Acquisition of iron from human transferrin by Porphyromonas gingivalis: a role for Arg- and Lys-gingipain activities

Porphyromonas gingivalis, a key causative agent of adult periodontitis, is known to produce a variety of virulence factors including proteases. The aim of this study was to evaluate the participation of Arg- and Lys-gingipain activities of P. gingivalis in the acquisition of iron from human transferrin and its subsequent utilization in growth. Iron-saturated transferrin was found to support the long-term growth of P. gingivalis. Our results indicated that P. gingivalis does not produce siderophore and does not possess ferric reductase and transferrin-binding activities. Incubating transferrin with P. gingivalis resulted in degradation of the protein, a step that may be critical for the acquisition of iron from transferrin. Spontaneous and site-directed mutants of P. gingivalis deficient in one or several proteases were used to demonstrate the key role of specific enzymes in degradation of transferrin and subsequent utilization for growth. The lack of both Arg- and Lys-gingipain activities (mutants M1 and KDP128) was associated with an absence of degradation of transferrin and the incapacity of bacteria to grow in the presence of transferrin as the sole source of iron. It was also found that the Lys-gingipain activity is more critical than the Arg-gingipain activity since the mutant KDP112 (deficient in Arg-gingipain A and B) could grow whereas the mutant KDP129 (deficient in Lys-gingipain) could not. The fact that growth of mutant KDP112 was associated with a lower final optical density and a generation time much longer compared with the parent strain suggests that the Arg-gingipain activity also participates in the acquisition of iron from transferrin. Selected inhibitors of cysteine proteases (TLCK, leupeptin and cathepsin B inhibitor II) were tested for their capacity to reduce or inhibit the growth of P. gingivalis under different iron conditions. All three inhibitors were found to completely inhibit growth of strain ATCC 33277 in a medium supplemented with transferrin as the source of iron. The inhibitors had no effects when the bacteria were grown in a medium containing hemin instead of transferrin. The ability of P. gingivalis to cleave transferrin may be an important mechanism for the acquisition of iron from this protein during periodontitis.     

表没食子儿茶素没食子酸酯对牙龈卟啉单胞菌细菌毒力抑制作用的体外研究

目的 研究表没食子儿茶素没食子酸酯(epigallocatechin-3-gallate,EGCG)对牙龈卟啉单胞菌(Por-phyromonas gingivalis,P.gingivalis)体外抑菌活性及EGCG对P.gingivalis诱导人牙龈成纤维细胞(human gingi-val fibroblasts...     

Inhibitory effect of green tea catechins on cysteine proteinases in Porphyromonas gingivalis

The purpose of this study was to examine the effects of catechins and their derivatives on the activities of Arg-gingipain (Rgp) and Lys-gingipain (Kgp) in Porphyromonas gingivalis. Catechin derivatives, which included (-)-epigallocatechin gallate, (-)-epicatechin gallate, (-)-gallocatechin gallate, and (-)-catechin gallate, significantly inhibited the Rgp activity. The 50% inhibitory concentrations (IC50s) of these catechin derivatives for Rgp ranged from 3 to 5 microm. While (-)-epigallocatechin and (-)-gallocatechin moderately inhibited Rgp activity (IC50s, 20 microm), (-) -epicatechin, (+)-catechin, and gallic acid were not effective, with IC50s greater than 300 microm. Further, some of the catechin derivatives tested also inhibited the Kgp activity, though to a lesser extent than inhibition of the Rgp activity. These findings suggest that green tea catechins may have the potential to reduce periodontal breakdown resulting from the potent proteinase activity of P. gingivalis.     

Studies on the aminopeptidase activities of Porphyromonas gingivalis

Porphyromonas gingivalis is an asaccharolytic bacterium that requires nitrogen substrates as carbon and energy sources. The aims of this study were to investigate the aminopeptidase activities of P. gingivalis and to evaluate the effect of aminopeptidase inhibitors on bacterial growth. Only arginine aminopeptidase and dipeptidyl aminopeptidase IV activities were detected. Experimental evidence was obtained suggesting that the Arg-gingipains of P. gingivalis can function as both an endopeptidase and an aminopeptidase. Firstly, the arginine aminopeptidase activity was found to be inhibited by leupeptin, a well-known inhibitor of Arg-gingipain activity. Secondly, a preparation of Arg-gingipain activity could hydrolyze the chromogenic substrate for arginine aminopeptidase. Lastly, a mutant of P. gingivalis constructed via gene disruption by use of suicide plasmids and deficient in both Arg-gingipain A and B was also devoid of arginine aminopeptidase activity. To investigate the key role of aminopeptidase activities in growth of P. gingivalis, aminopeptidase inhibitors were incorporated in the culture medium prior to inoculation. Bestatin and actinonin were the only ones to inhibit growth of P. gingivalis. Their mechanism of growth inhibition appears to be different but does not involve inhibition of the two major aminopeptidase activities (arginine aminopeptidase and dipeptidyl aminopeptidase IV).     

The prevalence and pathogenic differences of Porphyromonas gingivalis fimA genotypes in patients with aggressive periodontitis

BACKGROUND: The fimA gene, which encodes fimbrillin (FimA), is found in Porphyromonas gingivalis and has been classified into six genotypes based on nucleotide sequence. P. gingivalis that possesses the type II fimA gene is prevalent in adult periodontitis. OBJECTIVES: The aim of this study was to investigate the prevalence of P. gingivalis fimA genotypes in Japanese aggressive periodontitis patients, and to examine their virulence. METHODS: Subgingival plaque samples were obtained from 223 sites in 18 aggressive periodontitis patients and 95 sites in 22 periodontally healthy young adults. Actinobacillus actinomycetemcomitans, P. gingivalis and Tannerella forsythensis detection, determination of the fimA genotype in P. gingivalis, and the quantification of P. gingivalis were analyzed by polymerase chain reaction (PCR) methods. The proteolytic activities of the P. gingivalis fimA type I and fimA type II were also examined. RESULTS: In aggressive periodontitis patients, the most prevalent fimA genotype was the type II (46.7%), followed by the type Ib and type I, whereas in healthy subjects, the type I fimA was the only genotype detected. The number of P. gingivalis pathogens was the greatest in the type I fimA positive sites, and the frequency of coexisting A. actinomycetemcomitans and T. forsythensis was highest in the type II fimA positive sites in the aggressive periodontitis patients. Both the arginine-specific cysteine proteinase (Arg-gingipain) and lysine-specific cysteine proteinase (Lys-gingipain) activity of the P. gingivalis fimA type I strain were significantly higher than those of the fimA type II strains. CONCLUSIONS: These results suggest that differences in virulence exist among different fimA genotypes. Coadherence with other pathogens in P. gingivalis fimA type II-associated aggressive periodontitis and quantitative increases in P. gingivalis in fimA type I-associated aggressive periodontitis are related to this virulence.     

Inhibition of proteolytic, serpinolytic, and progelatinase-b activation activities of periodontopathogens by doxycycline and the non-antimicrobial chemically modified tetracycline derivatives

BACKGROUND: Tetracyclines, particularly doxycycline (Doxy), and their non-antimicrobial chemically-modified derivatives (CMTs) inhibit the activities of human matrix metalloproteinases (MMPs), and reduce the severity and progression of periodontal disease in animal models and humans. In this study, the effects of Doxy and CMT-1, -3, and -5 on proteolytic, serpinolytic, and progelatinase-B activation activities of potent periodontopathogens were studied. METHODS: The effect of Doxy and CMTs (0.5 to 50 microM) on proteolytic activities were investigated by incubating bacteria with chromogenic substrates or human serum albumin. A collagenolytic fraction of Porphyromonas gingivalis was used to evaluate the effect of these substances on collagenolytic (type I collagen) and serpinolytic (alpha1-proteinase inhibitor) activities. Lastly, the effect of Doxy on progelatinase-B (pro-MMP-9) activation by purified proteinases from P. gingivalis and Treponema denticola was investigated by SDS-PAGE/Western immunoblotting. RESULTS: Doxy and CMTs, except CMT-5 which lacks the structural elements required for cation chelation, inhibited Arg- and Lys-gingipain activities as well as collagenolytic activity of P. gingivalis. Doxy and CMTs did not markedly affect the chymotrypsin-like activity of T. denticola but inhibited its trypsin-like activity. In addition, degradation of human serum albumin by cells of P. gingivalis and T. denticola was strongly inhibited by Doxy and CMT-1. Doxy and CMT-1 also inhibited the inactivation of alpha1-proteinase inhibitor (serpinolytic activity) by a collagenolytic fraction of P. gingivalis. Lastly, Doxy prevented the latent to active conversion of human neutrophil progelatinase-B (pro-MMP-9) by Arg-gingipains A/B of P. gingivalis but not by the chymotrypsin-like proteinase of T. denticola. CONCLUSIONS: Data from this study suggest that Doxy and CMTs have the potential to inhibit the periodontopathogenic bacterial proteinases, which contribute to tissue destruction cascades during periodontitis directly and indirectly by triggering the host response.     

牙龈卟啉单胞菌脂多糖对人牙周膜成纤维细胞胶原吞噬作用的影响

目的研究牙周病致病菌牙龈卟啉单胞菌脂多糖（LPS）对体外培养的人牙周膜成纤维细胞（hPDLF）胶原吞噬作用的影响。方法将不同质量浓度的LPS加入体外培养的hPDLF 48 h后,采用荧光定位术和流式细胞技术检测hPDLF胶原吞噬率的变化。结果LPS导致hPDLF胶原吞噬率显著增加（P<0.05）。结论牙龈卟啉单胞菌脂多糖具有促进hPDLF吞噬胶原的作用,可能是牙周组织破坏机制之一。     

Antibacterial Activity of Curcumin Against Periodontopathic Bacteria

Background: Curcumin is a polyphenol extracted from root of turmeric and known to possess multifunctional properties, including antibacterial activity. Although previous studies have investigated the effects of curcumin on microorganisms, available knowledge on the effects of curcumin on periodontopathic bacteria is still limited. In this study, the antibacterial effect of curcumin on periodontopathic bacteria is investigated, particularly Porphyromonas gingivalis. Methods: Representative periodontopathic bacteria were cultured in media with and without various curcumin concentrations, and the optical density at 600 nm was measured for 60 hours. The inhibitory effect of curcumin on P. gingivalis Arg‐ and Lys‐specific proteinase (RGP and KGP, respectively) activities were assessed using spectrofluorophotometric assay. Analysis of biofilm formation by P. gingivalis with or without Streptococcus gordonii was conducted using confocal laser‐scanning microscopy (CLSM). Results: Curcumin inhibited the growth of P. gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Treponema denticola in a dose‐dependent manner. Bacterial growth was suppressed almost completely at very low concentrations of curcumin. Conversely, 100 μg/mL curcumin did not suppress the growth of Aggregatibacter actinomycetemcomitans. It also demonstrated inhibitory effects against RGP and KGP activities in a dose‐dependent manner. CLSM revealed that curcumin suppressed P. gingivalis homotypic and P. gingivalis–S. gordonii heterotypic biofilm formation in a dose‐dependent manner. A concentration of 20 μg/mL curcumin inhibited these P. gingivalis biofilm formations by >80%. Conclusion: Curcumin possesses antibacterial activity against periodontopathic bacteria and may be a potent agent for preventing periodontal diseases.     

Effects of Porphyromonas gingivalis on human gingival fibroblasts from healthy and inflamed tissues

Background and Objective:  This study compared the ability of human gingival fibroblasts (HGFs) isolated from healthy and inflamed gingival tissues to degrade collagen in the presence and absence of Porphyromonas gingivalis supernatant.
Material and Methods:  Human gingival fibroblasts were cultured from explants of 21 healthy and 21 inflamed periodontal tissues. The HGFs that grew out of the explants were seeded in the center of six-well plates coated with collagen in the presence and absence of 10% P. gingivalis supernatant. An inflamed and a healthy cell line were also evaluated with Arg-gingipain. After 6 days, Coomassie Blue was used to visualize the collagen cleavage.
Results:  The collagen was totally cleaved in 12 (aggressive) of the 21 cell lines isolated from the inflamed tissues in the presence of P. gingivalis . The remaining nine cell lines (non-aggressive) cleaved only the collagen underneath the cell colonies in the presence of P. gingivalis . Of the healthy tissues, five (aggressive) of the 21 cell lines cleaved all the collagen and 16 cell lines (non-aggressive) only cleaved the collagen underneath the cell colonies in the presence of P. gingivalis . All the collagen was cleaved by an aggressive cell line and only the collagen underneath the cell colonies was cleaved by a non-aggressive cell line in the presence of Arg-gingipain.
Conclusion:  The collagen in the wells was more readily cleaved by the inflamed than by the healthy cell lines, and the difference was statistically significant ( p  = 0.0278). Arg-gingipain gave identical results to the P. gingivalis supernatant.     

Effect of gamma-immunoglobulin on the asaccharolytic growth of Porphyromonas gingivalis

BACKGROUND AND OBJECTIVES: A minimal medium is indispensable for examining the growth properties of the asaccharolytic bacterium, Porphyromonas gingivalis. The purpose of the present study was to improve the widely used KGB medium to support good growth of P. gingivalis. MATERIAL AND METHODS: Growth of P. gingivalis (W50, W83, and ATCC33277) in a minimal medium was monitored by measuring the optical density of the culture during incubation. RESULTS: W50, W83, and ATCC33277 grew poorly with bovine serum albumin as the sole carbon and nitrogen source, and alpha-ketoglutarate had little or no effect on this poor growth. In contrast, FeCl3 improved the growth of W83 and ATCC33277; however, the use of a high concentration of FeCl3 elicited black pigmentation of the cells. Bovine gamma-immunoglobulin greatly recovered the growth defect. None of alpha-ketoglutarate, citrate, or trace metal ions, when used to supplement KGB medium, was required for growth. We determined the optimal conditions for growth, and developed a new simple minimal medium for P. gingivalis (GA medium). Growth of ATCC33277 in GA medium was dependent on gingipains; Arg-gingipains and Lys-gingipain contributed comparably to proliferation of the bacterium. CONCLUSION: These data indicate that GA medium is currently the most reliable minimal medium for examining the growth properties of P. gingivalis.     

Separation of the outer membrane and identification of major outer membrane proteins from Porphyromonas gingivalis

The outer membrane of Porphyromonas gingivalis, an oral strict anaerobe, was isolated by sucrose density gradient centrifugation. The outer membrane obtained by the differential detergent extraction method, previously reported, showed an essentially similar protein pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), confirming that the latter method is suitable for the study of outer membrane proteins in this organism. N-terminal amino acid sequence analysis revealed that major outer membrane proteins in this organism included Arg-gingipain, Lys-gingipain, RagA (a TonB-linked receptor), and putative porins that were homologous to Escherichia coli OmpA.     

Stimulation of Fusobacterium nucleatum biofilm formation by Porphyromonas gingivalis

BACKGROUND/AIMS: Bacterial infection is a major cause of periapical periodontitis. Eradication of these microorganisms from apical lesions is essential to the success of endodontic treatment. The aim of this study was to clarify the molecular interaction between Fusobacterium nucleatum, Porphyromonas gingivalis and other microorganisms associated with periapical periodontitis. METHODS: Microorganisms isolated from periapical lesions were inoculated into type-I collagen-coated polystyrene microtiter plates and maintained at 37 degrees C under anaerobic conditions for 2 days, after which, the quantity of organized biofilm on the plates was evaluated by crystal violet staining. Growth enhancement via soluble factor was evaluated by separated coculture using a 0.4-mum membrane filter. RESULTS: F. nucleatum exhibited strong adherence to type-I collagen-coated polystyrene microplates. Biofilm formation by F. nucleatum was significantly enhanced by P. gingivalis. It was complemented by compartmentalized coculture with P. gingivalis. Enhancement of biofilm formation by P. gingivalis was only slightly reduced by inactivation of its autoinducer-2-producing gene luxS. CONCLUSION: The results suggest that P. gingivalis enhances biofilm formation by F. nucleatum by releasing diffusible signaling molecules other than autoinducer-2.     

Porphyromonas gingivalis mediates the shedding and proteolysis of complement regulatory protein CD46 expressed by oral epithelial cells

Introduction:  Human cells express membrane-bound complement regulatory proteins to prevent complement-mediated autologous tissue damage. In this study, we hypothesized that Porphyromonas gingivalis , the major etiological agent of chronic periodontitis, causes the shedding or proteolysis of the complement regulatory protein CD46 expressed by oral epithelial cells.
Methods:  Oral epithelial cells were treated with a culture of P. gingivalis before measurement of membrane-bound and shed CD46 by enzyme-linked immunosorbent assay (ELISA). The effect of soluble recombinant CD46 on secretion of interleukin-8 (IL-8) by epithelial cells was evaluated by ELISA. The susceptibility of soluble recombinant CD46 to proteolytic degradation by cells and purified Lys-gingipain of P. gingivalis was investigated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis/western immunoblotting analysis.
Results:  Oral epithelial cells treated with a culture of P. gingivalis showed a lower reactivity with antibodies directed to CD46. ELISA revealed that such a treatment resulted in increased amounts of CD46 in the conditioned media suggesting that P. gingivalis caused the shedding of membrane-anchored CD46. Stimulation of epithelial cells with soluble recombinant CD46 induced IL-8 secretion in a dose-dependent manner. Whole cells and purified Lys-gingipain of P. gingivalis degraded recombinant CD46 in a dose-dependent manner.
Conclusion:  This study showed the ability of P. gingivalis to induce the shedding/ proteolysis of CD46 from the surface of oral epithelial cells. This may render host cells susceptible to the complement system and contribute to tissue damage and the inflammatory process in periodontitis.     

Genetic evidence for the relationship of Porphyromonas gingivalis cysteine protease and hemagglutinin activities

Cysteine protease and hemagglutinin activities of Porphyromonas gingivalis have been implicated as virulence factors in periodontitis. In addition, a close structural relationship between these factors has been suggested. In order to examine the molecular basis for such a relationship, we constructed an isogenic mutant. G-102, of P. gingivalis 381 deficient in Arggingipain cysteine protease activity. The mutant displayed not only reduced protease activity but also significantly reduced hemagglutination activity compared with the wild-type strain. Therefore, this investigation provided genetic evidence for the recently proposed structural relationship between Arg-gingipain and the hemagglutinin activity of P. gingivalis strains.     

尼古丁和美卡拉明对牙周致病微生物的影响

目的 探讨尼古丁和美卡拉明对主要牙周致病微生物生长及生物膜形成的影响.方法 采用二倍稀释法,研究尼古丁对牙龈卟啉单胞菌和伴放线放线杆菌的最低抑菌浓度(MIC)和最低杀菌浓度(MBC).通过酶标仪测定吸光度值,研究在1/16 MIC、1/8 MIC、1/4 MIC、1/2 MIC、MIC等不同浓度的尼古丁对这2种细菌生物膜形成的影响.结果 尼古丁对2种细菌的菌悬液的MIC均为8 mg·mL-1,MBC为16 mg·mL-1.尼古丁浓度逐渐增加时,生物膜吸光度值逐渐增大;继续增加浓度,吸光度值减小;在8 mg.mL-1时,吸光度值减小到最低值,最低生物膜抑制浓度为8 mg.mL-1.添加尼古丁受体拮抗剂美卡拉明组较对照组吸光度值高.结论 小剂量的尼古丁能增加牙龈卟啉单胞菌和伴放线放线杆菌的生物膜形成,浓度继续增加则抑制生物膜形成,加入美卡拉明后降低尼古丁浓度,更能明显观察到尼古丁对两种细菌生物膜的促进作用.     

Incidence and antimicrobial susceptibility of Porphyromonas gingivalis isolated from mixed endodontic infections

AIM: To investigate the prevalence of Porphyromonas gingivalis in root canals of infected teeth with periapical abscesses and to investigate the antimicrobial susceptibility of this species to some frequently prescribed antibiotics. METHODOLOGY: Samples were obtained from 70 root canals of abscessed teeth. Microbial sampling, isolation and bacterial identification were accomplished using appropriate culture methods for anaerobic species. The antimicrobial susceptibility of the 20 strains of P. gingivalis isolated was determined by using the E-test. The antimicrobial agents tested were amoxicillin, amoxicillin + clavulanate, azythromycin, benzylpenicillin, cephaclor, clindamycin, erythromycin, metronidazole and tetracycline. RESULTS: A total of 352 individual strains, belonging to 69 different species, were isolated. Eighty three percent of the strains were strict anaerobes and 47.5% of the isolated bacteria were Gram-negative. Porphyromonas gingivalis was found in 20 root canals and was most frequently found in symptomatic cases. Statistically, the presence of P. gingivalis was related to purulent exudates and pain on palpation (both P < 0.05). All P. gingivalis strains were sensitive to amoxicillin, amoxicillin + clavulanate, cephaclor, clindamycin, benzylpenicyllin, metronidazole and tetracycline. The lowest range of minimum inhibitory concentration (MIC) (0.026-0.125 microg mL(-1)) was observed against amoxicillin + clavulanate and clindamycin. The lowest MIC 90 was observed against clindamycin (0.064 microg mL(-1)). One strain was resistant to erythromycin and eight strains were resistant to azythromycin. CONCLUSION: Porphyromonas gingivalis pathogen is isolated with frequency from root canals of infected teeth with periapical abscesses. Amoxicillin, as well as amoxicillin-clavulanic acid and benzylpenicillin were effective against P. gingivalis.     

多孔HAPw/n-ZnO骨修复材料的抗菌机理及抗生物膜研究

目的:评估多孔羟基磷灰石晶须/纳米氧化锌(HAPw/n-ZnO)骨修复材料在抑制细菌生物膜中的作用,并探讨其抗菌机理。方法:分别建立金黄色葡萄球菌、变形链球菌、牙龈卟啉单胞菌生物膜体外模型。无光条件在相同的多孔HAPw/n-ZnO浓度梯度(0~2 g/L)下分别进行生物膜抑制试验与细胞内活性氧(ROS)检测,并利用扫描电子显微镜分析材料性能,未加入材料组作为对照。结果:无光条件下随着材料浓度递增,各浓度多孔HAPw/n-ZnO组(1.00、1.25、1.50、1.75、2.00 g/L)相对于对照组(0 g/L),3种细菌的生物膜形成量(A值)逐渐减少以及细胞内活性氧(ROS)水平(荧光强度)逐渐升高,差异有统计学意义(P＜0.05)。扫描电镜图片显示,复合材料组细菌黏附减少,生物膜形成受到抑制;细胞形态异常,细胞膜受损导致内容物泄露。结论:无光条件下多孔HAPw/n-ZnO材料对金黄色葡萄球菌、变形链球菌、牙龈卟啉单胞菌生物膜均有一定程度的抑制作用,其抗菌机理之一是活性氧(ROS)的形成。     

Effects of N‐acyl homoserine lactone analogues on Porphyromonas gingivalis biofilm formation

Asahi Y, Noiri Y, Igarashi J, Asai H, Suga H, Ebisu S. Effects of N‐acyl homoserine lactone analogues on Porphyromonas gingivalis biofilm formation. J Periodont Res 2009; doi: 10.1111/j.1600‐0765.2009.01228.x © 2009 John Wiley & Sons A/S Background and Objective: The gram‐negative anaerobic rod Porphyromonas gingivalis in oral biofilms is a primary etiological agent of periodontal disease. Biofilm formation of various gram‐negative bacteria is regulated by a quorum‐sensing circuit that relies on N‐acyl homoserine lactones (HSLs). Some synthetic N‐acyl HSL analogues act as quorum‐sensing inhibitors and suppress biofilm formation in Pseudomonas aeruginosa. Development of chemical control agents against oral biofilms is necessary, because until now, biofilms have been removed only by mechanical debridement. The present study investigated the effect of N‐acyl HSL analogues on P. gingivalis biofilm formation, with the aim of developing new drugs that inhibit oral biofilm formation. Material and Methods: A flow‐cell model was used for P. gingivalis biofilm formation. Seventeen synthetic N‐acyl HSL analogues were quantitatively assessed by spectrophotometry. The effects of three antagonistic compounds against P. gingivalis biofilm formation were further examined by confocal laser scanning microscopy, and investigated for primary attachment using spectrophotometry and phase contrast microscopy. Results: Ten out of 17 analogues affected P. gingivalis biofilm formation. Three out of 10 analogues significantly decreased biofilm‐forming cells (p < 0.05), and these biofilm structures were less well formed three‐dimensionally. There were no quantitative or qualitative differences in cell attachment between the control and the three analogue‐treated groups. Conclusion: Three synthetic N‐acyl HSL analogues inhibited biofilm formation in P. gingivalis. We suggest that these analogues influence the development stage of P. gingivalis biofilm formation.     

The effect of cocoa polyphenols on the growth, metabolism, and biofilm formation by Streptococcus mutans and Streptococcus sanguinis

The aim of this study was to determine if cocoa polyphenols could interfere with biofilm formation by Streptococcus mutans or Streptococcus sanguinis, and reduce acid production from sucrose by S. mutans. The antimicrobial activity of cocoa polyphenols was assessed against cariogenic (S. mutans) and health-associated (S. sanguinis) species by minimum inhibitory concentration assays. Cocoa polyphenol dimer, tetramer, and pentamer inhibited the growth of S. sanguinis, whereas the growth of S. mutans was unaffected. However, pretreatment of surfaces with cocoa polyphenol pentamer (35 microM) reduced biofilm formation by S. mutans at 4 and 24 h, whereas the effects on S. sanguinis were less consistent. In contrast, brief exposure of preformed biofilms to pentamer either had no significant effect or resulted in increased counts of S. mutans under certain conditions. Cocoa polyphenol pentamer (500 microM) significantly reduced the terminal pH, and inhibited the rate of acid production by S. mutans at pH 7.0. In conclusion, cocoa polyphenols can reduce biofilm formation by S. mutans and S. sanguinis, and inhibit acid production by S. mutans.