Anaerobe 27 (2014) 7e13

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Clinical microbiology

Effect of estradiol on planktonic growth, coaggregation, and biofilm formation of the Prevotella intermedia group bacteria Dareen Fteita a, *, Eija Könönen a, b, Eva Söderling a, Ulvi Kahraman Gürsoy a a b

Department of Periodontology, Institute of Dentistry, University of Turku, FI-20520 Turku, Finland Department of Infectious Disease Surveillance and Control, National Institute for Health and Welfare (THL), FI-00300 Helsinki, Finland

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 September 2013 Received in revised form 20 January 2014 Accepted 19 February 2014 Available online 1 March 2014

Alterations in the quantity and quality of biofilms at gingival margin are considered to play a role in the initiation and development of pregnancy-related gingivitis. Prevotella intermedia sensu lato is able to consume estradiol, the major sex hormone secreted during pregnancy, in the absence of vitamin K. The aim of the study was to examine the effect of estradiol on the planktonic growth, coaggregation, polysaccharide production, and biofilm formation of the P. intermedia group bacteria, namely P. intermedia, Prevotella nigrescens, and Prevotella pallens. In all experiments, the type strain (ATCC) and a clinical strain (AHN) of P. intermedia, P. nigrescens, and P. pallens were incubated with the concentrations of 0, 30, 90, and 120 nmol/L of estradiol. Planktonic growth was assessed by means of the colony forming unit method, while coaggregation and biofilm formation were assessed by spectrophotometric methods. In the determination of protein and polysaccharide levels, the Bradford and phenolesulfuric acid methods were used, respectively. P. pallens AHN 9283 and P. nigrescens ATCC 33563 increased their numbers at planktonic stage with increasing estradiol concentrations. In 48-h biofilm tests, elevated protein levels were found for both strains of P. intermedia, and the strains P. nigrescens ATCC 33563 and P. pallens AHN 9283 in the presence of estradiol. The P. intermedia strains also increased the levels of polysaccharide formation in the biofilm. Coaggregation of the P. intermedia group organisms with Fusobacterium nucleatum was enhanced only in P. intermedia AHN 8290. In conclusion, our in vitro experiments indicate that estradiol regulates planktonic growth, coaggregation, polysaccharide production, and biofilm formation characteristics of P. intermedia, P. nigrescens, and P. pallens differently. These results may, at least partly, explain the differences seen in their contribution to the pathogenesis of pregnancy-related gingivitis. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Sex hormone Prevotella Biofilm Coaggregation Planktonic Pregnancy

1. Introduction Pigmented Prevotella species are ubiquitous inhabitants of the oral cavity but also frequently involved in oral infections and infections of the female genital tract [33e35]. The Prevotella intermedia group bacteria are composed of P. intermedia, Prevotella nigrescens, and Prevotella pallens, which are pigmented gramnegative, anaerobic rods. P. intermedia can be found in subgingival biofilms of periodontally diseased sites [49]. Although P. nigrescens carries biochemical similarities with P. intermedia, it has been associated with both healthy and diseased conditions of

* Corresponding author. Tel.: þ358 40 9650024. E-mail addresses: dareen.fteita@utu.fi, [email protected] (D. Fteita), eija. kononen@utu.fi (E. Könönen), eva.soderling@utu.fi (E. Söderling), ulvi.gursoy@ utu.fi (U.K. Gürsoy). http://dx.doi.org/10.1016/j.anaerobe.2014.02.003 1075-9964/Ó 2014 Elsevier Ltd. All rights reserved.

the periodontium [9,12,39,50]. Since P. intermedia and P. nigrescens are biochemically similar, they should be classified as P. intermedia sensu lato, if molecular methods have not been used for their separation [43]. In 1998, P. pallens was described as a novel species in the P. intermedia group, found mainly in the periodontally healthy oral cavity [30,31]. P. pallens has also been detected as part of the complex but conserved bacterial population in the normal distal esophagus [44]. Pregnancy itself does not induce pathologic changes in gingival and periodontal tissues. Pregnancy gingivitis is categorized under plaque-induced gingival diseases, which are modified by systemic factors, i.e., hormonal changes by the endocrine system during pregnancy [2,22]. According to the literature, pregnancy gingivitis has a wide range of prevalence, varying between 35% [6,20] and 100% [22,36]. During pregnancy, placenta secretes estradiol, which is the most potent form of the female sex hormone estrogen. From the second

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trimester to term, the placenta replaces the corpus luteum in the production of estradiol and secretes it into serum [37]. Serum levels of estradiol during pregnancy reach their peak by the end of the third trimester when pregnant women produce about 20 mg of estradiol daily. Estradiol levels decrease dramatically to nonpregnant concentrations postpartum (2e3 days after delivery) [37]. Especially during the second trimester of pregnancy, there is an increase in gingival inflammation regardless of dental plaque levels at gingival margin [11,13,28,48,52]. It was recently shown that the estrogen level is one of the major determinants of the magnitude of gingival inflammation developed against microbial plaque during pregnancy [15]. Changes in the periodontium during pregnancy are not limited to alterations in the host response. With increasing estradiol levels in gingival crevicular fluid, which is an inflammatory exudate originating from serum, the subgingival microbiota shows a tendency towards gram-negative anaerobes instead of facultative gram-positive species. The proportion of P. intermedia sensu lato, in particular, then increases significantly in subgingival biofilms [14,28]. Human hormones can affect the growth of oral bacteria, e.g., anaerobic periodontitis-related bacteria [24]. Besides, it has been previously shown that P. intermedia sensu lato (formerly Bacteroides melaninogenicus subsp. intermedius) is able to use estrogen and progesterone as an essential source of growth instead of using vitamin K in steady circumstances [29]. Also, clinical studies have indicated positive correlations between the qualitative and quantitative increase of P. intermedia sensu lato [5] and, in particular, P. nigrescens [14] in subgingival biofilms and increased estrogen levels during pregnancy in periodontitis-free women [4,28,37]. Planktonic growth and biofilm formation characteristics of the P. intermedia group bacteria in the presence of estradiol are not yet defined. Therefore, the aim of our present in vitro study was to determine the effect of estradiol on the planktonic growth, coaggregation, and biofilm formation of P. intermedia, P. nigrescens, and P. pallens. 2. Material and methods 2.1. Bacterial strains and cultures The type strains and a clinical strain of each species (P. intermedia ATCC 25611T and AHN 8290, P. nigrescens ATCC 33563T and AHN 8293, and P. pallens NCTC 13042T and AHN 9283) were the 6 Prevotella strains used in this study. All strains were obtained from the collections of National Institute for Health and Welfare, Helsinki, Finland. Clinical strains of P. intermedia AHN 8290 and P. nigrescens AHN 8293 were isolated from periodontal pockets, while P. pallens AHN 9283 strain was isolated from saliva of a periodontally healthy subject. The identification of each strain has been described previously [32]. The type strain of Fusobacterium nucleatum (ATCC 25586T) was used in the coaggregation assay. In all experiments, the strains were grown on Brucella blood agar plates supplemented with hemin (5 mg/l) and vitamin K1 (10 mg/l) in an anaerobic chamber (Whitley A35 Anaerobic Workstation, Don Whitely Scientific Ltd., West Yorkshire, UK) with an atmosphere of 10% H2, 5% CO2, and 85% N2 at 37  C for 3e5 days. Afterwards, bacteria were transferred to Todd-Hewitt broth (Becton and Dickinson DifcoÔ and BBLÔ, USA) supplemented with 5 g/l yeast extracts, 750 mg/l cysteine, 5 mg/l hemin and 5 mg/l menadione (Sigma Chemical Co., St. Louis, MO, USA). 2.2. Preparation of saliva-coated 96-well plates Volunteers for saliva collection were healthy adults (one male and six females; age range 27e52 years) with no history of

medications or antibiotics during the preceding month. They were given paraffin wax to chew and saliva was collected in ice bathed test tubes for a maximum period of 10 min per subject. Saliva was pooled and underwent centrifugation of 12,000 g for 40 min. For pasteurization, saliva was transferred into a water bath of 60  C for 30 min, and another centrifugation of 12,000 g for 40 min was carried out to obtain clarified saliva stored at 4  C for further use. For saliva coating, 50 ml of clarified saliva was added to all control and test wells and kept in 37  C for 1 h. Afterwards, saliva was blotted out without rinsing and bacterial suspension and fresh culture media were added. 2.3. Preparation of estradiol suspensions Estradiol stock solution was prepared by dissolving a known amount of estradiol (Sigma, Poole, UK) in 70% ethanol to insure homogeneity, and then distilled water was added. From the stock solution, 3 test groups with estradiol concentrations of 30, 90, or 120 nmol/L were prepared. These concentrations were chosen as equivalent to the serum estradiol concentrations simulating the first, second, and third trimester of pregnancy [41]. Solution with no added estradiol was used as a control. 2.4. Planktonic growth For practical purposes we used optical density (OD) to adjust the cell concentration to be the same in the experiments. The OD of 0.5 at 490 nm of each strain was corresponded to the logarithmic colony forming unit (log CFU/ml) as follows: P. intermedia ATCC 25611 (9.83) and AHN 8290 (7.04), P. nigrescens ATCC 33563 (9.62) and AHN 8293 (6.04), P. pallens NCTC 13042 (8.04) and AHN 9283 (8.08). For planktonic growth tests, all bacterial strains were grown in broth for 2 days; bacteria were adjusted to the OD of 0.7 at 490 nm with a spectrophotometer (Shimadzu Biotech, Tokyo, Japan). A known amount of the optically adjusted bacteria were incubated with 0, 30, 90, or 120 nmol/L of estradiol suspensions in anaerobic conditions for 24 h. After incubation, 10 ml of bacterial suspension of each concentration were inoculated on Brucella agar plates and incubated in an anaerobic atmosphere for 3e5 days. Bacterial growth was counted as CFUs. 2.5. Coaggregation assay To test the coaggregation ability of the selected 6 Prevotella strains, the Kolenbrander’s method with slight modifications was used [27]. Briefly, Prevotella strains were grown in the culture media and F. nucleatum ATCC 25586 in brain heart infusion (BHI) broth (Difco, Detroit, Mich., USA) in anaerobic conditions. Overnight cultures of the tested strains were harvested by centrifugation at 12,000 g for 10 min, and after removing supernatants, cells were washed once with phosphate-buffered saline (PBS, pH 7.2). Another centrifugation at 12,000 g for 10 min took a place, and after removing supernatants, cells were resuspended in the coaggregation buffer (TriseHCl, pH 8.0), containing 0.1 mM CaCl2, 0.1 mM MgCl2, and 150 mM NaCl [27]. The bacterial suspensions were optically adjusted to 0.7 OD at 660 nm. Equal volumes of each optically adjusted Prevotella strain suspension and F. nucleatum suspension were added to plastic cuvettes, followed by estradiol stock solution of 0, 30, 90, or 120 nmol/L of estradiol to reach a total volume of 800 ml. Spectrometric readings were recorded immediately (0 h) (SHIMADZU UVevisible, BioSpec-mini, Kyoto, Japan), followed by the next readings at 30 min [51]. All experiments were done in triplicates. During the time intervals, the test cuvettes were kept covered with paraffin strips at 37  C.

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2.6. Biofilm formation Four test sets of a 96-well plate (each set of 5 wells) were coated with clarified saliva for 1 h at 37  C. Each bacterial strain was incubated overnight in culture media in an anaerobic chamber, and then optically adjusted into 0.7 OD at 490 nm. After supplying each well with a known amount of bacterial suspension, estradiol solution, and fresh culture media, the 96-well plate was incubated in anaerobic conditions for 48 h. 2.7. Determination of biofilm protein levels Protein produced by each Prevotella strain was determined by the Bradford method of protein determination in biological samples [19]. Briefly, culture media with the planktonic cells were removed from the wells where the biofilms were grown. The test wells of the 96-well plate were washed twice with PBS, and 0.2 N NaOH were loaded to each well. By adjusting the sonicator to power 80, each well was sonicated for 3 s, followed by heating the plate in a 600 W microwave oven for 20 s according to the protein determination using the microwave enhancement method but with a slight modification in the heating duration [1,23]. Afterwards, the Bradford Protein Assay (Bio-Rad, Hercules, USA) was added to each well and the plate was incubated for 5 min while shaking on the vortex. Absorbance was assessed based on the wavelength of 595 nm with the micro-plate reader. Six bovine serum albumin concentrations of 0, 50, 125, 250, 375, and 500 mg/ml (Sigmae Aldrich, USA) were used as standard values in the conversion of OD readings to protein concentrations. 2.8. Determination of biofilm polysaccharide levels Levels of biofilm polysaccharides were measured by the phenolesulfuric acid method [57]. After removing the culture medium containing planktonic cells and rinsing twice with PBS, 40 ml of de-ionized water, 40 ml of 5% phenol solution, followed by 200 ml of 95e97% sulfuric acid were added to the 96-well plate where the biofilms were grown. The plate was incubated at room temperature for 30 min. The amount of polysaccharide in biofilms was determined by measuring the absorbance at 490 nm with the micro-plate reader. Four different glucose concentrations of 0, 5, 10, 20, and 100 mg/ml were used as standard values in the conversion of OD readings to polysaccharide concentrations. 2.9. Statistical analysis Each experiment was performed at least in triplicate and every experiment was repeated at least twice at different time points. Day-to-day variability was checked by repeating each set of experiments twice and results were accepted when no significant difference between the two sets of experiments was noticed. Mean values, standard deviations, and the T-test to compare the test data with the control were the main statistical tools used in the current study. A p value of

Effect of estradiol on planktonic growth, coaggregation, and biofilm formation of the Prevotella intermedia group bacteria.

Alterations in the quantity and quality of biofilms at gingival margin are considered to play a role in the initiation and development of pregnancy-re...
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