Chapter 4: Isolation and identification of biofilm formers from dental plaque

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1 Chapter 4: Isolation and identification of biofilm formers from dental plaque

2 4.1 INTRODUCTION The resident plaque microflora of a healthy site within the mouth remains relatively stable with time (Marsh, 2006). This state of homeostasis is of great importance to oral health as it insures that potentially harmful species stay at low numbers, and that dental biofilm retains its protective function in terms of resistance to colonization by exogenous flora (Al-Hebshi & Skaug, 2006). An unprecedented accumulation of plaque in the absence of diligent oral hygiene results in a breakdown of microbial homeostasis leading to development of caries and other oral diseases (Marsh, 2009). Mutans streptococci, particularly S.mutans are often considered as the target organism when evaluating the efficacy of an oral care product. However, in the light of currently available literature regarding oral micro flora and their role in disease, there is a need to re-assess this stand. Although mutans streptococci are strongly implicated with caries, the association is not unique. Caries can occur in the apparent absence of these species due to other acidogenic, non-mutans streptococci, while it has also been reported that mutans streptococci can persist in the oral cavity without any evidence of detectable demineralization (Russel, 2009). Additionally, in vivo, the oral microbiota is involved in numerous inter-species physiological and nutritional interactions which may influence the efficacy of an anti-plaque agent. The anti-plaque agent maybe effective against a single target organism in vitro but this efficacy may not be replicated against the multi-species population, characteristic of the oral cavity (Wilson, 1996). In order to obtain a heterogeneous population of oral bacteria that closely reflect the oral biosystem, a test consortium of biofilm forming oral bacteria was prepared from isolates obtained from dental plaque samples of volunteers. The chapter discusses: Collection of the plaque samples from volunteers Isolation of bacteria from the plaque samples School of Science, SVKM s NMIMS (deemed-to-be) University Page 69

3 Screening the isolates for biofilm formers using a standardized biofilm formation assay Identification of biofilm formers obtained The test consortium thus prepared was used through the duration of the study. School of Science, SVKM s NMIMS (deemed-to-be) University Page 70

4 4.2 MATERIALS AND METHODS During the present investigations, chemicals used were of analytical grade purchased from Qualigens Fine Chemicals, Mumbai, India. Readymade dehydrated media was procured from Himedia Laboratories Pvt. Ltd., Mumbai. Culture Media All media in the study were prepared according to manufacturer s instructions. Compositions are listed wherever required. Weighed quantity of dehydrated media was suspended in a measured quantity of distilled water and mixed well. In case of solid media, the medium was heated in a microwave to dissolve the dehydrated powder completely. The media was sterilized by autoclaving at 121ºC under a pressure of 15 psi for 20 min. Sterilized medium was allowed to cooled and stored at 4 ºC until use. All media thus prepared were used within seven days. Brain Heart Infusion Medium Brain Heart Infusion (BHI) Medium is useful for cultivating a wide variety of microorganisms since it is a highly nutritive medium. It is employed especially to culture fastidious organisms. Table 4.1: Composition of Brain Heart Infusion agar Components g/l Calf brain infusion 200 Beef heart infusion 250 Proteose peptone 10 Dextrose 2 Sodium chloride 5 Disodium phosphate 2 Agar 15 Final ph ( at 25 C) 7.4±0.2 Blood Brain Heart Infusion Agar Blood agar bases are typically supplemented with 5-10% blood for use in isolation, cultivation and determination of hemolytic reactions of fastidious pathogenic microorganisms. School of Science, SVKM s NMIMS (deemed-to-be) University Page 71

5 Preparation of medium Sterile blood for the preparation of BHI blood agar was procured from the blood bank at Dr. B. Nanavati Hospital, Mumbai. Preparation and composition of BHI agar medium is provided in this chapter in Table 4.1. Sterile blood was added additionally to the cooled and molten sterile BHI agar medium to achieve final concentrations of 7% blood in the medium Collection of Plaque samples The plaque samples were collected from 10 healthy volunteers with no specific oral health issues after obtaining the necessary consent. A sample volunteer consent form has been included in the Appendices. The plaque samples were taken after a gap of 7-8 h since last brushing. The samples were collected with the help of a sterile cotton swab moistened with sterile saline from supra-gingival region of the tooth surface. The swab was immediately used to inoculate a sterile BHI agar plate. The plate was incubated at 37ºC for 24 h. Post incubation, the plates were observed for colonies that differed in terms of macroscopic colony characteristics Isolation of bacteria from plaque samples Colonies with visually distinguishable morphologies were selected, isolated on BHI agar plates and incubated for 24 h to obtain a pure culture of each isolate. The isolated colonies obtained after incubation were then streaked onto fresh BHI plates and BHI slants and maintained at 4ºC. The colony characteristics and hemolysis pattern on Blood BHI agar of each of the isolates were recorded. The isolates were further screened for their ability to form biofilms Selection of bacteria for preparation of test consortium a. Standardization of biofilm formation assay using acrylic tooth as substratum A biofilm formation assay was performed to ascertain the potential of the isolates obtained from dental plaque samples to form biofilms de novo when exposed to an experimental salivary pellicle using acrylic tooth as a substratum. Prior to evaluation School of Science, SVKM s NMIMS (deemed-to-be) University Page 72

6 of the isolates, the conditions for the assay were standardised with respect to the appropriate method of sterilization of substratum and the optimum incubation period required for adequate biofilm formation. Organisms Streptococcus mutans MTCC#890 and Streptococcus mitis MTCC#2696 were obtained from Institute of Microbial Technology (MTCC), Chandigarh, India. S. mutans and S. mitis were selected for the study since S. mitis represents the early settlers of a dental biofilm, often associated with the normal flora observed in a healthy mouth while Streptococcus mutans represents the late settlers of a dental biofilm and is cariogenic. S. mitis uses its hydrophobicity and lectin like interactions with the acquired enamel pellicle to initialise biofilm formation (Loesche,1986). S.mutans can survive anywhere in the oral cavity, since it is a facultative anaerobe. It ferments most sugars and sugar alcohols present in food to produce acids. S. mutans also produces insoluble extracellular polysaccharides from the metabolism of sucrose to enhance their adherence to the tooth surface. These properties have contributed to the cariogenicity of S. mutans (Islam et al, 2007). Preparation of inoculum Through the complete study, the inoculum was prepared in sterile Phosphate Buffered Saline (PBS). PBS was prepared as described in Mackie and MacCartney s Practical Medical Microbiology (Poxton and Brown, 1996). S. mutans and S. mitis were sub-cultured onto fresh BHI agar and incubated at 37 o C for 24 h in a candle jar so as to provide 3-5% CO 2 conditions. To prepare inoculum, colonies from an 18 h old culture of each strain on BHI agar were suspended in sterile PBS to obtain a suspension equivalent to the turbidity of McFarland s 0.5 standard, which corresponds to organisms/ml. McFarland s standard solution for comparison was prepared by adding 0.05 ml of 1% solution of barium chloride to 9.95 ml of 1% sulfuric acid (Brown and Poxton, 1996). School of Science, SVKM s NMIMS (deemed-to-be) University Page 73

7 Preparation of saliva Sterile saliva was collected by a method described by Rahim et al. (2006) with some modifications. Saliva was collected from a single volunteer by expectoration into a sterile ice-chilled container. The method was modified such that the saliva collected was centrifuged at 4,000g for 10 min to remove cellular debris as described by Ahn et al. (2008) instead of treating the saliva with 1,4- Dithio- D,L- threitol (DTT) and centrifuging at 864g for 30 min as described by Rahim et al. (2006). The supernatant was filter sterilized using sterile 0.22µ cellulose nitrate filter (Sartorius stedim biotech, Germany) and stored at - 20 C till further use. Culture Medium Brain Heart Infusion Broth with 1% Sucrose Table 4.2: Composition of Brain Heart Infusion Broth Components g/l Calf brain infusion 200 Beef heart infusion 250 Proteose peptone 10 Dextrose 2 Sodium chloride 5 Disodium phosphate 2 Final ph ( at 25 C) 7.4±0.2 Sucrose was added additionally to the prepared BHI Broth medium to achieve final concentrations of 1% sucrose in the medium so as to provide an additional fermentable carbohydrate source. Oral bacteria are known to metabolize sucrose to enhance their adherence and co-aggregation (Wan Nordini Hasnor et al., 2006). Substratum Acrylic teeth were used as the substratum for the biofilm formation assay. The acrylic teeth are shown in Figure 4.1. School of Science, SVKM s NMIMS (deemed-to-be) University Page 74

8 Standardization of sterilization technique for substratum Three possible methods of sterilization were evaluated, namely using 3% formaldehyde, 2% gluteraldehyde and Microwave irradiation (Pavan et al., 2005). Three acrylic teeth were exposed to one of the three sterilization techniques as follows: 1. Immersion in 3% formaldehyde for 1 h 2. Immersion in 2% gluteraldehyde for 1 h 3. Exposure to Microwave irradiation for 6 min Following exposure to sterilizing agent, the tooth was placed in a tube containing sterile nutrient broth and incubated for 24 h at room temperature. Post incubation the absence of turbidity in the tube indicated effective sterilization. Preparation of substratum for biofilm assay Acrylic tooth was sterilised using 2% gluteraldehyde and washed twice with sterile distilled water before use. School of Science, SVKM s NMIMS (deemed-to-be) University Page 75

9 Figure 4.1: Acrylic teeth used as substratum for the biofilm formation assay School of Science, SVKM s NMIMS (deemed-to-be) University Page 76

10 Preparation of 1% Crystal violet 1% Crystal violet was prepared as described in Text book of Medical Laboratory Technology (Godkar and Godkar, 2007). Standardisation of incubation period for development of biofilm 1 ml of sterile saliva was added to a tube containing sterile acrylic tooth and kept undisturbed for 90 min for conditioning following which, 3.95 ml of BHI broth with 1% (w/v) sucrose was added in each tube. The tube was inoculated with 0.05 ml of test consortium. Tubes were incubated at 37 o C for varying periods of incubation of 3 h, 5 h, 7 h. Post incubation the acrylic teeth were stained with 1% crystal violet for 10 min to allow visualization of biofilm on the tooth surface to determine the optimum period of incubation after which a confluent biofilm forms on the acrylic tooth surface b. Biofilm formation assay using acrylic tooth as substratum 1 ml of sterile saliva was added to a tube containing sterile tooth and kept undisturbed for 90 min for conditioning ml of BHI broth with 1% (w/v) sucrose was then added following which the tube was inoculated with 0.05 ml of test consortium. Negative control was set up in a similar manner by substituting 0.05 ml of test consortium of with 0.05 ml of PBS. The tubes were incubated for 7 h at 37 o C. Post incubation the acrylic teeth were stained with 1% crystal violet for 10 min to allow visualization of biofilm on the tooth surface Screening of dental plaque isolates for biofilm forming ability The isolates obtained were screened in pairs for their ability to form biofilms using biofilm formation assay described above. Care was taken to ensure that both the isolates that form a pair originated from the same volunteer. They were classified as strong/weak/non biofilm formers on comparison with the biofilm formed by the strains S.mutans MTCC# 890 and S. mitis MTCC#2696. Isolates that showed strong biofilm forming ability were included in consortium along with S. mutans and S. mitis Biochemical identification of isolates capable of biofilm formation The strong biofilm formers selected after screening were identified by conventional biochemical tests. School of Science, SVKM s NMIMS (deemed-to-be) University Page 77

11 Physical characterization i) Gram staining: Gram staining of the isolates was performed as described in Textbook of Medical Laboratory Technology (Godkar and Godkar, 2007) and the Gram natures of the isolates were recorded. ii) Colony morphology: Colony characteristics of overnight culture of the isolate on BHI agar media was observed and recorded. iii) Cell morphology: The Gram stained smears of the isolates were viewed microscopically under a magnification of 1000 X using the oil immersion lens to determine the morphological characteristics of the cells. Biochemical characterization Bacteria differ widely in their ability to metabolize carbohydrates and other compounds. For the purpose of identification these differences can be demonstrated by four different tests which are described below (Collee et al, 1996). 1) Test to check for the ability of isolates to ferment different carbohydrates 2) Tests for specific breakdown products 3) Tests for the ability of isolates to utilize certain proteins and amino acids 4) Tests to study the various enzymes produced by the isolates All the tests were performed as per standard techniques described in Mackie and McCartney s Practical Medical Microbiology (Collee et al, 1996; Ross, 1996) and Bergey s Manual of Systemic Bacteriology (Hardie, 1986). Preparation of Inoculum Suspensions of isolates were made using 18 h old culture of each strain on BHI agar with turbidity equivalent to the 0.5 Mac Farland s turbidity standard ( organisms/ml) which was kept constant for all the biochemical tests (Brown and Poxton, 1996). School of Science, SVKM s NMIMS (deemed-to-be) University Page 78

12 Requirements, preparations and methods for biochemical identification of the isolates i. Fermentation of carbohydrates and related compounds (Collee et al, 1996) The purpose of the tests is to detect the production of acid and gas or acid alone when a pure culture grows in the presence of the test compound. Sugars used in this test: Hexose : Glucose Dissacharides : Sucrose, Lactose, Trehalose, Cellobiose Trisachharide : Raffinose Polyhydric Alcohol : Mannitol, Sorbitol Indicator used Medium used : Andrade s indicator : 1.5% Peptone water Preparation of media 1.5 % peptone water was prepared and Andrade s indicator was added to it so as to achieve a resultant concentration of indicator in the medium as 1%. The medium was dispensed into tubes. An inverted Durham s Tube was introduced into each tube to detect gas production. During preparation and sterilization, it is to be ensured that the inverted Durham s Tube is devoid of any air bubbles. 10 % solutions of the above mentioned sugars were prepared in distilled water and autoclaved at 10 psi for 10 min. The sterile sugar solution was added to the individual tubes of peptone water under sterile conditions so as to obtain the resultant concentration of 1% sugar in each tube. Method: The sugar fermentation media were inoculated with the test organisms. 8 tubes corresponding to each sugar were inoculated for each isolate and incubated at 37 C for 24 h. Interpretation: Turbidity in the tubes indicated growth. Andrade s indicator will change colour as a result of the formation of acids during the fermentation resulting in the development of a pink colour in the medium while appearance of air bubble School of Science, SVKM s NMIMS (deemed-to-be) University Page 79

13 within the inverted Durham s tube indicated the production of gas during fermentation of sugars. ii. Tests for specific breakdown products Voges Proskauer Test (VP or Acetoin production test) The purpose of the test is to detect the production of acetoin by organisms as a breakdown product of carbohydrate fermentation. Many bacteria ferment carbohydrates with the production of acetyl methyl carbinol (CH3.CO.CHOH.CH3) or its reduction product 2, 3 butylene glycol (CH3.CHOH.CHOH.CH3). It can be detected by chemical methods (Collee et al,1996). Media used: Methyl Red Voges Proskauer (MR-VP) medium (Buffered Glucose Broth) Table 4.3: Composition of MR VP medium Components g/l Buffered peptone 7 Di potassium phosphate 5 Dextrose 5 Final ph at 25 C 6.9 ± 0.2 Method: Glucose phosphate broth was inoculated with the test organism and incubated at 37 o C for 24 h. Post incubation for 24 h, 1 ml of Omera s reagent (40 % KOH and 3 ml of a 5 % solution of α naphthol in absolute ethanol) was added and incubated at 37 o C for 30 min and then observed for colouration. Interpretation: A positive reaction is denoted by the development of an eosin pink colour after incubation. iii. Test for ability of the bacteria to utilize Arginine: The purpose is to see if the isolate can utilize the amino acid arginine. Use of arginine is accomplished by the enzyme arginine dihydrolase. The medium School of Science, SVKM s NMIMS (deemed-to-be) University Page 80

14 contained a small amount of glucose which the organism initially utilises. This causes the ph to drop which is indicated by a change in the colour of the medium from purple to yellow. Once the medium has been acidified, the enzyme arginine dihydrolase is activated causing the arginine in the medium to be utilised. Change in the colour of the medium from yellow back to purple indicates a positive test for arginine dihydrolase (Collee et al., 1996). Medium used: L-arginine dihydrolase medium Table 4.4: Composition of L-arginine dihydrolase medium Components g/l L-arginine monohydrochloride 5 Yeast extract 3 Glucose 1 Bromocresol purple Final ph (at 25 o C) 6.8 ± 0.2 Method: L-arginine dihydrolase medium was inoculated with test organisms and incubated at 37 C for h. The medium was observed for change in colour at 24 h, 48 h and 72 h. Interpretation: Appearance of a yellow colour in the medium indicates acid production during glucose fermentation. Positive reaction is denoted by the change in the colour of the medium from yellow back to purple indicative of decarboxylation. iv. Study of various enzymes produced by the isolates The isolates were also checked for the production of various enzymes such as Catalase Amylase School of Science, SVKM s NMIMS (deemed-to-be) University Page 81

15 Detection of catalase activity This test demonstrates the presence of Catalase, an enzyme that catalyzes the release of oxygen from hydrogen peroxide (Collee et al., 1996). Method: Test organisms were inoculated on BHI agar slant and incubated at 37 C for 24 h. Post incubation, 10% Hydrogen peroxide was added drop wise to cultured slant with substantial growth and observed for effervescence. Interpretation: Effervescence indicates a presence of enzyme Catalase. Detection of amylase activity Amylase enzyme acts on starch and converts it into simple sugars. Starch BHI agar was used to detect amylase production. Utilization of starch is detected by pouring iodine solution over the colonies. In presence of starch, iodine gives dark blue coloration (Collee et al., 1996). Medium used: Starch BHI agar with 10% starch solution Preparation of the medium Preparation and composition of BHI Agar medium is provided in this chapter in Table 4.1. To this autoclaved, cooled and molten medium, 10 ml of 10% starch solution (autoclaved separately at 10 psi for 10 min) was added and poured to prepare Starch BHI agar plates. Method: Plates were spot inoculated with test organisms and incubated at 37 C for 48 h. After incubation, each plate was flooded with iodine solution. Interpretation: Presence of amylolytic activity was indicated by clearance around the colonies against the blue back ground. v. Hydrolysis of Esculin in Bile- Esculin agar Esculin is a carbohydrate linked to an alcohol. Many organisms can hydrolyze esculin but very few can do so in the presence of bile. Bile Esculin agar is a selective and differential medium. It contains bile, which inhibits Gram-positive Staphylococcus spp., esculin and peptone act as nutrient sources, while ferric ammonium citrate acts a School of Science, SVKM s NMIMS (deemed-to-be) University Page 82

16 colour indicator. Organisms that do metabolize esculin produce esculetin as a byproduct. Esculetin reacts with ferric ammonium citrate to form a black coloration (Ross, 1996). Medium used: Bile Esculin agar Table 4.5: Composition of Bile Esculin agar Components g/l Meat extract 3 Peptone 5 Ox bile 10 Esculin 1 Ferric ammonium sulphate 0.5 Sodium chloride 5 Agar 15 Final ph (at 25 o C) 6.6 ± 0.2 Preparation of medium Solution A: Meat extract, peptone, sodium chloride and agar were dissolved in distilled water so as to obtain a final volume of 400 ml. Solution B: Ox bile was dissolved in distilled water so as to obtain a final volume of 400 ml. Solution C: Ferric ammonium citrate was dissolved in distilled water so as to obtain a final volume of 100 ml. Solution D: Esculin was dissolved in distilled water so as to obtain a final volume of 100 ml. Solutions A, B and C were mixed and autoclaved at 15 psi for 20 min. Solution D was filter sterilized and added to the autoclaved, cooled and molten medium and poured into sterile screw capped tubes to prepare Bile Esculin agar slants. School of Science, SVKM s NMIMS (deemed-to-be) University Page 83

17 Method: Bile esculin agar slants were inoculated with test organisms and incubated at 37 C for 48 h. Observe for change in colour of the medium at 24 h and 48 h. Interpretation: A black colouration more than halfway down the agar slant within 48 h was considered a positive reaction while partial or absence of black colouration of the agar slant denoted a negative reaction. vi. Salt tolerance The purpose of the test is to determine whether the test organisms can tolerate and multiply in the presence of high concentration of salt (Ross, 1996). Medium used: Brain heart infusion broth with 4% NaCl Brain heart infusion broth with 6.5% NaCl Preparation of medium Preparation and composition of BHI broth medium is provided in this chapter in Table 4.2. Sodium chloride was added additionally to achieve final concentrations of 4% NaCl and 6.5% NaCl in the medium and autoclaved. Method: Tubes containing BHI broth with 4% NaCl and 6.5% NaCl were inoculated with test organisms and incubated at 37 C for 48 h. Interpretation: Positive reaction is denoted by the presence of turbidity in the tubes indicative of growth, suggesting the test organisms are salt tolerant. vii. Production of extracellular polysaccharides The purpose of the test is to determine the ability of the test organism to metabolise sucrose for the synthesis of extracellular polysaccharides like glucans (e.g. Dextrans) and fructans (e.g. Levans) (Waitkins et al., 1980). Medium used: BHI agar with 5% Sucrose School of Science, SVKM s NMIMS (deemed-to-be) University Page 84

18 Preparation of the medium BHI agar was prepared as per the composition given in Table 4.1. Sucrose was added additionally to achieve final concentrations of 5% sucrose in the medium and autoclaved. Method: Plates were inoculated with test organisms and incubated at 37 C for 48 h. Interpretation: Some bacteria produce a levan as the extracellular polysaccharide. A positive reaction is denoted by the appearance of very slimy, mucoidal, runny or large gum drops colonies on the agar. Some bacteria may produce dextrans as the extracellular polysaccharide. A positive reaction is denoted by the presence of dry and adherent colonies which can be confirmed by attempting to pick the colony using a nichrome loop. The results obtained from the conventional biochemical tests were interpreted with reference to Bergey s Manual of Systemic Bacteriology (1986) Preparation of glycerol stocks Glycerol stocks of the isolated and identified biofilm formers were prepared to ensure their viability during longer periods of storage. A single colony of interest was picked and grown overnight in BHI broth. After incubation, purity of the culture was ensured by Gram staining. Glycerol stocks with 15% glycerol content were prepared as prescribed by Sambrook et al. (1989) with slight modification where in 87% glycerol was used instead of 100% glycerol ml of 87% glycerol was added to a sterile 1.5 ml eppendorf tube followed by 0.83 ml of overnight culture. The tube was vortex mixed and labeled with all pertinent information. The glycerol stocks were stored at -80ºC until further use. School of Science, SVKM s NMIMS (deemed-to-be) University Page 85

19 4.3 RESULTS AND DISCUSSION Isolation of bacteria from plaque samples 26 isolates differing in their colony morphologies were isolated from the plaque samples of 10 volunteers. Colony characteristics of the isolates are summarized in Tables More than 80 % of the dental plaque isolates were Gram positive in nature, predominantly as cocci in chains which is consistent with the literature regarding early biofilms (4-12 h biofilms) (Li et al., 2004b; Tahmourespour et al., 2010). Since samples of dental plaque were taken from volunteers after approximately 7-8 h of any oral hygiene practice, it has been reported that such early biofilm samples demonstrate the presence of 60 90% Streptococcus spp. that colonise the teeth within hours of brushing (Foster and Kolenbrander, 2004; Jakubovics et al. 2008). Oral streptococci are pioneer bacteria that initiate the formation of biofilms on tooth surfaces (Loo et al., 2000). The acellular, insoluble and membranous salivary pellicle is characterised by the presence of proteins such as alpha-amylase, proline-rich proteins and proline-rich glycoproteins, that many oral streptococci have the ability to bind to. This ability may confer an advantage on Streptococcus spp., thus justifying their recognition as early colonizers (Kreth et al., 2009; Hojo et al., 2009). Early colonizers are of great importance, because after adhering to the tooth surface, they provide attachment substrates for the subsequent colonizers and ultimately influence the succeeding stages of biofilm formation (Li et al., 2004b). School of Science, SVKM s NMIMS (deemed-to-be) University Page 86

20 Table 4.6: Colony characteristics of dental plaque isolates 1-5 Colony Characteristic Colony 1 Colony 2 Colony 3 Colony 4 Colony 5 Size Medium Small Small Small Medium Shape Circular Circular Circular Circular Circular Colour White Creamish Creamish Creamish White Consistency Smooth Butyrous Butyrous Butyrous Butyrous Opacity Opaque Translucent Translucent Translucent Opaque Surface Smooth Smooth Smooth Smooth Smooth Margin Entire Entire Entire Entire Entire Elevation Low Convex Low Convex Low Convex Low Convex Low Convex Gram Nature Gram positive cocci in clusters Budding Yeasts Gram ve coccobaccilli Gram positive cocci in chains Gram positive cocci in chains Haemolysis Pattern on Blood BHI agar γ Haemolytic γ Haemolytic γ Haemolytic γ Haemolytic β Haemolytic School of Science, SVKM s NMIMS (deemed-to-be) University Page 87

21 Table 4.7: Colony characteristics of dental plaque isolates 6-10 Colony Characteristic Colony 6 Colony 7 Colony 8 Colony 9 Colony 10 Size Small Medium Small Pinpoint Small Shape Circular Circular Circular Circular Circular Colour White Creamish White Creamish White Consistency Butyrous Butyrous Butyrous Butyrous Butyrous Opacity Opaque Translucent Opaque Translucent Translucent Surface Smooth Smooth Smooth Smooth Smooth Margin Entire Entire Entire Entire Entire Elevation Low Convex Low Convex Low Convex Low Convex Low Convex Gram Nature Gram positive Cocci in chains Gram positive Cocci in chains Gram ve Coccobacilli Gram positive Cocci in chains Gram ve Coccobacilli Haemolysis Pattern on Blood BHI agar γ Haemolytic γ Haemolytic β Haemolytic γ Haemolytic β Haemolytic School of Science, SVKM s NMIMS (deemed-to-be) University Page 88

22 Table 4.8: Colony characteristics of dental plaque isolates Colony Characteristic Colony 11 Colony 12 Colony 13 Colony 14 Colony 15 Size Small Medium Medium Pinpoint Pinpoint Shape Circular Circular Circular Circular Circular Colour Creamish Creamish White White White Consistency Butyrous Butyrous Butyrous Butyrous Butyrous Opacity Translucent Translucent Opaque Translucent Translucent Surface Smooth Smooth Smooth Smooth Smooth Margin Entire Entire Entire Entire Entire Elevation Low Convex Low Convex Low Convex Flat Low Convex Gram Nature Gram positive Cocci in chains Gram positive Cocci in chains Gram positive Cocci in clusters Gram positive Cocci in chains Gram positive bacilli with tapering ends Haemolysis Pattern on Blood BHI agar γ Haemolytic γ Haemolytic γ Haemolytic α Haemolytic α Haemolytic School of Science, SVKM s NMIMS (deemed-to-be) University Page 89

23 Table 4.9: Colony characteristics of dental plaque isolates Colony Characteristic Colony 16 Colony 17 Colony 18 Colony 19 Colony 20 Size Pinpoint Small Small Medium Small Shape Circular Circular Circular Circular Slightly Irregular Colour White Creamish Creamish Creamish Creamish Consistency Butyrous Butyrous Butyrous Butyrous Butyrous Opacity Translucent Opaque Opaque Translucent Translucent Surface Smooth Smooth Smooth Smooth Smooth Margin Entire Entire Entire Entire Entire Elevation Flat Low Convex Low Convex Low Convex Flat Gram Nature Gram positive Cocci in chains Gram positive Cocci in chains Gram positive Cocci in chains Gram positive Cocci in chains Gram positive Cocci in chains Haemolysis Pattern on Blood BHI agar α Haemolytic β Haemolytic α Haemolytic β Haemolytic α Haemolytic School of Science, SVKM s NMIMS (deemed-to-be) University Page 90

24 Table 4.10: Colony characteristics of dental plaque isolates Colony Characteristic Colony 21 Colony 22 Colony 23 Colony 24 Colony 25 Colony 26 Size Medium Pinpoint Small Medium Small Small Shape Circular Circular Irregular Circular Circular Circular Colour Creamish White White Yellow White Creamish Consistency Butyrous Butyrous Butyrous Butyrous Butyrous Butyrous Opacity Translucent Translucent Translucent Opaque Opaque Translucent Surface Smooth Smooth Smooth Smooth Smooth Smooth Margin Entire Irregular Irregular Entire Entire Entire Elevation Low Convex Flat Flat Low Convex Low Convex Flat Gram Nature Gram -ve Cocci Gram positive Cocci in chains Gram positive Cocci in chains Gram positive Cocci in chains Gram positive Cocci in chains Gram positive Cocci in chains Haemolysis Pattern on Blood BHI agar β Haemolytic α Haemolytic α Haemolytic γ Haemolytic γ Haemolytic γ Haemolytic School of Science, SVKM s NMIMS (deemed-to-be) University Page 91

25 Standardisation of parameters of biofilm formation assay on acrylic tooth surface Selection of Sterilization technique for substratum The acrylic tooth used as substratum for the biofilm formation assay were subjected to three different methods of sterilization namely immersion in 3% formaldehyde for 1 h, immersion in 2% gluteraldehyde for 1 h and exposure to microwave irradiation for 6 min. Sterilization by immersion in 2% gluteraldehyde for 1 h proved to be most effective in sterilization of the acrylic tooth as compared to the two alternative sterilization techniques used. This method of sterilization was used through the study. Gluteraldehyde has been reported to be effective in chemical sterilization of various types of dental material and prosthetics, especially those of a thermo labile nature (Jnanadev et al., 2011). Glutaraldehyde has a broad spectrum of activity against bacteria and their spores, fungi, viruses, etc. The mechanism of action involves strong associations with the outer layers of Gram positive and Gram negative bacteria, crosslinking of amino groups within the protein content of the cells as well as inhibition of transport processes into the cell (McDonell and Russel, 1999). Additionally, long term exposure of acrylic resin to gluteraldehyde does not significantly alter their surface properties, hence making it a suitable choice for sterilization and disinfection of dental prosthetics (Sharan et al., 2012). Optimization of incubation period for development of biofilm In order to ascertain the optimum incubation time required for confluent biofilm formation on acrylic tooth for the biofilm formation assay, biofilm was allowed to develop on the acrylic tooth for 3 h, 5 h and 7 h. The extent of biofilm formation at varying periods of incubation is depicted in Figure 4.2. School of Science, SVKM s NMIMS (deemed-to-be) University Page 92

26 Figure 4.2: Optimization of incubation period for confluent biofilm formation on acrylic tooth. (A) denotes biofilm obtained post 3 h incubation period; (B) denotes biofilm obtained post 5 h incubation period; (C) denotes biofilm obtained post 7 h incubation period. School of Science, SVKM s NMIMS (deemed-to-be) University Page 93

27 As seen in Figure 4.2, a confluent growth of biofilm adequate for study was obtained post 7 h incubation period. Hence, a 7 h incubation period was set for the biofilm formation assay. The development of biofilm begins within seconds of brushing with the formation of the acquired salivary pellicle that provides the binding sites required for colonization by different oral bacteria (Scannapieco et al., 1993). To simulate this phenomenon, the acrylic tooth was exposed to saliva for 90 min as a conditioning phase prior to inoculation with S. mutans and S. mitis to allow formation of the salivary pellicle. It has been reported, that biofilms on the tooth surface in vivo, within 8-12 h develop into a multi-layered structure (Liljemark and Bloomquist, 1996). When studied in vitro, a confluent biofilm adequate for study developed within 7 h of incubation. It is possible that the addition of sucrose into the nutrient medium as an additional fermentable carbohydrate source influenced biofilm formation by S. mutans and S.mitis allowing biofilm to develop within a shorter period of incubation (Wan Nordini Hasnor et al., 2006; Walsh 2006). Sucrose is metabolized by extracellular bacterial enzymes for the formation of extracellular polysaccharidesglucans and fructans in dental plaque. The formation of glucan and fructan is catalyzed by glucosyltransferase (GTF) and fructosyltransferase (FTF) respectively (Koo et al., 2006). Glucans play a role in plaque formation by facilitating bacterial attachment to the tooth surface while fructans contribute to the virulence of the biofilm by acting as binding sites for the adhesion thereby enhancing build up of plaque (Wan Nordini Hasnor et al., 2006). Screening of biofilm formers from dental plaque isolates and their biochemical identification Isolates obtained from the same volunteer were evaluated in pairs for their biofilm forming ability and classified as strong biofilm formers, weak biofilm formers or non biofilm formers on comparison with the biofilm formed by S. mutans MTCC#890 and S. mitis MTCC#2696 as shown in Figure 4.3. Results of screening for biofilm formers using biofilm formation assay have been summarized in Table School of Science, SVKM s NMIMS (deemed-to-be) University Page 94

28 0 h 0 h 0 h 7 h 7h 7 h a b c Figure 4.3: Classification of dental plaque isolates as non biofilm formers, weak biofilm formers and strong biofilm formers. Biofilm formed on acrylic tooth was visualized post staining with 1% Crystal violet. At 0 h, Negative control (C) and Test (T) showing no biofilm formation in Figures (a), (b) and (c). At 7h, note that in figure (a), no biofilm formation is observed on Test (T) indicative of a non biofilm former; in figure (b), a weak biofilm can be clearly observed on Test (T) indicative of a weak biofilm former; in figure (c), a confluent biofilm is observed on Test (T), indicative of a strong biofilm former. School of Science, SVKM s NMIMS (deemed-to-be) University Page 95

29 Table 4.11: Screening of dental plaque isolates for biofilm forming ability Isolate No. Source Biofilm Former 1 Volunteer 1-2 Volunteer 1-3 Volunteer 2-4 Volunteer Volunteer 4 + (w) 6 Volunteer Volunteer 5-8 Volunteer 5-9 Volunteer 5-10 Volunteer 5-11 Volunteer 4 + (w) 12 Volunteer 6 + (w) 13 Volunteer 2-14 Volunteer Volunteer Volunteer 8 + (w) 17 Volunteer 8 + (w) 18 Volunteer Volunteer 9 + (w) 20 Volunteer Volunteer 6-22 Volunteer Volunteer 9 + (w) 24 Volunteer 7-25 Volunteer 7-26 Volunteer 2 + Key: + denotes strong biofilm former; + (w) denotes weak biofilm former; - denotes non biofilm former Of the 26 isolates evaluated, Isolates 4, 6, 14, 15, 18 and 26 exhibited biofilm formation comparable to S. mutans MTCC#890 and S. mitis MTCC#2696 and were further biochemically identified using conventional tests. The tests performed and results obtained thereof are summarized in Tables 4.12 and School of Science, SVKM s NMIMS (deemed-to-be) University Page 96

30 Table 4.12: General characteristics of biofilm formers isolated from dental plaque samples Isolate 4 Isolate 6 Isolate 14 Isolate 15 Isolate 18 Isolate 26 Gram Nature Gram positive Gram positive Gram positive Gram positive Gram positive Gram positive Effect of presence Enhanced No Change No Change No Change No Change No Change of CO 2 growth Microscopic Morphology Ovoid cocci in short chains Spherical cocci in short chains Spherical cocci in long chains Pleomorphic nature Spherical cocci in long chains Spherical cocci in long chains Solid media: Rods with tapering ends in long chains. Haemolysis pattern on Blood BHI Agar Liquid media: Spherical cocci in long chains γ γ α α γ γ School of Science, SVKM s NMIMS (deemed-to-be) University Page 97

31 Table 4.13: Results of biochemical characterization of biofilm formers isolated from dental plaque samples Test Isolate 4 Isolate 6 Isolate 14 Isolate 15 Isolate 18 Isolate 26 Fermentation of carbohydrates: Acid production from Glucose Sucrose Raffinose Mannitol Sorbitol Lactose Cellobiose Trehalose Utilization of amino acids Argenine Production of Acetoin Extracellular polysaccharides Growth in 6.5% NaCl % NaCl Hydrolysis of Esculin Test for enzymes Catalase Amylase Key: + denotes Positive reaction; - denotes Negative reaction School of Science, SVKM s NMIMS (deemed-to-be) University Page 98

32 As per Bergey s manual of Systematic Bacteriology (Hardie, 1986), on the basis of the results obtained for conventional biochemical tests isolates 4, 14, 15 and 26 were identified up to species level as Streptococcus milleri, Streptococcus mitior, Streptococcus sanguinis and Streptococcus salivarius respectively while isolates 6 and 18 were identified up to genus level as Streptococcus spp. The six isolates were included with S. mutans MTCC#890 and S. mitis MTCC#2696 to prepare the test consortium used through the study. In spite of being obtained from intact plaque samples, only 23% of the dental plaque isolates obtained were strong biofilm formers. Kolenbrander et al. (1993) have reported that the interactions that facilitate biofilm formation are highly specific, which explains why all oral bacteria do not possess the ability to attach directly to surfaces in the oral cavity. Affinity for receptors on the tooth surface is an important factor that controls the identity and number of bacteria that initially attach to the tooth surface (Scannapieco, 1994). In the present study, all the six strong biofilm formers obtained belonged to Streptococcus spp. Highly specific interactions between Streptococcus spp. and multiple salivary components like low molecular weight salivary mucin, highly glycosylated proline rich glycoproteins, α amylase and proline rich peptides have been reported (Whittaker et al., 1996). These specific interactions may be responsible for their observed ability to form biofilms de novo when exposed to the experimental salivary pellicle on the acrylic tooth. The remaining 77% of the dental plaque isolates may be able to incorporate themselves into biofilms in vivo by means of co-aggregation/co-adhesion with organisms already attached to the substratum, but are unable to form biofilms de novo. School of Science, SVKM s NMIMS (deemed-to-be) University Page 99

33 Magnification: 1000X Figure 4.4: Biofilm forming members of test consortium as viewed microscopically under magnification of 1000 X post Gram staining. Isolate 4: Streptococcus milleri (A), Isolate 6: Streptococcus spp (B), Isolate 14: Streptococcus mitior (C), Isolate 15: Streptococcus sanguinis (D), Isolate 18: Streptococcus spp (E), Isolate 26: Streptococcus salivarius (F), Streptococcus mutans MTCC#890 (G) and Streptococcus mitis MTCC#2696 (H). School of Science, SVKM s NMIMS (deemed-to-be) University Page 100

34 The identified isolates, Streptococcus milleri, Streptococcus mitior, Streptococcus sanguinis and Streptococcus salivarius as well as S. mutans and S. mitis belong to viridans streptococci, a heterologous group of very poorly defined organisms belonging to the genus Streptococcus (Facklam, 1977; Waitkins et al., 1980). Viridans streptococci form a significant part of the flora of the human oral cavity and are associated with several disease conditions including dental caries, infective endocarditis and septicaemia, as well as purulent infections of oral and other sites (Beighton et al., 1991). The identification of viridans streptococci is a difficult undertaking due to lack of uniformity in cultural and biochemical characteristics and the existence of different nomenclature and classification schemes (Pearce et al., 1995). Efforts are being undertaken to develop better identification strategies using analytical methods like mass spectroscopy (Freidrichs et al., 2007). S. sanguinis, S. salivarius, S. mitis, S. milleri are normal commensal flora recognized as pioneer colonizers of the tooth surface while S. mutans colonizes the tooth surface comparatively later and possesses cariogenic properties (Pearce et al., 1995; Law et al., 2007; Kreth et al., 2008; Tamura, 2008; Heng et al., 2011; Ogawa et al., 2011). The test consortium thus prepared with the inclusion of these organisms does reflect, to an extent, the heterogeneity of the oral microflora observed in early biofilms. The evaluation of the anti-cariogenic potential of plant extracts against such a multispecies test consortium will aid in making predictions about the probable behaviour of the extracts in vivo. School of Science, SVKM s NMIMS (deemed-to-be) University Page 101