234 Volume 45 Number 4 December 2012 The p�� changes of artificial saliva after interaction with oralp�� changes of artificial saliva after interaction with oralchanges of artificial saliva after interaction with oral micropathogen Basri a. Gani,1 Cut Soraya,2 Sunnati,3 abdillah imron nasution,1 nurfal Zikri1 and rina rahadianur1 1 Department of Oral Biology 2 Department of Conservative Dentistry 3 Department of Periodontics 4 Dentistry Program Study, Medical Faculty, Universitas Syiah Kuala Banda Aceh - Indonesia abstract Backgorund: Saliva contains several protein elements, exocrine proteins and antibodies, such as lactoferrin, sIgA, peroxidase, albumin, polypeptides, and oligopeptides that contribute to the defense of oral mucosa and dental pellicle to prevent infection caused by oral micropathogen, such as Candida albicans, Streptococcus mutans and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans). Those micropathogens have a role to change salivary pH as an indicator of oral disease activities. Purpose: This study was aimed to analyze the changes of artificial saliva pH after interaction with S. mutans, C. albicans, and A. Actinomycetemcpmitans. Methods: The materials used in this study consist of S. mutans (ATCC 31987), C. albicans (ATCC 10231), A. actinomycetemcomitans (ATTC 702 358), and artificial saliva. To examine the pH changes of artificial saliva, those three microbiotas were cultured and incubated for 24 hours. results: The results showed that the interactions of S. mutans, C. albicans, and A. actinomycetemcomitans in the artificial saliva can change the salivary on neutral. There were no significant difference with the control treatment salivary pH 4, 5, 6, 8, and 9 (p>0.05). Similarly, there was also no significant difference when those three microorganism interacted each other in the artificial saliva (p<0.05). Conclusion: It can be concluded that the biological activity of S. mutans, C. albicans, and A. actinomycetemcomitans in artificial saliva can change the salivary pH into neutral. It indicates that those microbiotas mutually supported and cooperated in influencing the biological cycle of the oral cavity with salivary pH as an indicator. Key words: Salivary pH, Candida albicans, Streptococcus mutans, Aggregatibacter actinomycetemcomitans abstrak latar belakang: Saliva merupakan cairan eksokrin yang mengandung unsur protein dan antibodi seperti sIgA laktoferin peroksidase, albumin, polipeptida dan oligopeptida yang berperan pada pertahanan mukosa rongga mulut dan gigi guna mencegah infeksi oral mikropatogen seperti C. albicans, S. mutans, dan A. actinomycetemcpmitans. Patogenesis ketiga oral mikropatogen tersebut diawali dengan mempengaruhi perubahan pH saliva sebagai langkah invasi dan infeksi pada mukosa oral dan pelikel gigi. tujuan: Penelitian ini bertujuan untuk untuk mengetahui perubahan pH saliva buatan setelah diinteraksikan dengan S. mutans, C. albicans, dan A. Actinomycetemcpmitans. Metode: Materi penelitian ini berupa Streptococcus mutans strain ATCC 31987, Candida albicans strain ATCC 10231, Aggregatibacter actinomycetemcomitans strain ATTC 702358, dan saliva buatan. Untuk mengetahui perubahan pH saliva, maka ketiga mikrobiota tersebut dikultur dan untuk menguji perubahan pH saliva dilakukan uji interaksi ketiga mikroorganisme tersebut dalam saliva buatan selama 24 jam dengan pengaturan pH saliva sebagai indikator hasil penelitian. hasil: Hasil penelitian menunjukkan interaksi S. mutans, C. albicans, dan A. actinomycetemcomitans dalam saliva buatan mampu mereduksi perubahan pH saliva mengarah ke pH netral dengan kontrol perlakuan pH saliva 4, 5, 6, 8, dan pH 9 secara statistik tidak tidak menunjukkan perbedaan bermakna (p>0,05), begitu juga ketika dilakukan interakasi diantara masing-masing mikroorganisme tersebut dalam saliva buatan menunjukkan adanya perbedaan bermakna (p<0,05). Kesimpulan: Dapat disimpulkan bahwa aktivitas biologi S. Research Report 235Gani, et al.: The p�� changes of artificial saliva after interaction with oral micropathogenp�� changes of artificial saliva after interaction with oral micropathogenchanges of artificial saliva after interaction with oral micropathogen mutans, C. albicans, dan A. actinomycetemcomitans dalam saliva buatan mampu merubah pH Saliva sekaligus mempertahankan pH netral. Hal ini menggambarkan bahwa mikrobiota tersebut saling mendukung dan bekerjasama dalam mempengaruhi siklus biologi rongga mulut dengan pH saliva sebagai indikator. Kata kunci: pH Saliva, Candida albicans, Streptococcus mutans, Aggregatibacter actinomycetemcomitans Correspondence: Basri A. Gani: c/o: Bagian Biologi Oral, Program Studi Kedokteran Gigi, Fakultas Kedokteran Universitas Syiah Kuala Darussalam-Banda Aceh. 23111. Email: basriunoe@yahoo.com introduction As pathogens in oral cavity, Streptococcus mutans (S. mutans) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) are considered as the cause of dental caries and periodontitis.1,2 Although infection caused by them is different, but it is known that both of these bacteria support each other in pathogenic process, especially in biofilm formation of dental caries infection and periodontitis.3 These two micropathogens also play an important role on the activity of moleculer fungus, Candida albicans (C. albicans), as an agent of oral candidiasis infection. In alkaline condition, those two bacteria can facilitate C. albicans to form biofilms on oral mucosa,4,5 on the other hand, in acidic condition, C. albicans is precisely able to adapt to changes in the acidic environment of the oral cavity, that is affected by salivary pH.6 It means that the salivary pH can determine the pathogenicity of those micropathogens. Saliva is exocrine fluid containing water, approximately about 99%. Other supporting elements consist of an organic component involving sodium, calcium, potassium, magnesium, bicarbonate, chloride, rodanida and thiocynate (CNS), phosphate, potassium, and nitrate; meanwhile, inorganic components consist of amylase, peroxidase, maltase, albumin protein, kretinin, mucin, vitamin C, amino acids, lysozyme, lactic acid, and hormones, such as testosterone and salivary cortisol; besides, there are also sIgA antibodies, lactoferrin, polypeptides and oligopeptides that contribute to oral mucosal defense and dental pellicle.7, 8 The degree of acidity (pH) of saliva is affected by diet, stimulation of salivary secretion, and activity of oral microorganisms. Diet with high carbohydrate can either lead to a decrease in salivary pH, accelerate the demineralization of tooth enamel, or produce acid through glycolysis process that can lower the salivary pH into the critical one (5.5 to 5.2).9 In contrast, the alkaline properties of saliva can either neutralize the acidity of the mouth, reduce tooth decay, prevent the formation of plaque and calculus, or reduce the risk of periodontitis. Furthermore, calcium contained in saliva may play a role in tooth enamel remineralization.10 The changes of the biological properties of the saliva that are likely to affect oral biological abnormalities, such as xerostomia are caused by the imbalance of salivary pH regulation.11 In addition, the critical pH of saliva is also worsened by carbohydrate fermentation activities triggered by a number of pathogenic oral microorganisms, such as C. albicans, S. mutans, and A. actinomycetemcomitans.12 The changes of the salivary pH in oral soft tissues are often associated with dental caries, periodontitis, and oral candidiasis. The changes of the pH of acidic and alkaline saliva can trigger saliva viscosity and facilitate the fermentation of carbohydrates and salivary proteins that can cause the imbalance of the growth of those three micropathogens11 since S. mutans can survive only at the critical pH of 4.5-5.0,1 C. albicans can grow only at the pH of 4.5-6.5,13 while A. Actinomycetemcomitans can only grow better at the pH of 7-8.5.2 In addition to being considered as pathogens in the oral cavity infections, those three microorganisms also contribute to endocarditis infection.14 Antibacterial medicines can either accelerate the growth of fungi or provide the threat of chronic candidiasis oral infections. Thus, the pH of the oral cavity must be adjusted with salivary pH as an indicator and supporter of biological activity in oral cavity preventing the virulence factors of those three micropathogens from biofilm formation, colonization, invasion and infection of the host. Therefore, saliva specifically becomes the controler of pathogenic microorganism activities in oral cavity through salivary pH regulation in order to maintain the pH balance of the oral cavity. As a result, this study was aimed to analyze the changes of salivary pH (acidic and alkaline) after interaction with C. albicans, S. mutans, and A. actinomycetemcomitans as well as after those interaction with each other. Finally, the results of this study are expected to be able to prevent invasion and infection caused by oral micropathogens, mainly on dental caries, periodontitis, and oral candidiasis through salivary pH control in order to regulate oral biological and ecological cycles. materials and methods This study is an in-vitro laboratory experiment conducted in the Laboratory of Microbiology and Immunology, Faculty of Veterinary, Syiah Kuala University in Banda Aceh. Materials used in this study were a laboratory strain of S. mutans, ATCC 31 987, and a laboratory strain of C. albicans, ATCC 10231, obtained from the Laboratory of Microbiology, Faculty of Veterinary, Syiah Kuala University, as well as a laboratory strain of A. Actinomycetemcomitans, ATTC 702 358, 236 Dent. J. (Maj. Ked. Gigi), Volume 45 Number 4 December 2012: 234–238 obtained from the Laboratory of Oral Biology, Faculty of Dentistry, Universitas Indonesia. Those three pathogenic microorganisms were used as biological control or balance of acid and alkaline environment changes. Other materials used as artificial saliva were 0.702 g NaCl, 0.221 g KCN, 1.495 g NaHCO3, 1.153 g KCl, 1.100 g H2NCONH2, 0.213 g Na2HPO4, and 0.204 g KH2PO4 obtained from the Laboratory of Biochemistry, Faculty of Medicine, Universitas Indonesia. The artificial saliva was used as a reference for the interactions of those micropathogens, especially in acidic and alkaline environments. The various components of these materials were then analyzed with various forms of analysis approaches. The laboratory strains of A. actinomicetemcomitans and S. mutans were cultured by using treak late. The laboratory strain of S. mutans was cultured on Muller Hilton agaragargar (MHA) media, while A. actinomicetemcomitans using nutrient agar (NA) media. Each of those two bacteria wasutrient agar (NA) media. Each of those two bacteria wasagar (NA) media. Each of those two bacteria wasgar (NA) media. Each of those two bacteria was then put into anerobic jar and incubated at 370 C for 48 hours. For confirmatory test, those bacteria were stained. Afterwards, they were cultured in TSB liquid medium for 48 hours in order to become samples for analysis. On the other hand, the laboratory strain of C. albicans was taken about 1 Ose, and then cultured on selective Sabouraud dextrose agar (SDA) media using T scratchingdextrose agar (SDA) media using T scratchingextrose agar (SDA) media using T scratchingagar (SDA) media using T scratchinggar (SDA) media using T scratching technique. Next, it was incubated in an incubator for 24- 48 hours at 370 C. Colonies of C. albicans grown on SDA media were suspended by taking a colony of C. albicans from the SDA media, which was then put into peptone solution. The level of turbidity of C. albicans in the peptone solution was compared with Mc Farland solution (1.5 x 108 CFU/ml). S. mutans and A. actinomicetemcomitans, that have been cultured from TSB liquid medium, and C. albicans, that has been cultured in peptone, were respectively centrifuged at 2000 rpm for 5 min. Their residue was then collected and added with 15 ml PBS. Afterwards, 15 ml PBS was also added to the bacteria and C. albicans was added with 15 ml peptone solution. They were then vortexed and resuspended. Next, each of these microorganisms was taken and put into 2 ml of the artificial saliva prepared with pH of 4, 5, 6, 8, and 9. Afterwards, they were incubated for 72 hours, and every 24 hours the changes of the salivary pH were measured by using a pH meter, and the artificial saliva with normal pH was used as control. Finally, data obtained from the interaction between S. mutans, C. albicans and A. actinomycetemcpmitans in the artificial saliva were statistically analyzed with a normality test, Kolmogorov-Smirnov test, followed with repeatedrepeatedepeated measures and ANOVA test using SPSS software.easures and ANOVA test using SPSS software. results The results of this study were then reported in two forms of analysis, the analysis of the changes of the salivary pH after interacted with oral micropathogens (Table 1) and the analysis of the changes of the pH of the artificial saliva after interacted with oral micropathogen (Table 2). The pH of the saliva that has been interacted with S. mutans, C. albicans, and A. actinomycetemcomitans was measured 3 times during 24 hours. Each of the pH values presented in the table was the average value derived from the examintaions conducted three times. discussion Many researches on S. mutans which cause dental caries, A. actinomycetemcomitans which cause periodontitis, and C. albicans which cause oral candidiasis, have showed that they can cause oral health problems, one of which is related to saliva pH imbalance.15 This is due to the salivary pH that has an important role to regulate both metabolic activities of normal flora microbiotas and biological balance of oral cavity. Thus, the changes of the saliva pH can cause the table 1. The changes of the pH of the artificial saliva after interacted with S. mutans, C. albicans, and A. actinomycetemcomitans types of microorganism in the artificial saliva the changes of the salivary ph ph 4 ph 5 ph 6 ph 8 ph 9 S. mutans 5.79 5.25 6.38 7.38 7.53 C. albicans 4.71 5.59 6.42 7.16 7.29 A. actinomycetemcomitans 5.84 6.75 7.91 8.49 8.43 table 2. The changes of the pH of the artificial saliva after S. mutans, C. albicans, and A. actinomycetemcomitans interacted each other types of microorganism in the artificial saliva the changes of the salivary ph ph 4 ph 5 ph 6 ph 8 ph 9 S. mutans and C. albicans 6.76 6.93 7.07 8.18 8.19 S. mutans and A. actinomycetemcomitans 6.47 6.53 7.04 7.95 7.82 A. actinomycetemcomitans and C. albicans 7.32 7.23 7.25 7.27 7.34 237Gani, et al.: The p�� changes of artificial saliva after interaction with oral micropathogenp�� changes of artificial saliva after interaction with oral micropathogenchanges of artificial saliva after interaction with oral micropathogen normal flora microorganisms of the oral cavity evolved into a pathogen accelerating invasion, inflammation and infection of the host.16 The changes of the pH of the artificial saliva after interacted with S. mutans, C. albicans, and A. actinomycetemcomitans for 24 hours (Table 1) indicate that those three microorganisms have the properties of adaptation to its pH growth. Besides, it is also known that the saliva could also serve as a biological control for the activities of the oral microbiotas contained in it although in general the changes were not significantly different (p>0.05) from the control pH, so it is suspected that there was adjustment phase for the properties of each of these microorganisms. This nature will change as there is a change in the ecology and biology of the oral cavity caused by an imbalance triggered by various factors, such as disorders of hormonal system and oral cavity pH.17 Temperature factors, chemical factors, and psychological factors can also become the main determinant of the pathogenic properties of oral microbiota, such as S. mutans, C. albicans, and A. actinomycetemcomitans.18 This shows that the virulence properties of the three oral microbiotas will become active and pathogenic when influenced by those factors.19 It is known that the interactions between S. mutans and A. actinomycetemcomitans, and between C. albicans and A. actinomycetemcomitans as shown in table 2 were significant (p<0.05). It means that those oral micropathogens could lower the control pH 8 and 9 to neutral one (7.2 to 7.9). It is related with the bioactive components contained in the artificial saliva, that is possible to affect the changes of the pH caused by bacteria and fungi, while in the normal condition, sIgA and lactoferrin proteins have a role to inhibit the biological activity of the oral microbiota associated with the changes of the salivary pH.17 Interaction of S. mutans and C. albicans in the artificial saliva can actually make the pH more alkaline, specifically the control pH 4, 5, and 6. It can be assumed that the microorganisms, in addition to making the pH stable in an acidic environment, is also able to adapt to an alkaline environment, called the instability of acid.19 S. mutans and C. albicans are two microorganisms that always express virulence properties when the instability of acid occurs in the oral cavity, and their growth is still stable despite the critical acidic pH (3 to 4.5).1 The instability of acid can trigger and stimulate the virulence properties of the microorganisms to become more pathogenic, as well as can affect the biological properties of saliva.20 S. mutans not only have both aciduric and acidogenic properties by producing a dextran, which play a role in the formation of biofilm on the tooth surface before causing colonization, invasion and infection of the tooth enamel and chronic caries.21 Meanwhile, C. albicans can live in various acidic and alkaline pH as well as in neutral pH although at 4.5- 6.5 pH. The activity and expression of manoprotein of C. albicans are affected by changes in temperature and pH that can inhibit protein synthesis and reduce manan activity (cell wall protein) that affect the growth of C. albicans.22 As bacteria which are stable in alkaline environments, A. actinomycetemcomitans and C. albicans were able to make the pH of the artificial saliva neutral or alkaline (Table 2). This is because A. actinomycetemcomitans has strong adhesion properties and can colonize well in saliva, so the condition of the salivary pH can be controlled by the bacteria in order to balance the pH and the growth of C. albicans.23,24 In molecular level, S. mutans (39-864) molecules, played a role in attachment to the salivary protein, so the localization of acid products occured with a high concentration on the surface of tooth enamel. This acid will lower the pH of the oral cavity, so it can cause demineralization of the enamel.14 In cellular level, two proteins of the cell wall were hydrolyzed, such as fructose hydrolyzed by fructosyltransferase and dextran hydrolyzed by glucosyltransferase (GTF). This process aims to maintain the instability of the critical pH of GTF and glucan binding protein (GBP) as trigger protein to produce lactic acid either by S. mutans or by other bacteria that mediates colonization on tooth enamel, so glucose- dextran can lower saliva properties either as a protector or as antibacteria on tooth surface.25 Interactions among microbiotas in the oral cavity were either facilitated by components of salivary proteins or caused by interactions of molecule bondings, such as interactions between proteins, hydrophobicity bonding on cell surface, electrostatic bonding, and biofilm matrix protein molecule bonding that interacts with either C. albicans, S. mutans or A. actinomycetemcomitans. The bonding and interaction among those protein molecules then can form coaggregasion and cause adhesion among microorganisms.15 One function of the molecular interaction among the microbiotas is to maintain the pH balance of oral cavity, including salivary pH, as biology and ecology control of the oral cavity. The acidity of the saliva is also dependent on hydrogen ions contained in the solution. At alkaline pH, C. albicans unable to adapt because the increasing of cell wall polysaccharide reactiviting and components of salivary proteins, thereby affecting cell wall resistance and instability of the absorption of hydrogen ions caused by cytoplasma that can interfere energy supply as a results of cell lysis. 24 The research of oral candidiasis in denture users indicates that the interaction of C. albicans, Streptococcus Sp, and Actinomyces Sp in the saliva may decrease the population of C. albicans. In addition, this condition occurs as a result of the activity of lipopolysaccharide molecules produced by bacteria that are able to inhibit the formation of hyphae of C. albicans, so this condition then can facilitate the penetration of protein molecules of the bacterial surface into the cells of C. albicans.12, 2626 Therefore, this research showed that there was a close relation between the influence of oral activity of microphatogen and the changes of the pH of the artificial saliva since the results of this research provide information 238 Dent. J. (Maj. Ked. Gigi), Volume 45 Number 4 December 2012: 234–238 that in normal conditions, C. albicans, S. mutans, and A. Actinomycetemcomitans can consistently maintain pH of the oral cavity in order to maintain their stable life as normal floras, but they will change into pathogens when the salivary pH changes. This research also provides information that the salivary pH will change when there is biological imbalance of oral cavity, thus, it is necessary to balance the salivary pH as an attempt to control the growth of oral pathogenic microbiotas and to support ecological and biological activities in oral cavity. In other words, it can also be considered as an attempt to maintain the integrity of pathogenic bacteria as normal flora microorganisms, so it can prevent infection and oral diseases, such as dental caries, periodontitis and oral candidiasis. It can be concluded that biological activities of S. mutans, C. albicans, and A. actinomycetemcomitans in artificial saliva can change salivary pH as well as maintainsalivary pH as well as maintainlivary pH as well as maintain its neutral pH. This condition indicates that the microbiotas can support and co-operate each other in influencing the biological cycle of the oral cavity with saliva pH as an indicator. acknowledgments This study was financially supported by Universitas Syiah Kuala, Ministry of Education, in accordance with the Agreement of Research Assignment for �oung Lecturer, Number: 2159/H11/LK-PNBP/2011, Dated May 18, 2011. references 1. Gani BA. Acidogenic and aciduric properties of Streptococcus mutans as the bacteriostatic against oral microbiota pathogen. Cakradonya Dental Journal 2010; 2(1): 128–36. 2. Ardila CM, Alzate J, Guzmán IC. Relationship between Gram negative enteric rods, Aggregatibacter actinomycetemcomitans, and clinical parameters in periodontal disease. J Indian Soc Periodontol 2012; 16(1): 65–9. 3. Loesche W. Dental caries and periodontitis: contrasting two infections that have medical implications. Infect Dis Clin North Am. 2007; 21(2): 471–502. 4. Filoche S, Wong L, Sissons CH. Oral biofilms: Emerging concepts in microbial ecology. J Dent Res 2010; 89(1): 8–18. 5. Vílchez R, Lemme A, Ballhausen B, Thiel V, Schulz S, Jansen R, Sztajer H, Wagner-Döbler, I. Streptococcus mutans inhibits Candida albicans hyphal formation by the fatty acid signaling molecule trans- 2-decenoic Acid (SDSF). ChemBioChem 2010; 11: 1552–62. 6. Puy CL. The rôle of saliva in maintaining oral health and as an aid to diagnosis. Med Oral Patol Oral Cir Bucal 2006; 11: 449–55. 7. Dawes C. Salivary flow patten and the health of hard and soft oral tissues. J Am Dent Assoc 2008; 139: 185–245. 8. Damle SG, Vidya I, �adav R, Bhattal H, Loomba A. Quantitative deter m ination of inorganic constituents in saliva and their relationship with dental caries experience in children. Dentistry Journal 2012; 2(3): 131–5. 9. Ilie O, van Loosdrecht MCM, Picioreanu C. Mathematical modelling of tooth demineralization and pH profiles in dental plaque. Journal of Theoretical Biology 2012; 309: 159–75. 10. Vijayaprasad KE, Ravichandra KS, Vasa AA, Suzan S. Relation of salivary calcium, phosphorus and alkaline phosphatase with the incidence of dental caries in children. J Indian Soc Pedod Prev Dent 2010; 28: 156–61. 11. Lončar B, Stipetić MM, Baričević M, Risović D. The effect of low- level laser therapy on salivary glands in patients with xerostomia. Photomedicine and Laser Surgery 2011; 29(3): 171–5. 12. Morales DK, Hogan DA. Candida albicans interaction with bacteria in the context of human health and disease. Microbiol Mol Biol Rev 2010; 21: 245–345. 13. Gani BA. Diversity of Candida albicans virulence factor as the infectious recognizer. Cakradonya Dental Journal 2011, 3(1): 323–31. 14. Basri AG, Tanzil A, Mangundjaja S. Molecular aspect of the Streptococcus mutans virulence properties. Indonesian Journal of Dentistry 2006; 13(2): 107–14. 15. Taubman MA, Genco RJ, Hillman JD. The specific pathogen-free human: A new frontier in oral infectious disease research. Adv Dent Res 1989; 3(l): 58–68. 16. Hasturk H, Kantarci A, Thomas E, Dyke V. Oral inflammatory diseases and systemic inflammation: role of the macrophage. Front Immunol 2012; 3(118): 1–17. 17. Marcotte H, Lavoie MC. Oral microbial ecology and the role of salivary immunoglobulin A. Microbiol Mol Biol Rev 1998; 62(1): 71–109. 18. Fernanda GB, Camila FO. Amanda F, Cristina K, Vanderlei SB, Denise M P, Spolidório, Josimeri H, Carlos ADSC. In vitro effect of low-level laser therapy on typical oral microbial biofilms. Braz Dent J 2011; 22(6): 502–10. 19. Saha S, Tomaro-Duchesneau C, Malhotra M, Tabrizian M, Prakash S. Suppression of Streptococcus mutans and Candida albicans by Probiotics: an In vitro Study. Dentistry Journal 2012; 2(6): 141. 20. Niemi LZ. Host ligands and oral bacterial Adhesion: Studies on phosphorylated polypeptides and gp-340 in saliva and milk. Dissertation. Umeå University: Umeå Department of Odontology; 2010. p. 11–26. 21. Arthur RA, Cury AADC, Mattos-Graner RO, Rosalen PL, Vale GC, Leme AFP, Cury JA, Tabchoury CPM. Genotypic and phenotypic analysis of S. mutans isolated from dental biofilms formed in vivo under high cariogenic conditions. Braz Dent J 2011; 22(4): 267–74. 22. Kruppa M, Rachel R. Greene, Noss I, Douglas W. Lowman, Williams DL. Candida albicans increases cell wall mannoprotein, but not mannan, in response to blood, serum and cultivation at physiological temperature. Oxford: Oxford University Press; 2011. p. 1–28. 23. Ouhara K, Komatsuzawa H, Shiba H, Uchida �, Kawai T, Sayama K, Hashimoto K, Taubman MA, Kurihara H, Sugai M. Actinobacillus actinomycetemcomitans outer membrane protein 100 triggers innate immunity and production of β-defensin and the 18-kilodalton cationic antimicrobial protein through the fibronectin-integrin pathway in human gingival epithelial cells. Infection and Immunity 2006; 74(9): 5211–20. 24. Lima LL, Farias FF, Carvalho MAR, Alviano CS, Farias LM. Influence of abiotic factors on the bacteriocinogenic activity of Actinobacillus actinomycetemcomitans. Microbiology 2002; 153(4): 249–52. 25. Bikandi J, Moragues, MD, Quindos, G, Polonelli L, Ponton J. Influence enviromental pH on reactivity of Candida albicans With Salivary IgA. J Dent Rest 2000; 79: 1439. 26. Mayocchi K, Restelli, Alfredo M. Ultra structural study of the effect of Candida albicans on the surface of the temporary enamel acta microspora. American Dental Association 2009; 18: 453–4.