Introduction
Our objective was to determine whether components of fixed orthodontic appliances as received from the manufacturers and after exposure to the clinical environment are free from microbial contamination before clinical use. A pilot molecular microbiologic laboratory study was undertaken at a dental hospital in the United Kingdom.
Methods
A range of orthodontic materials “as received” from the manufacturers and materials “exposed” to the clinical environment were studied for bacterial contamination. After growth on blood-rich media, cultured bacteria were identified by 16S rDNA polymerase chain reaction amplification and sequence phylogeny.
Results
Bacteria were isolated from “as received” bands, archwires, and impression trays, but the level of contamination was low (0.5 × 10 1 to 1.825 × 10 2 CFU/mL −1 ). Various bacterial species were isolated from “clinic exposed” bands, archwires, impression trays, coil springs, and elastomeric modules, but the level of contamination was low (0.5 × 10 1 to 8.0 × 10 1 CFU/mL −1 ). The most commonly identified bacterial species was Staphylococcus epidermidis , followed by Kocuria , Moraxella , and Micrococcus species.
Conclusions
New materials “as received” from the manufacturers and those exposed to the clinical environment are not free from bacterial contamination before use in patients, but this contamination is low considering the potential for aerosol and operator contamination and could be considered insignificant. Further studies would be required to determine the level of risk that this poses.
Health professionals have a duty of care to their patients and must take appropriate precautions with respect to protecting patients and the team from the risks of cross-infection. There is a considerable cost implication to ensure that materials and instruments used in the dental surgery are free from contamination and to reduce the risk of cross-infection. An attempt has been made to improve the standard of decontamination in primary dental care in the United Kingdom by the introduction of the Health Technical Memorandum 01-05, which has brought dental decontamination and sterilization closer to the standards used in the United Kingdom in medical and hospital practices. This guidance was introduced because of concerns over the actual cross-infection regimens that were used in primary dental care practices after the Glennie Group report.
Guidance for dentists in the United Kingdom on infection control is provided by the British Dental Association, which incorporates the advice from the Department of Health document. The current advice is that “wherever possible, cleaning should be undertaken using an automated and validated washer-disinfector in preference to manual cleaning; a washer-disinfector includes a disinfection stage that renders instruments safe for handling and inspection.” Reusable equipment must therefore be thoroughly cleaned and sterilized (if appropriate) before reuse in other patients. New instruments must also be sterilized before use if they are not purchased in a sterile state.
Many instruments or new products in dentistry and orthodontics are often assumed to be sterile before use, although manufacturers’ packaging might not state this. The assumption of sterility can lead many practitioners to use these instruments and materials “as received” from the manufacturers without the necessary sterilization. If orthodontic materials are not provided sterile, should they be sterilized before use?
Until recently, there was no literature on the sterility of “as received” orthodontic products. Purmal et al investigated the sterility of “as received” molar tubes and identified 3 species of bacteria: Micrococcus luteus , Staphylococcus haemolyticus, and Acinetobacter calcoaceticus . The article does not state the numbers of colony-forming units (CFU) or whether all brackets were contaminated; this does not allow an evaluation of the extent of their contamination. A recent study investigated the reuse of tungsten carbide debonding burs (classified as reuseable items according to manufacturers) in hospital-based orthodontic departments. Although the purpose of the study was to investigate sterilization methods, worryingly, it was found that only 24% of departments correctly presterilized these burs before initial use (Decontamination in Primary Dental Care recommends that all reusable instruments should be sterilized before use).
In dentistry, investigations have been conducted into the sterility of dental burs and endodontic files “as received” from the manufacturers. Hauptman et al found, on 8 of 100 nonsterilized burs evaluated, bacterial growth after incubation. The bacteria identified were from the genus Bacillus . Examination of “as received” endodontic files showed that 13% of the sample investigated (150) was contaminated with bacteria; after sequencing, the bacteria included Paenibacillus amylolyticus , Paenibacillus polymyxa , Bacillus megaterium, and Staphylococcus epidermidis . This might be an underestimate of the contamination, since the experiment did not seek to detect or identify anaerobic bacteria. The files examined by this group were from a variety of manufacturers with only 7 of 15 stating the sterility of their products. Some did not disclose sterility information, whereas others stated “nonsterile” or “sterilize before use.” Practitioners can assume the sterility of these products when they are packaged in single blister packs. Although the types of bacteria identified in this study were not considered to be pathogenic, fungal species were identified. Some studies have shown fungal and nonoral microbial contamination in failed endodontic treatments. Morrison and Conrod examined the effectiveness of several techniques on cleaning used dental burs and endodontic files and found that in all techniques used, there was still bacterial growth after incubation. They extended their investigation to examine the sterility of the burs and files as received from the manufacturers and found that 42% and 45%, respectively, were contaminated with bacteria. They did not identify the bacterial species, but the study paints a worrying picture of the cleanliness of “as received” instruments and highlights a possible cross-infection risk associated with the use of these items. New items that were sterilized before the bacterial investigation were found to be sterile, with no bacterial growth.
Although all instruments used in orthodontics, as in dentistry, are sterilized before use, the same is not true for orthodontic archwires, brackets, bands, and impression trays. These are used “as received” from the manufacturers, often with the assumption that the level of hygiene in the manufacturing process and subsequent transportation is sufficient to allow for clinical use. Only 1 article was found about the cleanliness of orthodontic “as received” materials: molar tubes. With such products likely to be intimately associated with the oral tissues (eg, orthodontic bands impinging on the gingival sulcus and thus in contact with gingival crevicular fluid and the crevicular environment), there is a theoretical risk of cross-infection on placement. In addition, after placement, they might cause trauma to the oral tissues, such as the buccal mucosa, which could be a pathway for infection.
Dental health care professionals work closely with the general public and are subjected to potential contact with blood, saliva, secretions, and mucous membranes. As well as direct contact with patients, the instruments that are used in the dental surgery can increase our exposure. Dental hand pieces and ultrasonic scalers create aerosols that can remain suspended in the surgery air for several hours or even days depending on the size of the particles. Larger particles (50-100 μm) can be splattered across the dental surgery, whereas smaller particles can form an aerosol and be inhaled. Aerosols that contain microorganisms and their by-products are termed bio-aerosols. It has been shown that aerosols produced in dentistry are concentrated within a 60-cm radius, although a more recent study showed that significant contamination was found at much greater distances (>1.5 m) when high-speed rotating instruments were used. The authors also found a greater density of aerobic bacteria at these greater distances, although not statistically significantly different from those at closer distances. This has implications regarding the equipment stored on the surfaces of the dental surgery, because microbial contamination of the whole room is likely after the use of high-speed hand pieces, which might pose a cross-infection control risk. In addition to microbial spread, dental aerosols have also been shown to be able to carry blood and blood products into the air; these have potentially greater cross-infection risks in terms of the possible transmission of human immunodeficiency virus and hepatitis B and C viruses. All of this is in addition to the risks associated with aerosols contaminated as a result of bacterial growth in the dental unit’s water line.
To date, there have been few investigations of contamination of orthodontic materials “as received” from the manufacturers. We often assume sterility, and yet we have no evidence for this. Therefore, the aims of this study were to evaluate the bacterial load of various orthodontic items directly received from the manufacturers and also to evaluate the possible contamination, via aerosol or cross-contamination by personnel, of items in the orthodontic surgery environment.
Material and methods
This was a pilot microbiologic investigation conducted at Bristol Dental Hospital and the Oral Microbiology Department at Bristol University in the United Kingdom.
The study was divided into 2 parts. In part 1, we investigated contamination of “as received” orthodontic materials directly from the manufacturers. These items are listed in Table I and were obtained from newly arrived stock at the orthodontic department of the dental hospital. In part 2, we investigated possible contamination of new orthodontic materials ( Table I ) that had been stored in the dental clinic and had been exposed to the everyday clinical environment. Items from the clinic were transported in the containers in which they were normally stored in the clinic to reduce the risk of contamination from the investigators. All items were transported to the oral microbiology department for analysis. Five of each item were studied for contamination. This sample size was chosen because, at the time of this investigation, there were no data on potential contamination of orthodontic materials, and the size was deemed appropriate for a pilot study.
| Items tested | As received | Clinic exposed | Manufacturer | Packaging “as received” |
|---|---|---|---|---|
| 0.017 × 0.025-in beta-titanium alloy archwire | • | • | TP Orthodontics, LaPorte, Ind | Individual packets |
| 0.019 × 0.025-in stainless steel archwire | • | • | TP Orthodontics, LaPorte, Ind | 10 per packet |
| 0.014-in nickel-titanium archwire | • | • | TP Orthodontics, LaPorte, Ind | 10 per packet |
| Molar bands | • | • | 3M Unitek, Monrovia, Calif | 5 per packet |
| Stainless steel orthodontic brackets | • | • | Dentsply GAC, Bohemia, NY | 20 per packet |
| Coil spring (100-mm sample) | • | • | Rocky Mountain Orthodontics, Denver, Colo | Polythene bag |
| Impression trays | • | • | Ortho Technology, Tampa, Fla | Polythene bag, 100 units |
| Elastomeric modules (20 sampled) | • | • | TP Orthodontics, LaPorte, Ind | 10 per holder, 100 per bag |
| Elastomeric chart | – | • | In-house production | |
| 0.019 × 0.025-in stainless steel posted archwire | – | • | Dentsply GAC, Bohemia, NY | |
| Tungsten carbide burs | • | – | Dentsply GAC, Bohemia, NY | Individual sealed packets |
Items that were received directly from the manufacturers were taken from the new stock arrivals over a 6-month period to obtain a representative sample of each item from different batches of the manufacturing processes. The items that had been stored in the clinical environment were again sampled over a 6-month period. Items used in the clinic are constantly used and replaced; therefore, sampling at different times would represent what would occur in the normal clinical environment.
One milliliter of 10 m mol/L:1 m mol/L of tris-EDTA buffer solution was added to a sterile tube (Eppendorf, SARSTEDT Ltd, Leicester, United Kingdom), and each item was opened under laminar airflow and placed in the tube. If an item was too large (eg, archwire) to fit into the tube, it was cut into smaller pieces by using sterilized orthodontic instruments and an aseptic technique, with the investigator wearing nonlatex gloves cleaned with 70% ethanol. Items that could not be cut into smaller pieces (eg, impression trays) were thoroughly swabbed with sterile swabs (Fisher Scientific UK, Loughborough, United Kingdom) for 1 minute, and the swab was placed into the Eppendorf tube. The item and solution were vortexed by using a Whirlimixer (Fisons, Ipswich, United Kingdom) for 60 seconds to dislodge and equally distribute any contaminates on the specimen. We plated 100 μL of the sample onto blood-rich media blood agar base number 2 (Lab M Limited, Bury, United Kingdom) and fastidious anaerobe agar (Lab M Limited). Plated samples from each specimen were then cultured aerobically, in an oxygen-depleted candle jar, and in an anaerobic cabinet (Don Whitley Mark II; Don Whitley Scientific Limited, Shipley, United Kingdom) (nitrogen, hydrogen, and carbon dioxide at 8:1:1). All samples were incubated at 37°C for 5 days together with the uncontaminated controls. These were then visually assessed to detect and enumerate any bacterial colonies that indicated sample contamination. The numbers of colony forming units per milliliter (CFU/mL) present were determined. Individual colonies were replated and grown again on blood-enriched agar under the same conditions to determine colony type, to increase the bacterial numbers, and to ensure pure colonies. Resulting pure cultures were stored in 1 mL of brain heart infusion (BHI) 15% glycerol at –70°C. Different colonies were identified based on their phenotypic differences. Gram staining of subcultured bacteria was undertaken and compared with colony appearance to ensure that duplication was minimized.
Two methods were used to extract DNA from the stored bacterial samples: GenElute Bacterial Genomic DNA kit (Sigma-Aldrich, St. Louis, Mo) and GeneReleaser (BioVentures, Murfreesboro, Tenn). Extraction of DNA was performed according to the manufacturer’s instructions. The resultant 16S rRNA gene was amplified by using 27 forward (27F: AGA GTT TGA TCC TGG CTC AG) and 1492 reverse (1492R: TAC GGG TAC CTT GTT ACG ACT T) primers in a 50-μL reaction containing 1.25 units of Go Taq DNA polymerase (Promega, Southampton, United Kingdom), 10 μL of supplied buffer (final magnesium chloride concentration, 1.5 m mol/L), 0.2 m mol/L of dNTP, 1.0 m mol/L of primer, and approximately 20 μL of GenElute extracted template DNA or 5 μL of GeneReleaser extracted template DNA. Negative controls with polymerase chain reaction water and positive controls with previously validated bacterial DNA samples were included in all experiments. A touchdown protocol in a thermocycler (PTC-100TM; MJ Research, St. Bruno, Quebec, Canada) was used, with a predenaturation step of 94°C for 2 minutes, followed by 34 cycles of 94°C for 30 seconds, 56°C for 60 seconds, 72°C for 2 minutes each, and a final extension step of 72°C for 10 minutes, after which the temperature was held at 10°C. DNA samples were electrophoresed through 1% (weight/volume) agarose gels (AGTC Bioproducts, Hessle, United Kingdom) containing 0.75 μg/mL −1 of ethidium bromide in tris-borate EDTA buffer (Sigma-Aldrich) and were compared against the migration of a known 1-kilobase marker. Nucleic acids were visualized by using an ultraviolet transilluminator and EDAS image capture (Kodak, Rochester, NY). Before sequencing, the remaining polymerase chain reaction sample (40 μL) was purified by using the QIAquick PCR purification kit (Qiagen, Crawley, United Kingdom) according to the manufacturer’s instructions. The extracted DNA was sent to an external laboratory for DNA sequencing (Source BioScience, University of Oxford, Oxford, United Kingdom). The resultant sequence was then compared with existing databases of bacterial DNA sequences ( http://blast.ncbi.nlm.nih.gov ). This provided a likely identity of the bacterial species grown from the experiments.
Results
In part 1, investigation into the sterility of “as received” orthodontic materials, the bacterial counts were recorded by counting bacterial cultures from the investigated materials ( Table I ) grown in the different atmospheric conditions and on blood agar or fastidious anaerobic agar. The results are expressed as contaminants per milliliter of fluid used to remove bacteria from the sampled items and are shown in Table II . Five “as received” materials tested were not contaminated by culturable bacteria. However, 4 “as received” materials showed evidence of contamination—most notably the molar bands and the impression trays, which showed evidence of bacterial contamination on 2 of 5 samples. The overall numbers of contaminants were low; the highest number obtained was for molar band sample 1 at 3.65 × 10 2 CFU/mL −1 . Species identified from the bacterial sample from the items “as received from the manufacturer” are shown in Table III . The bacteria identified from these items were mainly environmental, but some oral bacterial species were cultivated from the molar bands; this was unexpected, since these items had not been previously used.
| Materials sampled | Bacterial counts (CFU/mL −1 ) | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | ||||||
| Clinic exposed | As received | Clinic exposed | As received | Clinic exposed | As received | Clinic exposed | As received | Clinic exposed | As received | |
| Beta-titanium alloy, 0.017 × 0.025 in | 0.5 × 10 1 | 0 | 0 | 1.5 × 10 1 | 0 | 0 | 0.5 × 10 1 | 0 | 0.5 × 10 1 | 0 |
| Stainless steel, 0.019 × 0.025 in | 0.5 × 10 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| Nickel-titanium, 0.014 in | 0 | 0 | 0 | 0 | 8.0 × 10 1 | 0 | 0.5 × 10 1 | 2.0 × 10 1 | 0 | 0 |
| Molar bands | 0.5 × 10 1 | 3.65 × 10 2 | 0.5 × 10 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0.5 × 10 1 |
| Brackets | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| Coil spring | 5.5 × 10 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| Impression trays | 0 | 0 | 0 | 0.5 × 10 1 | 0.5 × 10 1 | 0.5 × 10 1 | 2.5 × 10 1 | 0 | 0 | 0 |
| Elastomeric modules | 0.5 × 10 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0.5 × 10 1 | 0 |
| Elastomeric chart | 0.5 × 10 1 | – | 1.0 × 10 1 | – | 4.5 × 10 1 | – | 0.5 × 10 1 | – | 0 | – |
| Stainless steel, 0.019 × 0.025-in posted | 0 | – | 0 | – | 5.5 × 10 1 | – | 0 | – | 0 | – |
| Tungsten carbide burs | – | 0 | – | 0 | – | 0 | – | 0 | – | 0 |
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