Adherent Streptococci Research Articles

Evaluation of the antibacterial activity of Enamelast® and Fluor defender® fluoride varnishes against Streptococcus mutans biofilm: an in vitro study in primary teeth

PurposeThe aim of the current work was to compare the antibacterial activity of Enamelast® and Fluor defender® fluoride varnish on biofilm generation by Streptococcus mutans on extracted primary teeth.MethodsThirty-six primary molars were collected and sliced into seventy-two test model disks. All specimens were examined, and the cracked or broken ones were discarded. A total number of specimens (n = 54) were divided into two experimental analyses viz; biofilm formation (n = 27) and microscopic examination (n = 27). Specimens of each analysis were tested under different experimental conditions: a negative control group (n = 9), Fluor defender group (n = 9), and Enamelast group (n = 9). Following treatment, biofilms were generated by adherent Streptococcus mutans on the test model disks on three time intervals: 24 h (n = 3), 48 h (n = 3), and 72 h (n = 3) for each analysis. Then, for biofilm formation analysis, the biofilm was detected spectrophotometrically at 620 nm after being stained by crystal violet. For microscopical analysis, the surfaces of the test model disks were visualized by scanning electron microscopy (SEM), and each image was processed and analyzed using ImageJ software.ResultsAt 48 and 72 h, Enamelast® and Fluor defender®-treated group showed significantly (p < 0.001) slight adhered bacterial cells when compared with the negative control group as revealed by the absorbance and SEM. Compared with the Fluor defender®-treated group, the absorbance of the Enamelast®-treated group showed a significant (p < 0.001) increase by approximately 7- and 16.5-fold at 48 and 72 h, respectively. Similarly, SEM showed that the number of bacterial cells adhered to enamel surfaces in the Fluor defender®-treated group was significantly (p < 0.001) fewer than the Enamelast®-treated group by approximately 36.55% and 20.62% at 48 and 72 h after exposure, respectively.ConclusionWe conclude that the anti-biofilm activity of Fluor defender® against Streptococcus mutans was significantly (p < 0.001) greater than Enamelast® fluoride varnish. The use of Fluor defender® is encouraged as a preventive measure in children with the high risk of developing dental caries.

A dentifrice containing salivary enzymes and xylitol exhibits superior antimicrobial activity in vitro against adherent Streptococcus mutans compared to a chlorhexidine dentifrice.

Human saliva contains natural antimicrobial enzymes. In this in vitro study, we evaluate the antimicrobial activity of a dentifrice containing a salivary enzyme complex (SEC) with xylitol versus a standard 0.12% chlorhexidine (CHX) dentifrice. Adherent cells of Streptococcus gordonii, Streptococcus mutans, Actinomyces naeslundii, Fusobacterium nucleatum subsp polymorphum and Corynebacterium matruchotii were exposed to SEC-xylitol and CHX dentifrices for 2 mins and viable CFUs were enumerated. Exposure to the SEC-xylitol dentifrice resulted in a significant reduction in bacterial viability, which was greater than that shown by the CHX dentifrice, against all organisms tested. The SEC-xylitol dentifrice also exhibited greater antimicrobial activity against all organsims in well diffusion assays compared to CHX. Dentifrice activity was also evaluated against a three species community of Streptococcus gordonii, Streptococcus mutans and Corynebacterium matruchotii using bacterial live/dead stain. The SEC-xylitol dentifrice was at least as effective as CHX in removal of the multispecies community. The combination of SEC and xylitol generates a highly effective antimicrobial dentifrice with greater anti-bacterial activity than a standard 0.12% CHX formulations. SEC and xylitol combinations are worthy of further investigation for routine use and in the management of gingivitis and periodontal disease.

A Comparison of the Adaptive Response of Staphylococcus aureus vs. Streptococcus mutans and the Development of Chlorhexidine Resistance.

Antimicrobials with nonselective antibacterial efficacy such as chlorhexidine can be effective in reducing biofilm, but bear the risk of inducing resistance in specific bacteria. In clinical practice, bacteria such as Staphylococcus aureus have been found resistant to chlorhexidine, but other bacteria, including Streptococcus mutans, have largely remained susceptible to chlorhexidine despite its widespread use in oral healthcare. Here, we aim to forward a possible reason as to why S. aureus can acquire resistance against chlorhexidine, while S. mutans remains susceptible to chlorhexidine. Measurement of surface-enhanced fluorescence indicated that chlorhexidine caused gradual, but irreversible deformation to adhering green fluorescent S. aureus due to irreparable damage to the cell wall. Concurrently, the metabolic activity of adhering staphylococci was higher than of planktonic bacteria, suggesting efflux mechanisms may have been activated upon cell wall deformation, impeding the buildup of a high chlorhexidine concentration in the cytoplasm and therewith stimulating the development of chlorhexidine resistance in S. aureus. Exposure of S. mutans to chlorhexidine caused immediate, but reversible deformation in adhering streptococci, indicative of rapid self-repair of cell wall damage done by chlorhexidine. Due to cell wall self-repair, S. mutans will be unable to effectively reduce the chlorhexidine concentration in the cytoplasm causing solidification of the cytoplasm. In line, no increased metabolic activity was observed in S. mutans during exposure to chlorhexidine. Therewith, self-repair is suicidal and prevents the development of a chlorhexidine-resistant progeny in S. mutans.

Anti-adherence and anti-bacterial activities of Pistacia atlantica resin extract against strongly adherent Streptococcus mutans strains

The reduction of Streptococcus mutans from the oral cavity or its adherence to tooth surfaces can prevent or decrease the progression of caries. In this study, the antimicrobial and anti-adherence properties of Pistacia atlantica (P. atlantica) resin (Essential oil [EO] and methanolic extract [ME]) were investigated on S. mutans strains. In this in vitro experimental study, the growth rate, biofilm formation ability, and antibiotic susceptibility profile of S. mutans ATCC35668 and 3 strains isolated from caries lesions were studied. The EO and ME of P. atlantica resin were prepared. The anti-bacterial and anti-adherence properties of them were evaluated using microdilution and microplate adherence tests, respectively. The data were statistically analyzed using SPSS with one-way and two-way analysis variance. Direct comparisons between the groups were made using the Wilcoxon W-Mann-Whitney U-test. Statistical significance was set at P < 0.05. All target strains showed the same growth rate and antibiotic susceptibility profile and were found strongly adherent. Both EO and ME showed moderate anti-bacterial properties (growth reduction up to 47.1% and 39.1%, respectively) against S. mutans, while the anti-bacterial effect of EO was higher than ME, significantly (P < 0.05). In all tested concentrations, EO showed a significantly stronger anti-adherence activity (50%-80%) than ME. The results showed an anti-cariogenic effect of EO extracted from P. Atlantica resin. Considering that S. mutans adhesion is a necessary step in the beginning and progression of dental caries, this study can suggest the use of such extract in mouthwashes or toothpaste as an alternative agent for preventing bacterial attachment and biofilm formation.

Contribution of Adsorbed Protein Films to Nanoscopic Vibrations Exhibited by Bacteria Adhering through Ligand–Receptor Bonds

Bacteria adhering to surfaces exhibit nanoscopic vibrations that depend on the viscoelasticity of the bond. The quantification of the nanoscopic vibrations of bacteria adhering to surfaces provides new opportunities to better understand the properties of the bond through which bacteria adhere and the mechanisms by which they resist detachment. Often, however, bacteria do not adhere to bare surfaces but to adsorbed protein films, on which adhesion involves highly specific ligand-receptor binding next to nonspecific DLVO interaction forces. Here we determine the contribution of adsorbed salivary protein and fibronectin films to vibrations exhibited by adhering streptococci and staphylococci, respectively. The streptococcal strain used has the ability to adhere to adsorbed salivary proteins films through antigen I/II ligand-receptor binding, while the staphylococcal strain used adheres to adsorbed fibronectin films through a proteinaceous ligand-receptor bond. In the absence of ligand-receptor binding, electrostatic interactions had a large impact on vibration amplitudes of adhering bacteria on glass. On an adsorbed salivary protein film, vibration amplitudes of adhering streptococci depended on the film softness as determined by QCM-D and were reduced after film fixation using glutaraldehyde. On a relatively stiff fibronectin film, cross-linking the film in glutaraldehyde hardly reduced its softness, and accordingly fibronectin film softness did not contribute to vibration amplitudes of adhering staphylococci. However, fixation of the staphylococcus-fibronectin bond further decreased vibration amplitudes, while fixation of the streptococcus bond hardly impacted vibration amplitudes. Summarizing, this study shows that both the softness of adsorbed protein films and the properties of the bond between an adhering bacterium and an adsorbed protein film play an important role in bacterial vibration amplitudes. These nanoscopic vibrations reflect the viscoelasticity of the bacterial bond with a substratum and play important roles in bacterial adhesion, detachment and susceptibility to antimicrobials.

Streptococcus mutans and Streptococcus sobrinus biofilm formation and metabolic activity on dental materials

Objectives. To examine potential correlations between streptococcal biofilm formation and lactate production in streptococcal biofilms formed on the surface of dental materials with different surface characteristics. Materials and methods. Samples of a glass-ionomer cement (Ketac Molar) and a ceramic (Empress 2) were incubated with whole saliva and suspensions of Streptococcus mutans ATCC 25175 or Streptococcus sobrinus ATCC 33478 for initiating single-species biofilm formation for either 4 or 24 h. The relative amount of adherent, viable cells was determined using a Resazurin and a MTT assay. Metabolic activity was assessed by quantifying lactate production with a modification of the commercial Clinpro Cario L-Pop kit. Results. Both assays identified similar S. sobrinus biofilm formation on the two substrata; for S. mutans, the MTT test showed significantly fewer streptococci on the glass-ionomer cement than on the ceramic. Concerning metabolic activity, for S. sobrinus, significantly higher lactate production was observed for biofilms formed on the glass-ionomer cement in comparison to the ceramic, whereas similar values were identified for S. mutans. Conclusions. Within the limitations of the study, the results suggest that the pure amount of adherent streptococci does not a priori indicate the metabolic activity of the cariogenic bacteria organized in the respective biofilm. Thus, comparisons between the relative amount of adherent streptococci and their metabolic activity may allow for an improved understanding of the effect of dental material surfaces on the formation and metabolic activity of streptococcal biofilms.

Quantification of vital adherent Streptococcus sanguinis cells on protein-coated titanium after disinfectant treatment

The quantification of vital adherent bacteria is challenging, especially when efficacy of antimicrobial agents is to be evaluated. In this study three different methods were compared in order to quantify vital adherent Streptococcus sanguinis cells after exposure to disinfectants. An anaerobic flow chamber model accomplished initial adhesion of S. sanguinis on protein-coated titanium. Effects of chlorhexidine, Betadine®, Octenidol®, and ProntOral® were assessed by quantifying vital cells using Live/Dead BacLight™, conventional culturing and isothermal microcalorimetry (IMC). Results were analysed by Kruskal-Wallis one-way analysis of variance. Live/dead staining revealed highest vital cell counts (P < 0.05) and demonstrated dose-dependent effect for all disinfectants. Microcalorimetry showed time-delayed heat flow peaks that were proportioned to the remaining number of viable cells. Over 48 h there was no difference in total heat between treated and untreated samples (P > 0.05), indicating equivalent numbers of bacteria were created and disinfectants delayed growth but did not eliminate it. In conclusion, contrary to culturing, live/dead staining enables detection of cells that may be viable but non-cultivable. Microcalorimetry allows unique evaluation of relative disinfectant effects by quantifying differences in time delay of regrowth of remaining vital cells.

Invasion of Endothelial Cells by Tissue-invasive M3 Type Group A Streptococci Requires Src Kinase and Activation of Rac1 by a Phosphatidylinositol 3-Kinase-independent Mechanism

Streptococcus pyogenes can cause invasive diseases in humans, such as sepsis or necrotizing fasciitis. Among the various M serotypes of group A streptococci (GAS), M3 GAS lacks the major epithelial invasins SfbI/PrtF1 and M1 protein but has a high potential to cause invasive disease. We examined the uptake of M3 GAS into human endothelial cells and identified host signaling factors required to initiate streptococcal uptake. Bacterial uptake is accompanied by local F-actin accumulation and formation of membrane protrusions at the entry site. We found that Src kinases and Rac1 but not phosphatidylinositol 3-kinases (PI3Ks) are essential to mediate S. pyogenes internalization. Pharmacological inhibition of Src activity reduced bacterial uptake and abolished the formation of membrane protrusions and actin accumulation in the vicinity of adherent streptococci. We found that Src kinases are activated in a time-dependent manner in response to M3 GAS. We also demonstrated that PI3K is dispensable for internalization of M3 streptococci and the formation of F-actin accumulations at the entry site. Furthermore, Rac1 was activated in infected cells and accumulated with F-actin in a PI3K-independent manner at bacterial entry sites. Genetic interference with Rac1 function inhibited streptococcal internalization, demonstrating an essential role of Rac1 for the uptake process of streptococci into endothelial cells. In addition, we demonstrated for the first time accumulation of the actin nucleation complex Arp2/3 at the entry port of invading M3 streptococci.

The anti-adherence activity and bactericidal effect of microparticulate silver additives in composite resin materials

Objective Resin composite materials tend to accumulate microorganisms and dental plaque, which in turn may induce secondary caries around adhesive restorations. The aim of the present in vitro study was to evaluate the antibacterial activity of a resin composite material loaded with silver microparticles against Streptococcus mutans. Design Circular specimens (10.0 mm in diameter) of a resin composite matrix loaded with two different concentrations of a silver additive ( Comp0.3: 0.3%; Comp0.6: 0.6%) and one unloaded reference composite matrix ( Comp0: 0%) were made. Surface roughness R a was assessed by perthometer measurements and hydrophobicity according to water contact angles was determined by computerized image analysis. The specimens were incubated in a S. mutans suspension (1 h, 37 °C) and adhering streptococci were quantified by using a biofluorescence assay (Alamar blue/Resazurin). Additionally, the viability of adhering bacteria was assessed by live/dead cell labelling in combination with fluorescence microscopy. Results Statistically significant differences between the median water contact angles of Comp0 (66.3°), Comp0.3 (76.7°), and Comp0.6 (89.4°) were observed ( p < 0.001). A three- to fourfold higher amount of adhering S. mutans was found on reference Comp0 (12,093 relative fluorescence units) than on Comp0.3 (4258 rfu) and Comp0.6 (3292) ( p < 0.001 for both). Significantly higher percentages of dead cells than on Comp0 (0.5%) were found on Comp0.3 (6.1%) and on Comp0.6 (10.1%) ( p < 0.001 for both). Conclusions The addition of microparticulate silver to a resin composite material increased the surface hydrophobicity and reduced the number of adhering streptococci. Simultaneously it increased the percentage of dead and inactive cells on the composite surface. Thus, silver additives seem to demonstrate anti-adherence activity as well as a bactericidal effect.

Surface characterization of dental ceramics and initial streptococcal adhesion in vitro

Objectives The aim of this in vitro study was to investigate the surface properties of dental ceramic materials belonging to different ceramic classes, and to correlate the findings to the initial adherence of three oral streptococcal strains. Methods Rectangular specimens were prepared from different ceramic materials (glass/lithium disilicate glass/glass-infiltrated zirconia/partially sintered zirconia/hipped zirconia ceramic) and polished; surface roughness (Ra) was determined. Glass plates were used as a control. Specimens were incubated with phosphate-buffered saline or an artificial saliva (protein mixture; 2 h, 37 °C). Surface free energy ( γ t) and its polar ( γ p) and disperse ( γ d) contribution were determined prior to and after artificial saliva exposure. Uncoated and protein-coated specimens were incubated with Streptococcus gordonii DSMZ 6777, Streptococcus oralis DSMZ 20068 or Streptococcus sanguinis DSMZ 20068 suspension for 2.5 h at 37 °C ( n = 15 for each treatment and strain). Adherent streptococci were quantified fluorometrically. Results The lithium disilicate glass ceramic showed the highest values for Ra; the lowest values were found for the glass ceramic, the partially sintered zirconia and the hipped zirconia ceramic. Protein coating caused a significant increase in γ t and γ p, but not in the control material. The control material showed higher values for streptococcal adhesion than all ceramic materials. After protein coating, only slight and random differences in streptococcal adhesion were found between the various ceramic materials. Significance Dental ceramic materials show differences in terms of Ra, γ t and initial streptococcal adhesion; however, correlations between surface properties and streptococcal adhesion were poor.

Streptococcal adhesion to various luting systems and the role of mixing errors

Objective. This study aims at ranking various luting systems according to their susceptibility to adhering Streptococcus mutans and at evaluating the influence of incongruent mixing ratios on adhesion quantities. Material and Methods. Circular specimens measuring 8 mm in diameter were made of nine widely used dental cements—three of them mixed in different ratios—and then incubated with S. mutans. Adhering streptococci were quantified using a biofluorometric assay in combination with an automated plate reader for cell quantification. Surface roughness (Ra) was determined by perthometer measurements. Results. Meron plus revealed the highest Ra (0.90µm) and glass the lowest Ra (<0.01µm). In regular cement mixtures, the highest mean fluorescence intensities indicated the presence of many viable bacteria [Meron Plus (35,533 relative fluorescence units (rfu)), Maxcem (13,374 rfu), and Panavia F 2.0 (11,701 rfu)]. Moderate fluorescence intensities were found in Harvard (4,171 rfu), Ketac cem (3,766 rfu), Durelon (3,276 rfu), Calibra (3,259), Rely X Unicem (4,358 rfu), and Bifix SE (3,102 rfu). A medium correlation between Ra and S. mutans adhesion was found. Changes in regular cement proportions (powder/liquid and base/catalyst, respectively) had a significant influence on relative fluorescence intensities, which linearly increased with a higher proportion of liquid in Harvard and with a higher proportion of catalyst in Calibra and Maxcem.Conclusions. Various luting systems revealed considerable differences in their potential to adhere S. mutans. Variations from recommended cement proportions led to significant changes in the amount of adhering streptococci.

Streptococcal adhesion to novel low-shrink silorane-based restorative

Objectives The aim of the present study was to compare the susceptibility of one novel silorane-based and four widely used conventional methacrylate-based resin composites to adhere oral streptococci and to relate any differences to surface roughness, hydrophobicity and type of matrix. Methods Specimens of restoratives Filtek Silorane, Filtek Z250, Tetric EvoCeram, Quixfil and Spectrum TPH were prepared (10.0-mm diameter, 2.0-mm height). Surface roughness was assessed by perthometer measurements and hydrophobicity according to water contact angles was determined by computerized image analysis. Bacterial suspensions of Streptococcus oralis, Streptococcus sanguinis, Streptococcus gordonii and Streptococcus mutans were incubated for 2.5 h at 37 °C with 15 test specimens for each material and adhesion was quantified with fluorescence dye Alamar Blue/Resazurin and an automated multi-detection reader. Glass served as reference. Statistical analysis was performed using the Mann–Whitney U-test ( α = 0.05). Results Median roughness values of all composites ranged between 0.04 and 0.11 μm, median contact angles between 59.7° and 92.1°. Significantly lowest fluorescence intensities of all test materials were found on Filtek Silorane for three of four streptococcal strains. The fluorescence intensities of the four conventional methacrylate resin composites were significantly higher and comparable among each other. Significance When compared against four conventional methacrylate composite resins, a general significance to a lower quantity of adhering streptococci was found on the novel silorane-based composite resin, which might result from its increased hydrophobicity. The low adhesion potential of the silorane-based composite may potentially improve the longevity of direct fillings and reduce recurrent caries.

Host cell caveolae act as an entry-port for group A streptococci.

This study identified caveolae as an entry port for group A streptococci into epithelial and endothelial cells. Scanning electron microscopy as well as ultrathin sections of infected cells demonstrated accumulation of small omega-shaped cavities in the host cell membrane close to adherent streptococci. During invasion, invaginations were formed that subsequently revealed intracellular compartments surrounding streptococci. Caveolin-1 was shown to be present in the membrane of invaginations and the compartment membranes. These compartments were devoid of any classic endosomal/lysosomal marker proteins and can thus be described as caveosomes. Disruption of caveolae with methyl-beta-cyclodextrin and filipin abolished host cell invasion. Importantly, streptococci inside caveosomes avoid fusion with lysosomes. Expressing of SfbI protein on the surface of the non-invasive S. gordonii resulted in identical morphological alterations on the host cell as for S. pyogenes. Incubation of HUVEC cells with purified recombinant sole SfbI protein also triggered accumulation of cavity-like structures and formation of membrane invaginations. Tagged to colloidal gold-particles, SfbI protein was shown to cluster following membrane contact. Thus, our results demonstrate that host cell caveolae initiate the invasion process of group A streptococci and that the streptococcal invasin SfbI is the triggering factor that activates the caveolae-mediated endocytic pathway.

Active detachment of Streptococcus mutans cells adhered to epon–hydroxylapatite surfaces coated with salivary proteins in vitro

Although the formation of biofilms has been much studied, detachment of adherent cells from biofilms has been relatively neglected. Recent results have shown that adherent Streptococcus mutans cells can actively detach from epon–hydroxylapatite (EHA) rods conditioned with hog gastric mucin. The mechanisms for adherence and detachment of Strep. mutans cells in this system was uncertain. In the present study, resting Strep. mutans cells were used to form a simple monolayer on EHA rods coated with saliva and salivary agglutinin (SAG). Preliminary experiments defined the variables for conditioning EHA with saliva and SAG and establishing the adherence of Strep. mutans to the conditioned surfaces. The results showed that salivary proteins including SAG adsorbed rapidly to EHA and that a relatively stable Strep. mutans NG8 monolayer was formed within 60 min of incubation. The monolayers were subsequently used for detachment studies. The results showed that adherent Strep. mutans cells detached in a temperature-dependent manner and responded to the addition of a preparation of surface protein-releasing enzyme (SPRE) obtained from Strep. mutans in a dose-dependent fashion. The effect of the exogenous SPRE on detachment could be abrogated by pronase treatment. Two putative SPRE-defective mutants (A and E) were generated by Tn 917 mutagenesis. Both mutants possessed a single transposon insertion as demonstrated by Southern hybridization and appeared to be different from one another based on the hybridization patterns. Mutant A displayed an increased quantity of cell-surface antigen P1, an adhesin that interacts with SAG. At the same time mutant A was unable to release P1 and other high molecular-weight proteins from the cell surface. Mutant A detached at a significantly lower rate (21%) than the parent strain (37%) ( p=0.05). SPRE prepared from mutant A was unable to release Strep. mutans NG8 adherent cells as compared to SPRE obtained from the wild-type cells. Collectively, these results suggest that the detachment of Strep. mutans adherent cells formed on salivary protein-coated EHA was an active process mediated by the action of SPRE.

Influence of aeration of Candida albicans during culturing on their surface aggregation in the presence of adhering Streptococcus gordonii

Candida albicans surfaces are extremely sensitive to changes in growth conditions. In this study, adhesion to glass of aerated and non-aerated C. albicans ATCC 10261 in the presence and absence of adhering Streptococcus gordonii NCTC 7869 was determined in a parallel plate flow chamber. In addition, the influence of aeration on the yeast cell surface hydrophobicity, surface charge, and elemental cell surface composition was measured. S. gordonii adhering at the glass surface caused a reduction in the initial deposition rate of C. albicans, regardless of aeration. In a stationary end-point, only adhesion of non-aerated C. albicans was suppressed by the adhering S. gordonii. Non-aerated yeasts had a higher O/C elemental surface concentration ratio, indicative of cell surface polysaccharides, than aerated yeasts, at the expense of nitrogen-rich cell surface proteins. Both yeasts were essentially uncharged, but the nitrogen-rich cell surface of aerated yeasts had a slightly higher water contact angle than non-aerated yeasts. Summarizing, this study suggests that highly localized, hydrophobic cell surface proteins on C. albicans are a prerequisite for their interaction with adhering streptococci.

Influence of aeration of Candida albicans during culturing on their surface aggregation in the presence of adhering Streptococcus gordonii.

Candida albicans surfaces are extremely sensitive to changes in growth conditions. In this study, adhesion to glass of aerated and non-aerated C. albicans ATCC 10261 in the presence and absence of adhering Streptococcus gordonii NCTC 7869 was determined in a parallel plate flow chamber. In addition, the influence of aeration on the yeast cell surface hydrophobicity, surface charge, and elemental cell surface composition was measured. S. gordonii adhering at the glass surface caused a reduction in the initial deposition rate of C. albicans, regardless of aeration. In a stationary end-point, only adhesion of non-aerated C. albicans was suppressed by the adhering S. gordonii. Non-aerated yeasts had a higher O/C elemental surface concentration ratio, indicative of cell surface polysaccharides, than aerated yeasts, at the expense of nitrogen-rich cell surface proteins. Both yeasts were essentially uncharged, but the nitrogen-rich cell surface of aerated yeasts had a slightly higher water contact angle than non-aerated yeasts. Summarizing, this study suggests that highly localized, hydrophobic cell surface proteins on C. albicans are a prerequisite for their interaction with adhering streptococci.

Automatic enumeration of adherent streptococci or actinomyces on dental alloy by fluorescence image analysis

The aim of the present study was to develop an automated image analysis method to quantify adherence of Streptococcus sanguinis or Actinomyces viscosus on surfaces of a currently used dental alloy. Counting such bacterial strains was difficult because of their arrangement, thus S. sanguinis being a coccus arranged in chains or pairs, and A. viscosus a long complexly arranged polymorph rod. Direct counting of fluorescently stained adherent bacteria was done visually and with image analysis methods. To differentiate these two morphotypes, two programs were developed: (i) for streptococci, thresholding and selection of the object maxima, and (ii) for actinomyces, two step thresholding and processing of the characteristic points of the object skeletons. The triplicate enumerations for each bacterial strain were not significantly different ( p>0.005) and correlations between visual counting and automated counting were significant ( r=0.91 for S. sanguinis and r=0.99 for A. viscosus, p<0.0001). These rapid and reproducible methods, allowed us to count either cocci or rods, adherent on an inert substratum, in high density conditions.

Keratinocyte proinflammatory responses to adherent and nonadherent group A streptococci.

The gram-positive bacterium Streptococcus pyogenes (group A streptococcus) is the causative agent of a wide variety of suppurative infections of cutaneous tissues. Previous analyses have demonstrated that the M protein of S. pyogenes is an adhesin that directs the attachment of the streptococcus to keratinocytes in the skin. In this study, we have examined keratinocyte function in response to S. pyogenes and found that adherent versus nonadherent streptococci promote distinct patterns of expression of several proinflammatory molecules and keratinocyte cell fate. When analyzed by a quantitative reverse transcriptase PCR method, infection of cultured HaCaT keratinocytes with adherent, but not nonadherent, streptococci resulted in increased expression of mRNA for the cytokines interleukin-1alpha (IL-1alpha), IL-1beta, and IL-8 but neither infection induced expression of tumor necrosis factor alpha. In contrast, both adherent and nonadherent S. pyogenes induced expression of IL-6 and each promoted synthesis and release of prostaglandin E2 (PGE2). However, considerably greater levels of IL-6 expression were stimulated by adherent streptococci relative to nonadherent streptococci and the kinetics of PGE2 release in response to nonadherent streptococci was delayed compared to the response to adherent streptococci. Staining with the fluorescent probe ethidium homodimer-1 revealed that keratinocyte membranes were rapidly damaged upon infection with adherent streptococci but were not damaged by nonadherent streptococci. Finally, treatments which inhibited streptococcal metabolism completely blocked the ability of adherent streptococci to elicit responses. These data suggest that expression of an adhesin is a strategy used by S. pyogenes to modulate keratinocyte responses during infection of the skin and implicate additional streptococcal products in these signaling interactions.

Co-adhesion of oral microbial pairs under flow in the presence of saliva and lactose.

Co-aggregation (interactions between two suspended micro-organisms) between oral microbial pairs has been studied extensively and is believed to be an important factor in dental plaque formation. However, co-adhesion (interactions between suspended and already-adhering micro-organisms) may well be equally important. The aim of this paper was to determine the influence of saliva and lactose on the co-adhesion of streptococci (S. oralis 34 and S. sanguis PK1889) to actinomyces (A. naeslundii T14V-J1 or 5951) adhering on glass under flow from buffer and saliva in the absence and presence of lactose. The kinetics of co-adhesion as well as co-adhesion in a stationary end-point of co-aggregating and non-co-aggregating pairs was studied in a parallel plate flow chamber by analysis of the spatial arrangement of co-adhering micro-organisms as a function of time. For co-aggregating pairs, initial deposition rates of streptococci in the immediate vicinity of adhering actinomyces (local initial deposition rates) were up to 5 to 10 times higher than the non-local initial deposition rates in buffer and in saliva, respectively. In a stationary end-point of co-adhesion, 5 to 6 times more streptococci co-adhered with the adhering actinomyces than averaged over the entire substratum surface. A non-co-aggregating pair showed only minor preferential (co-)adhesion near the adhering actinomyces. Co-adhesion in buffer was fully lost when lactose was added. However, addition of lactose to saliva did not inhibit co-adhesion, but co-adhesion became more reversible. Detachment of micro-organisms from the substratum due to the passage of an air-liquid interface, as occurs in the oral cavity during eating, drinking, and speaking, was minimal when deposition was carried out from buffer to bare glass. Major detachment of streptococci adhering to the substratum occurred when adhesion was mediated through a salivary conditioning film on the glass, while detachment of adhering actinomyces and streptococci co-adhering with them remained low. It is suggested that, in the development of dental plaque, adhering actinomyces may act as strongholds for other micro-organisms, like streptococci, to adhere.

A quantitative method to study co-adhesion of microorganisms in a parallel plate flow chamber. II: Analysis of the kinetics of co-adhesion

Recently (J. Microbiological Methods 20, 289–305, 1994), it was suggested that dental plaque formation involved both interspecies binding between planktonic microorganisms (‘coaggregation’) as well as between planktonic and sessile microorganisms (‘co-adhesion’) and that co-adhesion could be measured by analyzing the spatial arrangements of co-adhering microorganisms in a stationary end point state. Limited information is available, however, on the kinetics of coaggregation and co-adhesion. The aim of this work was to develop a quantitative method to study the kinetics of co-adhesion and to determine the effects of mass transport conditions, like shear and concentration, on the kinetics of co-adhesion. The kinetics of co-adhesion of the coaggregating and non-coaggregating streptococci ( S. oralis J22 and S. sanguis PK1889) to glass with adhering A. naeslundii T14V-J1 cells, have been studied in a parallel plate flow chamber using real time image analysis. Initial local deposition rates in the vicinity of the Actinomyces were similar as observed on other regions over the substratum surface (‘non-local deposition rates’) for the non-co-adhering pair, but were up to 19 times higher for the co-adhering pair. Thus, whereas we have previously shown that co-adhesion in a stationary end point state can be quantified by radial pair distribution functions, this paper demonstrates that the tendency of coaggregating strains to co-adhere is also reflected in the kinetics of co-adhesion. Local deposition rates increased with increasing streptococcal concentration for the coaggregating pair. However, if it is attempted to increase the local deposition rate by increasing the shear, it was found that local deposition rates decreased, most likely because the interaction time between the adhering actinomyces and the flowing streptococci becomes too short. The high deposition rates for the coaggregating pair could not be explained on the basis of convective-diffusional mass transport towards the substratum, but required accounting for collisions between adhering actinomyces and streptococci flowing parallel to the surface.