The consistent application of hydrogen peroxide controls biofilm growth and removes Vermamoeba vermiformis from multi-kingdom in-vitro dental unit water biofilms.

The water systems inside a dental unit are known to be contaminated with a multi-kingdom biofilm encompassing bacteria, fungi, viruses and protozoa. Aerosolization of these micro-organisms can potentially create a health hazard for both dental staff and the patient. Very little is known on the efficacy of dental unit disinfection products against amoeba. In this study we have examined the effect of four different treatment regimens, with the hydrogen peroxide (H2O2) containing product Oxygenal, on an in-vitro multi-kingdom dental unit water system (DUWS) biofilm. The treatment efficacy was assessed in time using heterotrophic plate counts, the bacterial 16S rDNA, fungal 18S rDNA gene load and the number of genomic units for Legionella spp. the amoeba Vermamoeba vermiformis. The results indicated that a daily treatment of the DUWS with a low dose H2O2 (0.02% for 5 h), combined with a weekly shock dose (0.25% H2O2, 30 min) is necessary to reduce the heterotrophic plate count of a severely contaminated DUWS (>106 CFU.mL-1) to below 100 CFU.mL-1. A daily treatment with a low dose hydrogen peroxide alone, is sufficient for the statistically significant reduction of the total amount of bacterial 16S rDNA gene, Legionella spp. and Vermamoeba vermiformis load (p < 0.005). Also shown is that even though hydrogen peroxide does not kill the trophozoite nor the cysts of V. vermiformis, it does however result in the detachment of the trophozoite form of this amoeba from the DUWS biofilm and hereby ultimately removing the amoeba from the system.

The Highly Leukotoxic JP2 Genotype of Aggregatibacter actinomycetemcomitans Is Present in the Population of the West African Island, Sal in Cape Verde: A Pilot Study.

Aggregatibacter actinomycetemcomitans is strongly associated with severe periodontitis, possibly due to its production of a potent leukotoxin. A genetic variant, the JP2 genotype, was found to produce more leukotoxin than the wild type because of a mutation in the leukotoxin gene, and this genotype is frequently found in African populations. The aim of this study was to investigate whether this JP2 genotype can be found in a randomly selected group of inhabitants of Sal, Cape Verde. Twenty-nine adults between 20 and 59 years of age (58.6% female) participated, and information on their oral health and living standards was collected. An oral examination was performed for each participant, including DMF-T and CPI scores. Plaque and saliva samples were collected and transported to Europe, where DNA was isolated, and the concentration of A. actinomycetemcomitans and its JP2 genotype was determined using dedicated PCR analyses. All 29 plaque and 31% of the saliva samples harboured A. actinomycetemcomitans, and two participants were positive for the JP2 genotype. The presence of this JP2 genotype was not associated with either CPI or DMF-T. This pilot study is the first to describe the presence of the A. actinomycetemcomitans JP2 genotype in a Cape Verdean population living in the Cape Verde Islands, and the findings warrant further research.

Niacin Limitation Promotes Candida glabrata Adhesion to Abiotic Surfaces.

Candida glabrata is a prevalent fungal pathogen in humans, which is able to adhere to host cells and abiotic surfaces. Nicotinic acid (NA) limitation has been shown to promote the adherence of C. glabrata to human epithelial cells. Clinically, the elderly and hospitalized patients who are prone to C. glabrata-related denture stomatitis often suffer from vitamin deficiency. This study aimed to investigate C. glabrata adhesion to abiotic surfaces, including acrylic resin (a denture material) surfaces, cell surface hydrophobicity and adhesion gene expression. C. glabrata CBS138 was grown in media containing decreasing NA concentrations (40, 0.4, 0.04 and 0.004 µM). Adherence of C. glabrata to glass coverslips and acrylic resin was analyzed. C. glabrata adhesion to both surfaces generally increased with decreasing NA concentrations. The highest adhesion was found for the cells grown with 0.004 µM NA. The cell surface hydrophobicity test indicated that NA limitation enhanced hydrophobicity of C. glabrata cells. Quantitative PCR showed that of all adhesion genes tested, EPA1, EPA3 and EPA7 were significantly up-regulated in both 0.004 µM NA and 0.04 µM NA groups compared to those in the 40 µM NA group. No significant up- or down-regulation under NA limitation was observed for the other tested adhesion genes, namely AWP3, AWP4, AWP6 and EPA6. NA limitation resulted in increased expression of some adhesion genes, higher surface hydrophobicity of C. glabrata and enhanced adhesion to abiotic surfaces. NA deficiency is likely a risk factor for C. glabrata-related denture stomatitis in the elderly.

Long-Term Analysis of Resilience of the Oral Microbiome in Allogeneic Stem Cell Transplant Recipients.

Stem cell transplantation (SCT) is associated with oral microbial dysbiosis. However, long-term longitudinal data are lacking. Therefore, this study aimed to longitudinally assess the oral microbiome in SCT patients and to determine if changes are associated with oral mucositis and oral chronic graft-versus-host disease. Fifty allogeneic SCT recipients treated in two Dutch university hospitals were prospectively followed, starting at pre-SCT, weekly during hospitalization, and at 3, 6, 12, and 18 months after SCT. Oral rinsing samples were taken, and oral mucositis (WHO score) and oral chronic graft-versus-host disease (NIH score) were assessed. The oral microbiome diversity (Shannon index) and composition significantly changed after SCT and returned to pre-treatment levels from 3 months after SCT. Oral mucositis was associated with a more pronounced decrease in microbial diversity and with several disease-associated genera, such as Mycobacterium, Staphylococcus, and Enterococcus. On the other hand, microbiome diversity and composition were not associated with oral chronic graft-versus-host disease. To conclude, dysbiosis of the oral microbiome occurred directly after SCT but recovered after 3 months. Diversity and composition were related to oral mucositis but not to oral chronic graft-versus-host disease.

Oral hygiene in patients with motor neuron disease requires attention: A cross-sectional survey study.

AIMS: Motor Neuron Disease (MND) is a progressive neurodegenerative neuromuscular disease, which can progressively impair arm-hand function. Needs and barriers of MND patients and their caregivers in performing oral hygiene were studied. METHODS: An online survey was sent to 706 MND patients. The questions of the survey included self-reliance, self-reported oral health, and oral hygiene. The oral health-related quality of life (GOHAI-NL) and the subjective well-being (ALSAQ-5) were also measured. RESULTS: A total of 259 patients responded (36.7%), of which 71.9% stated not to be informed about the importance of maintaining good oral health by their MND treatment team. Moreover, 40.4% would like to receive help concerning oral hygiene from a dental professional. 19.8% were not satisfied about oral care as conducted by themselves or their caregivers. Patients who do not ask for support with their daily oral care had a significantly worse oral health-related quality of life compared to patients who do ask for support. CONCLUSIONS: The support for daily oral hygiene of MND patients and their barriers to requesting support needs more attention from both MND-treatment teams and general dental professionals.

16S rDNA sequencing and metadata of Dutch dental unit water.

Dental practices were approached to fill out a questionnaire on the infection control protocols in use to control biofilm growth in the dental unit and to send two types of water sample. Sampling of the dental units had to be performed prior to any infection control measures and on the second day of operation, to avoid residual effects of biofilm disinfection protocols performed in the weekend. Instructions were given on how to sample the units. Only samples, accompanied with a completed questionnaire and returned within two days by regular mail, were analysed. Samples were processed for heterotrophic plate counts, 16S (V4) rDNA microbiome sequencing and q-PCR for the concentration of bacterial 16S rDNA, fungal 18S rDNA, Legionella spp. and the presence of amoeba. The files contain the metadata needed to interpret and analyse the microbiome data. This dataset can be used by other scientists, members of infection control units, (trainee) bioinformaticians and policy makers. This dataset can provide leads to further unexplored parameters which could influence the microbial ecology of the dental unit.

The microbiological load and microbiome of the Dutch dental unit; 'please, hold your breath'.

Dental unit water systems are prone to biofilm formation. During use of the dental unit, clumps of biofilm slough off and can subsequently be aerosolized and inhaled by both patient and staff, potentially causing infections. The aim of this study was to determine the microbial load and microbiome of dental unit water, in the Netherlands, and the factors influencing these parameters. In total, 226 dental units were sampled and heterotrophic plate counts (HPC) were determined on the traditional effluent sample. Of all dental units, 61% exceeded the recommended microbiological guidelines of 100 colony forming units per milliliter. In addition, the microbiome, with additional q-PCR analysis for specific species, was determined on an effluent sample taken immediately after an overnight stagnancy period, in which the biofilm is in its relaxed state. These relaxed biofilm samples showed that each dental unit had a unique microbiome. Legionella spp., amoeba and fungi were found in 71%, 43% and 98% of all units, respectively. The presence of amoeba was positively associated with nine bacterial biomarkers and correlated positively with bacterial and fungal DNA and Legionella spp. concentrations, but not with HPC. Only when adhering to disinfection protocols, statistically significant effects on the microbial load and microbiome were seen. The relaxed biofilm sample, in combination with molecular techniques gives better insight in the presence of opportunistic pathogens when compared to the heterotrophic plate counts. Infection control measures should focus on biofilm analysis and control in order to guarantee patient safety.

Chlorine-based DUWL disinfectant leads to a different microbial composition of water derived biofilms compared to H2O2-based chemical disinfectants in vitro.

BACKGROUND: Biofilm formation in dental unit waterlines (DUWL) may lead to health risks for dental staff and patients. Therefore, dental unit waterlines need to be disinfected, for instance by using chemical disinfectants. However, the application of chemical disinfectants may lead to the selection of specific microorganisms. Therefore, the aim of our study was to assess the microbial composition of water-derived biofilms, after a continuous exposure to maintenance doses of commercially available chemical disinfectants, in vitro. METHODS: The AAA-model was used to grow water derived biofilms. The biofilms were subjected to the maintenance dose of each disinfectant. To determine the microbial composition, the V4 hypervariable region of the 16S rRNA gene was sequenced. The sequences were clustered in operational taxonomic units (OTUs). RESULTS: The bacterial composition of biofilms in all treatment groups differed significantly (PERMANOVA F = 4.441, p = 0.001). Pairwise comparisons revealed Anoxyl treated biofilms were significantly different from all groups (p = 0.0001). In the Anoxyl-treated biofilms, the relative abundance of Comamonadaceae and Sphingopyxis was high compared to the Dentosept, Green and Clean and Oxygenal groups. CONCLUSION: We concluded that exposure to low doses of the chlorine-based chemical disinfectant Anoxyl led to a substantially different composition of water derived biofilms compared to biofilms exposed to H2O2-based chemical disinfectants.

Efficiency of chemical versus mechanical disruption methods of DNA extraction for the identification of oral Gram-positive and Gram-negative bacteria.

OBJECTIVE: Clinical diagnostics often requires the detection of multiple bacterial species in limited clinical samples with a single DNA extraction method. This study aimed to compare the bacterial DNA extraction efficiency of two lysis methods automated with the MagNA-Pure LC instrument. The samples included five oral bacterial species (three Gram-positive and two Gram-negative) with or without human saliva background. METHODS: Genomic DNA (gDNA) was extracted from bacterial cultures by bead-beating lysis (BMP) or chemical lysis (MP), followed by automated purification and measurement by quantitative PCR. RESULTS: For pure bacterial cultures, the MP method yielded higher quantities of extracted DNA and a lower detection limit than the BMP method, except where the samples contained high numbers of Gram-positive bacteria. For bacterial cultures with a saliva background, no difference in gDNA extraction efficacy was observed between the two methods. CONCLUSIONS: The efficiency of a bacterial DNA extraction method is not only affected by the bacterial cell wall structure but also by the sample milieu. The MP method provided superior gDNA extraction efficiency when the samples contained a single bacterial species, whereas either of the BMP and MP methods could be applied with similar efficiencies to samples containing multiple species of bacteria.

An in-vitro dynamic flow model for translational research into dental unit water system biofilms.

Dental unit water systems (DUWS) provide an excellent environment for biofilm formation and can form a potential health risk for patients and staff. To control this biofilm formation, better understanding of the DUWS biofilm ecology is needed. Described is a newly developed in-vitro DUWS model which is easy to build, can be inoculated with different water sources and allows for sampling of both the effluent and biofilm. Unlike most models, a dynamic flow pattern, typical for a dental unit is used to provide water as a nutrient source. Microbial growth and composition were analyzed using heterotrophic plate counts (HPC) and 16S rDNA sequencing. Growth was reproducible in all models, reaching quasi-steady state at day 16 in the effluent (105-106 CFU∙mL-1) and day 23 in the biofilm (108 and 107 CFU∙cm-2) for non-potable and potable water, respectively. Principal component analysis of the microbial composition showed that biofilms originating from either non-potable or potable water were significantly different after 30 days of growth (n = 8, PERMANOVA, F = 35.6, p < .005). Treatment of the biofilms with 1000 ppm active chlorine showed a biological and statistical significant decrease in viable counts in the effluent phase to below the detection limit of 100 CFU∙mL-1. The HPC returned to pre-treatment levels within 14 days. Using this model results in inoculum dependent biofilms with a higher bacterial density compared to previously described models. The relative ease in which samples can be taken allows for the monitoring of antimicrobial disinfection efficacy on the effluent, biofilm and matrix.

Exploring the role of oral microorganisms in the pathogenesis of mucositis by assessing their impact on metabolic activity and reproductive capacity of epithelial cells in vitro.

PURPOSE: Clinical and in vitro studies showed selected oral microorganisms to be related to delayed wound healing and ulcerative oral mucositis. However, it is not known whether this effect is due to reduced metabolism and/or the reduced reproductive capacity of epithelial cells. Therefore, we studied the influence of the oral microorganisms Porphyromonas gingivalis, Candida glabrata, and Candida kefyr on cell metabolism and reproductive capacity of oral epithelial cells, aimed to further unravel the pathogenesis of oral mucositis. METHODS: Oral epithelial cells were exposed to different concentrations of P. gingivalis, C. glabrata, and C. kefyr as mono-infections or mixed together. An MTT assay was performed to determine the effect on cell metabolism. A clonogenic assay was used to study the effect on the reproductive capacity of oral epithelial cells. RESULTS: The metabolism of oral epithelial cells was reduced when the microorganisms were present in high concentrations: P. gingivalis at a multiplicity of infection (MOI) of 1000 and the Candida spp. at MOI 100. No statistical difference was observed in the ability of a single epithelial cell to grow into a colony of cells between control and P. gingivalis, C. glabrata, and C. kefyr, independent of the concentrations and combinations used. CONCLUSION: P. gingivalis, C. glabrata, and C. kefyr lowered the metabolic activity of oral epithelial cells in high concentrations, yet they did not influence the reproductive capacity of epithelial cells. Their impact on ulcerative oral mucositis is likely due to an effect on the migration, proliferation, and metabolism of epithelial cells.

Microbial changes in relation to oral mucositis in autologous hematopoietic stem cell transplantation recipients.

The aim of this prospective, two center study was to investigate the dynamics of the microbial changes in relation to the development of ulcerative oral mucositis in autologous SCT (autoSCT) recipients. Fifty-one patients were diagnosed with multiple myeloma and treated with high-dose melphalan followed by autoSCT. They were evaluated before, three times weekly during hospitalization, and three months after autoSCT. At each time point an oral rinse was collected and the presence or absence of ulcerative oral mucositis (UOM) was scored (WHO scale). Oral microbiome was determined by using 16S rRNA amplicon sequencing and fungal load by qPCR. Twenty patients (39%) developed UOM. The oral microbiome changed significantly after autoSCT and returned to pre-autoSCT composition after three months. However, changes in microbial diversity and similarity were more pronounced and rapid in patients who developed UOM compared to patients who did not. Already before autoSCT, different taxa discriminated between the 2 groups, suggesting microbially-driven risk factors. Samples with high fungal load (>0.1%) had a significantly different microbial profile from samples without fungi. In conclusion, autoSCT induced significant and reversible changes in the oral microbiome, while patients who did not develop ulcerative oral mucositis had a more resilient microbial ecosystem.

Candida and Porphyromonas gingivalis: the effect on wound closure in vitro.

Microorganisms play a role in oral mucositis after cancer therapy. The current study explored the hypothesis that Candida spp. alone and together with Porphyromonas gingivalis cause delayed healing of oral ulcerations due to the inhibition of wound closure. An in vitro scratch assay model was used to study the influence of viable and heat-killed Candida glabrata, Candida kefyr, and Candida albicans on cell migration of oral epithelial cells. Separately, the effect of conditioned medium of Candida spp. and the effect of a mixed infection of Candida spp. with P. gingivalis on wound closure was studied. In the presence of 10 viable C. glabrata or C. kefyr versus one epithelial cell, with a multiplicity of infection (MOI) of 10, the relative closure of the scratch was 26% and 17%, respectively. At a MOI of 1, this was 60% for C. glabrata and 78% for C. kefyr. The inhibition of oral epithelial cell migration challenged with either C. glabrata or C. kefyr together with P. gingivalis was stronger than the inhibition caused by one of both organisms separately. Candida spp. inhibit cell migration in vitro. A combination of Candida spp. and P. gingivalis inhibited cell migration more than either microorganism separately.

Calcium Hydroxide Treatment Does Not Alter the Susceptibility of Enterococcus faecalis Biofilms to Sodium Hypochlorite.

OBJECTIVE: To investigate the influence of calcium hydroxide (Ca(OH)2) on susceptibility to disinfection with sodium hypochlorite (NaOCl) of biofilm bacteria. METHODS: Monospecies biofilms of eight Enterococcus faecalis strains were subjected to a 2-h challenge with Ca(OH)2. After a recovery phase, the biofilms were treated with a concentration of NaOCl that was lower than the minimum inhibitory concentration. In a metabolic assay, the efficacy of NaOCl disinfection in Ca(OH)2-challenged biofilms and unchallenged biofilms was evaluated. The data were analyzed with Mann-Whitney U and Kruskall- Wallis tests. A P value of less than 0.05 was considered statistically significant. RESULTS: There were marginal differences in susceptibility to NaOCl among the E. faecalis strains. After the Ca(OH)2 challenge, seven strains remained equally susceptible to NaOCl disinfection whereas one strain became more resistant to NaOCl (P=0.03). CONCLUSION: After a Ca(OH)2 challenge, in general E. faecalis remained equally susceptible to disinfection with NaOCl.

Red and Green Fluorescence from Oral Biofilms.

Red and green autofluorescence have been observed from dental plaque after excitation by blue light. It has been suggested that this red fluorescence is related to caries and the cariogenic potential of dental plaque. Recently, it was suggested that red fluorescence may be related to gingivitis. Little is known about green fluorescence from biofilms. Therefore, we assessed the dynamics of red and green fluorescence in real-time during biofilm formation. In addition, the fluorescence patterns of biofilm formed from saliva of eight different donors are described under simulated gingivitis and caries conditions. Biofilm formation was analysed for 12 hours under flow conditions in a microfluidic BioFlux flow system with high performance microscopy using a camera to allow live cell imaging. For fluorescence images dedicated excitation and emission filters were used. Both green and red fluorescence were linearly related with the total biomass of the biofilms. All biofilms displayed to some extent green and red fluorescence, with higher red and green fluorescence intensities from biofilms grown in the presence of serum (gingivitis simulation) as compared to the sucrose grown biofilms (cariogenic simulation). Remarkably, cocci with long chain lengths, presumably streptococci, were observed in the biofilms. Green and red fluorescence were not found homogeneously distributed within the biofilms: highly fluorescent spots (both green and red) were visible throughout the biomass. An increase in red fluorescence from the in vitro biofilms appeared to be related to the clinical inflammatory response of the respective saliva donors, which was previously assessed during an in vivo period of performing no-oral hygiene. The BioFlux model proved to be a reliable model to assess biofilm fluorescence. With this model, a prediction can be made whether a patient will be prone to the development of gingivitis or caries.

Failed bonded interfaces submitted to microcosm biofilm caries development.

OBJECTIVES: This study aimed to evaluate the dentin wall carious lesion development of different composite-dentin interfaces in the presence of two adhesive bonding materials in the gaps, using a microcosm biofilm model. METHODS: Dentin samples were prepared (10.4mm(2)) and restored with a composite resin using two adhesive systems (etch-and-rinse and self-etch techniques). Different conditions with respect to composite-dentin interfaces were produced with a 200µm gap: failed bonded without ageing or after mechanical ageing, or non-bonded with or without the presence of adhesive material on the dentin wall. For cariogenic challenge, specimens were subjected to a biofilm microcosm model for 14days to create caries-like wall lesions. Before and after caries development, transverse wavelength-independent microradiography images were taken, and lesion depth and mineral loss were measured. Data were analysed with linear regression models (p<0.05). RESULTS: The composite-dentin interface conditions significant influenced the caries development: lesion development was reduced by the presence of the adhesive material on dentin wall, while lesion development was increased by the mechanical ageing (p=0.019). There was no difference between the adhesive materials (p values>0.05). CONCLUSION: Different composite-dentin interfaces influence wall lesion development in gaps, with the interfaces submitted to ageing showing less carious protection than those interfaces with the presence of adhesive covering the dentin. CLINICAL SIGNIFICANCE: The presence of adhesive bonding material in the gaps plays a role on the wall caries lesion development.

Proteomic analysis of hyperadhesive Candida glabrata clinical isolates reveals a core wall proteome and differential incorporation of adhesins.

Attachment to human host tissues or abiotic medical devices is a key step in the development of infections by Candida glabrata. The genome of this pathogenic yeast codes for a large number of adhesins, but proteomic work using reference strains has shown incorporation of only few adhesins in the cell wall. By making inventories of the wall proteomes of hyperadhesive clinical isolates and reference strain CBS138 using mass spectrometry, we describe the cell wall proteome of C. glabrata and tested the hypothesis that hyperadhesive isolates display differential incorporation of adhesins. Two clinical strains (PEU382 and PEU427) were selected, which both were hyperadhesive to polystyrene and showed high surface hydrophobicity. Cell wall proteome analysis under biofilm-forming conditions identified a core proteome of about 20 proteins present in all C. glabrata strains. In addition, 12 adhesin-like wall proteins were identified in the hyperadherent strains, including six novel adhesins (Awp8-13) of which only Awp12 was also present in CBS138. We conclude that the hyperadhesive capacity of these two clinical C. glabrata isolates is correlated with increased and differential incorporation of cell wall adhesins. Future studies should elucidate the role of the identified proteins in the establishment of C. glabrata infections.

Proteins and peptides in parotid saliva of irradiated patients compared to that of healthy controls using SELDI-TOF-MS.

BACKGROUND: Radiotherapy to the head and neck area damages the salivary glands. As a consequence hyposalivation may occur, but also the protein composition of saliva may be affected possibly compromising oral health. The aim of our study was to compare the relative abundance of proteins and peptides in parotid saliva of irradiated patients to that of healthy controls. METHODS: Using Lashley cups and citric acid, saliva from the parotid glands was collected from nine irradiated patients and ten healthy controls. The samples were analyzed with SELDI-TOF-MS using a NP20 and IMAC-30 chip in the molecular weight range of 1-30 kDa. RESULTS: On the NP20 chip 61 (out of 217) and on the IMAC-30 chip 32 (out of 218) peaks differed significantly in intensity between the saliva of the irradiated patients and healthy controls. 55 % of the significant peaks showed higher intensity and 45 % showed lower intensity in the saliva of irradiated patients. The peaks may represent, amongst others, the salivary proteins lysozyme, histatins, cystatin, protein S100 and PRP's. CONCLUSIONS: Large differences were found in the relative abundance of a wide range of proteins and peptides in the parotid saliva of irradiated patients compared to healthy controls.

Low methicillin-resistant Staphylococcus aureus carriage rate among Italian dental students.

We assessed methicillin-resistant Staphylococcus aureus (MRSA) carriage rate among dental students from an Italian university. A total of 157 subjects participated (67 preclinical students and 90 clinical students); samples were collected from the nose, mouth, and skin. Five preclinical students and 0 clinical students were MRSA-positive. Carriage rates were 3.2% (95% confidence interval [CI], 0.4%-6.0%) overall, 7.5% (95% CI, 1.2%-13.8%) in preclinical students and 0% in clinical students. There were 2 MRSA clusters among the preclinical students: 3 second-year and 2 first-year students, who sat close to one another in the classroom the day of the sample. MRSA carriage was not associated with dental health care. The pooled carriage rate among dental students was assessed to obtain a reliable figure of carriage rate unaffected by local conditions. The 4 published surveys were pooled, and the fixed-effects method was used. Among the 484 dental students, the pooled carriage rate was 4.1% (95% CI, 2.4%-5.8%).

Additional disinfection with a modified salt solution in a root canal model.

OBJECTIVES: The aim of this study is to investigate the disinfecting properties of a modified salt solution (MSS) and calcium hydroxide (Ca(OH)2) in a non-direct-contact ex-vivo model. METHODS: Seventy-four single-canal roots infected with Enterococcus faecalis were treated with 1% sodium hypochlorite (NaOCl) irrigation or with NaOCl irrigation with subsequent dressing with MSS or Ca(OH)2. After removal of the dressings, the roots were filled with bacterial growth medium and incubated for seven days to enable the surviving bacteria to repopulate the root canal lumen. Growth was determined by sampling the root canals with paper points before treatment (S1), after treatment (S2) and incubation after treatment (S3). The colony forming units were counted at S1 and S2. At S3, growth was determined as no/yes regrowth. The Kruskal-Wallis, McNemar and χ(2) test were used for statistical analyses. RESULTS: At S2, in the NaOCl group, growth was found in 5 of 19 root canals. After the removal of MSS or Ca(OH)2 bacteria were retrieved from one root canal in both groups. At S3, repopulation of the root canals had occurred in 14 of 19 roots after sole NaOCl irrigation, 6 of 20 roots after MSS-dressing and in 14 of 20 roots after Ca(OH)2-dressing. MSS was more effective in preventing regrowth than Ca(OH)2 (P=0.009). CONCLUSIONS: The modified salt solution prevented regrowth in roots which indicates that it can eliminate persistent bacteria. Dressing the root canals with Ca(OH)2 did not provide additional disinfection after NaOCl irrigation.