Hrev_master [page 18] [Healthcare in Low-resource Settings 2013; 1:e5] Microbial contamination of pumice used in dental laboratories Farzaneh Firoozeh,1 Mohammad Zibaei,2 Abolfazl Zendedel,3 Hushang Rashidipour,4 Aziz Kamran5 1Department of Microbiology and Immunology, School of Medicine, Kashan University of Medical Sciences, Kashan; 2Department of Parasitology and Mycology, School of Medicine, Lorestan University of Medical Sciences, Khorram Abad; 3Department of Internal Medicine, School of Medicine, Lorestan University of Medical Sciences, Khorram Abad; 4Department of Endodontics, Dental School, Lorestan University of Medical Sciences, Khorram Abad; 5Department of Public Health, Isfahan University of Medical Sciences, Isfahan, I.R. Iran Abstract Dental appliances as well as sending and receiving prosthesis from laboratories are potential sources of cross-contamination for technicians, dentists, patients and can transmit different infectious agents as well. This study was conducted to determine the types of the microorganisms in pumice powder and pumice slurry used in dental laboratory in order to eval- uate necessary disinfection control procedure in the dental settings. Twenty-four active dental laboratories of Khorram Abad participated in our study. Samples were randomly collected from prosthesis polishing containers in steril- ized condition and were immediately sent to microbiology laboratory. Specimens were cul- tured on selective bacterial and fungal media in order to determine the microorganisms. Both oral and non-oral bacteria were recovered from pumice samples as follows: Staphylococcus aureus (15.4%), Streptococcus viridance (10.8%), Bacillus cereus (18.7%), Pseudomonas aeruginosa (12.8%), Diphtheriods (7.3%), Enterobacter cloace (4.3%), Escherichia coli (13.1%), Klebsiella pneumonia (5.4%), and Acinetobacter spp. (12.2%). The isolated fungi included Candida albicans (36.7%), other yeasts (17.3%), Fusarium spp. (13.8%), Aspergilus spp. (22.4%) and Penicillium spp. (9.8%). This study showed that polishing pumices in the form of powder or slurry were contaminated with differ- ent oral and non-oral bacteria and also fungi. Therefore, the chance of cross-contamination still severely exists, and measures should be conducted to prevent the contamination of pre- disposed people such as technicians, dentists and patients. Introduction Cross-contamination is a serious problem in dentistry and may occur among dental staff and patients.1 Dental patients and dental per- sonnel (dentists, dental laboratory technicians and assistants) can be exposed to a wide vari- ety of pathogenic microorganisms in the blood and saliva, such as hepatitis B virus (HBV), hepatits C virus (HCV), HIV, pseudomonas, Acinetobacter, Diphteroids, Lactobacilli, Staphylococci, Streptococci, Mycobacterium and other microorganisms that colonize the oral cavity and respiratory tract. These organ- isms can be transmitted to dental settings through direct or indirect contact.2,3 Most recent literature has focused on cross- contamination of dental prostheses in the den- tal laboratory.4,5 In dental laboratories, pumice is used in prostheses polishing. The pumice – as the last step of prosthesis finishing – has been reported to be the greatest source of con- tamination and also a transmission potential source for infection.6,7 During prosthesis pol- ishing, contaminated aerosol particles spread and remain in the air for a long time causing high risks for both dental staff and patients. Aspiration and inhalation of these aerosols for elderly immunocompromised patients, patients with endocarditis and respiratory dis- ease is really hazardous.8,9 The bacteria, such as Acinetobacter, Pseudomonas and Moraxella, which are not part of normal oral flora, can cause serious dis- eases if passed to patients whose dentures are polished with contaminated material and to the technician by exposure to contaminated aerosol.10 The prosthesis contaminated by potentially pathogenic microorganisms such as Gram negative bacilli can cause serious diseases when it penetrates the oropharyngeal area and increases pneumonia incidence.7 Despite rig- orous need for sterilization and disinfection of dental instruments, prosthetic appliances do not receive adequate infection control.11 The sterilization has to be performed with suitable validated procedures so that the success of these procedures can be monitored and safety and health of patients, users, and other per- sons guaranteed.12 An earlier research from Shiraz area (Iran) reported the microorgan- isms isolated from pumice in dental laborato- ries.7 The aim of this research was to deter- mine the bacterial and fungal contaminations present in pumice powder and slurry used in Khorram Abad dental laboratories to evaluate the role of pumice in cross-contamination of dental laboratories. Materials and Methods Survey area Khorram Abad, the capital of Lorestan province is located in the south-west Iran, bor- dering with the provinces of Markazi, Hamedan, Kermanshah, Khuzestan, Ilam, and Isfahan. The estimated population of Khorram Abad is 540,000. The district covers an area of approximately 6233 km2. The study site (48°21’S, 30°43’W) is the largest city in Lorestan province. Sample collection This study was conducted between June and September 2012 in twenty-four dental labora- tories in Khorram Abad. Samples randomly col- lected were placed in sterile containers and immediately transferred to the microbiology laboratory for isolation of microorganisms. Preparation, cultivation and identi- fication Initially, 1 g of pumice was aseptically weighed and a suspension in 9 mL sterile nor- mal saline was prepared in a small test tube. Healthcare in Low-resource Settings 2013; volume 1:e5 Correspondence: Mohammad Zibaei, Department of Parasitology and Mycology, School of Medicine, Lorestan University of Medical Sciences, Khorram Abad, Iran. Tel. +98.661.6200133 - Fax: +98.661.6200149. E-mail: zibaeim@sums.ac.ir Key words: contamination, microbial, pumice, dental laboratory. Contributions: FF, conception and design of the study, critical revision of the article for important intellectual content; MZ, administrative, techni- cal or logistical support, final approval of the study, guarantor of integrity of the entire study; AZ, obtaining funding for the study; HR, provision of study material or patients, collection; AK, analysis and interpretation of data. Funding: the work was supported by the Vice Chancellor for research of Lorestan University of Medical Sciences, Khorram Abad, Iran. Conflict of interests: the authors declare no potential conflict of interests. Received for publication: 11 December 2012. Revision received: 13 February 2013. Accepted for publication: 16 February 2013. This work is licensed under a Creative Commons Attribution 3.0 License (by-nc 3.0). ©Copyright F. Firoozeh et al., 2013 Licensee PAGEPress, Italy Healthcare in Low-resource Settings 2013; 1:e5 doi:10.4081/hls.2013.e5 No n- co mm er cia l u se on ly [Healthcare in Low-resource Settings 2013; 1:e5] [page 19] the tubes were mixed for 30 sec. afterwards, 1 ml of the suspension was cultured on blood agar [automatic tank dewater (ATD); Antec International Ltd., Sudbury, UK] with 5% defib- rinated sheep blood cell for isolation of all bac- teria, on McConkey agar (MERCK KGaA, Darmstadt, Germany) for isolation of Gram negative bacteria, on manitol salt agar (MERCK KGaA) for isolation of Staphylococcus aureus, and on Sabouroud dextrose agar (HiMedia Laboratories Ltd., Mumbai, India) for detection of fungi. The cultured plates were incubated 24-48 h at 37°C for bacterial isola- tion and at 25°C for 2 weeks for fungi. The plates were checked daily for detection of microorganisms. Morphologically different bacterial and fungal colonies were subcul- tured, and isolated colonies were identified to genus and species levels using microscopic and macroscopic characters.7 In addition, coag- ulase, catalase, sugar fermentation test, KOH and hemolysis test were carried out according to the standard methods.13 To assure the steril- ity and reliability of the techniques, 24 non- treated pumices (with denture) for each dental laboratory were considered as the control group. Results Of the 72 samples collected from 24 dental laboratories, 16 (66.7%) dental laboratories were contaminated for microorganisms (Table 1). The isolated microorganisms from cultures of pumice samples collected from dental labora- tories in Khorram Abad are reported in Figures 1 (Gram positive bacteria) and 2 (Gram negative bacteria). The results indicated that the highest rate belonged to Bacillus cereus (18.7%) and the lowest one was Enterobacter cloace (4.3%). Candida albicans (36.7%) was the highest rate of isolated fungi and Penicillium spp. (9.8%) was the lowest (Figure 3). Discussion Pumice used as the last step in prosthesis polishing could be a potential source of con- tamination to dental laboratory technicians.10 It was shown that in patients with immune deficiency problems, dentures have higher lev- els of contamination,14,15 since most denture users are elderly people, so the risk of infec- tion is even higher. In this study, we found a great part of bacterial species from pumices. Most interestingly, this is consistent with report by Verran et al.,16 although they also iso- lated Micrococcus from pumice slurry. Results obtained in the present study revealed a strong oral and non-oral contami- nating source in polishing pumices. Most of the fungi and bacteria isolated in our study were not pathogenic in healthy people, but some of them, such as Staphylococcus aureus and Streptococcus viridans, can be harmful both for the immunoicompromised and elderly patients as well as for healthy people. Viridans streptococci are part of the oral cav- ity normal flora. The main significance of these bacteria relates to their ability to cause 30-40% of cases of subacute bacterial endo- carditis.14 Since the organisms are most abun- dant in the mouth, minor trauma may lead to their entry into the bloodstream and initiate of endocarditis especially in predisposed patients. Witt et al.8 notified a similar situa- tion. They found Streptococcus viridians in cul- tures of pumice from laboratories. When prosthesis is polished with pumice, Article Table 1. Microorganisms isolated from dental laboratories. No. Lab.Microorganisms S.vi. S.ae B.ce. P.ae Dipht. E.co. E.cl. K.pn. Acin. C.al. Other Fusa. Aspe. Peni. 1 + + - - - - - + - + - - - - 2 - + - + + - - - + + + - + + 3 - - - + + - + - + + + - + - 4 - - + + - - + - + + - + + - 5 - - + + - - + - + + + + - - 6 - + - + + + + - + + + + + - 7 - + - + + + - - + - - + + + 8 + + - + - - - + + - - + + + 9 - - + - + + - - - + - - - + 10 - - + - - - - - - + + - + - 11 - - + - - + - - - - + - + - 12 - - + - - + - - - + + - + - 13 - + - - - - - + - + - - + + 14 - - - - - - - + - + - - + - 15 - - - - + + - + - + + + - - 16 - + - + - + - - - - - + - - 17 - - - + + + - - - - - + - - 18 - - - - - + + - - - - + - + 19 - - - - + + + - - - - - + - 20 - - + + + - + - - - - - - - 21 + + - + - - + - - - - - - - 22 - + - - - - + - + + - - - + 23 - + - + - - - - + - - - - + 24 - - - + + - - - + + + - - + S.vi., Streptococcus viridance; S.ae., Staphylococcus aureus; B.ce., Bacillus cereus; P.ae., Pseudomonas aeruginosa; Dipht., Diphtheriods; E.co., Escherichia coli; E.cl., Enterobacter cloace; K.pn., Klebsiella pneumonia; Acin., Acinetobacter species; C.al., Candida albicans; Other, other yeasts; Fusa., Fusarium species; Aspe., Aspergilus species; Peni., Penicillium species. No n- co mm er cia l u se on ly [page 20] [Healthcare in Low-resource Settings 2013; 1:e5] contaminated aerosol particles of microorgan- isms such as Gram negative bacteria and fungi, are spread all around the laboratory. This could be a major source for different oral and non-oral infections. Several studies have reported isolation of Gram negative bacteria like Pseudomonas, Moraxella and Acinetobacter from pumice which can be trans- ferred to patients and dental laboratory staff by contaminated aerosols, and cause ocular and respiratory infection especially in persons with chronic respiratory disorders.16 The entry of Gram negative bacteria such as Escherichia coli, Enterobacter and Klebsiella into the blood of patients can cause a fatal infection especial- ly Gram negative septicemia in debilitated patients.17 Isolation of Gram negative bacteria in the current study is similar to that obtained by other studies.7 Fungi recovered from used pumice samples in the current study included Aspergillus, Fusarium, Penicillium and Candida that increased risk of fungal infection especially in persons who work for a long period of time in dental laboratories and have been exposed to fungal spores.18 Some reports support that Candida albicans belong to the normal physiol- ogy flora of mouth. It is able to grow in pumice and cause infections in humans.19 Besides, new studies have been conducted on viral infection transmission especially HBV and HIV in dental laboratories. Occupational infection of the dental laboratory technicians with HBV has been reported. The studies sug- gest that all healthcare workers working in dental laboratories should be vaccinated against hepatitis B virus.20 There are many studies that provide some additional information regarding prosthesis disinfection. A previous study in Brazil showed a transfer of microorganisms from patients prosthesis to sterile prosthesis and in most laboratories pumice was not changed or disin- fected between polishing procedures.10 Jagger et al.21 reported that about 6.1% of dental labo- ratories used disinfectants in the pumice and 92.9% did not disinfect the polishing instru- ment. A previous study has proven that pumice slurry freshly made up using disinfectants was reported to be free from most contaminations.8 Unfortunately, in the present study most of the laboratories did not used a disinfectant while working with pumice, however, it will be good to use such disinfection protocol to minimize the chance of infection among the dental labo- ratories technicians and patients. It is recom- mended to disinfect old or used dentures before starting any action. The technician should use sterilized gloves, disinfected pro- tecting glasses, oral masks, brushes and pol- ishing tools to polish prosthesis. Conclusions Polishing pumices are potential source of infection in dental laboratories when consider- ing the wide variety of microorganisms in the blood and saliva of patients. Following our study results, low temperature sterilization, such as gas or plasma sterilization, would allow optimal reduction in the number of path- ogenic bacteria. The use of sterile pumice or association of disinfectants with pumice for polishing the prosthesis, sterilization of con- tainers after each use with adding of an appro- priate disinfectant such as 0.2% chlorohexi- dine gluconate or 5% hypochlorite sodium to pumice could be effective and daily change of polishing paste is recommended to reduce the hazard of cross-contamination. However, no standard procedures actually exist. References 1. Kugel G, Perry RD, Ferrari M, Lalicata P. Disinfection and communication prac- tices: a survey of U.S. dental laboratories. J Am Dent Assoc 2000;131:786-92. 2. Al-Saadi AK. Bacterial cross-contamina- tion between clinic & dental laboratory during polishing procedure of complete denture. Mustansiria Dental Journal 2011;8:288-92. Available from: http://www. iasj.net/iasj?func=issueTOC&isId=1872& uiLanguage=en 3. Al-Kheraif AA, Mobarak FA. Infection con- trol practice in private dental laboratories in Riyadh. Saudi Dent J 2008;20:163-9. 4. Debattista N, Zarb M, Portelli JM. Bacterial cross-contamination between the dental clinic and laboratory during prosthetic treatment. Malta Med J 2010;22:17-9. 5. Parisi E, Glick M. Immune suppression and considerations for dental care. Dent Clin N Am 2003;47:709-31. 6. King AH, Matis B. Infection control of in- office dental laboratories. Dent Clin N Am 1991;35:415-26. 7. Vojdani M, Zibaei M. Frequency of bacteria and fungi isolated from pumice in dental laboratories. J Res Health Sci 2006;6:33-8. 8. Witt S, Hart P. Cross infection hazards associated with the use of pumice in den- tal laboratories. J Dent 1990;18:281-3. 9. Chris H, Miller C, John P. Infection control and management of hazardous materials for the dental team. 2nd ed. London: Mosby; 1998. 10. Agostinho AM, Miyoshi PR, Gnoatto N, et al. Cross-contamination in dental labora- tory through the polishing procedure of complete dentures. Braz Dent J 2004;15: 138-43. 11. Williams DW, Chamary N, Lewis MA, et al. Microbial contamination of removable prosthodontic appliance from laboratories and impact of clinical storage. Brit Dent J 2011;26:163-6. 12. Smith PN, Palenik CJ, Blanchard SB. Microbial contamination and the steriliza- tion/disinfection of surgical guides used in the placement of endosteal implants. Int J Oral Max Impl 2011;26:274-81. Article Figure 3. Fungal species isolated from pumice samples. Figure 1. Frequency of Gram positive bac- teria isolated from pumice samples of den- tal laboratories. Figure 2. Frequency of Gram negative bac- teria isolated from pumice samples of den- tal laboratories. No n- co mm er cia l u se on ly [Healthcare in Low-resource Settings 2013; 1:e5] [page 21] 13. Washigton W Jr, Allen S, Janda W, et al. Color atlas and Textbook of Diagnostic Microbiology. Philadelphia, PA: Lippincott Williams & Wilkins; 2006. 14. Henderson CW, Schwartz RS, Herbold ET, Mayhew RB. Evaluation of the barrier sys- tem, an infection control system for the dental laboratory. 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