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Acid Tolerance of Streptococcus Mutans Biofilms
Malmö högskola, Faculty of Odontology (OD).
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [sv]

Mikrobiologisk forskning har till stor del bedrivits genom att man odlat bakterier i näringslösningar och på agarplattor och sedan studera bakterierna på olika sätt. Detta sätt att odla bakterier överensstämmer inte särskilt bra med hur bakterier växer i verkligenheten. I naturen växer bakterier fastsittande på olika typer av ytor (tandemalj, epitel, stenar, plastytor etc.) i s.k. biofilmer. Det har visat sig att bakterier i biofilmer inte alls beter sig som bakterier som växer fritt i en näringslösning (planktonisk form). Biofilmbak-terier skiljer sig från planktoniska bakterier genom att de är mer resistenta mot antibiotika och biocider. De är mer stresståliga, de växer långsammare och de har ett annorlunda gen- och proteinuttryck. Sammantaget har biofilmbakterier andra egenskaper. På grund av detta, har fokus inom mikrobiologisk forskning det senaste decenniet riktats mot att studera biofilmer för att komma närmare bakteriernas verklighet. I munnen växer bakterierna i biofilmer. Bakterierna i munnen är nödvändiga för att god munhälsa men de är också orsak till två av världens vanligaste sjukdomar; karies och parodontit. Streptococcus mutans tillhör den normala munfloran men medverkar också till att karies utvecklas. Karies uppstår när det blir en obalans i det orala ekosystemet. När man äter sjunker pH i munnen pga att bakterierna i plack producerar syror när de bryter ner kolhydrater. Om man äter ofta kommer pH i plack att vara surt under längre perioder, vilket leder till en selektion av bakterier som är stresståliga och kan anpassa sig till den sura miljön. Detta resulterar i att en syratolerant mikroflora dominerar i placket. Dessa bakterier har i sin tur kapacitet att göra det ännu surare i placket under långa perioder. Detta kan leda till demineralisering av emaljen och upp-komst av karies. S. mutans anpassar sig till en sur miljö genom att inducera en syratoleransrespons (ATR) vid subletala pH-värden. Syftet med denna avhandling var att studera hur S. mutans beter sig när den växer i biofilmer och ta reda på mer om syratoleransresponsen i S. mutans biofilmer. Resultaten från avhandlingen visar att S. mutans bakterier som vuxit i en biofilm i tre dagar har ett annorlunda proteinuttryck än planktoniska bakterier och att förändringar i proteinuttryck sker redan efter två timmar av ytkontakt. Proteinuttrycket ändras efter hand som biofilmen växer och blir äldre. Biofilmbakterierna visade sig också vara många gånger mer syratoleranta än de planktoniska bakterierna och ytkontakt i sig var viktigt för den ökade syratole-ransen. Äldre biofilmer (3 dagar) var mer syratoleranta än unga biofilmer (3 timmar) vilket kan bero på att de utsatts för pH chocker under växt i biofilmen och därmed inducerat en ATR. Unga biofilmer inducerade en ATR när de utsattes för pH 5.5 i tre timmar och denna ATR kunde inhiberas genom tillsats av fluor. Det visade sig också att olika stammar av S. mutans var olika syra-toleranta och hade olika förmåga att inducera en ATR. Detta kan i praktiken innebära att människor med bakterier som är mer benägna att inducera en ATR löper större risk att utveckla karies. Genom att förhindra induceringen av ATR skulle man alltså kunna förhindra utvecklingen av karies.

Abstract [en]

In nature, bacteria are organized in surface-associated biofilm communities. Dental plaque is one of the most studied biofilms and harbours many different species including the star of this thesis Streptococcus mutans. S. mutans has been implicated in the aetiology of caries and has been studied extensively but most research has been carried out on liquid cultures (planktonic cells). Biofilm cells have been shown to differ in many ways from planktonic cells when it comes to protein and gene expression, growth rate, and stress tolerance. The overall aim of this thesis was to study S. mutans grown in biofilms of different ages (2 hours – 3 days) and to study the acid tolerance response (ATR) in biofilm cells. Biofilms were grown in chemostats, on glass slides and in flow-cell systems. To evaluate protein expression in cells, 14C-labelled proteins were separated by 2-dimensional gel eletrophoresis (2-DE) followed by autoradiography and computer-assisted image analysis. Acid killing experiments were conducted by exposing the cells to pH 3.0 for two hours and then counting the number of survivors by plating on blood agar. ATR-experiments were carried out in a similar way to the acid killing experiments except that the cells were first exposed to pH 5.5 for 2-3 hours and then exposed to pH 3.0-3.5 for 30 minutes to two hours. Surviving cells were counted by plating on blood agar but also examined using LIVE/DEAD® BacLightTM viability stain. Inhibition of the cell’s ability to induce an ATR was tested by exposing the cells to 0.5 M fluoride. Mature biofilm cells (3 days old) showed a different protein expression pattern compared to corresponding planktonic cells with the most prominent difference being the decreased expression of enzymes involved in carbohydrate catabolism. Contact with a surface induced changes in protein expression as quickly as two hours after surface adhesion. In these cells however, the proteins involved in carbohydrate catabolism were enhanced. Surface contact and growth in a biofilm lead to increased acid tolerance. In newly formed biofilm cells of S. mutans H7, 5% of the cells survived an acid shock of pH 3.0 for two hours compared to only 0.0004% of the planktonic cells. The mature biofilm cells were even more acid tolerant with 41.5% survivors after two hours at pH 3.0. Mature biofilms induced only a negligible ATR that did not enhance their survival significantly. Still, 20% of the proteins analysed had an altered expression after exposure to pH 5.5. In newly formed biofilm cells the ATR lead to decreased membrane damage at low pH as visualised by LIVE/DEAD® BacLightTM staining. Different strains showed different abilities to induce an ATR and it was also shown that this ATR could be inhibited by exposure to 0.5 M fluo-ride during the pH 5.5-adaptation period. The results of the thesis show that biofilm cells of S. mutans differ from their planktonic counterparts and the strains of S. mutans tested behave somewhat differently.

Place, publisher, year, edition, pages
Malmö University , 2007.
Keywords [en]
Acid tolerance, Streptococcus mutans, Biofilms, Acid stress, Fluoride
National Category
Dentistry
Identifiers
URN: urn:nbn:se:mau:diva-7704Local ID: 4702ISBN: 91-7104-295-4 (print)OAI: oai:DiVA.org:mau-7704DiVA, id: diva2:1404644
Note

Note: The papers are not included in the fulltext online

Available from: 2020-02-28 Created: 2020-02-28 Last updated: 2024-02-29Bibliographically approved
List of papers
1. Protein expression by planktonic and biofilm cells of Streptococcus mutans
Open this publication in new window or tab >>Protein expression by planktonic and biofilm cells of Streptococcus mutans
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2001 (English)In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 205, no 1, p. 139-146Article in journal (Refereed) Published
Abstract [en]

Streptococcus mutans, a major causal agent of dental caries, functions in nature as a component of a biofilm on teeth (dental plaque) and yet very little information is available on the physiology of the organism in such surface-associated communities. As a consequence, we undertook to examine the synthesis of proteins by planktonic and biofilm cells growing in a biofilm chemostat at pH 7.5 at a dilution rate of 0.1 h(-1) (mean generation time=7 h). Cells were incubated with (14)C-labelled amino acids, the proteins extracted and separated by two-dimensional electrophoresis followed by autoradiography and computer-assisted image analysis. Of 694 proteins analysed, 57 proteins were enhanced 1.3-fold or greater in biofilm cells compared to planktonic cells with 13 only expressed in sessile cells. Diminished protein expression was observed with 78 proteins, nine of which were not expressed in biofilm cells. The identification of enhanced and diminished proteins by mass spectrometry and computer-assisted protein sequence analysis revealed that, in general, glycolytic enzymes involved in acid formation were repressed in biofilm cells, while biosynthetic processes were enhanced. The results show that biofilm cells possess novel proteins, of as yet unknown function, that are not present in planktonic cells.

Place, publisher, year, edition, pages
Elsevier, 2001
Keywords
Animals, Bacterial Proteins, Biofilms, Culture Media, Dental Caries, Electrophoresis, Gel, Two-Dimensional, Humans, Image Processing, Computer-Assisted, Plankton, Streptococcus mutans
National Category
Dentistry
Identifiers
urn:nbn:se:mau:diva-15570 (URN)10.1111/j.1574-6968.2001.tb10937.x (DOI)000172471900021 ()11728728 (PubMedID)28589 (Local ID)28589 (Archive number)28589 (OAI)
Available from: 2020-03-30 Created: 2020-03-30 Last updated: 2024-05-23Bibliographically approved
2. Effect of acid shock on protein expression by biofilm cells of Streptococcus mutans
Open this publication in new window or tab >>Effect of acid shock on protein expression by biofilm cells of Streptococcus mutans
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2003 (English)In: 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.

Place, publisher, year, edition, pages
Blackwell, Oxford, 2003
Keywords
Cardiovascular disease, Bacteria, Micrococcales, Streptococcaceae, Shock, Protein synthesis, Biofilm, Protein, Streptococcus mutans
National Category
Dentistry
Identifiers
urn:nbn:se:mau:diva-6684 (URN)10.1016/S0378-1097(03)00693-1 (DOI)000186390900019 ()14592721 (PubMedID)2-s2.0-0142195881 (Scopus ID)7865 (Local ID)7865 (Archive number)7865 (OAI)
Available from: 2020-02-28 Created: 2020-02-28 Last updated: 2024-02-05Bibliographically approved
3. Protein expression by Streptococcus mutans during initial stage of biofilm formation
Open this publication in new window or tab >>Protein expression by Streptococcus mutans during initial stage of biofilm formation
2004 (English)In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 70, no 6, p. 3736-3741Article in journal (Refereed)
Abstract [en]

Cells growing on surfaces in biofilms exhibit properties distinct from those of planktonic cells, such as increased resistance to biocides and antimicrobial agents. In spite of increased interest in biofilms, very little is known about alterations in cell physiology that occur upon attachment of cells to a surface. In this study we have investigated the changes induced in the protein synthesis by contact of Streptococcus mutans with a surface. Log-phase planktonic cells of S. mutans were allowed to adhere to a glass slide for 2 h in the presence of a (14)C-amino acid mixture. Nonadhered cells were washed away, and the adhered cells were removed by sonication. The proteins were extracted from the nonadhered planktonic and the adhered biofilm cells and separated by two-dimensional gel electrophoresis followed by autoradiography and image analysis. Image analysis revealed that the relative rate of synthesis of 25 proteins was enhanced and that of 8 proteins was diminished > or =1.3-fold in the biofilm cells. Proteins of interest were identified by mass spectrometry and computer-assisted protein sequence analysis. Of the 33 proteins associated with the adhesion response, all but 10 were identified by mass spectrometry and peptide mass fingerprinting. The most prominent change in adhered cells was the increase in relative synthesis of enzymes involved in carbohydrate catabolism indicating that a redirection in protein synthesis towards energy generation is an early response to contact with and adhesion to a surface.

Place, publisher, year, edition, pages
American Society for Microbiology, 2004
Keywords
Bacterial Adhesion, Bacterial Proteins, Biofilms, Electrophoresis, Gel, Two-Dimensional, Glass, Humans, Image Processing, Computer-Assisted, Plankton, Streptococcus mutans
National Category
Dentistry
Identifiers
urn:nbn:se:mau:diva-6590 (URN)10.1128/AEM.70.6.3736-3741.2004 (DOI)000221981100071 ()15184181 (PubMedID)2-s2.0-2942528670 (Scopus ID)28593 (Local ID)28593 (Archive number)28593 (OAI)
Available from: 2020-02-28 Created: 2020-02-28 Last updated: 2024-02-05Bibliographically approved
4. Acid Tolerance of Biofilm Cells of Streptococcus mutans
Open this publication in new window or tab >>Acid Tolerance of Biofilm Cells of Streptococcus mutans
2007 (English)In: 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.

Place, publisher, year, edition, pages
American Society for Microbiology, 2007
Keywords
Fluoride, Biofilms, Streptococcus mutans, Acid tolerance
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:mau:diva-6945 (URN)10.1128/AEM.01049-07 (DOI)000249246700030 ()17630302 (PubMedID)2-s2.0-34548472841 (Scopus ID)4735 (Local ID)4735 (Archive number)4735 (OAI)
Available from: 2020-02-28 Created: 2020-02-28 Last updated: 2024-02-05Bibliographically approved

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