- Email: [email protected]
Microbial contamination of “as received” and “clinic exposed” orthodontic materials
ORIGINAL ARTICLE
Microbial contamination of “as received” and “clinic exposed” orthodontic materials Christopher S. Barker,a Valeria Soro,b David Dymock,c Jonathan R. Sandy,d and Anthony J. Irelande Leeds, Bristol, and Bath, United Kingdom 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 3 101 to 1.825 3 102 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 3 101 to 8.0 3 101 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. (Am J Orthod Dentofacial Orthop 2013;143:317-23)
H
ealth 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
a Senior specialty registrar in orthodontics, Leeds Dental Institute, Leeds, United Kingdom. b Research technician in microbiology, School of Oral and Dental Sciences, Bristol, United Kingdom. c Senior lecturer in oral microbiology, School of Oral and Dental Sciences, Bristol, United Kingdom. d Professor and honorary consultant in orthodontics, School of Oral and Dental Sciences, Bristol, United Kingdom. e Professor in orthodontics, School of Oral and Dental Sciences, Bristol, United Kingdom; consultant in orthodontics, Royal United Hospitals, Bath, United Kingdom. The authors report no commercial, proprietary, or financial interest in the products or companies described in this article. Research supported by a small research and development equipment grant from the Royal United Bath NHS Trust, Bath, United Kingdom. This research is part of the first author's thesis for the degree of DDS at the University of Bristol, Bristol, United Kingdom. Reprint requests to: Christopher S. Barker, Leeds Dental Institute, Clarendon Way, Leeds, LS2 9LU, United Kingdom; e-mail, [email protected]. Submitted, July 2012; revised and accepted, September 2012. 0889-5406/$36.00 Copyright Ó 2013 by the American Association of Orthodontists. http://dx.doi.org/10.1016/j.ajodo.2012.09.020
decontamination in primary dental care in the United Kingdom by the introduction of the Health Technical Memorandum 01-05,1 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.2-5 Guidance for dentists in the United Kingdom on infection control is provided by the British Dental Association,6 which incorporates the advice from the Department of Health document.1 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.”6 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 317
Barker et al
318
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 al7 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.8 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).1 In dentistry, investigations have been conducted into the sterility of dental burs and endodontic files “as received” from the manufacturers. Hauptman et al9 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.10 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 Conrod11 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
March 2013 Vol 143 Issue 3
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.7 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.12 Larger particles (50-100 mm) can be splattered across the dental surgery, whereas smaller particles can form an aerosol and be inhaled.13,14 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,15 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.16 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 crossinfection risks in terms of the possible transmission of human immunodeficiency virus and hepatitis B and C viruses.17 All of this is in addition to the risks associated
American Journal of Orthodontics and Dentofacial Orthopedics
Barker et al
with aerosols contaminated as a result of bacterial growth in the dental unit's water line.18-20 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 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
319
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 mL 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 bloodenriched 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-mL reaction containing 1.25 units of Go Taq DNA polymerase (Promega, Southampton, United Kingdom), 10 mL 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 mL of GenElute extracted template DNA or 5 mL 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)
American Journal of Orthodontics and Dentofacial Orthopedics
March 2013 Vol 143 Issue 3
Barker et al
320
Table I. “As received” and “clinic exposed” orthodontic materials investigated Items tested 0.017 3 0.025-in beta-titanium alloy archwire 0.019 3 0.025-in stainless steel archwire 0.014-in nickel-titanium archwire Molar bands Stainless steel orthodontic brackets Coil spring (100-mm sample) Impression trays Elastomeric modules (20 sampled) Elastomeric chart 0.019 3 0.025-in stainless steel posted archwire Tungsten carbide burs
As received
Clinic exposed
-
Packaging “as received” Individual packets 10 per packet 10 per packet 5 per packet 20 per packet Polythene bag
Manufacturer TP Orthodontics, LaPorte, Ind TP Orthodontics, LaPorte, Ind TP Orthodontics, LaPorte, Ind 3M Unitek, Monrovia, Calif Dentsply GAC, Bohemia, NY Rocky Mountain Orthodontics, Denver, Colo Ortho Technology, Tampa, Fla TP Orthodontics, LaPorte, Ind In-house production Dentsply GAC, Bohemia, NY Dentsply GAC, Bohemia, NY
Polythene bag, 100 units 10 per holder, 100 per bag
Individual sealed packets
indicates tested; - indicates not tested.
Table II. Mean total combined aerobic and anaerobic bacterial counts on orthodontic items Bacterial counts (CFU/mL 1) Sample 1 Materials sampled Beta-titanium alloy, 0.017 3 0.025 in Stainless steel, 0.019 3 0.025 in Nickel-titanium, 0.014 in Molar bands Brackets Coil spring Impression trays Elastomeric modules Elastomeric chart Stainless steel, 0.019 3 0.025-in posted Tungsten carbide burs
Sample 2
Clinic exposed 0.5 3 101
As received 0
0.5 3 101
0
Sample 3
Clinic As Clinic exposed received exposed 0 1.5 3 101 0 0
0
0 0 0 0 0 0.5 3 101 3.65 3 102 0.5 3 101 0 0 0 0 0 0 0 5.5 3 101 0 0 0 0.5 3 101 0 0 0 0.5 3 101 1.0 3 101 0.5 3 101 0 0 -
0
-
0
Sample 4
Sample 5
As Clinic As Clinic As received exposed received exposed received 0 0.5 3 101 0 0.5 3 101 0
0
0
0
0
0
0
0 0.5 3 101 2.0 3 101 0 0 8.0 3 101 0 0 0 0 0 0.5 3 101 0 0 0 0 0 0 0 0 0 0 0 0 0.5 3 101 0.5 3 101 2.5 3 101 0 0 0 0 0 0 0 0.5 3 101 0 1 1 4.5 3 10 0.5 3 10 0 0 0 5.5 3 101 -
0
-
0
-
0
Five samples of each item were analyzed. The elastomeric chart and the stainless steel 0.019 3 0.025-in posted archwire were sampled only as “clinic exposed,” and the tungsten carbide bur was sampled only “as received.”
agarose gels (AGTC Bioproducts, Hessle, United Kingdom) containing 0.75 mg/mL 1 of ethidium bromide in trisborate 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 mL) 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
March 2013 Vol 143 Issue 3
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
American Journal of Orthodontics and Dentofacial Orthopedics
Barker et al
321
Table III. Distribution of bacterial species identified
from cultures on “as received” items after 27F1492R 16S rRNA gene amplification and DNA sequencing Distribution of bacteria on items Species Streptococcus cristatus Streptococcus sp. Staphylococcus pasteuri Rothia aeria Actinomyces viscosus Corynebacterium durum Streptococcus sanguinis Neisseria bacilliformis Micrococcus yunnanensis, Micrococcus luteus Micrococcus luteus Staphylococcus warneri, Staphylococcus pasteuri, Psychrobacter pulmonis Staphylococcus sp.
Molar band
TMA archwire
NiTi archwire
Imp tray
TMA, Beta-titanium alloy; NiTi, nickel-titanium; Imp tray, impression tray; indicates presence of bacteria.
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 3 102 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. In part 2, investigation into the sterility of “benchtop exposed” orthodontic supplies, the bacterial counts were recorded by enumeration of the bacterial cultures from these materials (Table I) grown under the different conditions and multiplied to give the contamination per milliliter. These are shown in Table II. Nine “bench-top” materials tested showed evidence of contamination; however, the overall numbers of contaminants were low. The most consistently contaminated material was the “in-house” elastomeric color chart. Species identified from the bacterial samples of the “bench-top exposed” items are shown in Table IV. S epidermidis was the most commonly identified bacterial species on these materials. The item with the most species cultured was the “in-house” elastomeric color chart.
DISCUSSION
As a result of this research, we found that bacteria were present on items “as received from the manufacturers” (part 1). Therefore, these items are not sterile, and this confirms the findings of previous studies of dental burs,9 endodontic files,10 and orthodontic molar tubes.7 The levels of contamination were found to be low (0.5 3 101 to 3.65 3 102 CFU/mL 1) and can be considered to be inconsequential because these items are placed in the oral environment, which has about 5 3 108 bacterial cells per milliliter 1. Examination of the species of bacteria isolated in this experiment has shown that oral bacteria were cultured from unused items; this is interesting since the method of transmission must be related to hand hygiene or possible aerosols produced from the mouth when the items were packaged. One species identified from the molar band was Streptococcus sanguinis, a member of the viridans streptococci, which has been implicated as a causative agent in infective endocarditis.21 Although it is a resident microorganism in the oral flora in healthy people, it was identified on only 1 band and was not identified on any other bands subsequently tested. However, the presence of such bacteria has important implications, since the molar band comes into contact with the gingival crevice, and thus the bloodstream, and could be a potential area for cross-contamination. Evaluation of whether placement of an orthodontic band causes a clinically detectable bacteremia showed that this was not the case in a study in which blood cultures before placement and 30 seconds after placement were investigated.22 This study did not disclose whether the band was presterilized or used directly from the manufacturer. The manufacturers of molar bands state that these items are not produced in a sterile environment and recommend sterilization before initial use. Most species isolated in this investigation were commensal bacteria normally found on the skin; this would be expected for items that are not classified as sterile. As before, the levels at which the bacteria were present were low and can be considered to be low risk for both crosscontamination and the health of the recipient orthodontic patient. Because this was a pilot study into the contamination of items “as received from the manufacturers,” only a small sample was investigated to determine the extent of the clinical situation. Larger sample sizes and more extensive investigations into other manufacturers might be indicated to ensure the validity of these results. The sampling methodology allowed investigation of a variety of items over a 6-month period to obtain a representative sample of items available to orthodontists.
American Journal of Orthodontics and Dentofacial Orthopedics
March 2013 Vol 143 Issue 3
Barker et al
322
Table IV. Distribution of bacterial species identified from cultures on “clinic exposed” items after 27F-1492R ampli-
fication and DNA sequencing
Bacteria on clinic items Species Moraxella sp. Moraxella osloensis Micrococcus luteus Brevibacterium sanguinis Staphylococcus epidermidis Staphylococcus caprae Micrococcus yunnanensis Staphylococcus sp. Kocuria palustris
TMA
SS
NiTi
SS post
Coil
Band
Chart
Imp
O-ring
TMA, Beta-titanium alloy; NiTi, nickel-titanium; SS, stainless steel archwire; SS post, posted stainless steel archwire; Imp, impression tray; indicates presence of bacteria.
Investigation into the potential contamination of orthodontic items in the clinic (part 2) found some contamination. However, bacteria were not cultured on all items in each experiment; when they were present, the level of contamination again was low (0.5 3 101 to 8.0 3 101 CFU/mL 1). It is possible that some of this contamination was from the manufacturing process rather than directly from the clinic itself. The most common contaminant across all items investigated was S epidermidis, indicating that the most likely source of cross-contamination was skin contact. The most commonly contaminated item in this investigation was the elastomeric color chart. This item was investigated because cross-contamination might occur as the chart is passed between patient, orthodontist, and assistant. This problem of cross-contamination could be easily overcome by having a large chart in the waiting area for patients to choose their elastics before entering the clinic. Cross-contamination by oral bacteria does not seem to be the case, and it would seem that orthodontic materials left on the clinic work-top are sufficiently protected from aerosol dispersion of oral bacteria. We did not aim to determine time-dependent contamination rates of items exposed to the clinical environment because this does not reproduce clinical practice. In a busy orthodontic clinic, there is a rapid turnover of materials, with new items regularly replacing those used. It is worth considering the techniques used in this investigation. The method used to dislodge bacteria from the items under test—immersion in tris-EDTA buffer solution and vortexing for 60 seconds—might not have fully dislodged all adherent bacteria. This would have underestimated the bacterial contamination. Other investigators have used different methods to determine bacterial contamination. Hauptman et al,9 investigating dental burs, bathed them in Luria-Bertani broth under
March 2013 Vol 143 Issue 3
aerobic conditions. They were agitated at 200 rpm for 24 hours at 37 C. Once growth was identified, these were further cultured onto Luria-Bertani agar and then sent for bacterial identification by a commercial laboratory using fatty acid profiles and 16S rRNA gene alignment profiles. This method only assessed the aerobic conditions but might have had a greater ability to obtain growth from adherent bacteria. The study design by Roth et al10 into the sterility of “as received” endodontic files used a slightly different microbiologic technique in which the endodontic files were placed in BHI broth and incubated statically at 37 C for 72 hours. Turbid cultures were then cultured on BHI agar for a further 24 hours. The species present were identified by 16S rRNA gene sequencing. Although this could lead to underestimating the degree of contamination because only aerobic conditions were investigated, at least the item itself was immersed in the broth so that maximal growth would take place, regardless of adherence. Whitworth et al23 used a similar technique to the experiments reported here when they examined the contamination of used dental burs. These investigators used BHI broth as the medium in which to vortex the sample (used dental burs) for 90 seconds, followed by culture on agar. We investigated only the bacterial contamination of orthodontic materials, but this might ultimately underestimate the overall situation, since viruses and prions have not been investigated. CONCLUSIONS
These investigations show the potential areas of contamination that might cause harm in susceptible patients, such as immunocompromised patients. During everyday life, we come into contact with many microbes, some with the potential to be pathogenic; in most healthy people, this contact has little effect. This is especially true of
American Journal of Orthodontics and Dentofacial Orthopedics
Barker et al
the oral cavity, which has a well-established oral flora, and any additional ingested microbes must compete with the resident microflora to gain a niche before ultimately passing through the gastrointestinal tract. The extent to which the bacteria identified in these studies can have an effect on a patient is currently unknown. It is also unknown whether larger numbers of environmental contaminants have a greater effect than low levels of potentially pathogenic bacteria, especially among immunocompromised patients. Because of this, it would be wise to reduce the possible risks of using potentially contaminated items with additional presterilization procedures before use. Clarity on this matter might be obtained from the manufacturers, if they state the sterility of their items or advise presterilization. This study highlights the need for manufacturers to state the sterility of their products and for practitioners to ensure the sterility of materials before use and not to assume that all items are sterile because of individual packaging. REFERENCES 1. Department of Health. Health technical memorandum 01-05: decontamination in primary care dental practices. London, United Kingdom: Her Majesty's Stationary Office; 2009. 2. NHS Scotland. Survey of decontamination in general dental practice (2004). Available at: http://www.scotland.gov.uk/Publications/ 2004/11/20093/45208. Accessed on March 3, 2011. 3. Bagg J, Smith AJ, Hurrell D, McHugh S, Irvine G. Pre-sterilisation cleaning of re-usable instruments in general dental practice. Br Dent J 2007;202:E22. 4. Smith AJ, Bagg J, Hurrell D, McHugh S. Sterilization of reusable instruments in general dental practice. Br Dent J 2007; 203:E16. 5. Smith AJ, Hurrell D, Bagg J, McHugh S, Mathewson H, Henry M. A method for surveying instrument decontamination procedures in general dental practice. Br Dent J 2007;202:E20. 6. British Dental Association. Infection control in dentistry. Advice sheet A12; London, BDA, 2009. 7. Purmal K, Chin S, Pinto J, Yin WF, Chan KG. Microbial contamination of orthodontic buccal tubes from manufacturers. Int J Mol Sci 2010;11:3349-56.
323
8. Sheriteh Z, Hassan T, Sherriff M, Cobourne M. Decontamination procedures for tungsten carbide debonding burs: a crosssectional survey of hospital-based orthodontic departments. J Orthod 2010;37:174-80. 9. Hauptman JM, Golberg MB, Rewkowski CA. The sterility of dental burs directly from the manufacturer. J Esthet Restor Dent 2006;18: 268-71. 10. Roth TP, Whitney SI, Walker SG, Friedman S. Microbial contamination of endodontic files received from the manufacturer. J Endod 2006;32:649-51. 11. Morrison A, Conrod S. Dental burs and endodontic files: are routine sterilization procedures effective? J Can Dent Assoc 2009;75:39. 12. Micik RE, Miller RL, Mazzarella MA, Ryge G. Studies on dental aerobiology. I. Bacterial aerosols generated during dental procedures. J Dent Res 1969;48:49-56. 13. Miller RL, Micik RE, Abel C, Ryge G. Studies on dental aerobiology. II. Microbial splatter discharged from the oral cavity of dental patients. J Dent Res 1971;50:621-5. 14. Day CJ, Sandy JR, Ireland AJ. Aerosols and splatter in dentistry— a neglected menace? Dent Update 2006;33:601-2, 604-6. 15. Toroglu MS, Bayramoglu O, Yarkin F, Tuli A. Possibility of blood and hepatitis B contamination through aerosols generated during debonding procedures. Angle Orthod 2003;73:571-8. 16. Rautemaa R, Nordberg A, Wuolijoki-Saaristo K, Meurman JH. Bacterial aerosols in dental practice—a potential hospital infection problem? J Hosp Infect 2006;64:76-81. 17. Cristina ML, Spagnolo AM, Sartini M, Dallera M, Ottria G, Lombardi R, et al. Evaluation of the risk of infection through exposure to aerosols and spatters in dentistry. Am J Infect Control 2008;36:304-7. 18. Pankhurst CL, Coulter WA. Do contaminated dental unit waterlines pose a risk of infection? J Dent 2007;35:712-20. 19. Walker JT, Marsh PD. Microbial biofilm formation in DUWS and their control using disinfectants. J Dent 2007;35:721-30. 20. Coleman DC, O'Donnell MJ, Shore AC, Russell RJ. Biofilm problems in dental unit water systems and its practical control. J Appl Microbiol 2009;106:1424-37. 21. Knox KW, Hunter N. The role of oral bacteria in the pathogenesis of infective endocarditis. Aust Dent J 1991;36:286-92. 22. Lucas VS, Omar J, Vieira A, Roberts GJ. The relationship between odontogenic bacteraemia and orthodontic treatment procedures. Eur J Orthod 2002;24:293-301. 23. Whitworth CL, Martin MV, Gallagher M, Worthington HV. A comparison of decontamination methods used for dental burs. Br Dent J 2004;197:635-40.
American Journal of Orthodontics and Dentofacial Orthopedics
March 2013 Vol 143 Issue 3
Microbial contamination of “as received” and “clinic exposed” orthodontic materials Christopher S. Barker,a Valeria Soro,b David Dymock,c Jonathan R. Sandy,d and Anthony J. Irelande Leeds, Bristol, and Bath, United Kingdom 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 3 101 to 1.825 3 102 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 3 101 to 8.0 3 101 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. (Am J Orthod Dentofacial Orthop 2013;143:317-23)
H
ealth 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
a Senior specialty registrar in orthodontics, Leeds Dental Institute, Leeds, United Kingdom. b Research technician in microbiology, School of Oral and Dental Sciences, Bristol, United Kingdom. c Senior lecturer in oral microbiology, School of Oral and Dental Sciences, Bristol, United Kingdom. d Professor and honorary consultant in orthodontics, School of Oral and Dental Sciences, Bristol, United Kingdom. e Professor in orthodontics, School of Oral and Dental Sciences, Bristol, United Kingdom; consultant in orthodontics, Royal United Hospitals, Bath, United Kingdom. The authors report no commercial, proprietary, or financial interest in the products or companies described in this article. Research supported by a small research and development equipment grant from the Royal United Bath NHS Trust, Bath, United Kingdom. This research is part of the first author's thesis for the degree of DDS at the University of Bristol, Bristol, United Kingdom. Reprint requests to: Christopher S. Barker, Leeds Dental Institute, Clarendon Way, Leeds, LS2 9LU, United Kingdom; e-mail, [email protected]. Submitted, July 2012; revised and accepted, September 2012. 0889-5406/$36.00 Copyright Ó 2013 by the American Association of Orthodontists. http://dx.doi.org/10.1016/j.ajodo.2012.09.020
decontamination in primary dental care in the United Kingdom by the introduction of the Health Technical Memorandum 01-05,1 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.2-5 Guidance for dentists in the United Kingdom on infection control is provided by the British Dental Association,6 which incorporates the advice from the Department of Health document.1 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.”6 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 317
Barker et al
318
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 al7 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.8 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).1 In dentistry, investigations have been conducted into the sterility of dental burs and endodontic files “as received” from the manufacturers. Hauptman et al9 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.10 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 Conrod11 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
March 2013 Vol 143 Issue 3
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.7 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.12 Larger particles (50-100 mm) can be splattered across the dental surgery, whereas smaller particles can form an aerosol and be inhaled.13,14 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,15 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.16 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 crossinfection risks in terms of the possible transmission of human immunodeficiency virus and hepatitis B and C viruses.17 All of this is in addition to the risks associated
American Journal of Orthodontics and Dentofacial Orthopedics
Barker et al
with aerosols contaminated as a result of bacterial growth in the dental unit's water line.18-20 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 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
319
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 mL 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 bloodenriched 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-mL reaction containing 1.25 units of Go Taq DNA polymerase (Promega, Southampton, United Kingdom), 10 mL 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 mL of GenElute extracted template DNA or 5 mL 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)
American Journal of Orthodontics and Dentofacial Orthopedics
March 2013 Vol 143 Issue 3
Barker et al
320
Table I. “As received” and “clinic exposed” orthodontic materials investigated Items tested 0.017 3 0.025-in beta-titanium alloy archwire 0.019 3 0.025-in stainless steel archwire 0.014-in nickel-titanium archwire Molar bands Stainless steel orthodontic brackets Coil spring (100-mm sample) Impression trays Elastomeric modules (20 sampled) Elastomeric chart 0.019 3 0.025-in stainless steel posted archwire Tungsten carbide burs
As received
Clinic exposed
-
Packaging “as received” Individual packets 10 per packet 10 per packet 5 per packet 20 per packet Polythene bag
Manufacturer TP Orthodontics, LaPorte, Ind TP Orthodontics, LaPorte, Ind TP Orthodontics, LaPorte, Ind 3M Unitek, Monrovia, Calif Dentsply GAC, Bohemia, NY Rocky Mountain Orthodontics, Denver, Colo Ortho Technology, Tampa, Fla TP Orthodontics, LaPorte, Ind In-house production Dentsply GAC, Bohemia, NY Dentsply GAC, Bohemia, NY
Polythene bag, 100 units 10 per holder, 100 per bag
Individual sealed packets
indicates tested; - indicates not tested.
Table II. Mean total combined aerobic and anaerobic bacterial counts on orthodontic items Bacterial counts (CFU/mL 1) Sample 1 Materials sampled Beta-titanium alloy, 0.017 3 0.025 in Stainless steel, 0.019 3 0.025 in Nickel-titanium, 0.014 in Molar bands Brackets Coil spring Impression trays Elastomeric modules Elastomeric chart Stainless steel, 0.019 3 0.025-in posted Tungsten carbide burs
Sample 2
Clinic exposed 0.5 3 101
As received 0
0.5 3 101
0
Sample 3
Clinic As Clinic exposed received exposed 0 1.5 3 101 0 0
0
0 0 0 0 0 0.5 3 101 3.65 3 102 0.5 3 101 0 0 0 0 0 0 0 5.5 3 101 0 0 0 0.5 3 101 0 0 0 0.5 3 101 1.0 3 101 0.5 3 101 0 0 -
0
-
0
Sample 4
Sample 5
As Clinic As Clinic As received exposed received exposed received 0 0.5 3 101 0 0.5 3 101 0
0
0
0
0
0
0
0 0.5 3 101 2.0 3 101 0 0 8.0 3 101 0 0 0 0 0 0.5 3 101 0 0 0 0 0 0 0 0 0 0 0 0 0.5 3 101 0.5 3 101 2.5 3 101 0 0 0 0 0 0 0 0.5 3 101 0 1 1 4.5 3 10 0.5 3 10 0 0 0 5.5 3 101 -
0
-
0
-
0
Five samples of each item were analyzed. The elastomeric chart and the stainless steel 0.019 3 0.025-in posted archwire were sampled only as “clinic exposed,” and the tungsten carbide bur was sampled only “as received.”
agarose gels (AGTC Bioproducts, Hessle, United Kingdom) containing 0.75 mg/mL 1 of ethidium bromide in trisborate 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 mL) 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
March 2013 Vol 143 Issue 3
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
American Journal of Orthodontics and Dentofacial Orthopedics
Barker et al
321
Table III. Distribution of bacterial species identified
from cultures on “as received” items after 27F1492R 16S rRNA gene amplification and DNA sequencing Distribution of bacteria on items Species Streptococcus cristatus Streptococcus sp. Staphylococcus pasteuri Rothia aeria Actinomyces viscosus Corynebacterium durum Streptococcus sanguinis Neisseria bacilliformis Micrococcus yunnanensis, Micrococcus luteus Micrococcus luteus Staphylococcus warneri, Staphylococcus pasteuri, Psychrobacter pulmonis Staphylococcus sp.
Molar band
TMA archwire
NiTi archwire
Imp tray
TMA, Beta-titanium alloy; NiTi, nickel-titanium; Imp tray, impression tray; indicates presence of bacteria.
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 3 102 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. In part 2, investigation into the sterility of “benchtop exposed” orthodontic supplies, the bacterial counts were recorded by enumeration of the bacterial cultures from these materials (Table I) grown under the different conditions and multiplied to give the contamination per milliliter. These are shown in Table II. Nine “bench-top” materials tested showed evidence of contamination; however, the overall numbers of contaminants were low. The most consistently contaminated material was the “in-house” elastomeric color chart. Species identified from the bacterial samples of the “bench-top exposed” items are shown in Table IV. S epidermidis was the most commonly identified bacterial species on these materials. The item with the most species cultured was the “in-house” elastomeric color chart.
DISCUSSION
As a result of this research, we found that bacteria were present on items “as received from the manufacturers” (part 1). Therefore, these items are not sterile, and this confirms the findings of previous studies of dental burs,9 endodontic files,10 and orthodontic molar tubes.7 The levels of contamination were found to be low (0.5 3 101 to 3.65 3 102 CFU/mL 1) and can be considered to be inconsequential because these items are placed in the oral environment, which has about 5 3 108 bacterial cells per milliliter 1. Examination of the species of bacteria isolated in this experiment has shown that oral bacteria were cultured from unused items; this is interesting since the method of transmission must be related to hand hygiene or possible aerosols produced from the mouth when the items were packaged. One species identified from the molar band was Streptococcus sanguinis, a member of the viridans streptococci, which has been implicated as a causative agent in infective endocarditis.21 Although it is a resident microorganism in the oral flora in healthy people, it was identified on only 1 band and was not identified on any other bands subsequently tested. However, the presence of such bacteria has important implications, since the molar band comes into contact with the gingival crevice, and thus the bloodstream, and could be a potential area for cross-contamination. Evaluation of whether placement of an orthodontic band causes a clinically detectable bacteremia showed that this was not the case in a study in which blood cultures before placement and 30 seconds after placement were investigated.22 This study did not disclose whether the band was presterilized or used directly from the manufacturer. The manufacturers of molar bands state that these items are not produced in a sterile environment and recommend sterilization before initial use. Most species isolated in this investigation were commensal bacteria normally found on the skin; this would be expected for items that are not classified as sterile. As before, the levels at which the bacteria were present were low and can be considered to be low risk for both crosscontamination and the health of the recipient orthodontic patient. Because this was a pilot study into the contamination of items “as received from the manufacturers,” only a small sample was investigated to determine the extent of the clinical situation. Larger sample sizes and more extensive investigations into other manufacturers might be indicated to ensure the validity of these results. The sampling methodology allowed investigation of a variety of items over a 6-month period to obtain a representative sample of items available to orthodontists.
American Journal of Orthodontics and Dentofacial Orthopedics
March 2013 Vol 143 Issue 3
Barker et al
322
Table IV. Distribution of bacterial species identified from cultures on “clinic exposed” items after 27F-1492R ampli-
fication and DNA sequencing
Bacteria on clinic items Species Moraxella sp. Moraxella osloensis Micrococcus luteus Brevibacterium sanguinis Staphylococcus epidermidis Staphylococcus caprae Micrococcus yunnanensis Staphylococcus sp. Kocuria palustris
TMA
SS
NiTi
SS post
Coil
Band
Chart
Imp
O-ring
TMA, Beta-titanium alloy; NiTi, nickel-titanium; SS, stainless steel archwire; SS post, posted stainless steel archwire; Imp, impression tray; indicates presence of bacteria.
Investigation into the potential contamination of orthodontic items in the clinic (part 2) found some contamination. However, bacteria were not cultured on all items in each experiment; when they were present, the level of contamination again was low (0.5 3 101 to 8.0 3 101 CFU/mL 1). It is possible that some of this contamination was from the manufacturing process rather than directly from the clinic itself. The most common contaminant across all items investigated was S epidermidis, indicating that the most likely source of cross-contamination was skin contact. The most commonly contaminated item in this investigation was the elastomeric color chart. This item was investigated because cross-contamination might occur as the chart is passed between patient, orthodontist, and assistant. This problem of cross-contamination could be easily overcome by having a large chart in the waiting area for patients to choose their elastics before entering the clinic. Cross-contamination by oral bacteria does not seem to be the case, and it would seem that orthodontic materials left on the clinic work-top are sufficiently protected from aerosol dispersion of oral bacteria. We did not aim to determine time-dependent contamination rates of items exposed to the clinical environment because this does not reproduce clinical practice. In a busy orthodontic clinic, there is a rapid turnover of materials, with new items regularly replacing those used. It is worth considering the techniques used in this investigation. The method used to dislodge bacteria from the items under test—immersion in tris-EDTA buffer solution and vortexing for 60 seconds—might not have fully dislodged all adherent bacteria. This would have underestimated the bacterial contamination. Other investigators have used different methods to determine bacterial contamination. Hauptman et al,9 investigating dental burs, bathed them in Luria-Bertani broth under
March 2013 Vol 143 Issue 3
aerobic conditions. They were agitated at 200 rpm for 24 hours at 37 C. Once growth was identified, these were further cultured onto Luria-Bertani agar and then sent for bacterial identification by a commercial laboratory using fatty acid profiles and 16S rRNA gene alignment profiles. This method only assessed the aerobic conditions but might have had a greater ability to obtain growth from adherent bacteria. The study design by Roth et al10 into the sterility of “as received” endodontic files used a slightly different microbiologic technique in which the endodontic files were placed in BHI broth and incubated statically at 37 C for 72 hours. Turbid cultures were then cultured on BHI agar for a further 24 hours. The species present were identified by 16S rRNA gene sequencing. Although this could lead to underestimating the degree of contamination because only aerobic conditions were investigated, at least the item itself was immersed in the broth so that maximal growth would take place, regardless of adherence. Whitworth et al23 used a similar technique to the experiments reported here when they examined the contamination of used dental burs. These investigators used BHI broth as the medium in which to vortex the sample (used dental burs) for 90 seconds, followed by culture on agar. We investigated only the bacterial contamination of orthodontic materials, but this might ultimately underestimate the overall situation, since viruses and prions have not been investigated. CONCLUSIONS
These investigations show the potential areas of contamination that might cause harm in susceptible patients, such as immunocompromised patients. During everyday life, we come into contact with many microbes, some with the potential to be pathogenic; in most healthy people, this contact has little effect. This is especially true of
American Journal of Orthodontics and Dentofacial Orthopedics
Barker et al
the oral cavity, which has a well-established oral flora, and any additional ingested microbes must compete with the resident microflora to gain a niche before ultimately passing through the gastrointestinal tract. The extent to which the bacteria identified in these studies can have an effect on a patient is currently unknown. It is also unknown whether larger numbers of environmental contaminants have a greater effect than low levels of potentially pathogenic bacteria, especially among immunocompromised patients. Because of this, it would be wise to reduce the possible risks of using potentially contaminated items with additional presterilization procedures before use. Clarity on this matter might be obtained from the manufacturers, if they state the sterility of their items or advise presterilization. This study highlights the need for manufacturers to state the sterility of their products and for practitioners to ensure the sterility of materials before use and not to assume that all items are sterile because of individual packaging. REFERENCES 1. Department of Health. Health technical memorandum 01-05: decontamination in primary care dental practices. London, United Kingdom: Her Majesty's Stationary Office; 2009. 2. NHS Scotland. Survey of decontamination in general dental practice (2004). Available at: http://www.scotland.gov.uk/Publications/ 2004/11/20093/45208. Accessed on March 3, 2011. 3. Bagg J, Smith AJ, Hurrell D, McHugh S, Irvine G. Pre-sterilisation cleaning of re-usable instruments in general dental practice. Br Dent J 2007;202:E22. 4. Smith AJ, Bagg J, Hurrell D, McHugh S. Sterilization of reusable instruments in general dental practice. Br Dent J 2007; 203:E16. 5. Smith AJ, Hurrell D, Bagg J, McHugh S, Mathewson H, Henry M. A method for surveying instrument decontamination procedures in general dental practice. Br Dent J 2007;202:E20. 6. British Dental Association. Infection control in dentistry. Advice sheet A12; London, BDA, 2009. 7. Purmal K, Chin S, Pinto J, Yin WF, Chan KG. Microbial contamination of orthodontic buccal tubes from manufacturers. Int J Mol Sci 2010;11:3349-56.
323
8. Sheriteh Z, Hassan T, Sherriff M, Cobourne M. Decontamination procedures for tungsten carbide debonding burs: a crosssectional survey of hospital-based orthodontic departments. J Orthod 2010;37:174-80. 9. Hauptman JM, Golberg MB, Rewkowski CA. The sterility of dental burs directly from the manufacturer. J Esthet Restor Dent 2006;18: 268-71. 10. Roth TP, Whitney SI, Walker SG, Friedman S. Microbial contamination of endodontic files received from the manufacturer. J Endod 2006;32:649-51. 11. Morrison A, Conrod S. Dental burs and endodontic files: are routine sterilization procedures effective? J Can Dent Assoc 2009;75:39. 12. Micik RE, Miller RL, Mazzarella MA, Ryge G. Studies on dental aerobiology. I. Bacterial aerosols generated during dental procedures. J Dent Res 1969;48:49-56. 13. Miller RL, Micik RE, Abel C, Ryge G. Studies on dental aerobiology. II. Microbial splatter discharged from the oral cavity of dental patients. J Dent Res 1971;50:621-5. 14. Day CJ, Sandy JR, Ireland AJ. Aerosols and splatter in dentistry— a neglected menace? Dent Update 2006;33:601-2, 604-6. 15. Toroglu MS, Bayramoglu O, Yarkin F, Tuli A. Possibility of blood and hepatitis B contamination through aerosols generated during debonding procedures. Angle Orthod 2003;73:571-8. 16. Rautemaa R, Nordberg A, Wuolijoki-Saaristo K, Meurman JH. Bacterial aerosols in dental practice—a potential hospital infection problem? J Hosp Infect 2006;64:76-81. 17. Cristina ML, Spagnolo AM, Sartini M, Dallera M, Ottria G, Lombardi R, et al. Evaluation of the risk of infection through exposure to aerosols and spatters in dentistry. Am J Infect Control 2008;36:304-7. 18. Pankhurst CL, Coulter WA. Do contaminated dental unit waterlines pose a risk of infection? J Dent 2007;35:712-20. 19. Walker JT, Marsh PD. Microbial biofilm formation in DUWS and their control using disinfectants. J Dent 2007;35:721-30. 20. Coleman DC, O'Donnell MJ, Shore AC, Russell RJ. Biofilm problems in dental unit water systems and its practical control. J Appl Microbiol 2009;106:1424-37. 21. Knox KW, Hunter N. The role of oral bacteria in the pathogenesis of infective endocarditis. Aust Dent J 1991;36:286-92. 22. Lucas VS, Omar J, Vieira A, Roberts GJ. The relationship between odontogenic bacteraemia and orthodontic treatment procedures. Eur J Orthod 2002;24:293-301. 23. Whitworth CL, Martin MV, Gallagher M, Worthington HV. A comparison of decontamination methods used for dental burs. Br Dent J 2004;197:635-40.
American Journal of Orthodontics and Dentofacial Orthopedics
March 2013 Vol 143 Issue 3