Antimicrobial effects of dental luting glass ionomer cements on Streptococcus mutans.

OBJECTIVE: To reduce secondary caries, glass ionomer luting cements are often used for cementing of indirect restorations. This is because of their well-known antimicrobial potential through the release of fluoride ions. The aim of this in vitro study was to investigate the antimicrobial effect of five dental luting cements which were based on glass ionomer cement technology. METHODS: Five different glass ionomer based luting cements were tested for their antimicrobial effects on Streptococcus mutans in two different experimental setups: (i) determination of colony-forming units (CFUs) in a plate-counting assay; (ii) live/dead staining (LDS) and fluorescence microscopy. All experiments were conducted with or without prior treatment of the materials using sterilized human saliva. Antimicrobial effects were evaluated for adherent and planktonic bacteria. Bovine enamel slabs (BES) were used as negative control. BES covered with 0.2% chlorhexidine (CHX) served as positive control. RESULTS: Each of the tested materials significantly reduced the number of initially adhered CFUs; this reduction was even more pronounced after prior incubation in saliva. Antimicrobial effects on adherent bacteria were confirmed by live-dead staining. CONCLUSION: All five luting cements showed an antimicrobial potential which was increased by prior incubation with human saliva, suggesting an enhanced effect in vivo.

Antibiotic resistance and capacity for biofilm formation of different bacteria isolated from endodontic infections associated with root-filled teeth.

INTRODUCTION: To date, a variety of microbial species have been isolated from endodontic infections. However, endodontic clinical bacterial isolates have not been sufficiently characterized with regard to their capacity for antibiotic resistance and biofilm formation. In this study, antibiotic resistance and biofilm formation of 47 different aerobic and anaerobic bacterial isolates, belonging to 32 different species previously isolated from infected filled root canals, were studied. METHODS: Antibiotic sensitivity to 11 antibiotics including penicillin G, amoxicillin, clindamycin, gentamicin, vancomycin, tetracycline, doxycycline, fosfomycin, rifampicin, ciprofloxacin, and moxifloxacin was tested using the standardized Etest method (Bio Merieux, Marcy-1'Etoile, France). The antibiotic sensitivity of 4 control strains was also estimated in parallel. Additionally, the capacity to form biofilms was quantified using the microtiter plate test. RESULTS: Different aerobic and anaerobic bacterial species were either resistant against a number of antibiotics or showed high minimal inhibitory concentrations against clinically relevant antibiotics. Five aerobic and 2 anaerobic isolates, including Enterococcus faecalis, Streptococcus mutans, Lactobacillus fermentum, Actinomyces naeslundii, Actinomyces viscosus, Prevotella buccae, and Propionibacterium acidifaciens, were characterized as being high biofilm producers, whereas 8 aerobic and 3 anaerobic isolates were found to be moderate biofilm producers. Most isolates with resistance or markedly high minimal inhibitory concentration values were also either moderate biofilm producers or high biofilm producers. CONCLUSIONS: These results suggest that the clinical significance of endodontic infections could include that they serve as a reservoir for antibiotic resistance. Furthermore, endodontic treatment should consider the adhesion and biofilm formation by a variety of bacteria.

Bacterial and Candida albicans adhesion on different root canal filling materials and sealers.

INTRODUCTION: Microbial adhesion and subsequent biofilm formation on endodontic root canal filling materials and sealers lead to survival of microorganisms in treated root canals and subsequently to endodontic treatment failures. The present study focused on initial microbial adhesion to different endodontic filling materials. METHODS: The following endodontic biomaterials were tested: AH-Plus, Tubli Seal, gutta-percha, Real Seal SE, EndoREZ, Apexit Plus, GuttaFlow, and dentin. Samples of each material were prepared. Bovine dentin samples were used as a control. The initial adhesions of salivary bacteria as well as the subsequent single species were quantified by determination of colony-forming units (CFUs) and visualized by scanning electron microscopy and confocal microscopy (CLSM): Enterococcus faecalis, Streptococcus mutans, Streptococcus sanguis, Candida albicans, and Prevotella nigrescens. RESULTS: Initially adherent microorganisms could be detected and microscopically visualized on each of the materials tested. Considering the values of the CFUs and the covering grade as detected by CLSM, there were significant differences among the materials. Fewer bacteria tended to adhere to Apexit Plus, whereas Real Seal SE and the widely used gutta-percha showed the highest number of adherent bacteria. This tendency was not detected for C. albicans. CONCLUSIONS: Endodontic microorganisms have a high affinity to root canal filling materials and sealers, especially to gutta-percha. Because of this high level of bacterial adhesion, subsequent biofilm formation on these materials could be suggested as leading to the persistence of microorganisms in root canals.

Targeted immobilisation of lysozyme in the enamel pellicle from different solutions.

Mouthwashes containing protective enzymes are required especially for patients suffering from xerostomia. The present study aimed to investigate the possibilities of modulating the immobilisation of lysozyme in the in situ pellicle layer. In situ formed pellicles were incubated in vitro for 10 min with various enzymatic buffer solutions containing lysozyme and additive enzymes such as transglutaminase or trypsin as well as polyphenolic compounds (cistus tea). After the rinses, the pellicle samples were incubated in collected whole saliva or in desorption solutions for 0, 20 and 40 min and the enzyme activities were measured. Furthermore, accumulation of lysozyme in the pellicle was visualised in ultrathin sections of the pellicle using the gold immunolabelling technique and transmission electron microscopy. Hen egg white lysozyme was accumulated in the in situ pellicle tenaciously. Up to 2.8-fold higher activities than in controls were observed. The addition of transglutaminase did not enhance the immobilisation of lysozyme activity, whereas the polyphenolic compound had no adverse effect. Accumulation of lysozyme in the acquired pellicle was confirmed by gold immunolabelling. Targeted and tenacious immobilisation of lysozyme in the acquired pellicle is possible. Poylphenolic compounds might be a relevant additive for mouthwashes containing lysozyme.

Food-borne enterococci integrate into oral biofilm: an in vivo study.

INTRODUCTION: Enterococci, particularly Enterococcus faecalis, are still a primary concern in endodontic infections. To date, enterococci have been considered to be only transiently present in the oral cavity. The aim of this study was to examine whether different enterococci from food are able to reside in oral biofilm. METHODS: Six healthy volunteers wore dental splints loaded with enamel slabs. After 3 days, the volunteers consumed cheese containing enterococci. The fate of the enterococci was analyzed by culture technique and 16S rRNA gene sequencing. All isolates were characterized genotypically by macrorestriction analysis (SmaI) and pulsed-field gel electrophoresis. E. faecalis was also analyzed by using fluorescent in situ hybridization (FISH). RESULTS: E. faecalis, E. faecium, E. avium, and E. durans were detected in the initial biofilm after 2 hours, as well as in the 5-day-old oral biofilm. E. faecalis, E. faecium, and E. avium isolated from the initial biofilm and from the 5-day-old biofilm, as well as those isolated from cheese, showed genetic homogeneity. E. faecium and E. avium had integrated into a pre-existing 3-day-old biofilm. No genetic similarity between E. durans strains isolated from cheese and those from the initial and 5-day-old oral biofilm was detected. E. faecalis was also detected in the oral biofilm by using FISH. CONCLUSIONS: Food-borne enterococci, particularly E. faecalis, might not only be transient but could also survive in the oral biofilm and become a source for endodontic infections. Moreover, genotypic analysis is required to study the source of oral enterococci.

Visualization of initial bacterial colonization on dentine and enamel in situ.

Bacterial colonization of dentine is of high relevance in cariology, endodontology and periodontology. The aim of the present in situ study was to establish recent methods for visualization and quantification of initial bacterial adherence to dentine in comparison to enamel. For this purpose, bovine enamel and dentine slabs were fixed on buccal sites of individual upper jaw splints worn by 6 subjects for 30min, 120min and 360min, respectively. Adherent bacteria on the slabs were visualized and quantified with DAPI-staining (4',6-diamidino-2-phenylindole) and fluorescence in situ hybridization (FISH) of streptococci and eubacteria using the CLSM (confocal laser scanning microscopy) as well as an epifluorescence microscope. In addition, the number of colony forming units was quantified after desorption. Representative samples were processed for SEM (scanning electron microscopy) and TEM (transmission electron microscopy). All methods clearly indicated that a significantly higher number of bacteria adhered to dentine than to enamel. Furthermore, the amount of bacteria on the dentine increased with increasing oral exposure time, but remained rather constant on the enamel. The CLSM allowed visualization of bacteria in the dentinal tubules. Bacteria were found preferentially at the openings of the dentine tubules, but were distributed randomly on the enamel. In conclusion, the adopted methods are suitable for visualization and quantification of bacterial adhesion to dentine. Even the initial bacterial colonization of dentine is much more pronounced than bacterial adherence to the enamel.

Polyphenolic beverages reduce initial bacterial adherence to enamel in situ.

OBJECTIVES: Polyphenols are antibacterial and anti-oxidative natural agents. The present in situ study aimed to investigate the effect of different polyphenolic beverages on initial bacterial adherence to enamel in the oral cavity. METHODS: Initial biofilm formation was performed on bovine enamel specimens mounted buccally on individual upper jaw splints and carried by six subjects. After 1 min of pellicle formation, oral rinses with black tea, green tea, grape juice, Cistus tea or red wine were performed for 10 min. Afterwards the slabs were carried for another 19 or 109 min, respectively. Samples exposed to the oral fluids for 30 and 120 min served as controls. Following intraoral exposure, the slabs were rinsed with saline solution. The amount of adherent bacteria was determined with DAPI-staining (4',6-diamidino-2-phenylindole) and with fluorescence-in situ hybridization (FISH) of eubacteria and streptococci. RESULTS: Rinses with all beverages reduced the amount of detectable bacteria. Lowest number of adherent bacteria was found following rinses with red wine, Cistus tea and black tea as measured with DAPI (up to 66% reduction of adherent bacteria vs. controls). Also FISH revealed significant impact of most tested beverages. CONCLUSIONS: Rinses with certain polyphenolic beverages as well as consumption of these foodstuffs may contribute to prevention of biofilm induced diseases in the oral cavity.

Characterisation of lysozyme activity in the in situ pellicle using a fluorimetric assay.

Lysozyme is among the most protective enzymes in the pellicle layer. The aim of the present study was to establish a precise fluorimetric assay for determination and characterisation of lysozyme activity immobilised in the initial in situ formed pellicle. For in situ pellicle formation, bovine enamel slabs were fixed on maxillary splints and carried by six subjects for different times (3, 30 min) on buccal and palatal sites. The enzymatic assay was based on hydrolysis of cell walls from Micrococcus lysodeicticus linked to a fluorogenic substance. When the substrate is hydrolysed, a fluorescing product is released. Furthermore, the effects of chlorhexidine and black tea on lysozyme in the in situ pellicle were investigated. The fluorimetric method allowed direct determination of the enzyme activity with the slab inside the well of a microtiter plate. The mean immobilised activity over all samples amounted to 68.67 +/- 27.35 U/cm(2) (desorbed activity = 46.76 +/- 21.18 U/cm(2)). The enzyme activity exposed at the pellicles' surfaces increased in a time-dependant manner and showed a Michaelis-Menten kinetic. Chlorhexidine and black tea reduced lysozyme activity of the in situ pellicle significantly. After rinsing with tea or chlorhexidine, V(max) was reduced, whereas K(m) remained unaffected indicating a negative allosteric effect of the V type. The fluorimetric method is appropriate for determination of pellicle lysozyme activity. The influence of effectors on immobilised lysozyme activity can be monitored.

Effects of Cistus-tea on bacterial colonization and enzyme activities of the in situ pellicle.

OBJECTIVES: Polyphenols are expected to have antibacterial properties. Cistus is a tea rich in polyphenols. The aim of the present in situ study was to investigate the effect of Cistus-tea on the pellicle and on the initial oral biofilm. METHODS: For in situ pellicle formation and initial biofilm formation, bovine enamel slabs were fixed on maxillary splints and carried by four subjects at buccal sites for up to 2 h. Bacteria present in 120-min pellicles were determined with DAPI-staining and fluorescence in situ hybridization with and without a 10 min rinse with Cistus-tea performed 1 min after incorporation of the slabs. In addition, amylase, lysozyme, glucosyltransferase and peroxidase activities immobilised in the pellicle layer were measured before and after rinsing for 10 min with Cistus-tea. RESULTS: The amount of bacteria detected in the 120-min biofilm was reduced significantly, if a 10 min rinse with Cistus-tea was performed one min after insertion of the enamel slabs. DAPI-staining yielded 13.2+/-3.5 for controls and 6.5+/-1.1 x 10(4) bacteria/cm(2), if a rinse with Cistus-tea was applied. Lysozyme, amylase and glucosyltransferase activities immobilised in the pellicle were not affected following a rinse with Cistus-tea. However, peroxidase activity was reduced significantly. CONCLUSIONS: Cistus-tea may be used to reduce the initial bacterial adhesion in the oral cavity.

Detection and activity of peroxidase in the in situ formed enamel pellicle.

AIM: Peroxidase is the main salivary antioxidant. The aim of the present study was to detect and to characterise peroxidase in the in situ enamel pellicle. METHODS: Bovine enamel slabs were fixed on maxillary splints and carried by six subjects for different times (3, 30 and 120 min) on buccal and palatal sites. Pellicle bound peroxidase activity was determined fluorimetrically using 2',7'-dichlorofluorescin as a substrate. The peroxidase molecules present in the pellicle were visualised with the gold-immunolabelling technique and evaluated by TEM. Furthermore, effects of polyphenols and hydrogen peroxide on peroxidase and its enzymatic activity were examined. RESULTS: All pellicles which were tested revealed peroxidase activity and labelled peroxidase molecules were detected in all samples. The numbers of gold-labelled peroxidase molecules detectable in cross-sections of the pellicles were correlated significantly with the pellicle formation time. After 3 min, 0.50+/-1.01 labelled molecules were detected (30 min: 1.42+/-1.98; 120 min: 4.15+/-4.13, ANOVA, p<0.001). The mean immobilised peroxidase activity exposed at the surface amounted to 24.4+/-27.7 mU/cm2; no continuous increase of activity with formation time was found. Hydrogen peroxide and polyphenolic beverages inactivated peroxidase activity of the pellicle. Despite these inhibiting effects, considerable amounts of peroxidase molecules were still detectable by gold-immunolabelling. After contact with the inhibiting agents in situ, peroxidase activity of the pellicle reconstituted slowly. CONCLUSION: Peroxidase activity is present in the pellicle already after 3 min of formation time, but is inhibited by the substrate and polyphenolic beverages.

Plasmid-mediated quinolone resistance in isolates obtained in german intensive care units.

Screening of 703 isolates of Enterobacteriaceae, obtained from 34 German intensive care units (ICUs), revealed qnr-positive, integron-containing isolates of Enterobacter sp. and Citrobacter freundii from four patients in 2 German ICUs. This is one of the first reports of qnr-positive strains obtained from patients in Europe.

Discrimination of Klebsiella pneumoniae and Klebsiella oxytoca phylogenetic groups and other Klebsiella species by use of amplified fragment length polymorphism.

Bacteria of the genus Klebsiella are opportunistic pathogens responsible for an increasing number of multiresistant infections in hospitals. The two clinically and epidemiologically most important species, Klebsiella pneumoniae and K. oxytoca, have recently been shown to be subdivided into three and two phylogenetic groups, respectively. The aim of this study was an in depth evaluation of the amplified fragment length polymorphism (AFLP) genetic characterization method for epidemiological and phylogenic analyzes of Klebsiella isolates. First, we investigated the variability of AFLP patterns for Klebsiella strains within and between different outbreaks. Second, by use of carefully characterized phylogenetically representative strains, we examined whether different Klebsiella species and phylogenetic groups can be discriminated using AFLP. Twenty-four strains originating from seven presumed outbreaks and 31 non-associated strains were investigated. The AFLP fingerprints of all epidemiologically associated strains showed three or fewer fragment differences, whereas unrelated strains differed by at least four fragments. Cluster analysis of the AFLP data revealed a very high concordance with the phylogenetic assignation of strains based on the gyrA sequence and ribotyping data. The species K. pneumoniae, K. oxytoca, K. terrigena and the possibly synonymous pair K. planticola/K. ornithinolytica each formed a separate cluster. Similarly, strains of the phylogenetic groups of K. pneumoniae and K. oxytoca fell into their corresponding clusters, with only two exceptions. This study provides a preliminary cut-off value for distinguishing epidemiologically non-related Klebsiella isolates based on AFLP data; it confirms the sharp delineation of the recently identified phylogenetic groups, and demonstrates that AFLP is suitable for identification of Klebsiella species and phylogenetic groups.