significant difference in proportions of aciduric microorganisms in different levels in the lesion. Approximately 38% (superficial), 5% (centre) and 38% (cavity floor) of the total cultivable microbiota was able to grow at pH 5.5, a pH value critical for demineralisation of dentine. The acid-tolerant streptococcal isolates included mutans streptococci, S. anginosus, S. constellatus and a group of unidentified streptococci. Lactate consuming taxa were found in one case, but only at pH 5.5. Conclusion: There was no significant difference in numbers of microorganisms on different levels in the lesion. Composition of the dentine caries microflora differs from lesion to lesion. The dentine caries microflora consortia differ from lesion to lesion. A common property of these bacteria was acid tolerance. Approved by the ethical committee Lund University. Funded by Faculty grants.

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  • 27.
    Leo, Fredrik
    et al.
    Genovis AB, Lund, Sweden..
    Svensäter, Gunnel
    Malmö University, Faculty of Odontology (OD).
    Lood, Rolf
    Lund Univ, Fac Med, Dept Clin Sci Lund, Div Infect Med, Lund, Sweden..
    Wickström, Claes
    Malmö University, Faculty of Odontology (OD).
    Characterization of a highly conserved MUC5B-degrading protease, MdpL, from Limosilactobacillus fermentum2023In: Frontiers in Microbiology, E-ISSN 1664-302X, Vol. 14, article id 1127466Article in journal (Refereed)
    Abstract [en]

    MUC5B is the predominant glycoprotein in saliva and is instrumental in the establishment and maintenance of multi-species eubiotic biofilms in the oral cavity. Investigations of the aciduric Lactobacillaceae family, and its role in biofilms emphasizes the diversity across different genera of the proteolytic systems involved in the nutritional utilization of mucins. We have characterized a protease from Limosilactobacillus fermentum, MdpL (Mucin degrading protease from Limosilactobacillus) with a high protein backbone similarity with commensals that exploit mucins for attachment and nutrition. MdpL was shown to be associated with the bacterial cell surface, in close proximity to MUC5B, which was sequentially degraded into low molecular weight fragments. Mapping the substrate preference revealed multiple hydrolytic sites of proteins with a high O-glycan occurrence, although hydrolysis was not dependent on the presence of O-glycans. However, since proteolysis of immunoglobulins was absent, and general protease activity was low, a preference for glycoproteins similar to MUC5B in terms of glycosylation and structure is suggested. MdpL preferentially hydrolyzed C-terminally located hydrophobic residues in peptides larger than 20 amino acids, which hinted at a limited sequence preference. To secure proper enzyme folding and optimal conditions for activity, L. fermentum incorporates a complex system that establishes a reducing environment. The importance of overall reducing conditions was confirmed by the activity boosting effect of the added reducing agents L-cysteine and DTT. High activity was retained in low to neutral pH 5.5-7.0, but the enzyme was completely inhibited in the presence of Zn2+. Here we have characterized a highly conserved mucin degrading protease from L. fermentum. MdpL, that together with the recently discovered O-glycanase and O-glycoprotease enzyme groups, increases our understanding of mucin degradation and complex biofilm dynamics.

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  • 28.
    Lima, Bruno P.
    et al.
    Univ Minnesota, Sch Dent, Dept Diagnost & Biol Sci, Minneapolis, MN 55455 USA..
    Davies, Julia R
    Malmö University, Faculty of Odontology (OD).
    Wickström, Claes
    Malmö University, Faculty of Odontology (OD).
    Johnstone, Karen F.
    Univ Minnesota, Sch Dent, Dept Diagnost & Biol Sci, Minneapolis, MN 55455 USA..
    Hall, Jeffrey W.
    Univ Minnesota, Sch Dent, Dept Diagnost & Biol Sci, Minneapolis, MN 55455 USA..
    Svensäter, Gunnel
    Malmö University, Faculty of Odontology (OD).
    Herzberg, Mark C.
    Univ Minnesota, Sch Dent, Dept Diagnost & Biol Sci, Minneapolis, MN 55455 USA..
    Streptococcus gordonii Poised for Glycan Feeding through a MUC5B-Discriminating, Lipoteichoic Acid-Mediated Outside-In Signaling Circuit2022In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 204, no 6, article id e00118-22Article in journal (Refereed)
    Abstract [en]

    Many oral bacteria employ cell wall-anchored adhesins to bind to the salivary films coating the teeth and mucosal surfaces. Surface binding prevents clearance and facilitates catabolism of salivary film glycoproteins. We asked whether Streptococcus gordonii adhesin expression changes in response to surface salivary cues using a eukaryote-like, outside-in recognition and signaling circuit. To determine whether the cues were discriminated, S. gordonii was tested during cell adhesion and biofilm formation on a MUC5B-rich or lower-molecular-mass salivary fraction or an uncoated abiotic surface. Cells were recovered and analyzed for differences in gene expression and proteins in cell wall fractions. In salivary-free conditions, planktonic S. gordonii presented three prominent cell wall LPXTG-motif proteins, SGO_1487, SGO_0890, and MbpA (mucin-binding protein A; SGO_0707). During biofilm formation on MUC5B-coated surfaces, MbpA, a MUC5B-binding protein, and key genes in the tagatose and quorum-sensing pathways were strongly promoted. The response to MUC5B required the two-component system (TCS), streptococcal regulator of adhesins sensor and regulator (SraSR, SGO_1180/81), lipoteichoic acid (LTA), and the homologous paired adhesins, SspA and SspB (SspAB). LTA appears to link the outside signal (MUC5B) to intramembrane SraSR. Tagatose pathway gene expression may poise cells to metabolize MUC5B glycans and, with a quorum-sensing gene (luxS), may direct formation of a consortium to facilitate glycan cross-feeding by S. gordonii. We now show that a Gram-positive bacterium discriminates specific surface environmental cues using an outside-in signaling mechanism to apparently optimize colonization of saliva-coated surfaces. IMPORTANCE All organisms throughout the tree of life sense and respond to their surface environments. To discriminate among mucosal surface environmental cues, we report that Streptococcus gordonii recognizes a high-molecular-weight mucin glycoprotein, MUC5B, using the paired adhesins SspAB and lipoteichoic acid; the latter bridges the outside signal to an intramembrane two-component system to transcriptionally regulate a MUC5B-specific adhesin and genes that may facilitate glycan catabolism. All organisms throughout the tree of life sense and respond to their surface environments. To discriminate among mucosal surface environmental cues, we report that Streptococcus gordonii recognizes a high-molecular-weight mucin glycoprotein, MUC5B, using the paired adhesins SspAB and lipoteichoic acid; the latter bridges the outside signal to an intramembrane two-component system to transcriptionally regulate a MUC5B-specific adhesin and genes that may facilitate glycan catabolism.

  • 29. Lima, Bruno P
    et al.
    Kho, Kelvin
    Nairn, Brittany L
    Davies, Julia R
    Malmö University, Faculty of Odontology (OD).
    Svensäter, Gunnel
    Malmö University, Faculty of Odontology (OD).
    Chen, Ruoqiong
    Steffes, Amanda
    Vreeman, Gerrit W
    Meredith, Timothy C
    Herzberg, Mark C
    Streptococcus gordonii type I lipoteichoic acid contributes to surface protein biogenesis2019In: mSphere, E-ISSN 2379-5042, Vol. 4, no 6Article in journal (Refereed)
    Abstract [en]

    Lipoteichoic acid (LTA) is an abundant polymer of the Gram-positive bacterial cell envelope and is essential for many species. Whereas the exact function of LTA has not been elucidated, loss of LTA in some species affects hydrophobicity, biofilm formation, and cell division. Using a viable LTA-deficient strain of the human oral commensal Streptococcus gordonii, we demonstrated that LTA plays an important role in surface protein presentation. Cell wall fractions derived from the wild-type and LTA-deficient strains of S. gordonii were analyzed using label-free mass spectroscopy. Comparisons showed that the abundances of many proteins differed, including (i) SspA, SspB, and S. gordonii 0707 (SGO_0707) (biofilm formation); (ii) FtsE (cell division); (iii) Pbp1a and Pbp2a (cell wall biosynthesis and remodeling); and (iv) DegP (envelope stress response). These changes in cell surface protein presentation appear to explain our observations of altered cell envelope homeostasis, biofilm formation, and adhesion to eukaryotic cells, without affecting binding and coaggregation with other bacterial species, and provide insight into the phenotypes revealed by the loss of LTA in other species of Gram-positive bacteria. We also characterized the chemical structure of the LTA expressed by S. gordonii Similarly to Streptococcus suis, S. gordonii produced a complex type I LTA, decorated with multiple d-alanylations and glycosylations. Hence, the S. gordonii LTA appears to orchestrate expression and presentation of cell surface-associated proteins and functions.IMPORTANCE Discovered over a half-century ago, lipoteichoic acid (LTA) is an abundant polymer found on the surface of Gram-positive bacteria. Although LTA is essential for the survival of many Gram-positive species, knowledge of how LTA contributes to bacterial physiology has remained elusive. Recently, LTA-deficient strains have been generated in some Gram-positive species, including the human oral commensal Streptococcus gordonii The significance of our research is that we utilized an LTA-deficient strain of S. gordonii to address why LTA is physiologically important to Gram-positive bacteria. We demonstrate that in S. gordonii, LTA plays an important role in the presentation of many cell surface-associated proteins, contributing to cell envelope homeostasis, cell-to-cell interactions in biofilms, and adhesion to eukaryotic cells. These data may broadly reflect a physiological role of LTA in Gram-positive bacteria.

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  • 30.
    Lindh, Liselott
    et al.
    Malmö högskola, Faculty of Odontology (OD).
    Glantz, Per-Olof
    Svensäter, Gunnel
    Malmö högskola, Faculty of Odontology (OD).
    Arnebrant, Thomas
    Malmö högskola, Faculty of Health and Society (HS).
    Adsorption from human saliva: time dependence and major components2002In: Journal of Dental Research Conferens;81, 2002Conference paper (Refereed)
    Abstract [en]

    Objectives: The aims of the present study were to investigate the adsorption from human whole saliva to solid surfaces in terms of dependence of adsorption time and surface wettability, to investigate pellicle elutability with buffer and sodium dodecyl sulphate (SDS) and finally to identify major components involved. Methods: Time resolved in situ ellipsometry was used to determine the adsorbed amounts and adsorption rates from human whole saliva onto pure (hydrophilic) and methylated (hydrophobized) silica surfaces. Two-dimensional gel electrophoresis was used to identify salivary components in the secretions as well as major components in pellicles. Results: The results demonstrated that on hydrophobic surfaces the initial adsorption was rapid and a plateau was reached, whereas on hydrophilic ones a continuous increase was observed during the time course of experiments. Contrary to what was expected, it was found that buffer rinsing removed less material after short adsorption times on hydrophobic surfaces, whereas less time dependence was observed on hydrophilic ones. After SDS exposure a minor fraction remained adsorbed after 15 minutes of adsorption, while a complete removal of the adsorbed film was observed after 2 hours of adsorption on hydrophobic surfaces. On hydrophilic surfaces a minor fraction remained adsorbed after both 15 minutes and 2 hours of adsorption. The two-dimensional gel electrophoresis revealed the presence of more than 50 proteins, with a molecular mass below 200 kDa present in whole saliva. Of these proteins only a few components were detected in the fraction eluted by SDS. Conclusions: We conclude that the different desorbability upon buffer rinsing and addition of SDS indicate that adsorbed proteins have varying binding strengths to the two types of surfaces. The time dependence observed and the compositional analysis show that the adsorbed pellicle undergoes conformational and/or compositional changes.

  • 31. Ljunggren, Stefan
    et al.
    Bengtsson, Torbjörn
    Karlsson, Helen
    Starkhammar Johansson, Carin
    Palm, Eleonor
    Nayeri, Fariba
    Ghafouri, Bijar
    Davies, Julia
    Malmö University, Faculty of Odontology (OD).
    Svensäter, Gunnel
    Malmö University, Faculty of Odontology (OD).
    Lönn, Johanna
    Modified lipoproteins in periodontitis: a link to cardiovascular disease?2019In: Bioscience Reports, ISSN 0144-8463, E-ISSN 1573-4935, Vol. 39, no 3Article in journal (Refereed)
    Abstract [en]

    There is a strong association between periodontal disease and atherosclerotic cardiovascular disorders. A key event in the development of atherosclerosis is accumulation of modified lipoproteins within the arterial wall. We hypothesise that patients with periodontitis have an altered lipoprotein profile towards an atherogenic form. Therefore, the present study aims at identifying modifications of plasma lipoproteins in periodontitis. Lipoproteins from ten female patients with periodontitis and gender- and age-matched healthy controls were isolated by density-gradient ultracentrifugation. Proteins were separated by 2D gel-electrophoresis and identified by map-matching or by nano-LC followed by MS. Apolipoprotein (Apo) A-I (ApoA-I) methionine oxidation, Oxyblot, total antioxidant capacity and a multiplex of 71 inflammation-related plasma proteins were assessed. Reduced levels of apoJ, phospholipid transfer protein, apoF, complement C3, paraoxonase 3 and increased levels of α-1-antichymotrypsin, apoA-II, apoC-III were found in high-density lipoprotein (HDL) from the patients. In low-density lipoprotein (LDL)/very LDL (VLDL), the levels of apoL-1 and platelet-activating factor acetylhydrolase (PAF-AH) as well as apo-B fragments were increased. Methionine oxidation of apoA-I was increased in HDL and showed a relationship with periodontal parameters. α-1 antitrypsin and α-2-HS glycoprotein were oxidised in LDL/VLDL and antioxidant capacity was increased in the patient group. A total of 17 inflammation-related proteins were important for group separation with the highest discriminating proteins identified as IL-21, Fractalkine, IL-17F, IL-7, IL-1RA and IL-2. Patients with periodontitis have an altered plasma lipoprotein profile, defined by altered protein levels as well as post-translational and other structural modifications towards an atherogenic form, which supports a role of modified plasma lipoproteins as central in the link between periodontal and cardiovascular disease (CVD).

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  • 32.
    Neilands, J
    et al.
    Malmö högskola, Faculty of Odontology (OD).
    Svensäter, G
    Malmö högskola, Faculty of Odontology (OD).
    Acid tolerance in streptococcal biofilm cells (Malmö)2008Conference paper (Other academic)
  • 33.
    Neilands, J
    et al.
    Malmö högskola, Faculty of Odontology (OD).
    Svensäter, G
    Malmö högskola, Faculty of Odontology (OD).
    Acid tolerance in streptococcal biofilm cells (Toronto)2008Conference paper (Other academic)
  • 34.
    Neilands, Jessica
    et al.
    Malmö University, Faculty of Odontology (OD).
    Davies, Julia R
    Malmö University, Faculty of Odontology (OD).
    Bikker, Floris J
    Svensäter, Gunnel
    Malmö University, Faculty of Odontology (OD).
    Parvimonas micra stimulates expression of gingipains from Porphyromonas gingivalis in multi-species communities2019In: Anaerobe, ISSN 1075-9964, E-ISSN 1095-8274, Vol. 55, p. 54-60Article in journal (Refereed)
    Abstract [en]

    Dental biofilms are complex ecosystems containing many bacterial species that live in mutualistic relationships. These interactions can profoundly affect the virulence properties of the community. In this study we investigated how the production of gingipains, virulence factors from Porphyromonas gingivalis important in periodontal disease, was affected by other commonly found members of the sub-gingival microbiome. To mimic the subgingival microbiome, multispecies consortia (P. gingivalis, Fusobacterium nucleatum, Actinomyces naeslundii, Streptococus oralis, Streptococcus mitis, Streptococcus gordonii and Streptococcus cristatus, with or without Parvimonas micra) as well as dual species consortia (P. gingivalis with P. micra, S. oralis or F. nucleatum) were constructed and maintained anaerobically in 10% serum for up to seven days. The number of P. gingivalis was determined by plating on Brucella agar and the gingipain specific fluorogenic substrate BikKam-10 was used to investigate gingipain activity. The effect of secreted products from P. micra on gingipain activity was investigated by adding supernatants from P. micra to P. gingivalis cultures. The most prominent secreted proteins in the supernatant were identified using mass spectrometry. P. gingivalis was unable to grow in serum, either alone or in the presence of S. oralis or F. nucleatum. In contrast, with P. micra growth was significantly enhanced and this was associated with an increase in gingipain activity. In the multi-species consortia, the presence of P. micra caused a 13-fold increase in gingipain activity. Exposure of P. gingivalis to supernatants from P. micra for 24 hours caused a 3-fold increase in gingipain activity. This effect was reduced by 43% after heat-treatment of the supernatant. Two dimensional gel electrophoresis revealed that several of the most prominent proteins in the P. micra supernatant were glycolytic enzymes. The results from this study suggests that gingipains are produced in response to a P. micra derived signaling molecule that is most likely a protein. This is the first time it has been shown that P. micra can affect P. gingivalis virulence properties. This is likely to be of significance for the development of be of periodontitis since these two microorganisms are often found together in the subgingival biofilm.

  • 35.
    Neilands, Jessica
    et al.
    Malmö högskola, Faculty of Odontology (OD).
    Petersson, Lars Gunnar
    Beighton, David
    Svensäter, Gunnel
    Malmö högskola, Faculty of Odontology (OD).
    Fluoride-supplemented milk inhibits acid tolerance in root caries biofilms2012In: Caries Research, ISSN 0008-6568, E-ISSN 1421-976X, Vol. 46, no 2, p. 156-160Article in journal (Refereed)
    Abstract [en]

    In this study we investigated the effect of fluoride on plaque acid tolerance. The test group consumed 200 ml of milk supplemented with 5 mg F/l as NaF once a day, the milk control group drank 200 ml of unsupplemented milk, and the no-milk control group did not consume milk in this manner. Plaque samples were taken at baseline and after 15 months. The proportion of acid-tolerant bacteria in plaque was estimated using LIVE/DEAD® BacLight™ staining after exposure to pH 3.5 for 2 h. The fluoride group showed a statistically significant decrease in plaque acid tolerance compared to baseline. This study shows that daily intake of fluoride in milk reduces plaque acid tolerance.

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  • 36.
    Neilands, Jessica
    et al.
    Malmö högskola, Faculty of Odontology (OD).
    Svensäter, Gunnel
    Malmö högskola, Faculty of Odontology (OD).
    Acid Tolerance in Oral Biofilms2007Conference paper (Other (popular science, discussion, etc.))
  • 37.
    Neilands, Jessica
    et al.
    Malmö högskola, Faculty of Odontology (OD).
    Svensäter, Gunnel
    Malmö högskola, Faculty of Odontology (OD).
    Acid Tolerance of Biofilm Cells of Streptococcus mutans2007In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 73, no 17, p. 5633-5638Article in journal (Refereed)
    Abstract [en]

    Streptococcus mutans, a member of the dental plaque community, has been shown to be involved in the carious process. Cells of S. mutans induce an acid tolerance response (ATR) when exposed to sublethal pH values that enhances their survival at a lower pH. Mature biofilm cells are more resistant to acid stress than planktonic cells. We were interested in studying the acid tolerance and ATR-inducing ability of newly adhered biofilm cells of S. mutans. All experiments were carried out using flow-cell systems, with acid tolerance tested by exposing 3-h biofilm cells to pH 3.0 for 2 h and counting the number of survivors by plating on blood agar. Acid adaptability experiments were conducted by exposing biofilm cells to pH 5.5 for 3 h and then lowering the pH to 3.5 for 30 min. The viability of the cells was assessed by staining the cells with LIVE/DEAD BacLight viability stain. Three-hour biofilm cells of three different strains of S. mutans were between 820- and 70,000-fold more acid tolerant than corresponding planktonic cells. These strains also induced an ATR that enhanced the viability at pH 3.5. The presence of fluoride (0.5 M) inhibited the induction of an ATR, with 77% fewer viable cells at pH 3.5 as a consequence. Our data suggest that adhesion to a surface is an important step in the development of acid tolerance in biofilm cells and that different strains of S. mutans possess different degrees of acid tolerance and ability to induce an ATR.

  • 38.
    Neilands, Jessica
    et al.
    Malmö University, Faculty of Odontology (OD).
    Svensäter, Gunnel
    Malmö University, Faculty of Odontology (OD).
    Boisen, Gabriella
    Malmö University, Faculty of Odontology (OD).
    Robertsson, Carolina
    Malmö University, Faculty of Odontology (OD).
    Wickström, Claes
    Malmö University, Faculty of Odontology (OD).
    Davies, Julia R
    Malmö University, Faculty of Odontology (OD).
    Formation and Analysis of Mono-species and Polymicrobial Oral Biofilms in Flow-Cell Models2023In: Bacterial Pathogenesis: Methods and Protocols,, Springer, 2023, p. 33-52Chapter in book (Refereed)
    Abstract [en]

    The oral microbiota, which is known to include at least 600 different bacterial species, is found on the teethand mucosal surfaces as multi-species communities or biofilms. The oral surfaces are covered with a pellicleof proteins absorbed from saliva, and biofilm formation is initiated when primary colonizers, which expresssurface adhesins that bind to specific salivary components, attach to the oral tissues. Further developmentthen proceeds through co-aggregation of additional species. Over time, the composition of oral biofilms,which varies between different sites throughout the oral cavity, is determined by a combination ofenvironmental factors such as the properties of the underlying surface, nutrient availability and oxygenlevels, and bacterial interactions within the community. A complex equilibrium between biofilm communities and the host is responsible for the maintenance of a healthy biofilm phenotype (eubiosis). In the faceof sustained environmental perturbation, however, biofilm homeostasis can break down giving rise todysbiosis, which is associated with the development of oral diseases such as caries and periodontitis.In vitro models have an important part to play in increasing our understanding of the complex processesinvolved in biofilm development in oral health and disease, and the requirements for experimental system,microbial complexity, and analysis techniques will necessarily vary depending on the question posed. In thischapter we describe some current and well-established methods used in our laboratory for studying oralbacteria in biofilm models which can be adapted to suit the needs of individual users. 

  • 39.
    Neilands, Jessica
    et al.
    Malmö högskola, Faculty of Odontology (OD).
    Wickström, Claes
    Malmö högskola, Faculty of Odontology (OD).
    Kinnby, Bertil
    Malmö högskola, Faculty of Odontology (OD).
    Davies, Julia R
    Malmö högskola, Faculty of Odontology (OD).
    Hall, Jan
    Friberg, Bertil
    Malmö högskola, Faculty of Odontology (OD).
    Svensäter, Gunnel
    Malmö högskola, Faculty of Odontology (OD).
    Bacterial profiles and proteolytic activity in peri-implantitis versus healthy sitesBacterial profiles and proteolytic activity in peri-implantitis versus healthy sites2015In: Anaerobe, ISSN 1075-9964, E-ISSN 1095-8274, Vol. 35, p. 28-34Article in journal (Refereed)
    Abstract [en]

    Peri-implantitis is a biofilm-induced destructive inflammatory process that, over time, results in loss of supporting bone around an osseointegrated dental implant. Biofilms at peri-implantitis sites have been reported to be dominated by Gram-negative anaerobic rods with a proteolytic metabolism such as, Fusobacterium, Porphyromonas, Prevotella and Tannerella, as well as anaerobic Gram-positive cocci. In this study, we hypothesized that protease activity is instrumental in driving bone destruction and we therefore compared the microbial composition and level of protease activity in samples of peri-implant biofluid (PIBF) from 25 healthy subjects (H group) and 25 subjects with peri-implantitis (PI group). Microbial composition was investigated using culture techniques and protease activity was determined using a FITC-labelled casein substrate. The microbial composition was highly variable in subjects both in the H and PI groups but one prominent difference was the prevalence of Porphyromonas/Prevotella and anaerobic Gram positive cocci which was significantly higher in the PI than in the H group. A subgroup of subjects with peri-implantitis displayed a high level of protease activity in the PIBF compared to healthy subjects. However, this activity could not be related to the presence of specific bacterial species. We propose that a high level of protease activity may be a predictive factor for disease progression in peri-implantitis. Further longitudinal studies are however required to determine whether assessment of protease activity could serve as a useful method to identify patients at risk for progressive tissue destruction.

  • 40.
    Neilands, Jessica
    et al.
    Malmö högskola, Faculty of Odontology (OD).
    Wilkins, Joanna C
    Beighton, David
    Wrzesinski, Krzysztof
    Fey, Stephen J
    Mose-Larsen, Peter
    Hamilton, Ian R
    Svensäter, Gunnel
    Malmö högskola, Faculty of Odontology (OD).
    Effect of acid shock on protein expression by biofilm cells of Streptococcus mutans2003In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 227, no 2, p. 287-293Article in journal (Refereed)
    Abstract [en]

    Streptococcus mutans is a component of the dental plaque biofilm and a major causal agent of dental caries. Log-phase cells of the organism are known to induce an acid tolerance response (ATR) at sub-lethal pH values ( approximately 5.5) that enhances survival at lower pH values such as those encountered in caries lesions. In this study, we have employed a rod biofilm chemostat system to demonstrate that, while planktonic cells induced a strong ATR at pH 5.5, biofilm cells were inherently more acid resistant than such cells in spite of a negli-gible induction of an ATR. Since these results suggested that surface growth itself triggered an ATR in biofilm cells, we were interested in comparing the effects of a pH change from 7.5 to 5.5 on protein syn-thesis by the two cell types. For this, cells were pulse labeled with [(14)C]-amino acids following the pH change to pH 5.5, the proteins extracted and separated by two-dimensional (2D) electrophoresis fol-lowed by autoradiography and computer-assisted image analysis. A comparison between the cells incubated at pH 5.5 and the control biofilm cells revealed 23 novel proteins that were absent in the control cells, and 126 proteins with an altered relative rate of synthesis. While the number of changes in protein expression in the biofilm cells was within the same range as for planktonic cells, the magnitude of their change was significantly less in biofilm cells, supporting the observa-tion that acidification of biofilm cells induced a negligible ATR. Mass spectrometry and computer-assisted protein sequence analysis revealed that ATR induction of the planktonic cells resulted in the downregula-tion of glycolytic enzymes presumably to limit cellular damage by the acidification of the external environment. On the other hand, the gly-colytic enzymes in control biofilm cells were significantly less down-regulated and key enzymes, such as lactate dehydrogenase were upregulated during pH 5.5 incubation, suggesting that the enhanced acid resistance of biofilm cells is associated with the maintenance of pH homeostasis by H+ extrusion via membrane ATPase and increased lactate efflux.

  • 41. Nylander, Åsa
    et al.
    Svensäter, Gunnel
    Malmö högskola, Faculty of Odontology (OD).
    Senadheera, Dilani B.
    Cvitkovitch, Dennis G.
    Davies, Julia R
    Malmö högskola, Faculty of Odontology (OD).
    Persson, Karina
    Structural and functional analysis of the N-terminal domain of the Streptococcus gordonii adhesin Sgo07072013In: PLOS ONE, E-ISSN 1932-6203, Vol. 8, no 5, article id e63768Article in journal (Refereed)
    Abstract [en]

    The commensal Streptococcus gordonii expresses numerous surface adhesins with which it interacts with other microorganisms, host cells and salivary proteins to initiate dental plaque formation. However, this Gram-positive bacterium can also spread to non-oral sites such as the heart valves and cause infective endocarditis. One of its surface adhesins, Sgo0707, is a large protein composed of a non-repetitive N-terminal region followed by several C-terminal repeat domains and a cell wall sorting motif. Here we present the crystal structure of the Sgo0707 N-terminal domains, refined to 2.1 Å resolution. The model consists of two domains, N1 and N2. The largest domain, N1, comprises a putative binding cleft with a single cysteine located in its centre and exhibits an unexpected structural similarity to the variable domains of the streptococcal Antigen I/II adhesins. The N2-domain has an IgG-like fold commonly found among Gram-positive surface adhesins. Binding studies performed on S. gordonii wild-type and a Sgo0707 deficient mutant show that the Sgo0707 adhesin is involved in binding to type-1 collagen and to oral keratinocytes.

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  • 42. Ohlin, Acke
    et al.
    Mattsson, Emma
    Mörgelin, Matthias
    Davies, Julia R
    Malmö University, Faculty of Odontology (OD).
    Svensäter, Gunnel
    Malmö University, Faculty of Odontology (OD).
    Corvec, Stephane
    Tengvall, Pentti
    Riesbeck, Kristian
    Titanium granules pre-treated with hydrogen peroxide inhibit growth of bacteria associated with post-operative infections in spine surgery2018In: European spine journal, ISSN 0940-6719, E-ISSN 1432-0932, Vol. 27, no 10, p. 2463-2468Article in journal (Refereed)
    Abstract [en]

    PURPOSE: Post-operative infections are relatively common after posterior spine surgery, and there are several observations reflecting different infection complications related to various metals implanted. Here, we selected an array of different bacterial species that are often found in infections associated with orthopaedic implants and tested for inhibition by hydrogen peroxide-treated titanium (Ti-peroxy). METHODS: To study the possibility of using Ti-peroxy as an antimicrobial prophylaxis, we developed a protocol for standardized susceptibility testing of bacteria. RESULTS: Importantly, we found that the resulting Ti-peroxy was highly antimicrobial against all aerobic species tested, among others, Staphylococcus aureus and Pseudomonas aeruginosa. Proteus mirabilis was slightly more resistant than, for example, Klebsiella pneumoniae and enterococci. In contrast, anaerobic bacteria Cutibacterium acnes and Parvimonas micra were equally susceptible compared to staphylococci. CONCLUSIONS: Our findings suggest that the Ti-peroxy is a promising perioperative antimicrobial strategy that may be highly effective for prevention of post-operative infections. We therefore suggest application of hydrogen peroxide to implants prior to implantation. These slides can be retrieved under Electronic supplementary material.

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  • 43.
    Pihl, M
    et al.
    Malmö högskola, Faculty of Odontology (OD).
    Arnebrant, Thomas
    Malmö högskola, Faculty of Health and Society (HS).
    Kinnby, B
    Malmö högskola, Faculty of Odontology (OD).
    Svensäter, Gunnel
    Malmö högskola, Faculty of Odontology (OD).
    Peritoneal dialysis catheter biofilms.2008Conference paper (Other academic)
  • 44.
    Pihl, M
    et al.
    Malmö högskola, Faculty of Odontology (OD).
    Svensäter, G
    Malmö högskola, Faculty of Odontology (OD).
    Biofilms on catheters2007Conference paper (Other (popular science, discussion, etc.))
  • 45.
    Pihl, Maria
    et al.
    Malmö högskola, Faculty of Odontology (OD).
    Arvidsson, Anna
    Skepö, Marie
    Nilsson, Martin
    Givskov, Michael
    Tolker-Nielsen, Tim
    Svensäter, Gunnel
    Malmö högskola, Faculty of Odontology (OD).
    Davies, Julia R
    Malmö högskola, Faculty of Odontology (OD).
    Biofilm formation by Staphylococcus epidermidis on peritoneal dialysis catheters and the effects of extracellular products from Pseudomonas aeruginosa.2013In: Pathogens and Disease, E-ISSN 2049-632X, Vol. 67, no 3, p. 192-198Article in journal (Refereed)
    Abstract [en]

    Biofilm formation by Staphylococcus epidermidis is a cause of infections related to peritoneal dialysis (PD). We have used a PD catheter flow-cell model in combination with confocal scanning laser microscopy and atomic force microscopy to study biofilm formation by S. epidermidis. Adherence to serum-coated catheters was four times greater than to uncoated ones, suggesting that S. epidermidis binds to serum proteins on the catheter surface. Pseudomonas aeruginosa biofilm supernatant interfered with the formation of a serum protein coat thereby reducing the capacity for biofilm formation in S. epidermidis. Supernatants from ΔpelA, ΔpslBCD and ΔrhlAB strains of P. aeruginosa showed no differences from the wild-type supernatant indicating that the effect on serum coat formation was not due to rhamnolipids or the PelA and PslBCD polysaccharides. Supernatant from P. aeruginosa also dispersed established S. epidermidis biofilms. Supernatants lacking PelA or PslBCD showed no differences from the wild type but that from a ΔrhlAB strain, showed reduced, but not abolished, capacity for dispersal. This suggests that rhamnolipids are involved but not wholly responsible for the effect. Thus, supernatants from P. aeruginosa contain promising substances for the prevention and treatment of biofilm infections, although further work is required to identity more active components.

  • 46.
    Pihl, Maria
    et al.
    Malmö högskola, Faculty of Odontology (OD).
    Chávez de Paz, Luis Eduardo
    Schmidtchen, Artur
    Svensäter, Gunnel
    Malmö högskola, Faculty of Odontology (OD).
    Davies, Julia
    Malmö högskola, Faculty of Odontology (OD).
    Effects of clinical isolates of Pseudomonas aeruginosa on Staphylococcus epidermidis biofilm formation2010In: FEMS Immunology and Medical Microbiology, ISSN 0928-8244, E-ISSN 1574-695X, Vol. 59, no 3, p. 504-512Article in journal (Refereed)
    Abstract [en]

    Pseudomonas aeruginosa is often found in chronic infections, including cystic fibrosis lung infections and those related to chronic wounds and venous ulcers. At the latter sites, P. aeruginosa can be isolated together with Staphylococcus epidermidis, and we have therefore explored the effect of clinical isolates and laboratory strains of P. aeruginosa strains on colonisation by S. epidermidis in dual-species biofilms. Biofilm formation was assayed using 16S rRNA fluorescence in situ hybridization and confocal laser scanning microscopy. Amongst the six P. aeruginosa strains tested, one particular strain, denoted 14:2, exerted a significant inhibitory effect and even after six hours, S. epidermidis levels in dual-species biofilms were reduced by more than 85% compared to biofilms without P. aeruginosa. Interestingly strain 14:2 was found to be negative for classical virulence determinants including pyocyanin, elastase and alkaline protease. Therefore, we suggest that less virulent phenotypes of P. aeruginosa which may develop over time in chronic infections, could counteract colonisation by S. epidermidis, ensuring persistence and dominance by P. aeruginosa in the host micro-habitat. Further studies are required to explain the inhibitory effect on S. epidermidis, although, extracellular polysaccharides produced by P. aeruginosa might play a role in this phenomenon.

  • 47.
    Pihl, Maria
    et al.
    Malmö högskola, Faculty of Odontology (OD).
    Davies, Julia
    Malmö högskola, Faculty of Odontology (OD).
    Chávez de Paz, Luis Eduardo
    Svensäter, Gunnel
    Malmö högskola, Faculty of Odontology (OD).
    Differential effects of Pseudomonas aeruginosa on biofilm formation by different strains of Staphylococcus epidermidis.2010In: FEMS Immunology and Medical Microbiology, ISSN 0928-8244, E-ISSN 1574-695X, Vol. 46, no 3, p. 439-446Article in journal (Refereed)
    Abstract [en]

    Pseudomonas aeruginosa and Staphylococcus epidermidis are common opportunistic pathogens associated with medical device-related biofilm infections. 16S rRNA fluorescence in situ hybridization and confocal scanning laser microscopy were used to study these two bacteria in dual-species biofilms. We have shown that fresh isolates of S. epidermidis form biofilms more avidly on polymer surfaces than laboratory strains. In dual-species biofilms the presence of P. aeruginosa reduced biofilm formation by S. epidermidis, although the different clinical isolates differed in their susceptibility to this effect. The most resistant fresh isolate coexisted with P. aeruginosa for up to 18 hours and was also resistant to the effects of culture supernatant from P. aeruginosa biofilms which caused dispersal of other S. epidermidis strains from established biofilms. Thus, different strains of S. epidermidis differed in their capacity to withstand the action of P. aeruginosa, with some fresh isolates being better equipped than laboratory strains to coexist in biofilms with P. aeruginosa. Our data suggest that where S. epidermidis and P. aeruginosa are present on abiotic surfaces such as medical devices, S. epidermidis biofilm-formation can be inhibited by P. aeruginosa through two mechanisms: disruption by extracellular products, possibly polysaccharides, and, in the later stages, by cell lysis.

  • 48.
    Pihl, Maria
    et al.
    Malmö högskola, Faculty of Odontology (OD).
    Davies, Julia
    Malmö högskola, Faculty of Odontology (OD).
    Johansson, Ann-Cathrine
    Svensäter, Gunnel
    Malmö högskola, Faculty of Odontology (OD).
    Bacteria on catheters in patients undergoing peritoneal dialysis2013In: Peritoneal Dialysis International, ISSN 0896-8608, E-ISSN 1718-4304, Vol. 33, no 1, p. 51-59Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Peritonitis is the leading cause of morbidity for peritoneal dialysis (PD) patients, and microbial biofilms have previously been identified on catheters from infected patients. However, few studies of catheters from patients without clinical signs of infection have been undertaken. The aim of the present study was to investigate the extent to which bacteria are present on catheters from PD patients with no symptoms of infection. ♢ METHODS: Microbiologic culturing under aerobic and anaerobic conditions and confocal laser scanning microscopy were used to determine the distribution of bacteria on PD catheters from 15 patients without clinical signs of infection and on catheters from 2 infected patients. The 16S rRNA gene sequencing technique was used to identify cultured bacteria. ♢ RESULTS: Bacteria were detected on 12 of the 15 catheters from patients without signs of infection and on the 2 catheters from infected patients. Single-species and mixed-microbial communities containing up to 5 species were present on both the inside and the outside along the whole length of the colonized catheters. The bacterial species most commonly found were the skin commensals Staphylococcus epidermidis and Propionibacterium acnes, followed by S. warneri and S. lugdunensis. The strains of these micro-organisms, particularly those of S. epidermidis, varied in phenotype with respect to their tolerance of the major classes of antibiotics. CONCLUSIONS: Bacteria were common on catheters from patients without symptoms of infection. Up to 4 different bacterial species were found in close association and may represent a risk factor for the future development of peritonitis in patients hosting such micro-organisms.

  • 49. Rabe, Per
    et al.
    Twetman, Svante
    Kinnby, Bertil
    Malmö högskola, Faculty of Odontology (OD). Malmö högskola, Biofilms Research Center for Biointerfaces.
    Svensäter, Gunnel
    Malmö högskola, Faculty of Odontology (OD). Malmö högskola, Biofilms Research Center for Biointerfaces.
    Davies, Julia R
    Malmö högskola, Faculty of Odontology (OD). Malmö högskola, Biofilms Research Center for Biointerfaces.
    Effect of fluoride and chlorhexidine digluconate mouthrinses on plaque biofilms2015In: The Open Dentistry Journal, E-ISSN 1874-2106, Vol. 9, p. 106-111Article in journal (Refereed)
    Abstract [en]

    Objective. To develop a model in which to investigate the architecture of plaque biofilms formed on enamel surfaces in vivo and to compare the effects of anti-microbial agents of relevance for caries on biofilm vitality. Materials and Methodology : Enamel discs mounted on healing abutments in the pre-molar region were worn by three subjects for 7 days. Control discs were removed before subjects rinsed with 0.1% chlorhexidine digluconate (CHX) or 0.2% sodium fluoride (NaF) for 1 minute. Biofilms were stained with Baclight Live/Dead and z-stacks of images created using confocal scanning laser micoscopy. The levels of vital and dead/damaged bacteria in the biofilms, assessed as the proportion of green and red pixels respectively, were analysed using ImageTrak(®) software. Results : The subjects showed individual differences in biofilm architecture. The thickness of the biofilms varied from 28-96µm although cell density was always the greatest in the middle layers. In control biofilms, the overall levels of vitality were high (71-98%) especially in the area closest to the enamel interface. Rinsing with either CHX or NaF caused a similar reduction in overall vitality. CHX exerted an effect throughout the biofilm, particularly on the surface of cell clusters whereas NaF caused cell damage/death mainly in the middle to lower biofilm layers. Conclusion : We describe a model that allows the formation of mature, undisturbed oral biofilms on human enamel surfaces in vivo and show that CHX and NaF have a similar effect on overall vitality but differ in their sites of action.

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  • 50.
    Robertsson, Carolina
    et al.
    Malmö University, Faculty of Odontology (OD).
    Svensäter, Gunnel
    Malmö University, Faculty of Odontology (OD).
    Blum, Zoltan
    Malmö University, Faculty of Health and Society (HS), Department of Biomedical Science (BMV).
    Jakobsson, Magnus E.
    Lund Univ, Dept Immunotechnol, Lund, Sweden..
    Wickström, Claes
    Malmö University, Faculty of Odontology (OD).
    Proteomic response in Streptococcus gordonii DL1 biofilm cells during attachment to salivary MUC5B2021In: Journal of Oral Microbiology, ISSN 2000-2297, E-ISSN 2000-2297, Vol. 13, no 1, article id 1967636Article in journal (Refereed)
    Abstract [en]

    Background Salivary mucin MUC5B seems to promote biodiversity in dental biofilms, and thereby oral health, for example, by inducing synergistic 'mucolytic' activities in a variety of microbial species that need to cooperate for the release of nutrients from the complex glycoprotein. Knowledge of how early colonizers interact with host salivary proteins is integral to better understand the maturation of putatively harmful oral biofilms and could provide key insights into biofilm physiology. Methods The early oral colonizer Streptococcus gordonii DL1 was grown planktonically and in biofilm flow cell systems with uncoated, MUC5B or low-density salivary protein (LDP) coated surfaces. Bacterial cell proteins were extracted and analyzed using a quantitative mass spectrometry-based workflow, and differentially expressed proteins were identified. Results and conclusions Overall, the proteomic profiles of S. gordonii DL1 were similar across conditions. Six novel biofilm cell proteins and three planktonic proteins absent in all biofilm cultures were identified. These differences may provide insights into mechanisms for adaptation to biofilm growth in this species. Salivary MUC5B also elicited specific responses in the biofilm cell proteome. These regulations may represent mechanisms by which this mucin could promote colonization of the commensal S. gordonii in oral biofilms.

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