The effect of Parvimonas
micra on gingipain activity in
different strains of
Porphyromonas gingivalis
Elin Langeveld
Charlotte Åhman-Persson
Supervisor: Jessica Neilands
Malmö University
Master in Odontology
Faculty of Odontology
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ABSTRACT
Aim: The aim of this study was to experimentally determine if Parvimonas micra increase
growth and gingipain activity in different strains of Porphyromonas gingivalis. An increase in gingipain activity could indicate that P. micra induces pathogenic expression of P. gingivalis.
Material and method: Five strains of P. gingivalis 1A, 16A, 50A, 33F and W50 was
cultivated together with P. micra while control samples contained only P. gingivalis. Activity of arginine gingipain (Rgp) and lysine gingipain (Kgp) was measured using Bikkam-16 respectively Bikkam-14. Bacterial growth was determining by measuring the optical density. Results: The experiments show an increased growth of some strains (1A, 16A and 50A) of P. gingivalis when grown with P. micra, one showed moderate growth (33F) and one showed no growth (W50).
The gingipain activity (Rgp) increased in P. gingivalis strains (1A and 16A) in the presence of P. micra but did not increase in other strains (W50, 33F and 50A)
The gingipain activity (Kgp) increased in one P. gingivalis strains (1A) when mixed with P. micra and did not increase in others (16A, W50, 33F and 50A).
Conclusion: The results from this study indicates that P. micra effect the growth and
gingipain activity in certain strains of P. gingivalis but not all.
SAMMANFATTNING
Syfte: Syftet med denna studie var att experimentellt bestämma om Parvimonas micra ökar
tillväxt och gingipainaktiviteten hos olika stammar av Porphyromonas gingivalis. En ökning av gingipainaktiviteten indikerar att P. micra skulle kunna öka virulensen i P. gingivalis.
Material och metod: Fem stammar av P. gingivalis 1A, 16A, 50A, 33F och W50 odlades
med P. micra och jämfördes med kontrollen som endast innehöll P. gingivalis. Arginin gingipain (Rgp) och lysin gingipain (Kgp) aktivitet mättes med hjälp av Bikkam-16 respektive Bikkam-14. Bakterietillväxt mättes genom att mäta optisk densitet.
Resultat: Experimenten visade en ökad tillväxt av stammarna 1A, 16A och 50A när de
odlades med P. micra, 33F visade måttlig tillväxt och W50 ingen tillväxt.
Rgp aktiviteten ökade i vissa P. gingivalis-stammar (1A och 16A) i närvaro P. micra men ökade inte i andra (W50, 33F och 50A)
Kgp aktiviteten ökade i P. gingivalis lA i närvaro av P. micra men ingen ökning skedde i de andra stammarna (16A, W50, 33F och 50A).
Slutsats: Resultaten från denna studie visade att P. micra påverkar tillväxten och
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INDEX
INTRODUCTION ... 4
Aim ... 8
MATERIAL AND METHODS ... 9
Material ... 9 Method ... 9 RESULTS ... 10 DISCUSSION ... 13 Method discussion ... 13 Result discussion ... 14 Conclusion ... 15 REFERENCES ... 16
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1. INTRODUCTION
1.1 Periodontitis
The disease periodontitis develops due to a disturbed balance between the host's immune system and the biofilm accumulated around the gingival margin (1). This interaction will result in a breakdown of the periodontal tissue, starting with loss of connective tissue attachment and, when progressing, alveolar bone loss (1,2). Tissue breakdown is caused predominantly by the immune response of the host but is driven by the presence of
microorganisms (1) even so research shows that poor oral hygiene does not always lead to disease. From the individuals taking part in a longitudinal study 11% showed no periodontal lesions, even if no conventional oral hygiene was performed. 8% showed a periodontal disease with rapid progression. The main group 81% showed approximately 4 mm of tissue breakdown at the age of 35 (3). Epidemical studies give a prevalence of 10-15% regarding periodontitis resulting in a substantial tissue breakdown and tooth loss (4).
There are many risk factors that predisposes to periodontitis. Cigarette smoking is shown to be a major influenceable risk factor for getting periodontitis and contributing to its progress. Smokers show more tooth loss, higher rate of progression and less positive results after treat-ment both nonsurgical and surgical. A higher prevalence and severity of periodontitis is seen among patients with a long duration of diabetes mellitus especially when the diabetes is uncontrolled. Due to chronic inflammation periodontitis effects diabetes negatively by increasing insulin resistance. A low socioeconomic status has also shown correlation with periodontitis. There is also a high genetic factor that predisposes to periodontal disease and its progression (4). The presence of certain bacteria, such as Porphyromonas gingivalis,
Aggregatibacter actinomycetemcomitans and Tannerella forsythia, has also been considered a
risk factor for periodontitis. Although certain bacteria are more associated with disease their presence in the oral environment is not sufficient to develop disease (1).
In the healthy gingiva, polymorphonuclear neutrophils (PMNs) are present, they act as an important defense against microorganisms. When the lesion starts PMNs as well as macrophages and lymphocytes are increasing in number. The main lymphocyte in the established lesion is the B-cell. Immunologic cell invasion is triggered by lipoteichoic acid (LTA) and lipopolysaccharides (LPS) from the cell wall of microorganisms, recognized by toll-like receptors (TLR) found on host cells e.g. PMNs. The microorganisms form a biofilm on the tooth surface and are therefore continuously triggering the immune cells whose clearing attempts are failing. Production of complement factors C3a and C5a leads to an increased vascular permeability, which in turn leads to an increasing gingival crevicular fluid (GCF). Immune cells release cytokines such as tumor necrosis factor-alpha (TNF-α),
interleukin 1 (IL-1) and prostaglandin E2 (PGE2), cytokines increase the inflammation answer. Matrix metallo-proteinases (MMPs) are enzymes produced by fibroblasts, epithelial cells, macrophages and PMNs when triggered by cytokines; these are very important enzymes involved in tissue degradation. When connective tissue is degraded a periodontal pocket develops which will result in a junctional epithelium extending along the root surface
covering the areas of lost periodontal attachment. Bone loss takes place when osteoblasts via RANK ligand (RANKL) activate osteoclasts. The osteoclast starts resorbing bone when RANKL binds to the osteoclast receptor RANK. IL-1 takes part in regulation of RANKL production and thus affects bone resorption (1). Described inflammatory cell activity gives the symptoms found in periodontitis; bleeding on probing, gingival recession, pocket formation and tooth mobility (5).
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1.2 Keystone hypothesis
There have been different hypotheses regarding the origin of periodontitis. In the late 19th
century, when identification of bacteria was not possible, the “Non-specific plaque hypothesis” stated that it was the load of bacteria that led to disease in the oral cavity. Disease would develop if the number of bacteria would be higher than the capacity from e.g. saliva to neutralize them. The only way to prevent disease was to mechanically remove plaque (6). This hypothesis could not explain though why some individuals with poor oral hygiene and large amounts of plaque did not develop bone loss (1).
In the mid 20th century new techniques for culturing and identifying bacteria were developed
and made it possible to identify specific bacteria from patients suffering from periodontitis. This led, in the 1970’s, to a new hypothesis the “specific plaque hypothesis”. The belief that specific bacteria always would cause periodontitis, made researchers focus on the
development and search for antibiotics to treat oral disease e.g. chlorhexidine (6). However, it was later discovered that that these specific pathogens also could be detected in healthy individuals this gave rise to the “updated non-specific plaque hypothesis”, stating that all bacteria contributes to gingivitis/periodontitis but in different ways e.g. biofilm formation, evasion from the immune system and/or pro-inflammatory. This could explain why gingivitis always affected those with poor oral hygiene but would not always lead to periodontitis (6). Inspired by previous hypotheses, in 1994 professor Philip Marsh presented the “Ecological plaque hypothesis”, stating that disease occurs as a result of an ecological imbalance in the microbiota, (6,7). Specifically, the hypothesis states that changes in the oral environment will lead to the selection of pathogenic species and that all species with relevant traits can
contribute to the disease process. Changes in temperature, pH, nutrients, amount of
phagocytotic cells, oxygen etc. will promote growth of certain bacteria, already existing in small amount in the oral cavity, benefitting from these changes. These bacteria are by natural reasons more pathogenic, and due to a non-successful clearance inflammation will be induced (1,7).
Recently a new hypothesis has been proposed, regarding the pathogenesis of periodontitis, the “Keystone hypothesis”. In nature we can find different keystone species. What characterises them is not that they come in large amounts or dominance but that they substantially influence their ecosystem. If we could find a keystone microbe that affects the oral ecosystem either in maintaining host-microbe balance or by causing the imbalance leading to disease that would be of great interest. These findings could result in new treatment options and diagnostic tools. There is therefore an inquiry for more studies and research regarding identification of bacteria that have a keystone impact in the periodontal pocket (8).
A keystone-pathogen is a certain microbe that effects the microbial population of the
periodontal pocket to a dysbiotic state, giving an increasing quantity and altered composition of the microbiota leading to inflammation. The keystone pathogen therefore plays a crucial role in development of disease (8). P. gingivalis does not show a high capacity to induce inflammation; as a matter of fact the LPS of P. gingivalis has been shown to have a low ability to trigger cells of the immune system. P. gingivalis even reduces the inflammation due to the synthetization of Lipid A, an antagonist to TLR of immune cells (9). P. gingivalis also inhibits epithelial cells from secreting the proinflammatory chemokine interleukin-8, this phenomenon is called “local chemokine paralysis” (10). Even if P. gingivalis show these seemingly anti-inflammatory characteristics studies show that P. gingivalis, in a low abundance, inoculated in mice with commensal microbiota can induce periodontitis. The inoculation of P. gingivalis also led to a rise of the bacterial load and a change of species in
6 the biofilm. P. gingivalis was also inoculated in germ free mice where it did not induce bone loss, giving that P. gingivalis alone does not cause disease and that other bacteria from the oral microbiota are needed for disease development (11). P. gingivalis is shown, even in small amounts, to have a significant effect on its environment. P. gingivalis has therefore been proposed to be a “keystone” pathogen (8). P. gingivalis alters the microbial composition and load to a more dysbiotic one but exactly how P. gingivalis influences its environment is not entirely understood (1). It has been shown that P. gingivalis affects the bacterial environment indirectly by impairing the host defense e.g. inhibition of IL-8 with is needed for PMN
recruitment , but P. gingivalis probably also can affect the commensal microbiota directly (8). Gingipains are proteases secreted from P. gingivalis that initiate the inflammatory reactions leading to tissue breakdown. Recent studies show that Parvimonas micra can induce
gingipain activity from P. gingivalis (SUB1) thus P. micra seems to affect the keystone pathogen instead of the other way around (12).
1.3 Microbial interaction
Most bacteria are dependent on coexisting and interacting with other microbes. Bacteria interact both physically and chemically One of the most important microbial interaction is the formation of a biofilm (1,13). Most oral bacteria are dependent on the formation of a
polymicrobial biofilm to be able to survive and grow, this is also the case for the bacteria in the periodontal pocket. Bacteria adhering to a biofilm have been shown to alter protein and gene expressions, resulting in a different phenotype (1,13). Multiple phenotypes of one
species can be observed during formation of biofilms (14). In P. gingivalis 18% of its genome differs in expression when comparing planktonic and biofilm bound phenotypes. In a biofilm condition P. gingivalis produce less protective proteins, probably indicating a less stressful environment (15). The biofilm is not formed randomly but is well organized. Bacteria have tools to adhere to receptors in the biofilm such as adhesins or fimbriae (12, 14). In early biofilm formation genes promoting biofilm formation is upregulated (15). Biofilm formation gives aggregation of species that stimulates interactions like food chains, cell-cell signalling and gene transfer (16). The ability for bacteria to communicate via signalling substances such as peptides are key functions when forming biofilm. Loss of signalling substances results in diminished or altered biofilm formation (15). A biofilm with microbial balance, due to
interactions between bacteria, is of great importance for oral health. The balance can easily be disturbed by host factors such as poor oral hygiene, defects in the immune system,
xerostomia, inadequate diet etc. (1).
Bacterial interactions can be synergistic and mutualistic or antagonistic and competitive (5) An example of synergistic interaction is when early bacterial colonizers consume oxygen which creates microenvironments with low redox potential where anaerobic late colonizers can adhere to the biofilm (17). Nutritionally microorganisms benefit from each other when metabolic end-product from one bacterium can be consumed by others. Microorganisms also cooperate nutritionally when degrading molecules to consumable pieces. The normal
microbiota interacts antagonistic by hindering growth and multiplication of more pathogenic species. Interactions, such as competition of nutrients and secretion of antagonistic
substances, create a balanced environment beneficial for the host (1). Studies have shown that commensal oral bacteria such as Actinomyces naeslundii, Streptococcus intermedius and Streptococcus oralis etc. can reduce gingipain activity and inhibit growth of P. gingivalis. These bacteria can, by producing acid and/or hydrogen peroxide, reduce the growth of P. gingivalis leading to thatthese commensal bacteria in doing so, also may break the progress of periodontitis (18).
7 When periodontal disease evolves, there will be an environmental change in the periodontal pocket e.g. pH, nutrition etc. Pathogens involved in disease have to be able to withstand and probably profit from these changes. These pathogens also have to be able to induce tissue breakdown and evade the immune system. With this many different elements to take in consideration it is possible to assume that the bacteria work synergistically also when orchestrating disease, this phenomenon is called pathogenic synergy (13,19). A recent study has shown significant increase of P. gingivalis (SUB1) growth as well as increased gingipain activity when cultivated together with P. micra in serum. A reason for the increased gingipain activity was probably due to a signalling protein derived from P. micra concluding that P. micra when present has a synergistic effect on P. gingivalis growth and virulence expression (12). Other synergistic examples shown is that gingipains from P. gingivalis indicate induced growth of T. forsythia in multi-species biofilm and that gingipain affect how Treponema denticola cells are structured in the biofilm (20).
1.4 Porphyromonas gingivalis
P. gingivalis is an anaerobic, Gram-negative, proteolytic bacterium. It exhibits several virulence factors making it possible for the bacteria to evade cells from the immune system and trigger the immune response. It triggers the immune response either directly by inducing the release of cytokines, such as IL-1β and TNF-α or indirectly by increasing the bacterial load. An increased immune response leads to higher levels of GCF that contains hemin-derived iron and peptides, which is a nutritional source for P. gingivalis (6, 20).
One of the virulence factors helping P. gingivalis to avoid the immune cells is its ability to invade different host cells, when inside the cell it is still viable and can replicate (22). P. gingivalis possesses fimbriae (FimA) that bind to Beta1- integrin on the epithelial cell, the interaction results in internalization. As P. gingivalis is in the cell, it can cause an accelerated proliferation of the epithelial cell (22). FimA on P. gingivalis can bind with TLR on
macrophages, this gives a signal from the host cell, promoting P. gingivalis to be internalized (23). P. gingivalis is also able to produce a protective capsule, known as CPS, preventing it from getting phagocytized (1). P. gingivalis possesses LPS. LPS are recognized by TLR on the host cells and stimulates these cells to produce proinflammatory cytokines such as IL-1β 1α and TNF-α. The LPS from P. gingivalis does not stimulate cytokine production as strongly as LPS from other gram-negative bacteria. The LPS of P. gingivalis can stimulate TLR2, these TLR may even act inflammatory (22). As stated, P. gingivalis has
anti-inflammatory abilities giving altered composition of the microbiota. Changes in the oral microenvironment will affect P. gingivalis to express either pro-inflammatory or anti-inflammatory virulence factors (22).
1.4.1 P. gingivalis strain variation
Bacteria are classified into different species, species can be divided in different serotypes and there are different strains of the same species/serotypes. Strains of the same species can show different traits (2), giving some strains of P. gingivalis that are more virulent than others (18). A variation of fimA genotypes has been shown between strains of P. gingivalis. When
comparing strains from different fimA genotypes a distinct difference in biofilm structure is shown (24). P. gingivalis also show a strain dependent LPS variation. Results from in vitro studies have showed a variation of biologic response from macrophages to LPS when comparing LPS from two strains of P. gingivalis (25).
1.4.2
Gingipains
8 proteases that P. gingivalis secretes are the endopeptidases; gingipains (26). There are two main types of gingipains: arginine specific (RgpA and RgpB) and lysine specific (Kgp), referring to which amino acid it cleaves on the protein (20, 21).
Rgp gingipains can degrade cytokines, immunoglobulins and complement factors. The result from cleaving complement factor C5 is an increased recruitment of PMNs (22). The cellular invasion gives an increased level of granular proteinases probably leading to more tissue breakdown, and thus more nutrition for P. gingivalis. When gingipains cleave complement factor C3, a preopsonin, P. gingivalis is prevented from getting phagocytized. Gingipains Rgp and Kgp together increase the vascular permeability leading to more GCF, which is a nutrition source for P. gingivalis. Both Rgp and Kgp gingipain may in different ways affect the
coagulation process. Studies shows that Kgp can degrade fibrinogen and by doing so making the blood non-clottable thus increasing bleeding from the periodontal pocket (27).
1.5 Parvimonas micra
P. micra is an anaerobic gram-positive coccus found in the oral microbiota (2) and frequently found in polymicrobial infections. P. micra is associated with periodontal dysbiosis as well as endodontic infections. P. micra can be found in two different morphological colonies, rough and smooth. A difference in functions between the morphological colonies has not yet been reported. Further P. micra can be divided into at least five different genotypes but it has not yet been reported any differences in virulence factors. P. micra also shows different
phenotypes with different protease activity; collagenolytic activity, elastolytic activity and hemolytic activity (28).
1.7 Aim
The aim of this study was to experimentally determine if P. micra increases growth and gingipain activity on different strains of P. gingivalis. An increase in gingipain activity can indicate that P. micra induces the pathogenic expression of P. gingivalis.
1.8 Hypothesis
A previous study has shown that P. micra affects both growth and gingipain activity in P. gingivalis. Therefore, it is likely that P. micra also enhances growth and gingipain activity in other strains of P. gingivalis.
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2. MATERIAL AND METHOD
2.1 Strains and growth conditions
Five different strains of P. gingivalis were used in this study. P. gingivalis 1A, 16A, 50A, 33F were all isolates from periodontal pockets from individuals with periodontitis. Reference strain W50 was a kind gift from professor Torbjörn Bengtsson, Örebro University, Sweden. Morphologically smooth P. micra were isolated from a patient with periimplantitis. Bacteria was stored in milk at -80 °C until used.
Prior to the start of the experiment P. micra and P. gingivalis strains; 1A, 16A, 50A, 33F and W50, were cultivated on pre-reduced Brucella agar, in an anaerobic environment (10% H2,
5% CO2 in N2) for 7 days.
The bacterial colonies were then separately suspended in prereduced 10% heat-inactivated equine serum (Håtunalab, Bro, Sweden). In each suspension, absorbance was measured to an optical density between 0.1 and 0.15, at wavelength 600 nm (OD600).
For each strain of P. gingivalis equal volumes of P. micra suspension and P. gingivalis suspension were mixed. Control suspensions containing only P. gingivalis were also made for each strain. All suspensions (mixed and controls) were incubated anaerobically for 7 days after which the optical density (OD600) and gingipain activity was measured.
2.2 Gingipain activity measurements
After incubation for seven days the proteolytic activity was measured in mixed suspensions and in controls, by using fluorescent substrates BikKam-16 and BikKam-14 (PepScan Presto B.V., Lelystad, The Netherlands) which measure Arg-gingipain (Rgp) activity and Lys-gingipain (Kgp) activity respectively. 50 µL bacterial suspension was added to 2 µL BikKam-16 respectively Bikkam-14 in a 96-well plate (NUNC, ThermoFisher Scientific, Roskilde, Denmark) Fluorocent units were measured every minute for 10 minutes giving RFU/min in the fluorescence reader: BMG Clariostar plate reader (BMG Labtech, Offenburg, Germany) (using excitation and emission wavelengths of 488 and 538nm, respectively).
The experiments were performed in duplicates on at least three separate occasions and the mean value of all occasions for OD600 and mean value of duplicates for RFU/min was
calculated.
2.3 Statistical method
Statistical calculations were made with paired one-sided Student's t-test or Mann-Whitney U test, using Prism 5 for Mac OSX (Graphpad Inc, La Jolla, CA, USA).
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3. RESULTS
3. 1 The effect of P. micra on growth of P. gingivalis in serum
To investigate the effect of P. micra on growth of P. gingivalis in serum the different strains were incubated with or without P. micra. In the absence of P. micra no growth, e.i increase in optical density (OD600), was observed for any of the P. gingivalis strains included and in the
majority of the cases the optical density was less than at the start of the experiment indicating a reduction in the number of bacterial cells in the suspension. However, in samples with P. micra growth was observed for the P. gingivalis strains 1A, 16A and 50A. Moderate to no growth was observed for P. gingivalis strains 33F and W50 respectively. This data indicates that the ability of P. micra to stimulate growth varies between different strains of P.
gingivalis. (Fig 1)
Fig 1. Mean value of OD600 in mixed samples with the different strains of P. gingivalis and P. micra measured at the start of the experiment and after seven days of incubation.
3.2 The effect of P. micra on Rgp activity of P. gingivalis in serum
To investigate if P. micra had an effect on the Rgp activity of P. gingivalis in serum, the Rgp activity was measured using fluorescence substrate BikKam-16 in mixed suspensions
containing P. gingivalis and P. micra and controls containing only P. gingivalis.
Suspensions of P. gingivalis strains 1A or 16A coincubated with P. micra showed high Rgp activity with a mean value of 734 and 1859 RFU/min respectively. This was for both strains a three-fold increase compared to controls without P. micra. P. gingivalis strains W50, 33F and 50A coincubated with P. micra showed low to very low Rgp activity (50, 90 and 158
RFU/min). This meant a slight to no increase of Rgp activity in P. gingivalis strains W50 and 33F in mixed suspension compared with controls. A slight decrease of Rgp activity was seen in P. gingivalis strain 50A when cultivated with P. micra compared to controls. (Fig. 2)
W50+Pm 1A+Pm 50A+Pm 16A+Pm 33f+Pm 0 0,05 0,1 0,15 0,2 0,25 0,3 0,35 0,4 0,45 OD 60 0 Day 0 Day 7
11 This data suggests that P. micra’s ability to stimulate Rgp activity varies between different strains of P. gingivalis.
Fig 2. The difference in Rgp activity (RFU/min) between control suspensions and suspensions with P. gingivalis and P. micra. Each graph shows results from one strain and each line represents one separate experiment.
3.3 The effect of P. micra on Kgp activity of P. gingivalis in serum
To study if P. micra has an effect on the Kgp activity of P. gingivalis cultured in serum, the Kgp activity was measured using fluorescence substrate BikKam-14 (RFU/min) in mixed suspensions containing P. gingivalis and P. micra with controls containing only P. gingivalis. Suspensions of P. gingivalis strain 1A coincubated with P. micra showed high Kgp activity, (331 RFU/min). This was almost a 5-fold increase compared to controls without P. micra.
12 Suspensions of P. gingivalis 33F, 50A, W50 coincubated with P. micra showed very low Kgp activity (47, 77, 7 RFU/min respectively) and no increase in gingipain activity in the presence of P. micra. Unlike Rgp activity Kgp activity in suspensions of P. gingivalis 16 A and P. micra showed varying results and a mean activity of 217 RFU/min. (Fig. 3)
This data indicates that the ability of P. micra to stimulate Kgp activity varies between different strains of P. gingivalis.
Fig 3. The difference in Kgp activity (RFU/min) between control suspensions and suspensions with P. gingivalis and P. micra. Each graph showing results from respectively strain and each line represents a separate experiment.
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4. DISCUSSION
4.1 Method discussion
It has previously been demonstrated that P. micra affects the production of gingipains from P. gingivalis SUB 1 (11). In this study we examined the effect of P. micra on five other strains of P. gingivalis
Samples with P. gingivalis and P. micra coincubated in serum where compared to controls. This should give a valid view on how the strains of P. gingivalis differ; in the ability to grow in the presence of P. micra, as well as the effect of P. micra on gingipain activity. The result shows how these strains affect each other in the planktonic phase. As stated before P. gingi-valis changes genome expressions when in a biofilm. If our samples had been cultivated as a biofilm it is possible that the result could have been different and given a more realistic result as to how P. micra and P. gingivalis interact in a periodontal pocket. However, when it comes to Kgp gene expression,transcript level studies show no difference between P. gingivalis grown in a biofilm or in planktonic phase (20). This lack of difference has not been proven for Rgp gene expression; therefore, one can still not rule out that there could be differences when grown in biofilm.
Each experiment reports the mean from two duplicates. Using duplicates is a way to prevent operator differences in pipette and suspension techniques from giving an inaccurate result. Still if the measurements from the same two duplicates differ considerably the use of the mean from these two could be a misleading result why the use of triplicate in each run would possibly been better since any outliers could then have been omitted. However, in our tests the result from duplicates did not differ considerably. All experiments were in advance decided to be repeated at a minimum of three times on three separate occasions to give a more reliable result. A majority of the results were not statistically significant (p>0.05) although clear dif-ferences between test and control were seen. If the number of experiments had been increased the results would possibly have been significant. With some strains, P. gingivalis 1A and 50A, we performed more experiments (6 and 4 respectively). A decision was made to also present the result from these extra experiments, in doing so giving significant results for strain 1A supporting the hypothesis that more significant result might have been possible if experi-ment numbers were increased. The reason for added experiexperi-ments from strain 1A and 50A was due to repetition of experiments with all strains when initially the results from strains 16A, 33F and W50 unexpectedly did not show any effect of P. micra. The choice to present these extra results was made owing to the fact not knowing which results to exclude and still get a truthful view. The use of three experiments with duplicates is a standard method in microbio-logic research. Presenting the result as RFU/min gives a clear view on the level of gingipain activity in each sample.
Gingipain activity can be measured in different ways and traditionally BANA-test were used which detects trypsin-like activity in the samples. BANA- enzyme test is used for detection of P. gingivalis, T. denticola and T. forsythia that share the ability of hydrolyzing the trypsin substrate BANA. The test uses a synthetic peptidase and can only show the presence of these pathogens when there are at least 104 cells (29). In this study the fluorogenic substrate
Bik-Kam was used. BikBik-Kam is a peptide substrate that indicates gingipain activity. When cleaved by P. gingivalis enzymes BikKam becomes fluoroscent. BikKam-14 detects Kgp-activity and BikKam-16 detects Rgp-activity (30). The detection limit of BikKam14 and BikKam16 corre-sponds to the amount of gingipain produced by P. gingivalis in concentrations between 107
14 CFU/ml to 108 CFU/ml (30). This corresponds to the start concentration of the bacterial
sus-pension 0.1 OD600 (11). Bikkams have been shown to be specific to gingipain and P. micra
cannot break down Bikkam. Therefore, it is likely that the results from this study really reflect gingipain activity.
4.2 Result discussion
Results from these experiments show a difference between how P. micra affect the gingipain activity of the five strains of P. gingivalis. In experiments with P. gingivalis strains 1A and 16A an increase of Rgp activity was measured compared to e.g. W50 where no gingipain ac-tivity was measured. Similar studies have shown that P. micra, due to a protein it produces, has an increasing effect on gingipain activity of P. gingivalis (12). Studies also show that strains of P. gingivalis show strain dependent differences in genotype as well as in phenotype in FimA and LPS expressions (24,25) Therefore it is not unlikely that different P. gingivalis genotypes will react differently when cultivated together with P. micra.
Results from measuring optical density showed that P. gingivalis is dependent on P. micra to multiply. The different strains where affected differently by the presence of P. micra. Strains 1A and 16A showed an increased bacterial growth compared to strains W50 and 33F showing almost no growth. This indicates that strain 1A and P. micra has an increased synergistic in-teraction compared to P. micra and strain W50 and 33F. Strains W50 and 33F cultivated with P. micra showed very low growth, this can indicate a less developed synergistic interaction. P. gingivalis and P. micra are anerobic bacteria and do not tolerate an aerobic environment. However, both organisms can tolerate short exposure times to oxygen for example when be-ing handled outside the anaerobic chamber for suspension preparation in this case. Since P. gingivalis does not grow on its own in serum, no growth in the presence of P. micra is more difficult to interpret since it cannot be ruled out that no growth could be due to a too long ex-posure to oxygen while preparing the bacterial suspensions and the bacteria therefore being dead at the beginning of the experiment. Adding the bacteria to a rich growth medium could have been a good positive control but was not employed for any experiment in this study. A relationship was seen between a high optical density and a high gingipain activity. This re-lationship can be due to that gingipain activity increases as the number of P. gingivalis pro-ducing gingipains increases. It can also be due to that P. micra affect P. gingivalis to produce more gingipains. This has previously been shown for P. gingivalis SUB1 where the same amount of P. gingivalis gave rise to different gingipain activity in the presence of P. mi-cra.(12) Therefore the higher optical density could be due to growth of P. micra and not P. gingivalis. This thought is also supported by the keystone hypothesis that P. gingivalis affects the composition of the microbiota in its environment. To plate the bacterial suspension onto Brucella agar and count the number of different bacteria or identify the number of bacteria in the suspension using PCR would have given an answer to this but was not performed in this study. However, since an increase in optical density was connected to an increase in gingipain activity it is likely that the increase in optical density at least partly was due to increased growth of P. gingivalis. It has also been stated that increased Rgp activity is a good indicator as to measure growth of P. gingivalis (25). Previous studies have also shown that P. gingivalis cells had a 100-fold increase when cultivated seven days with P. micra, supporting the hy-pothesis that increased gingipain activity is a reflection of increased number of P. gingivalis. Whichever the case is, if it is P. micra or P. gingivalis growing or both, it is clear that P. mi-cra increases the virulence in some strains of P. gingivalis by increasing gingipain activity. This raises an interesting question; which bacterium is the keystone pathogen in the
patho-15 genic synergism between P. gingivalis and P. micra? The keystone pathogen is, as stated be-fore, affecting the microbiota of the periodontal pocket resulting in a dysbiotic state which in-creases the quantity and alters the composition of the normal microbiota. More research is needed to investigate if there are other keystone pathogens present in a periodontal pocket (1). These experiments only show how this specific strain of smooth P. micra affects these strains of P. gingivalis. In future studies it would be interesting to study different strains of P. micra both rough and smooth since they seem to differ in their properties (28). It is complex to ex-plain and understand how bacteria interact and affect each other seen to the multiple different phenotypes, genotypes, growth conditions etc. More studies must be performed to get a better understanding of the role of P. micra in development of periodontitis. It is anyhow important to increase the knowledge of bacterial interaction and the factors that are increasing virulence in pathogens, this understanding may be used to find new and improved ways to detect pa-tients with the risk of developing periodontitis. If relevant risk markers can be obtained, they would help dentists in identifying patients before they develop disease, early preventions could be put in which might prevent the actual disease.
The bacteria used in this study had been collected from routine paper point samples from gin-gival pockets in patients suffering from periodontitis, the bacteria had been separated from each other and cultivated. After this step the bacteria cannot be traced to a certain individual and therefore ethical approval is not needed for studies of this kind.
This study has shown on a wider understanding on how different genotypes of P. gingivalis interacts with P. micra. When more in vitro studies have been done in this field, it is assuma-ble the next step is in vivo experiments to get a fuller understanding on bacterial interactions and host response. This study shows that the likelihood of developing periodontitis may de-pend on which strain of P. gingivalis is residual in the gingival pocket as well as the composi-tion of the microbiota present. The difference of Rgp- and Kgp activity in different strains of P. gingivalis could explain why people with similar composition of oral microorganism show difference in tissue breakdown when it comes to periodontitis. In the future, after more stud-ies, this understanding could lead to the development of biomarkers used to detect people in risk of developing periodontitis. Periodontal diseases affect the quality of life negative due to e.g the loss of teeth, which reduces functions such as chewing food and speaking, it also has a social affect due to changes in appearance. Therefore, biomarkers would be of great value when it comes to helping people in risk of developing periodontitis by making it possible to put in early preventions.
5. CONCLUSION
The results from this study show that the growth and gingipain stimulating effect of P. micra on P. gingivalis differs between different strains of P. gingivalis. Some strains were affected and others not. Although it is too early to draw such conclusions from a small experimental study like this, this could suggest that two individuals with P. gingivalis subgingivally could differ in their likelihood to develop periodontitis since the gingipain activity could differ de-pending on P. gingivalis strain and presence of P. micra.
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REFERENCES
1. Lang NP, Lindhe J. Clinical Periodontology and Implant Dentistry, 2 Volume Set, 6th Edition. In: Clinical Periodontology and Implant Dentistry. 2015:
2. Marsh P, Martin M. Oral Microbiology. Oral Microbiology. 1992.
3. Löe H, Anerud A, Boysen H, Morrison E. Natural history of periodontal disease in man: Rapid, moderate and no loss of attachment in Sri Lankan laborers 14 to 46 years of age. J Clin Periodontol 1986;
4. Kinane DF, Stathopoulou PG, Papapanou PN. Periodontal diseases. Nature Reviews Disease Primers. 2017.
5. Peters BM, Jabra-Rizk MA, O’May GA, William Costerton J, Shirtliff ME. Polymicrobial interactions: Impact on pathogenesis and human disease. Clinical Microbiology Reviews. 2012.
6. Rosier BT, De Jager M, Zaura E, Krom BP. Historical and contemporary hypotheses on the development of oral diseases: Are we there yet? Frontiers in Cellular and Infection Microbiology. 2014.
7. Marsh PD. Are dental diseases examples of ecological catastrophes? Microbiology. 2003.
8. Hajishengallis G, Darveau RP, Curtis MA. The keystone-pathogen hypothesis. Nature Reviews Microbiology. 2012.
9. Darveau RP, Pham TTT, Lemley K, Reife RA, Bainbridge BW, Coats SR, et al. Porphyromonas gingivalis lipopolysaccharide contains multiple lipid A species that functionally interact with both toll-like receptors 2 and 4. Infect Immun 2004; 10. Darveau RP, Belton CM, Reife RA, Lamont RJ. Local Chemokine Paralysis, a Novel
Pathogenic Mechanism for Porphyromonas gingivalis. Infect Immun 1998;
11. Hajishengallis G, Liang S, Payne MA, Hashim A, Jotwani R, Eskan MA, et al. Low-abundance biofilm species orchestrates inflammatory periodontal disease through the commensal microbiota and complement. Cell Host Microbe 2011;
12. Neilands J, Davies JR, Bikker FJ, Svensäter G. Parvimonas micra stimulates
expression of gingipains from Porphyromonas gingivalis in multi-species communities. Anaerobe 2019;
13. Marsh PD. Dental plaque: Biological significance of a biofilm and community life-style. In: Journal of Clinical Periodontology. 2005.
14. Brogden KA, Guthmiller JM, Taylor CE. Human polymicrobial infections. Lancet. 2005.
15. Wright CJ, Burns LH, Jack AA, Back CR, Dutton LC, Nobbs AH, et al. Microbial interactions in building of communities. Mol Oral Microbiol 2013;
16. Marsh PD, Zaura E. Dental biofilm: ecological interactions in health and disease. J Clin Periodontol 2017;
17. Kolenbrander PE, Palmer RJ, Rickard AH, Jakubovics NS, Chalmers NI, Diaz PI. Bacterial interactions and successions during plaque development. Periodontology 2000. 2006.
18. Tenorio EL, Klein BA, Cheung WS, Hu LT. Identification of interspecies interactions affecting Porphyromonas gingivalis virulence phenotypes. J Oral Microbiol 2011; 19. van Steenbergen TJM, van Winkelhoff AJ, de Graaff J. Pathogenic synergy: mixed
infections in the oral cavity. Antonie Van Leeuwenhoek 1984;
20. Bao K, Belibasakis GN, Thurnheer T, Aduse-Opoku J, Curtis MA, Bostanci N. Role of Porphyromonas gingivalis gingipains in multi-species biofilm formation. BMC
Microbiol 2014;
21. Mysak J, Podzimek S, Sommerova P, Lyuya-Mi Y, Bartova J, Janatova T, et al. Porphyromonas gingivalis: Major periodontopathic pathogen overview. Journal of Immunology Research. 2014.
17 22. Bostanci N, Belibasakis GN. Porphyromonas gingivalis: An invasive and evasive
opportunistic oral pathogen. FEMS Microbiology Letters. 2012.
23. Costalonga M, Herzberg MC. The oral microbiome and the immunobiology of periodontal disease and caries. Immunol Lett 2014;
24. Kuboniwa M, Amano A, Inaba H, Hashino E, Shizukuishi S. Homotypic biofilm structure of Porphyromonas gingivalis is affected by FimA type variations. Oral Microbiol Immunol 2009;
25. Shapira L, Champagne C, Van Dyke TE, Amar S. Strain-dependent activation of monocytes and inflammatory macrophages by lipopolysaccharide of Porphyromonas gingivalis. Infect Immun 1998;
26. Sheets SM, Robles-Price AG, McKenzie RME, Casiano CA, Fletcher HM. Gingipain-dependent interactions with the host are important for survival of Porphyromonas gingivalis. Frontiers in Bioscience. 2008.
27. Genco CA, Potempa J, Mikolajczyk‐Pawlinska J, Travis J. Role of Gingipains R in the Pathogenesis of Porphyromonas gingivalis‐Mediated Periodontal Disease. Clin Infect Dis 1999;
28. Ota-Tsuzuki C, Alves Mayer MP. Collagenase production and hemolytic activity
related to 16S rRNA variability among Parvimonas micra oral isolates. Anaerobe 2010; 29. de Andrade JA, Feres M, de Figueiredo LC, Salvador SL, Cortelli SC. The ability of
the BANA Test to detect different levels of P. gingivalis, T. denticola and T. forsythia. Braz Oral Res 2010;
30. Kaman WE, Galassi F, De Soet JJ, Bizzarro S, Loos BG, Veerman ECI, et al. Highly specific protease-based approach for detection of porphyromonas gingivalis in diagnosis of periodontitis. J Clin Microbiol 2012;
