Initial catabolism of sorbitol in Actinomyces naeslundii and Actinomyces viscosus

The initial steps of sorbitol catabolism were studied in 4 strains of Actinomyces naeslundii and Actinomyces viscosus that had been isolated from human dental plaque. Cell-free extracts were prepared from cells grown in the presence of either sorbitol, xylitol or glucose. The extracts from all strains grown on sorbitol had nicotinamide adenine dinucleotide-linked dehydrogenase activities for sorbitol and xylitol and reduced nicotinamide adenine dinucleotide-linked reductase activities for fructose and xylulose. Two of the strains also exhibited these activities when grown in the presence of xylitol, and all glucose-grown cells lacked them. The results indicate that sorbitol metabolism in oral actinomyces involve oxidation of sorbitol to fructose by an inducible enzyme, nicotinamide adenine dinucleotide-linked sorbitol dehydrogenase. This step is followed by the phosphorylation of fructose with guanosine triphosphate as a main phosphoryl donor. Thus, the initial catabolic pathway of sorbitol in A. naeslundii and A. viscosus is different from those described for other oral bacteria.     

The role of the succinate pathway in sorbitol fermentation by oral Actinomyces viscosus and Actinomyces naeslundii

The sorbitol fermentation by Actinomyces viscosus and Actinomyces naeslundii was studied with washed sorbitol-grown cells. The fermentation was followed by titration of acids produced at pH 7.0 under anaerobic conditions. Metabolic end-products and intracellular levels of NAD, NADH and glycolytic intermediates during the fermentation were also analyzed. Cell extracts were examined for certain enzyme activities. Bicarbonate was required for acid production from sorbitol and from a mixture of glucose and sorbitol. Malate and fumarate could also support the acid production of A. viscosus. The main end-products were succinate and lactate but not ethanol. Cell extracts showed no activities of alcohol and aldehyde dehydrogenases, but they had activities of malate dehydrogenase and fumarate reductase. In the absence of bicarbonate, malate or fumarate, the intracellular NADH/NAD ratio increased and the levels of 3- and 2-phospho-glycerate and phosphoenolpyruvate decreased. The results indicate that oral sorbitol-fermenting actinomyces lack the ethanol pathway that can contribute to NADH oxidation. To maintain intracellular redox balance during anaerobic sorbitol fermentation, these bacteria can oxidize surplus NADH through a succinate pathway.     

Effects of pH on the glucose and lactate metabolisms by the washed cells of Actinomyces naeslundii under anaerobic and aerobic conditions

Effects of pH on the glucose and lactate metabolism by the washed cells of Actinomyces naeslundii genospecies 1 and 2 under anaerobic and aerobic conditions were studied. The rate of acid production from glucose was the highest at pH 7.0 and decreased as the pH lowered to 4.5, irrespective of atmospheric conditions. The anaerobic end-product in the absence of bicarbonate was mainly lactate, while in the presence of bicarbonate the rate of acid production increased 1.8-2.5 times with the production of formate, acetate and succinate in addition to lactate. Under aerobic conditions, the cells produced acids from glucose along with oxygen consumption and the end-product was mainly acetate. In contrast to the glucose metabilism, the cells produced base from lactate along with oxygen consumption. The rates of base production and oxygen consumption were the highest at pH 5.5. The end-products from lactate were acetate and pyruvate. These results indicate that oral actinomyces has a various activity of glucose and lactate metabolism at a wide range of environmental pH and suggest its flexibility in surviving in dental plaque, where the environmental factors fluctuate.     

尿素水解对内氏放线菌增殖及耐酸力的影响

目的通过研究尿素水解对内氏放线菌的生长和耐酸能力的影响,了解尿素水解对内氏放线菌的生理作用。方法比较内氏放线菌利用尿素或其它物质作为氮源的生长浊度;采用耐酸实验研究尿素水解与细菌耐酸能力的关系。结果与其他口腔中常见氮源物质相比,尿素可以促进内氏放线菌生长,获得更高的A值;在临床牙菌斑能够检测到的pH4.0~7.0范围内,尿素水解可以提高内氏放线菌的耐酸性,在pH3.0,尿素水解对细菌没有保护作用。结论尿素水解可以促进内氏放线菌生长,提高酸性环境中细菌的耐酸能力,增强细菌的生存竞争力。     

镍铬合金析出的镍离子对内氏放线菌糖代谢和尿素代谢的影响

目的研究镍铬合金析出的镍离子对内氏放线菌糖代谢和尿素代谢的影响。方法分别用含0.260mg/L和0.625mg/L镍离子的BHI培养基厌氧培养内氏放线菌WVU45型菌株,计算pH的变化值,检测乳酸脱氢酶(LDH)含量,测定尿素酶活性。结果镍离子对内氏放线菌产酸和产乳酸脱氢酶的作用不明显(P>0.05),对尿素酶活性有促进作用(P<0.05)。结论镍铬合金析出的镍离子可以增加内氏放线菌尿素酶活性,促进尿素代谢。     

Actinomyces naeslundii fimbrial protein Orf977 shows similarity to a streptococcal adhesin

The nucleotide sequence of the chromosomal DNA, upstream of Actinomyces naeslundii T14V fimbrial gene fimA, was determined. One open reading frame (orf977) encoding 977 amino acids was found, preceded by a gene homologous to elongation factor TU. Database searches revealed that Orf977 was homologous to CshA, a Streptococcus gordonii protein involved in cell adhesion. Previous studies had already determined two genes in the type 2 fimbrial gene cluster of A. naeslundii T14V: the structural subunit fimA, and orf365 with unknown function, followed by ribosomal genes. This study completes the type 2 fimbrial gene cluster sequence.     

Preparation and characterization of an Actinomyces naeslundii aggregation factor that mediates coaggregation with Porphyromonas gingivalis

Intergeneric coaggregation is responsible for the complexity of the microbiota in human dental plaque and is believed to be important in the initial bacterial colonization of the human oral cavity. Actinomyces naeslundii, an early colonizer of the tooth surface, may enhance subsequent colonization by Porphyromonas gingivalis which is associated with adult periodontitis. The purpose of this study was to isolate and characterize the A. naeslundii aggregation factor (AnAF) that mediates coaggregation with P. gingivalis. AnAF was isolated from A. naeslundii sonic extract (SE) by gel nitration on a Sephacryl S-400HR, by hydrophobic interaction chromatography on a HiTrap Octyl Sepharose 4FF, and by ion exchange chromatography on a HiTrap Q. The specific activity increased 12-fold with a yield of 2.5%. SDS-PAGE analysis of AnAF revealed a protein band of high molecular weight in excess of 200 kDa. Carbohydrate was detected as the only material coinciding with the protein band, indicating that the AnAF was a glycoprotein. Immunoblotting analysis indicated that AnAF directly bound to P. gingivalis cells. AnAF was sensitive to sodium metaperiodate treatment but not to heat or protease treatments. These results suggest that the AnAF carbohydrate component mediated coaggregation with P. gingivalis cells. AnAF also inhibited coaggregation with other periodontal disease-associated bacteria such as Prevotella intermedia, Fusobacterium nucleatum, Capnocytophaga ochracea, but not streptococci.     

Actinomyces naeslundii fimbrial protein Orf977shows similarity to a streptococcal adhesin

The nucleotide sequence of the chromosomal DNA, upstream of Actinomyces naeslundii T14V fimbrial gene fimA, was determined. One open reading frame (orf977) encoding 977 amino acids was found, preceded by a gene homologous to elongation factor TU. Database searches revealed that Orf977 was homologous to CshA, a Streptococcus gordonii protein involved in cell adhesion. Previous studies had already determined two genes in the type 2 fimbrial gene cluster of A. naeslundii T14V: the structural subunit fimA, and orf365 with unknown function, followed by ribosomal genes. This study completes the type 2 fimbrial gene cluster sequence.     

老年人根面菌斑内氏放线菌临床分离株基因型多样性分析

目的分析老年人根面菌斑中内氏放线菌临床株的基因型多样性,探讨内氏放线菌基因型与根面龋的关系。方法选择老年根面龋患者20例设为根面龋组,无根面龋老年人20例设为无龋组。根面龋组以暴露的无龋根面和根面龋损部位为取样位点,无龋组以暴露的根面为取样位点,刮取菌斑进行临床株的分离鉴定,并利用基因外重复回文序列聚合酶链反应（REP-PCR）分析内氏放线菌基因型的多样性。结果2组共分离出内氏放线菌299株,选择156株进行REP-PCR分析,分离出61个不同的基因型。根面龋组无龋根面分离的57株内氏放线菌有25个基因型,根面龋损部位分离的34株有25个基因型,无龋组分离的65株有26个基因型：内氏放线菌基因型存在多样性。单个取样位点的基因型数目存在差异（P<0.05）。结论多种基因型的内氏放线菌参与了根面龋的发生。     

内氏放线菌尿素酶对牙菌斑生物膜酸碱平衡调节作用的初步研究

目的：初步探讨在牙菌斑生物膜天然环境中,内氏放线菌尿素酶能否发挥高效尿素水解反应,以及尿素水解对口腔环境中pH值的调节作用。方法：通过酶促反应动力学实验寻找内氏放线菌尿素水解的最适条件．监测尿素水解调节细菌产酸后的pH值变化。采用SPSS软件包,对酶促动力学实验数据进行线性回归与相关分析。结果：内氏放线菌尿素酶米氏常数Km=7．5mmol／L,在口腔中正常尿素浓度3-10mmol／L范围内,内氏放线菌尿素酶催化活性大约保持在最大活性的20％-63％;内氏放线菌尿素酶最适pH值=6．5,但是在牙菌斑临界pH5．0,尿素酶活性仍保持40％的最大活性;在口腔正常尿素浓度范围内,内氏放线菌尿素水解产物中和细菌产酸后,pH值不会下降到牙菌斑临界pH5．0以下。结论：在牙菌斑生物膜中,内氏放线菌尿素酶可以发挥高效尿素水解反应．尿素水解对口腔环境pH值具有明显的调节作用。     

Antimicrobial activity of varying concentrations of sodium hypochlorite on the endodontic microorganisms Actinomyces israelii, A. naeslundii, Candida albicans and Enterococcus faecalis

AIM: To determine the resistance of microorganisms associated with refractory endodontic infections to sodium hypochlorite used as a root canal irrigant. METHODOLOGY: Two strains each of Actinomyces naeslundii, Candida albicans and Enterococcus faecalis were tested as late logarithmic phase inocula, against sodium hypochlorite adjusted to 0.5, 1.0, 2.5 and 5.25% w/v. Contact times used were 0, 10, 20, 30, 60 and 120 s. In the case of E. faecalis, additional experiments used contact times of 1.0, 2.0, 5.0, 10.0 and 30.0 min. Anti-microbial action was halted by sodium thiosulphate addition. Survivors were measured primarily using viable counts on drop plates. Additionally, pour plates were used to count low colony-forming units (cfu) and dilutions to 10(-6) were used to count high cfu. RESULTS: All concentrations of NaOCl lowered cfu below the limit of detection after 10 s in the case of A. naeslundii and C. albicans. However, E. faecalis proved to be more resistant to NaOCl. Using 0.5% NaOCl for 30 min reduced cfu to zero for both strains tested. This compares with 10 min for 1.0%, 5 min for 2.5% and 2 min for 5.25% (P < 0.001). Regression analysis for the dependent variable log(e)(count + 1) with log(e)(time + 1) and concentration as explanatory variables gave rise to a significant interaction between time and concentration (P < 0.001). CONCLUSION: The published association of E. faecalis with refractory endodontic infection may result, at least partially, from high resistance of this species to NaOCl. This does not appear to be the case with A. naeslundii or C. albicans.     

Mechanisms of inhibition by fluoride of urease activities of cell suspensions and biofilms of Staphylococcus epidermidis, Streptococcus salivarius, Actinomyces naeslundii and of dental plaque

BACKGROUND/AIMS: Fluoride is known to be a potent inhibitor of bacterial ureases and can also act in the form of hydrofluoric acid as a transmembrane proton conductor to acidify the cytoplasm of intact cells with possible indirect, acid inhibition of urease. Our research objectives were to assess the inhibitory potencies of fluoride for three urease-positive bacteria commonly found in the mouth and to determine the relative importance of direct and indirect inhibition of ureases for overall inhibition of intact cells or biofilms. METHODS: The experimental design involved intact bacteria in suspensions, mono-organism biofilms, cell extracts, and dental plaque. Standard enzymatic assays for ammonia production from urea were used. RESULTS: We found that ureolysis by cells in suspensions or mono-organism biofilms of Staphylococcus epidermidis, Streptococcus salivarius or Actinomyces naeslundii was inhibited by fluoride at plaque levels of 0.1-0.5 mm in a pH-dependent manner. The results of experiments with the organic weak acids indomethacin and capric acid, which do not directly inhibit urease enzyme, indicated that weak-acid effects leading to cytoplasmic acidification are also involved in fluoride inhibition. However, direct fluoride inhibition of urease appeared to be the major mechanism for reduction in ureolytic activity in acid environments. Results of experiments with freshly harvested supragingival dental plaque indicated responses to fluoride similar to those of S. salivarius with pH-dependent fluoride inhibition and both direct and indirect inhibition of urease. CONCLUSION: Fluoride can act to diminish alkali production from urea by oral bacteria through direct and indirect mechanisms.     

Genetic diversity of Actinomyces naeslundii genospecies 2 in mother-child pairs

Actinomyces naeslundii genospecies 2 (gsp-2) are members of the autochthonous oral flora. Chromosomal DNA fingerprinting (CDF) with SmaI revealed extensive genetic diversity among A. naeslundii gsp-2 strains within individual mothers and children. There was a low prevalence of genotype match among A. naeslundii gsp-2 strains between all mother and child pairs.     

Isolation and characterization of a 180-kiloDalton salivary glycoprotein which mediates the attachment of Actinomyces naeslundii to human buccal epithelial cells

The adherence of Actinomyces naeslundii to human buccal mucosa is mediated by specific interactions between the bacterial cell surface fimbriae and complementary β-linked galactoside receptors on the epithelial cell surface. The buccal mucosa and the bacteria that colonize its surface are constantly bathed in saliva. Several salivary components are thought to play an important role in modulating adhesive interactions between oral bacteria and the buccal epithelium. We have observed that pretreatment of isolated buccal epithelial cells (BEC) with human parotid saliva increased the attachment of three different strains of A. naeslundii . By employing affinity chromatography, ion-exchange and high-pressure liquid Chromatographic techniques we have isolated a 180 kDa A. naeslundii -binding salivary glycoprotein (An-SGP). This salivary glycoprotein was capable of mediating separate but specific binding interactions with A. naeslundii and BEC. Pretreatment of BEC with increasing amounts of An-SGP resulted in a corresponding increase in the attachment of A. naeslundii . The adherence of A. naeslundii to An-SGP-coated BEC is sensitive to the same inhibitors previously shown to block adherence of A. naeslundii to uncoated BEC, namely lactose and galactosyl-binding lectins. When a solubilized extract of freshly isolated and washed BEC was reacted on a Western blot with antibodies to An-SGP, a prominent 180 kDa immunoreactive band was detected. Furthermore, the immunoreactive component was demonstrated on the BEC surface when assayed by immunofluorescence using An-SGP-specific antibodies, suggesting that An-SGP or a protein structurally and immunologically identical to the isolated glycoprotein is present on BEC.     

Effects of short‐chain fatty acids on Actinomyces naeslundii biofilm formation

Actinomyces naeslundii is an early colonizer and has important roles in the development of the oral biofilm. Short‐chain fatty acids (SCFA) are secreted extracellularly as a product of metabolism by gram‐negative anaerobes, e.g. Porphyromonas gingivalis and Fusobacterium nucleatum; and the SCFA may affect biofilm development with interaction between A. naeslundii and gram‐negative bacteria. Our aim was to investigate the effects of SCFA on biofilm formation by A. naeslundii and to determine the mechanism. We used the biofilm formation assay in 96‐well microtiter plates in tryptic soy broth without dextrose and with 0.25% sucrose using safranin stain of the biofilm monitoring 492 nm absorbance. To determine the mechanism by SCFA, the production of chaperones and stress‐response proteins (GrpE and GroEL) in biofilm formation was examined using Western blot fluorescence activity with GrpE and GroEL antibodies. Adding butyric acid (6.25 mm ) 0, 6 and 10 h after beginning culture significantly increased biofilm formation by A. naeslundii, and upregulation was observed at 16 h. Upregulation was also observed using appropriate concentrations of other SCFA. In the upregulated biofilm, production of GrpE and GroEL was higher where membrane‐damaged or dead cells were also observed. The upregulated biofilm was significantly reduced by addition of anti‐GroEL antibody. The data suggest biofilm formation by A. naeslundii was upregulated dependent on the production of stress proteins, and addition of SCFA increased membrane‐damaged or dead cells. Production of GroEL may physically play an important role in biofilm development.