Update on infectious risks associated with dental unit waterlines

Modern dental chair units consist of a network of interconnected narrow-bore plastic tubes called dental unit waterlines (DUWLs). The water delivered by these DUWLs acts as both a coolant for a range of instruments and an irrigant during dental treatments. The quality of water is of considerable importance because both patients and dental team are regularly exposed to water and aerosols generated by dental equipment. Studies have demonstrated that DUWLs provide a favourable environment for microbial proliferation and biofilm formation, and that water is consequently often contaminated with high densities of various microorganisms (bacteria, fungi, protozoa, viruses). The presence of high levels of microbial contamination may be a health problem for dentists and patients, especially those who are immunocompromised. The current status of knowledge on microbial contamination of DUWLs is presented, with an emphasis on the infectious risk associated with DUWLs and on the various approaches for disinfecting and protecting DUWLs.     

Aerosolization of mycobacteria and legionellae during dental treatment: low exposure despite dental unit contamination

Dental unit waterlines (DUWL) support growth of a dense microbial population that includes pathogens and hypersensitivity-inducing bacteria, such as Legionella spp. and non-tuberculous mycobacteria (NTM). Dynamic dental instruments connected to DUWL generate aerosols in the work environment, which could allow waterborne pathogens to be aerosolized. The use of the real-time quantitative polymerase chain reaction (qPCR) provides a more accurate estimation of exposure levels compared with the traditional culture approach. Bioaerosol sampling was performed 13 times in an isolated dental treatment room according to a standardized protocol that included four dental prophylaxis treatments. Inhalable dust samples were taken at the breathing zone of both the hygienist and patient and outside the treatment room (control). Total bacteria as well as Legionella spp. and NTM were quantified by qPCR in bioaerosol and DUWL water samples. Dental staff and patients are exposed to bacteria generated during dental treatments (up to 4.3 E + 05 bacteria per m(3) of air). Because DUWL water studied was weakly contaminated by Legionella spp. and NTM, their aerosolization during dental treatment was not significant. As a result, infectious and sensitization risks associated with legionellae and NTM should be minimal.     

In vitro modeling of dental water line contamination and decontamination

The contamination of dental unit water lines (DUWL) is an emerging concern in dentistry. The aim of this study was to use an in vitro DUWL to model microbial contamination and evaluate the decontamination efficacy of tetraacetylethylenediamine (TAED) solutions. A DUWL biofilm model used to simulate clinical conditions was used to generate a range of biofilms in DUWL. Three distinct biofilms were generated: (1) biofilm from water, (2) biofilm from a mix of water + contaminating human commensal bacteria, (3) biofilm from water with contaminating oral bacteria added after biofilm formed. The contaminating oral species used were Streptococcus oralis, Enterococcus faecalis and Staphylococcus aureus. Decontamination by simple water flushing or flushing with TAED was evaluated (2, 5 and 10 min intervals). The DUWL tubes were split and samples were plated onto a range of media, incubated and bacteria enumerated. Water flushing did not reduce the number of microorganisms detected. Bacteria were not detected from any of the TAED sampling points for any of the biofilm types tested. Interestingly, if contamination was introduced to new DUWL along with the waterborne species a biofilm was formed containing only the waterborne species. If however, an existing biofilm was present before the introduction of "contaminating" bacteria then these could be detected in the biofilm. This implies that if the DUWL are new or satisfactorily cleaned on a regular basis then the associated cross-contamination aspects are reduced. In conclusion, TAED provides effective control for DUWL biofilms.     

Monitoring Dental-Unit-Water-Line Output Water by Current In-office Test Kits

The importance of monitoring contamination levels in the output water of dental-unit-water-lines (DUWLs) is essential as they are prone to developing biofilms that may contaminate water that is used to treat patients, with opportunistic pathogens such as species of Legionella, Pseudomonas and others. Dentists and practice staff are also at risk of being infected by means of cross-infection due to aerosols generated from DUWL water. The unit of measurement for the microbial contamination of water by aerobic mesophilic heterotrophic bacteria is the colony-forming unit per millilitre (cfu/ml) of water. The UK has its own guidelines set by the Department of Health for water discharged from DUWL to be between 100 and 200 cfu/ml of water. The benchmark or accepted standard laboratory test is by microbiological culture on R2A agar plates. However, this is costly and not convenient for routine testing in dental practices. A number of commercial indicator tests are used in dental surgeries, but they were not developed for the dental market and serve only to indicate gross levels of contamination when used outside of the manufacturer’s recommended incubation period. The aim of this article is to briefly review the universal problem of DUWL contamination with microbial biofilms and to update dental professionals on the availability of currently available commercial in-office monitoring systems for aerobic mesophilic heterotrophic bacteria and to discuss their limitations for testing water samples in assuring compliance with recommended guidelines.     

Establishing a laboratory model of dental unit waterlines bacterial biofilms using a CDC biofilm reactor

In this study, a laboratory model to reproduce dental unit waterline (DUWL) biofilms was developed using a CDC biofilm reactor (CBR). Bacteria obtained from DUWLs were filtered and cultured in Reasoner’s 2A (R2A) for 10 days, and were subsequently stored at ?70°C. This stock was cultivated on R2A in batch mode. After culturing for five days, the bacteria were inoculated into the CBR. Biofilms were grown on polyurethane tubing for four days. Biofilm accumulation and thickness was 1.3 × 10⁵ CFU cm^?2 and 10–14 μm respectively, after four days. Bacteria in the biofilms included cocci and rods of short and medium lengths. In addition, 38 bacterial genera were detected in biofilms. In this study, the suitability and reproducibility of the CBR model for DUWL biofilm formation were demonstrated. The model provides a foundation for the development of bacterial control methods for DUWLs.     

Effect of Biocides on Biofilm Bacteria from Dental Unit Water Lines

Microbial biofilm formation in dental unit water lines (DUWL) is a phenomenon that has been recognized for nearly four decades. Water delivered by DUWL can harbor high numbers of bacteria, including opportunistic pathogens. Biofilms on tubing within DUWL may serve as a reservoir for these microorganisms and should therefore be controlled. In this study, the effects of eight biocides were monitored on DUWL biofilms individually and in combination by epifluorescence microscopy and total viable counts (TVC). The effects of sodium dodecyl sulphate (SDS), hydrogen peroxide (H₂O₂), sodium hypochlorite (NaOCl), phenol (Phe), Tween 20 (Tw 20), ethylenediaminetetraacetic acid (EDTA), chlorohexidine gluconate (CHX), and povidine iodine (PI) were tested on DUWL biofilms alone and in combination. PI was found to have negligible effects on biofilm removal either applied alone or in combined form with CHX. Applying all biocides simultaneously did not completely eliminate viable bacteria nor did they remove biofilm. Overall, when combined, the biocides performed better than singly applied products. The most effective biocides were NaOCl and Phe (both alone and in combination).     

Assessment of inhibitory effects of fluoride-coated tubes on biofilm formation by using the in vitro dental unit waterline biofilm model

This study aimed to establish an in vitro model to simulate biofilms formed in dental unit waterlines (DUWLs) and to investigate the ability of polyvinylidene fluoride (PVDF)-coated tubes to inhibit biofilm formation using this model. The water and biofilm samples were obtained from DUWLs which had been clinically used for 2.5 years, and the predominant bacteria were identified. A conventional polyurethane tube was incubated for 24 to 96 h in the mixed flora of isolated bacteria, and the optimal incubation conditions to simulate a clinically formed biofilm were determined by observation with a scanning electron microscope. Biofilm formation on a PVDF-coated tube was observed using this in vitro model, and the adherence of different bacterial species to conventional and PVDF-coated tubes was assessed. Sphingomonas paucimobilis, Acinetobacter haemolytics, and Methylobacterium mesophilicum were predominantly isolated from contaminated DUWLs. Incubation of the polyurethane tube with the mixed flora containing these three species for 96 h resulted in the formation of a mature biofilm similar to the one clinically observed. The PVDF-coated tube was significantly less adhesive to all three bacterial species than the polyurethane tube (P < 0.05 by the Mann-Whitney U test), and the attachment of small amounts of rods was observed even after incubation with the mixed flora for 96 h. In conclusion, an in vitro biofilm model was obtained by using a mixed flora of bacteria isolated from DUWLs, and the PVDF-coated tube was found to be effective in preventing biofilm formation using this model.     

Pseudomonas aeruginosa and Achromobacter sp. Clonal Selection Leads to Successive Waves of Contamination of Water in Dental Care Units

Dental care unit waterlines (DCUWs) consist of complex networks of thin tubes that facilitate the formation of microbial biofilms. Due to the predilection toward a wet environment, strong adhesion, biofilm formation, and resistance to biocides, Pseudomonas aeruginosa, a major human opportunistic pathogen, is adapted to DCUW colonization. Other nonfermentative Gram-negative bacilli, such as members of the genus Achromobacter, are emerging pathogens found in water networks. We reported the 6.5-year dynamics of bacterial contamination of waterlines in a dental health care center with 61 dental care units (DCUs) connected to the same water supply system. The conditions allowed the selection and the emergence of clones of Achromobacter sp. and P. aeruginosa characterized by multilocus sequence typing, multiplex repetitive elements-based PCR, and restriction fragment length polymorphism in pulsed-field gel electrophoresis, biofilm formation, and antimicrobial susceptibility. One clone of P. aeruginosa and 2 clones of Achromobacter sp. colonized successively all of the DCUWs: the last colonization by P. aeruginosa ST309 led to the closing of the dental care center. Successive dominance of species and clones was linked to biocide treatments. Achromobacter strains were weak biofilm producers compared to P. aeruginosa ST309, but the coculture of P. aeruginosa and Achromobacter enhanced P. aeruginosa ST309 biofilm formation. Intraclonal genomic microevolution was observed in the isolates of P. aeruginosa ST309 collected chronologically and in Achromobacter sp. clone A. The contamination control was achieved by a complete reorganization of the dental health care center by removing the connecting tubes between DCUs.     

Antibiofilm activity of sodium bicarbonate, sodium metaperiodate and SDS combination against dental unit waterline-associated bacteria and yeast

Aim: To determine the effect of sodium bicarbonate (SB), sodium metaperiodate (SMP) and sodium dodecyl sulfate (SDS) combination on biofilm formation and dispersal in dental unit waterline (DUWL)-associated bacteria and yeast. Methods and Results: The in vitro effect of SB, SMP and SDS alone and in combination on biofilm formation and dispersal in Pseudomonas aeruginosa, Klebsiella pneumoniae, Actinomyces naeslundii, and Candida albicans was investigated using a 96-well microtitre plate biofilm assay. The combination showed a broad-spectrum inhibitory effect on growth as well as biofilm formation of both gram-negative and gram-positive bacteria, and yeast. In addition, the SB + SMP + SDS combination was significantly more effective in dispersing biofilm than the individual compounds. The combination dispersed more than 90% of P. aeruginosa biofilm whereas the commercial products, Oxygenal 6, Sterilex Ultra, and PeraSafe showed no biofilm dispersal activity. Conclusion: The composition comprising SB, SMP, and SDS was effective in inhibiting as well as dispersing biofilms in DUWL-associated bacteria and yeast. Significance and Impact of the Study: This study shows that a composition comprising environmentally friendly and biologically safe compounds such as SB, SMP, and SDS has a potential application in reducing DUWL-associated acquired infections in dental clinics.     

Isolate-Specific Effects of Patulin, Penicillic Acid and EDTA on Biofilm Formation and Growth of Dental Unit Water Line Biofilm Isolates

Dental unit water line (DUWL) contamination by opportunistic pathogens has its significance in nosocomial infection of patients, health care workers, and life-threatening infections to immunocompromized persons. Recently, the quorum sensing (QS) system of DUWL isolates has been found to affect their biofilm-forming ability, making it an attractive target for antimicrobial therapy. In this study, the effect of two quorum-sensing inhibitory compounds (patulin; PAT, penicillic acid; PA) and EDTA on planktonic growth, AI-2 signalling and in vitro biofilm formation of Pseudomonas aeruginosa, Achromobacter xylosoxidans and Achromobacter sp. was monitored. Vibrio harveyi BB170 bioassay and crystal violet staining methods were used to detect the AI-2 monitoring and biofilm formation in DUWL isolates, respectively. The V. harveyi BB170 bioassay failed to induce bioluminescence in A. xylosoxidans and Achromobacter sp., while P. aeruginosa showed AI-2 like activity suggesting the need of some pretreatments prior to bioassay. All strains were found to form biofilms within 72 h of incubation. The QSIs/EDTA combination have isolate-specific effects on biofilm formation and in some cases it stimulated biofilm formation as often as it was inhibited. However, detailed information about the anti-biofilm effect of these compounds is still lacking.     

Detection of DNA from periodontal pathogenic bacteria in biofilm obtained from waterlines in dental units

Direct person-to-person transmission of periodontal bacteria through saliva has recently been widely reported and dental units have been demonstrated to retract saliva from patients under treatment and to release it into the mouths of subjects undergoing the next operation. In this study the presence of a group of periodontal pathogenic bacteria inside waterlines in dental units was investigated using polymerase chain reaction (PCR) based methods. Briefly, 18 dental units of three different manufacturers were studied. Dental units were divided into two groups according to their prevalent use in routine practice. The first group consisted of nine dental units characterized by the frequent use of high-speed dental hand-pieces directly inside the mouth and in contact with patients' saliva. The second group, as a control, consisted of nine dental units where high-speed dental hand-pieces were not in use. A one cm section of the waterline tubing connected to the high-speed hand-piece was removed from each dental unit to evaluate the presence of DNA of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Bacteroides forsythus, Treponema denticola. Two specimens were positive for Prevotella intermedia DNA. All the positive results were from samples obtained from dental units categorised in the first group. These findings clearly suggest that dental units have the potential to transmit periodontal pathogens. Manufacturers should be invited to design dental units that incorporate automated devices to disinfect DUWLs between patients with minimal effort by dental staff.     

Formation of the Dermal Chromatophore Unit (DCU) in the Tree Frog Hyla Arborea

In the tadpole of the tree frog Hyla arborea, the color of the dorsal skin was dark brown. Dermal melanophores, xanthophores, and iridophores were scattered randomly under the subepidermal collagen layer (SCL). After metamorphosis, the dorsal color of the animal changed to green and the animal acquired the ability of dramatic color change, demonstrating that the dermal chromatophore unit (DCU) was formed at metamorphosis. Fibroblasts invaded the SCL and divided it into two parts: the stratum spongiosum (SS) and the stratum compactum (SC). The activity of collagenase increased at metamorphosis. The fibroblasts appeared to dissolve the collagen matrix as they invaded the SCL. Then, three types of chromatophores migrated through the SCL and the DCU was formed in the SS. The mechanism how the three types of chromatophores were organized into a DCU is uncertain, but different migration rates of the three chromatophore types may be a factor that determines the position of the chromatophores in the DCU. Almost an equal number of each chromatophore type is necessary to form the DCUs. However, the number of dermal melanophores in the tadpoles was less than the number of xanthophores and iridophores. It was suggested that epidermal melanophores migrated to the dermis at metamorphosis and developed into dermal melanophores. This change may account for smaller number of dermal melanophores available to form the DCU_s.     

细菌生物膜与龋齿相关性的研究进展

牙菌斑是由多种微生物在牙面上沉积,有机基质互相集聚、交联而形成的生物膜结构,生物膜中微生物相互依存、相互竞争,构成了复杂的微生态关系。牙菌斑生物膜的形成是导致龋齿重要过程。本文综述了细菌生物膜与龋齿发生的关系,以期为龋齿的预防与治疗提供新的思路。     

Evaluation of ultrafiltration cartridges for a water sampling apparatus

Aims:  To determine the efficiency of various ultrafiltration cartridges (UFC) in concentrating test micro-organisms from drinking water.
Methods and Results:  Replicate drinking water samples from three potable water supplies were dosed with Bacillus anthracis Sterne, Francisella tularensis LVS, Yersinia pestis CO92, bacteriophages MS2 and phi-X174, and Cryptosporidium parvum. The test micro-organisms were dosed together in 100 l of water, which was then recirculated through one of five different UFC until the retentate volume was reduced to c. 500 ml. The micro-organisms were assayed before and after ultrafiltration concentration and per cent recoveries were calculated. There were nine statistically significant differences among pairs of filters out of a possible 180 different combinations of UFC, test micro-organisms, and water types.
Conclusions:  No filter consistently performed better or worse than the others for each test micro-organism in all water samples tested.
Significance and Impact of the Study:  This study provides performance data on the ability of several different UFC to concentrate a panel of test micro-organisms from three sources of potable water. Water utilities and first responders may use these data when selecting UFC for use in emergency response protocols. This study also provides additional data as to the efficacy of ultrafiltration for recovering bacteria, virus-like particles, and protozoan oocysts from water samples.     

Control of microbial contamination in dental unit water systems using tetra-sodium EDTA

Aim:  To examine the efficacy of tetra-sodium EDTA in controlling microbial contamination of dental unit water systems (DUWS).
Methods and Results:  Ten dental units were treated once a week with either 4% or 8% tetra-sodium EDTA for four or two consecutive weeks, respectively. Before treatment, 43% and 60% of the water samples from the air/water triple syringe and high-speed hand-pieces, respectively, exceeded the American Dental Association (ADA) guidelines of 200 CFU ml⁻¹ water during a 6-week baseline period. After each weekend treatment, the levels of microbial contamination in all DUWS fell significantly ( P  < 0·001) to below the ADA guideline. By the end of the week, microbial counts in the outflowing water had returned to baseline levels indicating a transient effect of single doses of tetra-sodium EDTA, and the need for multiple applications. The biofilms were virtually eliminated after a single weekend treatment.
Conclusions:  Tetra-sodium EDTA is effective in controlling microbial contamination in DUWS.
Significance and Impact of the Study:  Inexpensive, effective and safe products for reducing the microbial load of water from DUWS are needed to meet ADA and other national guidelines. Tetra-sodium EDTA can significantly reduce microbial biofilms and bacterial counts in outflowing water, and is compatible for use in DUWS.     

Detection of Legionella spp. by fluorescent in situ hybridization in dental unit waterlines

AIMS: To confirm the presence of viable Legionella spp. in dental unit waterlines (DUWL) using fluorescent in situ hybridization (FISH) and compare this method with culture approach and also to validate the utility of an enrichment to increase FISH sensitivity. METHODS AND RESULTS: Water samples from 40 dental units were analysed. Three different techniques for detecting Legionella spp. were compared: (i) culture approach, (ii) direct FISH and (iii) FISH with a previous R2A medium enrichment (R2A/FISH). The FISH detection was confirmed by PCR. The use of the direct FISH does not improve significantly the detection of legionellae when compared with the culture. On the contrary, when R2A/FISH was performed, sensitivity was, respectively, two- and threefold higher than that with the direct FISH and culture approach. Using R2A/FISH, 63% of water samples analysed showed a contamination by legionellae. CONCLUSIONS: Legionellae detection by direct FISH and R2A/FISH in dental unit water is possible but is more rapid and more sensitive (R2A/FISH) than the culture approach. SIGNIFICANCE AND IMPACT OF THE STUDY: R2A/FISH showed that several pathogens present in DUWL are viable but may not be culturable. Unlike PCR, R2A/FISH is designed to detect only metabolically active cells and therefore provides more pertinent information on infectious risk.     

Reduction of saliva-promoted adhesion of Streptococcus mutans MT8148 and dental biofilm development by tragacanth gum and yeast-derived phosphomannan

The aim of this study was to investigate materials which reduce saliva-promoted adhesion of Streptococcus mutans onto enamel surfaces, and their potential in preventing dental biofilm development. The effects of hydroxyapatite (HA) surface pretreatment with hydrophilic polysaccharides on saliva-promoted S. mutans adhesion in vitro and de novo dental biofilm deposition in vivo were examined. Saliva-promoted adhesion of S. mutans MT8148 was significantly reduced by pretreatment of the HA surface with tragacanth gum (TG) and yeast-derived phosphoglycans. Extracellular phosphomannan (PM) from Pichia capsulata NRRL Y-1842 and TG reduced biofilm development on lower incisors in plaque-susceptible rats when administered via drinking water at concentrations of 0.5% and 0.01%, respectively. The inhibitory effect of TG on de novo dental biofilm formation was also demonstrated when administered via mouthwash in humans. It is concluded that TG and yeast-derived PM have the potential for use as anti-adherent agents and are effective in reducing de novo dental biofilm formation.     

Atmospheric pressure plasma: a high-performance tool for the efficient removal of biofilms

Introduction

The medical use of non-thermal physical plasmas is intensively investigated for sterilization and surface modification of biomedical materials. A further promising application is the removal or etching of organic substances, e.g., biofilms, from surfaces, because remnants of biofilms after conventional cleaning procedures are capable to entertain inflammatory processes in the adjacent tissues. In general, contamination of surfaces by micro-organisms is a major source of problems in health care. Especially biofilms are the most common type of microbial growth in the human body and therefore, the complete removal of pathogens is mandatory for the prevention of inflammatory infiltrate. Physical plasmas offer a huge potential to inactivate micro-organisms and to remove organic materials through plasma-generated highly reactive agents.

Method

In this study a Candida albicans biofilm, formed on polystyrene (PS) wafers, as a prototypic biofilm was used to verify the etching capability of the atmospheric pressure plasma jet operating with two different process gases (argon and argon/oxygen mixture). The capability of plasma-assisted biofilm removal was assessed by microscopic imaging.

Results

The Candida albicans biofilm, with a thickness of 10 to 20 µm, was removed within 300 s plasma treatment when oxygen was added to the argon gas discharge, whereas argon plasma alone was practically not sufficient in biofilm removal. The impact of plasma etching on biofilms is localized due to the limited presence of reactive plasma species validated by optical emission spectroscopy.     

The culturable bacteria isolated from organic-rich black shale potentially useful in biometallurgical procedures

Aims:  The aim of this study was the isolation and characterization of micro-organisms from Lubin copper mine potentially useful in biotechnology of metal recovery from copper bearing black shale.
Methods and Results:  Eight bacterial strains were isolated from black shale ore. Phylogenetic analysis based on 16S rRNA gene homology showed that five strains belonged to the γ-Proteobacteria, one to the Firmicutes and two to the Actinobacteria. The ability of the isolates to transform bituminous shale and use them as carbon and energy sources, as well as high resistance to metals and metalloids, esterase and lipase activities, assimilation of organic acids, degradation of phenanthrene and siderophores production were shown.
Conclusions:  The indigenous bacteria exhibited a broad range of physiological properties related to geochemical parameters of the examined environment and potentially useful in biometallurgical procedures.
Significance and Impact of the Study:  The results have yielded new insights into the microbiology of black shale. It can be suggested that isolated micro-organisms might play a role in the geochemical cycle of carbon and metals occurring in the organic fraction of black shale ore and might be of potential use in biotechnological procedures for the copper recovery and other valuable metals from tailings containing black shale as well as organic rich ore.     

Measuring psychosocial variables that predict older persons’oral health behaviour

The importance of recognising psychosocial characteristics of older people that influence their oral health behaviours and the potential success of dental procedures is discussed. Three variables and instruments developed and tested by the author and colleagues are presented. A measure of perceived importance of oral health behaviours has been found to be a significant predictor of dental service utilization in three studies. Self-efficacy regarding oral health has been found to be lower than self-efficacy regarding general health and medication use among older adults, especially among non-Western ethnic minorities. The significance of self-efficacy for predicting changes in caries and periodontal disease is described. Finally, a measure of expectations regarding specific dental procedures has been used with older people undergoing implant therapy. Studies with this instrument reveal that patients have concerns about the procedure far different than those focused on by dental providers. All three instruments can be used in clinical practice as a means of understanding patients’values, perceived oral health abilities, and expectations from dental care. These instruments can enhance dentist-patient rapport and improve the chances of successful dental outcomes for older patients.