S. Kneist

Acid production by oral strains of Candida albicans and lactobacilli.

Both Candida albicans and lactobacilli are common colonizers of carious lesions in children and adolescents. The purpose of this study is to compare the velocity of acid production between C. albicans and several Lactobacillus species at different pH levels and concentrations of glucose. Washed, pure resting-cell suspensions were obtained by culturing a total of 28 oral isolates comprising the species C. albicans, Lactobacillus rhamnosus, Lactobacillus paracasei paracasei, Lactobacillus paracasei tolerans and Lactobacillus delbrueckii lactis. Acid production from glucose was determined at a constant pH of 7.0, 5.5, 5.0 and 4.0 by repeated titrations with NaOH in an automated pH-stat system. Acid formation rates of yeast and lactobacilli proved to be similar at both neutral and low pH, while in a moderately acidic environment C. albicans produced less acid than the lactobacilli. Ion chromatographic analysis of the cell-free medium after titration revealed pyruvate to be the predominant organic acid anion secreted by C. albicans. The proportion of organic acids to overall acid production by the yeast was below 10% at neutral conditions, in contrast to 42–66% at pH 4.0. Compared to lactobacilli, yeast required a concentration of glucose that was about 50 times higher to allow acid production at half the maximum speed. Considering the clinical data in the literature about the frequency and proportions of microorganisms present in early childhood caries lesions, the contribution of oral lactobacilli as well as C. albicans to overall microbial acid formation appears to be important.

Comparison of the efficacies of disinfectants to control microbial contamination in dental unit water systems in general dental practices across the European union

ABSTRACT Water delivered by dental unit water systems (DUWS) in general dental practices can harbor high numbers of bacteria, including opportunistic pathogens. Biofilms on tubing within DUWS provide a reservoir for microorganisms and should be controlled. This study compared disinfection products for their ability to meet the American Dental Associations guideline of <200 CFU · ml−1 for DUWS water. Alpron, BioBlue, Dentosept, Oxygenal, Sanosil, Sterilex Ultra, and Ster4Spray were tested in DUWS (n = 134) in Denmark, Germany, Greece, Ireland, The Netherlands, Spain, and the United Kingdom. Weekly water samples were tested for total viable counts (TVCs) on yeast extract agar, and, where possible, the effects of products on established biofilm (TVCs) were measured. A 4- to 5-week baseline measurement period was followed by 6 to 8 weeks of disinfection (intermittent or continuous product application). DUWS water TVCs before disinfection ranged from 0 to 5.41 log CFU · ml−1. Disinfectants achieved reductions in the median water TVC ranging from 0.69 (Ster4Spray) to 3.11 (Dentosept) log CFU · ml−1, although occasional high values (up to 4.88 log CFU · ml−1) occurred with all products. Before treatment, 64% of all baseline samples exceeded American Dental Association guidelines, compared to only 17% following commencement of treatment; where tested, biofilm TVCs were reduced to below detectable levels. The antimicrobial efficacies of products varied (e.g., 91% of water samples from DUWS treated with Dentosept or Oxygenal met American Dental Association guidelines, compared to 60% of those treated with Ster4Spray). Overall, the continuously applied products performed better than those applied intermittently. The most effective products were Dentosept and Oxygenal, although Dentosept gave the most consistent and sustained antimicrobial effect over time.

Fluorescence‐controlled Er:YAG laser for caries removal in permanent teeth: a randomized clinical trial

The aim of this randomized clinical study was to compare the efficacy of a fluorescence-controlled erbium-loaded yttrium aluminum garnet (Er:YAG) laser with conventional bur treatment for caries therapy in adults. Twenty-six patients with 102 carious lesions were treated using either the Er:YAG laser, at threshold levels of 7, 8, 9, and 10 [U], or rotary burs. Both techniques were applied to each lesion at separate locations. After treatment, dentine samples were obtained using a carbide bur. The viable counts of Streptococcus mutans (SM) and lactobacilli (LB) [expressed as colony-forming units (log10 CFUs)], treatment time, pain, vibration, and sound intensity were determined. The median numbers of CFUs for SM and LB were not statistically different between laser and bur treatment at threshold levels 7 and 8 [U]. At threshold levels 9 and 10 [U], the median number of CFUs for LB [1.11 (range: 0.00-2.04)] were significantly higher following laser treatment than following bur treatment [0.30 (range: 0.00-0.60)]. The results indicate that treatment with a fluorescence-controlled Er:YAG laser at threshold levels of 7 and 8 removed caries to a level similar to that achieved using conventional bur treatment, with clinically irrelevant amounts of remaining bacteria. Although more time consuming, laser treatment provided higher patient comfort than bur treatment.

Shotgun mass mapping of Lactobacillus species and subspecies from caries related isolates by MALDI‐MS

A taxonomical study of 90 isolates of lactobacilli isolated from soft and hard carious dentine of 70 deciduous molars is presented. The Lactobacillus strains were determined by shotgun mass mapping (SMM). This method based on MALDI‐MS analysis of Lactobacillus isolates treated with trypsin followed by database comparison against a library of mass spectra derived from 20 reference strains. The SMM method allowed to discriminate different Lactobacillus subspecies. The method was used to analyse Lactobacillus isolates of unknown identity derived from carious dentine. Application of the SMM method to isolates from hard carious dentine revealed a nearly similar distribution of L. paracasei ss paracasei (29%), L. paracasei ss tolerans (32%) and L. casei ss rhamnosus (23%) as dominant subspecies. On the other hand, samples derived from soft carious dentine showed a clear bias only to L. paracasei ss paracasei (60%), whereas L. paracasei ss tolerans (14%) and L. casei ss rhamnosus (12%) were clear minorities. Compared to existent methods, SMM has unique potential for the analysis of Lactobacillus strains on subspecies level.

Suppression of caries-related microorganisms in dentine lesions after short-term chlorhexidine or antibiotic treatment.

This study investigated the efficiency of a chlorhexidine varnish and an antibiotic paste in suppressing the cultivable microflora of deep dentine cavities in a stepwise excavation procedure. Subsequent to enamel preparation and removal of the central biomass, infected dentine was sampled from the cavity floor. Ten cavities each were either covered with the 1% chlorhexidine- and 1% thymol-containing varnish Cervitec (CE), the demeclocycline hydrocortisone-containing ointment Ledermix (LE) or received no treatment as control (CO). A compomer composite was used as intermediate restorative. Cavities were reassessed after 6 weeks and again dentine samples were microbiologically investigated for total viable counts, mutans streptococci and lactobacilli. After 6 weeks a significant reduction of the total viable counts was observed in the LE group (p = 0.011) compared to the control, whereas no differences were found in the CE group (p > 0.05). Mutans streptococci were rarely recovered at baseline and after 6 weeks. Compared to the CO group counts of lactobacilli were significantly reduced in the CE and LE groups (p < 0.05). Lactobacillus species were frequently recovered at baseline and after 6 weeks of observation. Lactobacillus rhamnosus was the predominant species in all samples investigated. Application of CE or LE resulted in reduced counts of lactobacilli after a period of 6 weeks. Although none of the materials completely eliminated the viable microorganisms, the use of LE was more effective than CE in reducing the total anaerobic microorganisms associated with carious dentine.

Cariogenic Effects of Probiotic Lactobacillus rhamnosus GG in a Dental Biofilm Model

Probiotic bacteria have been suggested to inhibit Streptococcus mutans (SM) and thus prevent dental caries. However, supporting evidence is weak and probiotic species might be cariogenic themselves. Thus, we compared and combined the probiotic Lactobacillus rhamnosus GG (LGG) with SM and analysed the resulting mineral loss (ΔZ) in dental tissues. We simulated three biofilm compositions (SM, LGG, SM × LGG), two lesion sites (smooth enamel, dentin cavity) and two nutrition supply frequencies (twice/day, 6 times/day) in a multi-station, continuous-culture biofilm model. A total of 240 bovine enamel and dentin samples were cut, polished and embedded. All experimental procedures were performed in independent duplicates, with 10 samples being allocated to each group for each experiment (final sample size n = 20/group). Biofilms were cultured on the specimens and supplied with 2% sucrose medium and artificial saliva in consecutive pulses. After 10 days, ΔZ and bacterial numbers were assessed. SM × LGG biofilms caused significantly increased ΔZ compared with SM or LGG biofilms (p < 0.01, Mann-Whitney test), and ΔZ was significantly increased in dentin cavities compared with smooth enamel lesions (p < 0.01). Bacterial numbers did not significantly differ between biofilms of different species (p > 0.05, ANOVA). Frequent nutrition supply significantly increased bacterial numbers (p < 0.01). Biofilms in dentin cavities compared to smooth enamel harboured significantly more bacteria (p < 0.05). LGG induced mineral loss especially in dentin cavities and under highly cariogenic conditions. LGG did not have inhibitory effects on SM, but rather contributed to the caries process in vitro.

Streptococcus sobrinus in children and its influence on caries activity.

Aim: This was to study the longitudinal assessment of caries activity of Streptococcus sobrinus (SS) positive children during their mixed dentition. Methods: The occurrence of mutans streptococci (MS) in plaque and saliva was determined in a representative sample of 55 children aged 8 to 12 years over a period of 4 years. A total of 708 bacterial strains was isolated which were identified as MS or SS. Caries activity (ΔD1−4MFS) as well as plaque and gingival inflammation were recorded. Results: During the period of observation 52 of the 55 children harboured MS; 12 of these children were SS positive. SS was not permanently detectable and 3 of the children were MS and SS negative. SS was not found without the presence of MS. Children that were infected with both SS and MS showed a slightly higher increase in caries compared with children that were infected exclusively by MS (ΔD1,2MFS 6.2 vs. 3.0 and ΔD3,4MFS 5.3 vs. 3.8) over the period of 4 years. An SS infection accelerated the increase of ΔD3,4MFS significantly by a factor of 4 one year after its detection, whereas the ΔD1,2MFS was 3 times as high during the period of infection. Conclusion: The findings suggest that an SS infection represents an important additional risk factor for dental caries due to its obvious aggravating of caries activity.

Peroxidase Reaction as a Parameter for Discrimination of Streptococcus mutans and Streptococcus sobrinus

425 strains of mutans streptococci and 12 reference strains were investigated by membrane fatty acid spectra (MFAS) and peroxidase reaction (PR) after aerobic and anaerobic incubation. 423 strains were identified as Streptococcus mutans. The remaining 2 strains were identified as Streptococcus sobrinus. The PR of 29 strains was doubtful; immediately after anaerobic incubation a negative PR changed into a slightly positive PR. To test the diagnostic value of PR the strains were additionally investigated by means of species–specific polymerase chain reactions (PCR). The species–specific PCRs were developed on the basis of the respective genes of 16S rRNA of the pathogens S. mutans and S. sobrinus. Specificity and sensitivity were tested on reference strains (n = 17) and negative control strains (n = 39). The results of this investigation showed that an anaerobic incubation regime could lead to false–positive (S. mutans) or false–negative (S. sobrinus) PR. The 425 MS strains were classified as either S. mutans (n = 420) or S. sobrinus (n = 5). The findings on the reference strains required a reclassification of S. mutans V 100 into S. sobrinus V 100. Summarising, it is possible now to differentiate strains of mutans streptococci by MFAS and PR after aerobic incubation.

Diversity of Lactobacillus species in deep carious lesions of primary molars

AIM: This was to determine the prevalence of Lactobacilli (LB) species in different stages of caries progression and are considered as secondary invaders of existing carious lesions and specialists for caries progression. METHODS: Carious dentine samples were collected from 70 primary molars (M) during step-wise (S1, S2: n = 35 M) or one-step (O1: n = 35 M) caries treatment and after 11 months of temporary restorations (S3, O2). LB were identified by selected physiological and biochemical characteristics, ratio of lactic acid isomers, electrophoretic mobilities of lactic acid dehydrogenases, and shotgun mass mapping by MALDI mass spectrometry. RESULTS: LB were isolated from 46% of soft dentine samples (S1). The prevalence of LB from hard dentine collected during caries excavation (O1) reached 34%, after 8 weeks of temporary filling (S2) 11%, and 9% each after 11 months of temporary restoration (S3, O2). The mean total bacterial counts (cfu) of soft dentine (S1) were 3.6 × 105. From hard dentine during caries excavation (O1 ) 4.4×104 cfu were calculated, at S2 3.7 × 103 cfu, at S3 0.1 × 103 cfu, and at O2 1.8 × 103 cfu. The percentages of LB in the cfu for LB positive dentine samples were for S1 / S2 / S3 / O1 / O2: 60% (16 M)/34% (4 M)/54% (3 M)/57% (9 M), and 64% (3 M). Five LB species were identified from carious dentine: L. paracasei subsp. paracasei, L. paracasei subsp. tolerans, L. rhamnosus, L. gasseri, and L. alimentarius. CONCLUSIONS: While L. rhamnosus and L. paracasei subsp. paracasei occurred in all caries progression stages, the other species were found only sporadically. L. paracasei subsp. paracasei and L. rhamnosus might be the specialists of the LB in carious progression.

Inhibition of Streptococcus mutans Growth and Biofilm Formation by Probiotics in vitro

To exert anticaries effects, probiotics are described to inhibit growth and biofilm formation of cariogenic bacteria such as Streptococcus mutans (SM). We screened 8 probiotics and assessed how SM growth or biofilm formation inhibition affects cariogenicity of probiotic-SM mixed-species biofilms in vitro. Growth inhibition was assessed by cocultivating probiotics and 2 SM strains (ATCC 20532/25175) on agar. Probiotics were either precultured before SM cultivation (exclusion), or SM precultured prior to probiotic cultivation (displacement). Inhibition of SM culture growth was assessed visually. Inhibition of SM biofilm formation on bovine enamel was assessed using a continuous-flow short-term biofilm model, again in exclusion or displacement mode. The cariogenicity of mixed-species biofilms of SM with the most promising growth and biofilm formation inhibiting probiotic strains was assessed using an artificial mouth model, and enamel mineral loss (ΔZ) was measured microradiographically. We found limited differences in SM growth inhibition in exclusion versus displacement mode, and in inhibition of SM 20532 versus 25175. Results were therefore pooled. Lactobacillus acidophilus LA-5 inhibited significantly more SM culture growth than most other probiotics. L. casei LC-11 inhibited SM biofilm formation similarly to other alternatives but showed the highest retention of probiotics in the biofilms (p < 0.05). Mineral loss from SM monospecies biofilms (ΔZ = 9,772, 25th/75th percentiles: 6,277/13,558 vol% × µm) was significantly lower than from mixed-species SM × LA-5 biofilms (ΔZ = 24,578, 25th/75th percentiles: 19,081/28,768 vol% × µm; p < 0.01) but significantly higher than from SM × LC-11 biofilms (ΔZ = 4,835, 25th/75th percentiles: 263/7,865 vol% × µm; p < 0.05). Probiotics inhibiting SM culture growth do not necessarily reduce the cariogenicity of SM-probiotic biofilms. Nevertheless, SM biofilm formation inhibition may be relevant in the reduction of cariogenicity.