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Microbiological analysis of dental casts stored long-term
Journal of the World Federation of Orthodontists 2 (2013) e165ee168
Contents lists available at ScienceDirect
Journal of the World Federation of Orthodontists journal homepage: www.jwfo.org
Research
Microbiological analysis of dental casts stored long-term Simone Gallão a, *, Antonio Carlos Pizzolitto b, y, Lourdes Santos-Pinto c, Ary dos Santos-Pinto d, Kurt Faltin Jr. e, f, Lídia Parsekian Martins g a
Private Practice, Ribeirão Preto, São Paulo, Brazil Professor, Department of Clinical Analysis, School of Pharmaceutical Sciences, Universidade Estadual Paulista, São Paulo, Brazil Professor, Department of Orthodontics and Pediatric Dentistry, Araraquara Dental School, Universidade Estadual Paulista, Araraquara, São Paulo, Brazil d Professor, Department of Orthodontics and Pediatric Dentistry, Araraquara Dental School, University Estadual Paulista, Araraquara, São Paulo, Brazil e Professor and Department Chair, Department of Orthodontics, School of Dentistry, Universidade Paulista, São Paulo, Brazil f Invited Professor, Department of Facial Orthopedics, University of Ulm, Ulm, Germany g Professor and Department Chair, Department of Orthodontics and Pediatric Dentistry, Araraquara Dental School, Universidade Estadual Paulista, Araraquara, São Paulo, Brazil b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 April 2013 Received in revised form 19 June 2013 Accepted 23 July 2013 Available online 22 October 2013
Background: The aim of this study was to evaluate the presence of bacteria and fungi on dental plaster casts stored long-term. Methods: Forty-four pairs of dental casts stored since 1980e1981 were used. The material for microbiological analysis was collected with a swab and cultivated in brain-heart infusion cultures and Sabouraud dextrose broth for subsequent plating and colony identification. Results: Bacterial growth such as coagulase-negative Staphylococcus, Micrococcus sp., Bacillus sp., and nonfermenting gram-negative bacillus were present in 25% of the samples, whereas fungal growth such as Cladophialophora sp., Trichosporon beigelii, Aspergillus flavus, and Aureobasidium pullulans were found in 5.7%. Conclusions: There was growth of opportunistic bacteria and fungi, posing no risk to healthy people. Ó 2013 World Federation of Orthodontists.
Keywords: Bacteria Fungi Plaster cast
1. Introduction Plaster casts are widely used for diagnosis, planning, and manufacturing orthodontic appliances. Microorganisms, particularly in dental casts stored long-term, have been little investigated. Studies have been aimed at contamination and disinfection of the casts, but microorganisms they can also be present by the record bases. From the professional and legal points of view, it is crucial to develop effective means of disinfecting this material. In the presence of fungal infection, symptoms such as fatigue and irritation in the eyes, nose, and throat seem to be correlated with odor [1]. Streptococcus sp. and Staphylococcus sp., including methicillin-resistant Staphylococcus aureus (MRSA), Candida sp., and Pseudomonas aeruginosa, have been detected in plaster casts, with some microorganisms being potentially pathogenic [2]. Staphylococcus aureus is an important pathogen because of its virulence; antimicrobial resistance; and association with various diseases, including fatal systemic infections, intoxication, cutaneous infectious, and opportunistic diseases [3]. * Corresponding author: Rua Sete de Setembro, 668 ap 141, CEP 14010-180 Ribeirão Preto, São Paulo, Brazil. E-mail address: [email protected] (S. Gallão). y In memorian. 2212-4438/$ e see front matter Ó 2013 World Federation of Orthodontists. http://dx.doi.org/10.1016/j.ejwf.2013.07.003
Dental casts are subject to several forms of contamination, ranging from direct contact with a patient’s saliva to procedures involving measurement, planning, manufacture, laboratory sending, and storage. The present study was aimed at investigating the contamination of plaster casts stored long-term as well as the types of microorganisms involved and their pathogenicity in order to prevent cross-contamination.
2. Methods and materials The sample consisted of plaster dental casts of White individuals aged 12 to 17 years. The research ethics committee at the Universidade Estadual PaulistaeAraraquara (São Paulo, Brazil) approved the study protocol (no. 30/09). Forty-four pairs of dental plaster casts were used for study, totaling 88 bacteriologic and mycologic cultures. The casts had been stored since 1980e1981, remaining within plastic containers kept in wooden cabinets. Other practitioners had already manipulated them under unknown aseptic conditions. The samples had been coated to make them impermeable, resistant, and bright. The coating solution was 200 g of grated coconut soap dissolved in 1 L of hot water plus ½ teaspoon of borax. After cooling, the samples remained immersed for 12 hours and then were
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S. Gallão et al. / Journal of the World Federation of Orthodontists 2 (2013) e165ee168
Table 1 Quantity and percentage of fungi and bacteria isolated from the 88 microbiologically tested maxillary and mandibular samples of plaster casts (N ¼ 88)* Organism Bacteria Coagulase-negative Staphylococci Gram-negative nonfermenting bacilli Bacillus sp. Micrococcus sp. Fungi Aspergillus flavus Cladophialophora sp. Trichosporon beigelii Aureobasidium sp.
Maxillary
Mandibular
5 3 2 1
7 1 1 2
(5.7%) (3.4%) (2.3%) (1.1%)
1 (1.1%) 1 (1.1%) 1 (1.1%) 0
(8.0%) (1.1%) (1.1%) (2.3%)
1 (1.1%) 0 0 1 (1.1%)
Data are presented as number (%) of samples. * Sixty-one samples (69.3%) showed no growth in the cultures.
removed from the solution to dry naturally for 24 hours and polished. The microbiological analysis involved cultures for evaluating contamination of the casts. A sterile swab, soaked in phosphate buffer solution of pH 7.0, was rubbed over the whole area of the plaster cast to collect material for analysis. Procedure gloves were sterilized with 70% ethanol for each material collection. The swabs of each sample were placed in test tubes containing brain-heart infusion medium, incubated at 37 C, and maintained sequentially in Sabouraud glucose broth at room temperature for up to 30 days. The test tubes were examined for growth daily, and cultures presenting a darkish color were then submitted for microscopic examination following Gram staining. Next, they were placed in a medium favorable for screening isolated strains. The cultures in liquid medium were plated in brain-heart infusion agar and Sabouraud glucose agar for isolation and identification of the colonies. Media were prepared according to instructions from Difco Laboratories, Inc. (Detroit, Michigan). The plates were wrapped with polyvinyl chloride film and kept in a dark room at a temperature ranging from 2 to 8 C until their use. After being opened, the plates were used for 1 week at the same temperature. The plates were incubated at 35 to 37 C for bacteria, and at room temperature for fungi, both for 15 days. All cultures were examined in terms of growth and then identified. After incubation, some plates exhibited isolated colonies morphologically examined using Gram staining and identified as either fungi or bacteria through biochemicalephysiologic methods [4]. For the identification of gram-negative bacilli, the cultures used were: triple sugar iron agar for screening and evaluating the sugar metabolism; MacConkey agar for evaluating the presence of Enterobacteriaceae; blood agar for evaluating virulence factor (hemolytic capacity); and, for evaluating the growth of Pseudomonas sp., cetrimide agar. 3. Results and discussion Based on the plaster dental casts studied, the following fungi were isolated: Cladophialophora sp. (after 4-day incubation); Trichosporon beigelii (after 5-day incubation); and Aspergillus flavus and Aureobasidium pullulans (after 15-day incubation). As for the bacteria, coagulase-negative Staphylococcus (after 2-, 3-, and 5-day incubation); Micrococcus sp. (after 3- and 15-day incubation); Bacillus sp. (after 4-day incubation); and gram-negative nonfermenting bacilli (after 1-, 2-, 12-, and 15-day incubation) were isolated from the samples. Of the bacteria identified in the group of gram-positive microorganisms, Micrococcus sp. and coagulase-negative Staphylococcus were found, but with no evidence of Staphylococcus aureus. Gram-
positive rods with central spores, belonging to the genus Bacillus, were also identified by their microscopic characteristics. The isolated gram-negative bacilli were sequentially seeded over four cultures. In the triple sugar iron agar medium, no glucose fermentation by these bacilli was observed. In the MacConkey agar, four samples of gram-negative bacilli belonging to the glucose nonfermenting group were seeded, but no species of Enterobacteriaceae were isolated. In blood agar, the bacilli presented no virulence characteristics (hemolytic action). Finally, no growth of Pseudomonas sp. was detected in cetrimide agar. Bacterial and fungal growths were detected in 25% and 5.7% of the samples, respectively (Table 1). The identification of microorganisms in plaster dental casts as well as the determination of their pathogenic potential are important in determining the risk to professional, dental workers and patients. Although the plaster casts evaluated had been made impermeable with borax, an antifungal and bacteriostatic agent, the time that elapsed after the process and the way the plaster casts were stored may have contributed to the contamination found [5]. In addition to the possible cross-contamination resulting from manipulation, one can expect that some of the isolated microorganisms came from the oral cavity. In fact, Candida sp. (75%), Cladosporium sp. (65%), Aureobasidium sp. (50%), Saccharomycetales sp. (50%), Aspergillus sp. (35%), Fusarium sp. (30%), and Cryptococcus sp. (20%) were detected in saliva [6]. It is improbable that microorganisms had lived on gypsum cast long-term; however, the microorganisms exist in natural water and in human skin and mouth, so the contamination could have occurred during manipulation for study analysis. Preventive and therapeutic advances have prolonged life expectancy. However, individuals with chronic diseases may have immunosuppression from the disease or from the medications prescribed, and immunosuppression may cause free-living microorganisms to become pathogenic. The literature has reported several situations of contamination involving the microorganisms found in the present study. Some Cladophialophora sp. are pathogenic or neurotropic; can cause pheohyphomycosis infections, cerebral abscess, chromoblastomycosis, and contamination after transplantation, affecting mostly immunosuppressed individuals despite occurring in healthy patients; and can develop rapidly and be fatal [7e12]. Trichosporon beigelii can cause white piedra, a mycosis affecting the hair shaft of any body part, occurring mainly in South America [13,14]. Two cases of death have been described [15]. Aspergillus flavus is a common airborne pathogen and has been found in chronic granulomatous sinusitis, keratitis, cutaneous aspergillosis, infectious wounds, nondermatophytic onychomycosis, and osteomyelitis followed by trauma and inoculation. It produces aflatoxin, a natural toxin that is potentially cancerous [16,17]. Fungal infection with Candida sp., Aspergillus sp., and even Aureobasidium pullulans have been found in bone marrow transplant recipients [18]. Aureobasidium pullulans is common in tropical and subtropical regions; there are reports of keratomycosis, meningitis, splenic abscess, maxillary abscess, pulmonary mycosis, septicemia, opportunistic infections, cutaneous mycosis, and pheohyphomycosis [19,20]. For the genus Staphylococcus, coagulase-negative Staphylococcus has been found as a major pathogen in hospitals, and its occurrence is increasing with the use of prostheses, being more evident in immunocompromised patients [21,22]. Some Micrococcus sp. are usually found in the skin. It is thought that these bacteria are temporary residents; they are frequently found in the exposed skindface, arms, hands and legsdbut infectious diseases can be caused opportunistically in
S. Gallão et al. / Journal of the World Federation of Orthodontists 2 (2013) e165ee168
immunocompromised patients [4,23]. Those microorganisms likely infected the casts during manipulation. The majority of Bacillus sp. pose no pathogenic risk, except for Bacillus anthracis [24]. Due to the great number of gram-negative Bacillus sp. and the complexity involved in their identification, gram-negative bacilli have been excluded in the presence of the more significant microorganisms. Escherichia coli that indicate fecal contamination, followed by Pseudomonas aeruginosa (nonfermenting), considered pathogenic, were not observed in this sample [4,25]. The gramnegative bacilli isolated in the present study may be of free-living bacteria or opportunistic pathogens. Plaster casts remain contaminated even after 4 hours after being handled. Bacterial contamination decreased after the casts had been washed with water for 20 seconds, and even more when they were scrubbed with soap and tap water or soaked in 2% glutaraldehyde [26]. In this study, despite the 32-year storage duration, contamination was observed, but the casts were manipulated without the use of infection-control procedures. In the literature, disinfectant solutions mixed with water and then added to the plaster mixture were evaluated in terms of effectiveness. Povidoneeiodine eliminated microorganisms within 1 hour [27], and sodium hypochlorite was effective only after 24 hours at a 1:5 dilution, whereas 2% glutaraldehyde was the most effective disinfectant [28] after the plaster was immersed in solution for 20 minutes [26]. With regard to the physical properties of the plaster casts, it was observed that povidoneeiodine promoted changes in the compressive strength, and that the 1% sodium hypochlorite negatively changed the properties of the material, whereas 2% glutaraldehyde had a less adverse effect but is clinically toxic [28,29]. Chlorhexidine at 2% promoted no significant changes in the casts [29]. Determining the interactions between disinfectant solutions and the components in each new material being launched in the market will require further compatibility tests [30,31]. Microwave sterilization (1, 5, or 20 minutes) was not effective in plaster casts contaminated with Bacillus subtilis [32]. Disinfection was achieved following 5-minute exposure. The exception was a sample in which gram-positive bacilli were found but considered common in the environment. The dimensional stability of the samples was not affected, and no adverse effects were observed macroscopically [33,34]. The American Dental Association recommends cleaning and disinfecting impressions as soon as possible after removal from the patient’s mouth before the drying of blood or other bioburden can occur, because dental impression materials can act as vectors, transmitting an amount of microorganisms sufficient to stay viable within a plaster cast for 7 days [35e37]. After 32 years of storage, it is unlikely that the presence of the microorganisms in this sample was from the impression material. However, the casts’ manipulation with no personal-protection equipment could have contributed to the contamination found. Compliance with good practice is less than ideal, and education in impression disinfection and infection-control measures is necessary to ensure the health and safety of dental workers and patients [38,39]. 4. Conclusions To avoid the occurrence of cross-infection during manipulation of plaster casts, the use of both disinfection and individual protective equipment is recommended. Another alternative might be to replace the plaster casts with digital models if measured by scientifically proven computing systems. Fungi and bacteria were found in plaster casts stored long-term. In general, the microorganisms identified did not exhibit
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pathogenic potential in healthy individuals. However, they may represent a risk to patients who take antibiotics, corticoids, or immunosuppressors long-term. Acknowledgments The author thanks Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (www.capes.gov.br) for financial support during his PhD program at the School of Dentistry of Araraquara, Universidade Estadual Paulista, São Paulo, Brazil. Special appreciation goes to Prof. Kurt Faltin, Jr. from the Orthodontics and Pediatric Dentistry Department, Universidade Paulista, São Paulo, Brazil, who generously provided the sample for this research. References [1] Samson RA. Occurrence of moulds in modern living and working environments. Eur J Epidemiol 1985;1:54e61. [2] Egusa H, Watamoto T, Abe K, et al. An analysis of the persistent presence of opportunistic pathogens on patient-derived dental impressions and gypsum casts. Int J Prosthodont 2008;21:62e8. [3] Lowy FD. Staphylococcus aureus infections. N Engl J Med 1998;339:520e32. [4] Murray PR, Baron EJ, Jorgensen JH, Landry ML, Pfaller MA. Manual of clinical microbiology. 9th ed. Washington, DC: American Society of Microbiology; 2007. [5] Meers PD, Chow CK. Bacteriostatic and bactericidal actions of boric acid against bacteria and fungi commonly found in urine. J Clin Pathol 1990;43: 484e7. [6] Ghannoum MA, Jurevic RJ, Mukherjee PK, et al. Characterization of the oral fungal microbiome (mycobiome) in healthy individuals. PLoS Pathog 2010;6: e1000713. [7] Revankar SG, Sutton DA, Rinaldi MG. Primary central nervous system phaeohyphomycosis: a review of 101 cases. Clin Infect Dis 2004;38:206e16. [8] Li DM, de Hoog GS. Cerebral phaeohyphomycosisda cure at what lengths? Lancet Infect Dis 2009;9:376e83. [9] Borkar SA, Sharma MS, Rajpal G, et al. Brain abscess caused by Cladophialophora bantiana in an immunocompetent host: need for a novel cost-effective antifungal agent. Indian J Med Microbiol 2008;26:271e4. [10] Queiroz-Telles F, Esterre P, Perez-Blanco M, et al. Chromoblastomycosis: an overview of clinical manifestations, diagnosis and treatment. Med Mycol 2009;47:3e15. [11] Dupont C, Duong TA, Mallet S, et al. Unusual presentation of chromoblastomycosis due to Cladophialophora carrionii in a renal and pancreas transplant recipient patient successfully treated with posaconazole and surgical excision. Transpl Infect Dis 2010;12:180e3. [12] Sharma A, Hazarika NK, Gupta D. Chromoblastomycosis in sub-tropical regions of India. Mycopathologia 2010;169:381e6. [13] Lee JW, Melcher GA, Rinaldi MG, Pizzo PA, Walsh TJ. Patterns of morphologic variation among isolates of Trichosporon beigelii. J Clin Microbiol 1990;28:2823e7. [14] Roselino AM, Seixas AB, Thomazini JA, Maffei CM. An outbreak of scalp white piedra in a Brazilian children day care. Rev Inst Med Trop São Paulo 2008;50:307e9. [15] Lopes JO, Alves SH, Benevenga JP, Rosa AC, Gomez VC. Trichosporon beigelii peritonitis associated with continuous ambulatory peritoneal dialysis. Rev Inst Med Trop São Paulo 1994;36:121e3. [16] Hedayati MT, Pasqualotto AC, Warn PA, Bowyer P, Denning DW. Aspergillus flavus: human pathogen, allergen and mycotoxin producer. Microbiology 2007;153:1677e92. [17] Moreno G, Arenas R. Other fungi causing onychomycosis. Clin Dermatol 2010;28:160e3. [18] Joshi A, Singh R, Shah MS, Umesh S, Khattry N. Subcutaneous mycosis and fungemia by Aureobasidium pullulans: a rare pathogenic fungus in a post allogeneic BM transplant patient. Bone Marrow Transplant 2010;45:203e4. [19] Rinaldi MG. Phaeohyphomycosis. Dermatol Clin 1996;14:147e53. [20] Hawkes M, Rennie R, Sand C, Vaudry W. Aureobasidium pullulans infection: fungemia in an infant and a review of human cases. Diagn Microbiol Infect Dis 2005;51:209e13. [21] Rupp ME, Archer GL. Coagulase-negative staphylococci: pathogens associated with medical progress. Clin Infect Dis 1994;19:231e43 quiz 244e5. [22] von Eiff C, Peters G, Heilmann C. Pathogenesis of infections due to coagulasenegative staphylococci. Lancet Infect Dis 2002;2:677e85. [23] Kloos WE, Musselwhite MS. Distribution and persistence of Staphylococcus and Micrococcus species and other aerobic bacteria on human skin. Appl Microbiol 1975;30:381e5. [24] Logan NA. Bacillus species of medical and veterinary importance. J Med Microbiol 1988;25:157e65. [25] Martino R, Gomez L, Pericas R, et al. Bacteraemia caused by non-glucosefermenting gram-negative bacilli and Aeromonas species in patients with haematological malignancies and solid tumours. Eur J Clin Microbiol Infect Dis 2000;19:320e3.
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[26] Mitchell DL, Hariri NM, Duncanson Jr MG, Jacobsen NL, McCallum RE. Quantitative study of bacterial colonization of dental casts. J Prosthet Dent 1997;78: 518e21. [27] Abdelaziz KM, Combe EC, Hodges JS. The effect of disinfectants on the properties of dental gypsum: 1. Mechanical properties. J Prosthodont 2002;11: 161e7. [28] Ivanovski S, Savage NW, Brockhurst PJ, Bird PS. Disinfection of dental stone casts: antimicrobial effects and physical property alterations. Dent Mater 1995;11:19e23. [29] Lucas MG, Arioli-Filho JN, Nogueira SS, Batista AU, Pereira RP. Effect of incorporation of disinfectant solutions on setting time, linear dimensional stability, and detail reproduction in dental stone casts. J Prosthodont 2009;18:521e6. [30] Fracaro GB, Juchem C, Correa AM, Samuel SMW. The influence of peracetic acid immersion on the reproduction of details and compatibility with the elastomers with plaster [in Portuguese]. Revista Odonto Ciência 2007;22: 61e5. [31] Kotsiomiti E, Tzialla A, Hatjivasiliou K. Accuracy and stability of impression materials subjected to chemical disinfectionda literature review. J Oral Rehabil 2008;35:291e9.
[32] Davis DR, Curtis DA, White JM. Microwave irradiation of contaminated dental casts. Quintessence Int 1989;20:583e5. [33] Berg E, Nielsen O, Skaug N. High-level microwave disinfection of dental gypsum casts. Int J Prosthodont 2005;18:520e5. [34] Berg E, Nielsen O, Skaug N. Efficacy of high-level microwave disinfection of dental gypsum casts: the effects of number and weight of casts. Int J Prosthodont 2007;20:463e4. [35] Kohn WG, Collins AS, Cleveland JL, et al. Guidelines for infection control in dental health-care settingse2003. MMWR Recomm Rep 2003;52(RR-17):1e61. [36] Correia-Sousa J, Tabaio AM, Silva A, et al. The effect of water and sodium hypochlorite disinfection on alginate impressions. Rev Port Estomatol Med Dent Cir Maxillofac 2013;54:8e12. [37] Huizing KL, Palenik CJ, Setcos JC, Sheldrake MA, Miller CH. Method of evaluating the antimicrobial abilities of disinfectant-containing gypsum products. QDT Yearbook 1994;17:172e6. [38] Almortadi N, Chadwick RG. Disinfection of dental impressionsdcompliance to accepted standards. Br Dent J 2010;209:607e11. [39] Marya CM, Shukla P, Dahiya V, Jnaneswar A. Current status of disinfection of dental impressions in Indian dental colleges: a cause of concern. J Infect Dev Ctries 2011;5:776e80.
Contents lists available at ScienceDirect
Journal of the World Federation of Orthodontists journal homepage: www.jwfo.org
Research
Microbiological analysis of dental casts stored long-term Simone Gallão a, *, Antonio Carlos Pizzolitto b, y, Lourdes Santos-Pinto c, Ary dos Santos-Pinto d, Kurt Faltin Jr. e, f, Lídia Parsekian Martins g a
Private Practice, Ribeirão Preto, São Paulo, Brazil Professor, Department of Clinical Analysis, School of Pharmaceutical Sciences, Universidade Estadual Paulista, São Paulo, Brazil Professor, Department of Orthodontics and Pediatric Dentistry, Araraquara Dental School, Universidade Estadual Paulista, Araraquara, São Paulo, Brazil d Professor, Department of Orthodontics and Pediatric Dentistry, Araraquara Dental School, University Estadual Paulista, Araraquara, São Paulo, Brazil e Professor and Department Chair, Department of Orthodontics, School of Dentistry, Universidade Paulista, São Paulo, Brazil f Invited Professor, Department of Facial Orthopedics, University of Ulm, Ulm, Germany g Professor and Department Chair, Department of Orthodontics and Pediatric Dentistry, Araraquara Dental School, Universidade Estadual Paulista, Araraquara, São Paulo, Brazil b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 April 2013 Received in revised form 19 June 2013 Accepted 23 July 2013 Available online 22 October 2013
Background: The aim of this study was to evaluate the presence of bacteria and fungi on dental plaster casts stored long-term. Methods: Forty-four pairs of dental casts stored since 1980e1981 were used. The material for microbiological analysis was collected with a swab and cultivated in brain-heart infusion cultures and Sabouraud dextrose broth for subsequent plating and colony identification. Results: Bacterial growth such as coagulase-negative Staphylococcus, Micrococcus sp., Bacillus sp., and nonfermenting gram-negative bacillus were present in 25% of the samples, whereas fungal growth such as Cladophialophora sp., Trichosporon beigelii, Aspergillus flavus, and Aureobasidium pullulans were found in 5.7%. Conclusions: There was growth of opportunistic bacteria and fungi, posing no risk to healthy people. Ó 2013 World Federation of Orthodontists.
Keywords: Bacteria Fungi Plaster cast
1. Introduction Plaster casts are widely used for diagnosis, planning, and manufacturing orthodontic appliances. Microorganisms, particularly in dental casts stored long-term, have been little investigated. Studies have been aimed at contamination and disinfection of the casts, but microorganisms they can also be present by the record bases. From the professional and legal points of view, it is crucial to develop effective means of disinfecting this material. In the presence of fungal infection, symptoms such as fatigue and irritation in the eyes, nose, and throat seem to be correlated with odor [1]. Streptococcus sp. and Staphylococcus sp., including methicillin-resistant Staphylococcus aureus (MRSA), Candida sp., and Pseudomonas aeruginosa, have been detected in plaster casts, with some microorganisms being potentially pathogenic [2]. Staphylococcus aureus is an important pathogen because of its virulence; antimicrobial resistance; and association with various diseases, including fatal systemic infections, intoxication, cutaneous infectious, and opportunistic diseases [3]. * Corresponding author: Rua Sete de Setembro, 668 ap 141, CEP 14010-180 Ribeirão Preto, São Paulo, Brazil. E-mail address: [email protected] (S. Gallão). y In memorian. 2212-4438/$ e see front matter Ó 2013 World Federation of Orthodontists. http://dx.doi.org/10.1016/j.ejwf.2013.07.003
Dental casts are subject to several forms of contamination, ranging from direct contact with a patient’s saliva to procedures involving measurement, planning, manufacture, laboratory sending, and storage. The present study was aimed at investigating the contamination of plaster casts stored long-term as well as the types of microorganisms involved and their pathogenicity in order to prevent cross-contamination.
2. Methods and materials The sample consisted of plaster dental casts of White individuals aged 12 to 17 years. The research ethics committee at the Universidade Estadual PaulistaeAraraquara (São Paulo, Brazil) approved the study protocol (no. 30/09). Forty-four pairs of dental plaster casts were used for study, totaling 88 bacteriologic and mycologic cultures. The casts had been stored since 1980e1981, remaining within plastic containers kept in wooden cabinets. Other practitioners had already manipulated them under unknown aseptic conditions. The samples had been coated to make them impermeable, resistant, and bright. The coating solution was 200 g of grated coconut soap dissolved in 1 L of hot water plus ½ teaspoon of borax. After cooling, the samples remained immersed for 12 hours and then were
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Table 1 Quantity and percentage of fungi and bacteria isolated from the 88 microbiologically tested maxillary and mandibular samples of plaster casts (N ¼ 88)* Organism Bacteria Coagulase-negative Staphylococci Gram-negative nonfermenting bacilli Bacillus sp. Micrococcus sp. Fungi Aspergillus flavus Cladophialophora sp. Trichosporon beigelii Aureobasidium sp.
Maxillary
Mandibular
5 3 2 1
7 1 1 2
(5.7%) (3.4%) (2.3%) (1.1%)
1 (1.1%) 1 (1.1%) 1 (1.1%) 0
(8.0%) (1.1%) (1.1%) (2.3%)
1 (1.1%) 0 0 1 (1.1%)
Data are presented as number (%) of samples. * Sixty-one samples (69.3%) showed no growth in the cultures.
removed from the solution to dry naturally for 24 hours and polished. The microbiological analysis involved cultures for evaluating contamination of the casts. A sterile swab, soaked in phosphate buffer solution of pH 7.0, was rubbed over the whole area of the plaster cast to collect material for analysis. Procedure gloves were sterilized with 70% ethanol for each material collection. The swabs of each sample were placed in test tubes containing brain-heart infusion medium, incubated at 37 C, and maintained sequentially in Sabouraud glucose broth at room temperature for up to 30 days. The test tubes were examined for growth daily, and cultures presenting a darkish color were then submitted for microscopic examination following Gram staining. Next, they were placed in a medium favorable for screening isolated strains. The cultures in liquid medium were plated in brain-heart infusion agar and Sabouraud glucose agar for isolation and identification of the colonies. Media were prepared according to instructions from Difco Laboratories, Inc. (Detroit, Michigan). The plates were wrapped with polyvinyl chloride film and kept in a dark room at a temperature ranging from 2 to 8 C until their use. After being opened, the plates were used for 1 week at the same temperature. The plates were incubated at 35 to 37 C for bacteria, and at room temperature for fungi, both for 15 days. All cultures were examined in terms of growth and then identified. After incubation, some plates exhibited isolated colonies morphologically examined using Gram staining and identified as either fungi or bacteria through biochemicalephysiologic methods [4]. For the identification of gram-negative bacilli, the cultures used were: triple sugar iron agar for screening and evaluating the sugar metabolism; MacConkey agar for evaluating the presence of Enterobacteriaceae; blood agar for evaluating virulence factor (hemolytic capacity); and, for evaluating the growth of Pseudomonas sp., cetrimide agar. 3. Results and discussion Based on the plaster dental casts studied, the following fungi were isolated: Cladophialophora sp. (after 4-day incubation); Trichosporon beigelii (after 5-day incubation); and Aspergillus flavus and Aureobasidium pullulans (after 15-day incubation). As for the bacteria, coagulase-negative Staphylococcus (after 2-, 3-, and 5-day incubation); Micrococcus sp. (after 3- and 15-day incubation); Bacillus sp. (after 4-day incubation); and gram-negative nonfermenting bacilli (after 1-, 2-, 12-, and 15-day incubation) were isolated from the samples. Of the bacteria identified in the group of gram-positive microorganisms, Micrococcus sp. and coagulase-negative Staphylococcus were found, but with no evidence of Staphylococcus aureus. Gram-
positive rods with central spores, belonging to the genus Bacillus, were also identified by their microscopic characteristics. The isolated gram-negative bacilli were sequentially seeded over four cultures. In the triple sugar iron agar medium, no glucose fermentation by these bacilli was observed. In the MacConkey agar, four samples of gram-negative bacilli belonging to the glucose nonfermenting group were seeded, but no species of Enterobacteriaceae were isolated. In blood agar, the bacilli presented no virulence characteristics (hemolytic action). Finally, no growth of Pseudomonas sp. was detected in cetrimide agar. Bacterial and fungal growths were detected in 25% and 5.7% of the samples, respectively (Table 1). The identification of microorganisms in plaster dental casts as well as the determination of their pathogenic potential are important in determining the risk to professional, dental workers and patients. Although the plaster casts evaluated had been made impermeable with borax, an antifungal and bacteriostatic agent, the time that elapsed after the process and the way the plaster casts were stored may have contributed to the contamination found [5]. In addition to the possible cross-contamination resulting from manipulation, one can expect that some of the isolated microorganisms came from the oral cavity. In fact, Candida sp. (75%), Cladosporium sp. (65%), Aureobasidium sp. (50%), Saccharomycetales sp. (50%), Aspergillus sp. (35%), Fusarium sp. (30%), and Cryptococcus sp. (20%) were detected in saliva [6]. It is improbable that microorganisms had lived on gypsum cast long-term; however, the microorganisms exist in natural water and in human skin and mouth, so the contamination could have occurred during manipulation for study analysis. Preventive and therapeutic advances have prolonged life expectancy. However, individuals with chronic diseases may have immunosuppression from the disease or from the medications prescribed, and immunosuppression may cause free-living microorganisms to become pathogenic. The literature has reported several situations of contamination involving the microorganisms found in the present study. Some Cladophialophora sp. are pathogenic or neurotropic; can cause pheohyphomycosis infections, cerebral abscess, chromoblastomycosis, and contamination after transplantation, affecting mostly immunosuppressed individuals despite occurring in healthy patients; and can develop rapidly and be fatal [7e12]. Trichosporon beigelii can cause white piedra, a mycosis affecting the hair shaft of any body part, occurring mainly in South America [13,14]. Two cases of death have been described [15]. Aspergillus flavus is a common airborne pathogen and has been found in chronic granulomatous sinusitis, keratitis, cutaneous aspergillosis, infectious wounds, nondermatophytic onychomycosis, and osteomyelitis followed by trauma and inoculation. It produces aflatoxin, a natural toxin that is potentially cancerous [16,17]. Fungal infection with Candida sp., Aspergillus sp., and even Aureobasidium pullulans have been found in bone marrow transplant recipients [18]. Aureobasidium pullulans is common in tropical and subtropical regions; there are reports of keratomycosis, meningitis, splenic abscess, maxillary abscess, pulmonary mycosis, septicemia, opportunistic infections, cutaneous mycosis, and pheohyphomycosis [19,20]. For the genus Staphylococcus, coagulase-negative Staphylococcus has been found as a major pathogen in hospitals, and its occurrence is increasing with the use of prostheses, being more evident in immunocompromised patients [21,22]. Some Micrococcus sp. are usually found in the skin. It is thought that these bacteria are temporary residents; they are frequently found in the exposed skindface, arms, hands and legsdbut infectious diseases can be caused opportunistically in
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immunocompromised patients [4,23]. Those microorganisms likely infected the casts during manipulation. The majority of Bacillus sp. pose no pathogenic risk, except for Bacillus anthracis [24]. Due to the great number of gram-negative Bacillus sp. and the complexity involved in their identification, gram-negative bacilli have been excluded in the presence of the more significant microorganisms. Escherichia coli that indicate fecal contamination, followed by Pseudomonas aeruginosa (nonfermenting), considered pathogenic, were not observed in this sample [4,25]. The gramnegative bacilli isolated in the present study may be of free-living bacteria or opportunistic pathogens. Plaster casts remain contaminated even after 4 hours after being handled. Bacterial contamination decreased after the casts had been washed with water for 20 seconds, and even more when they were scrubbed with soap and tap water or soaked in 2% glutaraldehyde [26]. In this study, despite the 32-year storage duration, contamination was observed, but the casts were manipulated without the use of infection-control procedures. In the literature, disinfectant solutions mixed with water and then added to the plaster mixture were evaluated in terms of effectiveness. Povidoneeiodine eliminated microorganisms within 1 hour [27], and sodium hypochlorite was effective only after 24 hours at a 1:5 dilution, whereas 2% glutaraldehyde was the most effective disinfectant [28] after the plaster was immersed in solution for 20 minutes [26]. With regard to the physical properties of the plaster casts, it was observed that povidoneeiodine promoted changes in the compressive strength, and that the 1% sodium hypochlorite negatively changed the properties of the material, whereas 2% glutaraldehyde had a less adverse effect but is clinically toxic [28,29]. Chlorhexidine at 2% promoted no significant changes in the casts [29]. Determining the interactions between disinfectant solutions and the components in each new material being launched in the market will require further compatibility tests [30,31]. Microwave sterilization (1, 5, or 20 minutes) was not effective in plaster casts contaminated with Bacillus subtilis [32]. Disinfection was achieved following 5-minute exposure. The exception was a sample in which gram-positive bacilli were found but considered common in the environment. The dimensional stability of the samples was not affected, and no adverse effects were observed macroscopically [33,34]. The American Dental Association recommends cleaning and disinfecting impressions as soon as possible after removal from the patient’s mouth before the drying of blood or other bioburden can occur, because dental impression materials can act as vectors, transmitting an amount of microorganisms sufficient to stay viable within a plaster cast for 7 days [35e37]. After 32 years of storage, it is unlikely that the presence of the microorganisms in this sample was from the impression material. However, the casts’ manipulation with no personal-protection equipment could have contributed to the contamination found. Compliance with good practice is less than ideal, and education in impression disinfection and infection-control measures is necessary to ensure the health and safety of dental workers and patients [38,39]. 4. Conclusions To avoid the occurrence of cross-infection during manipulation of plaster casts, the use of both disinfection and individual protective equipment is recommended. Another alternative might be to replace the plaster casts with digital models if measured by scientifically proven computing systems. Fungi and bacteria were found in plaster casts stored long-term. In general, the microorganisms identified did not exhibit
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pathogenic potential in healthy individuals. However, they may represent a risk to patients who take antibiotics, corticoids, or immunosuppressors long-term. Acknowledgments The author thanks Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (www.capes.gov.br) for financial support during his PhD program at the School of Dentistry of Araraquara, Universidade Estadual Paulista, São Paulo, Brazil. Special appreciation goes to Prof. Kurt Faltin, Jr. from the Orthodontics and Pediatric Dentistry Department, Universidade Paulista, São Paulo, Brazil, who generously provided the sample for this research. References [1] Samson RA. Occurrence of moulds in modern living and working environments. Eur J Epidemiol 1985;1:54e61. [2] Egusa H, Watamoto T, Abe K, et al. An analysis of the persistent presence of opportunistic pathogens on patient-derived dental impressions and gypsum casts. Int J Prosthodont 2008;21:62e8. [3] Lowy FD. Staphylococcus aureus infections. N Engl J Med 1998;339:520e32. [4] Murray PR, Baron EJ, Jorgensen JH, Landry ML, Pfaller MA. Manual of clinical microbiology. 9th ed. Washington, DC: American Society of Microbiology; 2007. [5] Meers PD, Chow CK. Bacteriostatic and bactericidal actions of boric acid against bacteria and fungi commonly found in urine. J Clin Pathol 1990;43: 484e7. [6] Ghannoum MA, Jurevic RJ, Mukherjee PK, et al. Characterization of the oral fungal microbiome (mycobiome) in healthy individuals. PLoS Pathog 2010;6: e1000713. [7] Revankar SG, Sutton DA, Rinaldi MG. Primary central nervous system phaeohyphomycosis: a review of 101 cases. Clin Infect Dis 2004;38:206e16. [8] Li DM, de Hoog GS. Cerebral phaeohyphomycosisda cure at what lengths? Lancet Infect Dis 2009;9:376e83. [9] Borkar SA, Sharma MS, Rajpal G, et al. Brain abscess caused by Cladophialophora bantiana in an immunocompetent host: need for a novel cost-effective antifungal agent. Indian J Med Microbiol 2008;26:271e4. [10] Queiroz-Telles F, Esterre P, Perez-Blanco M, et al. Chromoblastomycosis: an overview of clinical manifestations, diagnosis and treatment. Med Mycol 2009;47:3e15. [11] Dupont C, Duong TA, Mallet S, et al. Unusual presentation of chromoblastomycosis due to Cladophialophora carrionii in a renal and pancreas transplant recipient patient successfully treated with posaconazole and surgical excision. Transpl Infect Dis 2010;12:180e3. [12] Sharma A, Hazarika NK, Gupta D. Chromoblastomycosis in sub-tropical regions of India. Mycopathologia 2010;169:381e6. [13] Lee JW, Melcher GA, Rinaldi MG, Pizzo PA, Walsh TJ. Patterns of morphologic variation among isolates of Trichosporon beigelii. J Clin Microbiol 1990;28:2823e7. [14] Roselino AM, Seixas AB, Thomazini JA, Maffei CM. An outbreak of scalp white piedra in a Brazilian children day care. Rev Inst Med Trop São Paulo 2008;50:307e9. [15] Lopes JO, Alves SH, Benevenga JP, Rosa AC, Gomez VC. Trichosporon beigelii peritonitis associated with continuous ambulatory peritoneal dialysis. Rev Inst Med Trop São Paulo 1994;36:121e3. [16] Hedayati MT, Pasqualotto AC, Warn PA, Bowyer P, Denning DW. Aspergillus flavus: human pathogen, allergen and mycotoxin producer. Microbiology 2007;153:1677e92. [17] Moreno G, Arenas R. Other fungi causing onychomycosis. Clin Dermatol 2010;28:160e3. [18] Joshi A, Singh R, Shah MS, Umesh S, Khattry N. Subcutaneous mycosis and fungemia by Aureobasidium pullulans: a rare pathogenic fungus in a post allogeneic BM transplant patient. Bone Marrow Transplant 2010;45:203e4. [19] Rinaldi MG. Phaeohyphomycosis. Dermatol Clin 1996;14:147e53. [20] Hawkes M, Rennie R, Sand C, Vaudry W. Aureobasidium pullulans infection: fungemia in an infant and a review of human cases. Diagn Microbiol Infect Dis 2005;51:209e13. [21] Rupp ME, Archer GL. Coagulase-negative staphylococci: pathogens associated with medical progress. Clin Infect Dis 1994;19:231e43 quiz 244e5. [22] von Eiff C, Peters G, Heilmann C. Pathogenesis of infections due to coagulasenegative staphylococci. Lancet Infect Dis 2002;2:677e85. [23] Kloos WE, Musselwhite MS. Distribution and persistence of Staphylococcus and Micrococcus species and other aerobic bacteria on human skin. Appl Microbiol 1975;30:381e5. [24] Logan NA. Bacillus species of medical and veterinary importance. J Med Microbiol 1988;25:157e65. [25] Martino R, Gomez L, Pericas R, et al. Bacteraemia caused by non-glucosefermenting gram-negative bacilli and Aeromonas species in patients with haematological malignancies and solid tumours. Eur J Clin Microbiol Infect Dis 2000;19:320e3.
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