Bacterial contamination in dental unit waterlines (DUWLs) was evaluated by molecular techniques in addition to the conventional culture method. Water samples (n=8) from DUWLs were investigated for heterotrophic bacteria by culture method using R2A agar. The selected bacterial antibiotic-resistance genes and Legionella species-specific 16SrDNA were identified by PCR. The profiles of bacterial contamination in DUWLs were further identified by PCR-DGGE. In this study, no antibiotic-resistant or Legionella genes were detected. Polycyclic aromatic hydrocarbon-degrading bacterium, Novosphingobium sp. was the most prevalent in DUWLs. Conventional PCR and PCR-DGGE were shown to be potentially useful for monitoring of bacterial contamination in DUWLs

Kokeguchi, Susumu

Matsuyama, Miwa

Tamaki, Naofumi

Watanabe, Akari

English

Tokushima University Institutional Repository

Short CommuniCationNew Microbiologica, 39, 2, 143-145, 2016, ISN 1121-7138Molecular analysis for bacterial contamination  in dental unit water lines Akari Watanabe1, Naofumi Tamaki2, Miwa Matsuyama1, Susumu Kokeguchi31Department of Oral Health Care and Rehabilitation, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan; 2Department of Preventive Dentistry, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan; 3Department of Oral Microbiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences,  Okayama University, Okayama, JapanDental chair units are equipped with a narrow-bore, flex-ible, plastic tubing called dental unit waterlines (DUWLs) that supply water to all dental instruments (air/water sy-ringe, turbine, rinsing equipment) (O’Donnell et al., 2011). Bacterial contamination is often observed in the complex dental unit. Therefore, biofilms containing multiple bacte-rial species easily form within DUWLs (Cobb et al., 2002). Bacterial numbers per ml in DUWLs can reach several mil-lion overnight (Depaola et al., 2002). Heavy microbial con-tamination of DUWLs is thought to be the result of biofilm formation within small-bore plastic tubes used to supply water to dental equipment (Pankhurst et al., 1998). Oppor-tunistic bacteria such as Legionella pneumophila and Pseu-domonas aeruginosa have previously been revealed in bio-films within DUWLs (Petti et al., 2004). In addition, Staph-ylococcus sp. isolated from DUWLs are resistant to several antibiotics (Lancellotti et al., 2007; Güngör et al., 2014). Thus, bacterial contamination in DUWLs is a recent grow-ing concern in dentistry. Several human pathogens in the environment can change to a “viable but non-culturable” state and are not detectable by culture methods (Oliver 2005; Su et al., 2013). The aim of this study was to mon-itor bacterial contamination in DUWLs using molecular techniques based on bacterial 16S ribosomal RNA gene (16SrDNA) and antibiotic-resistance genes.In this study, bacterial contamination in the 8 DUWLs lo-cated at a dental hospital in Japan were investigated by molecular techniques in addition to the conventional cul-Corresponding author:Akari WatanabeE-mail: akari.watanabe@tokushima-u.ac.jp©2016 by EDIMES - Edizioni Internazionali Srl. All rights reservedture method. Water samples were collected at the end of the working day, after 3.5 days, and at the beginning of the working day before use. After appropriate dilutions, the water samples were plated on R2A agar (DAIGO®, Ni-hon Pharmaceutical Co. Ltd, Tokyo, Japan), selective for heterotrophic bacteria and incubated at 25°C for 7 days, to obtain total bacterial counts in terms of colony form-ing units (CFU) per ml. Abundant white and off-white col-onies were observed on the R2A agar plates, from each DUWLs sample. The average bacterial count at end of the working day, for each DUWL water sample was as follows:1) 390 CFU/ml,2) 3,020 CFU/ml,3) 3,780 CFU/ml,4) 552 CFU/ml,5) 435 CFU/ml,6) 175 CFU/ml,7) 1,245 CFU/ml,8) 3,320 CFU/ml.Thus, only 3 chair units were found to meet the Centers for Disease Control and Prevention (CDC) recommended water quality standard in terms of the maximum permis-sible level of contamination by heterotrophic bacteria in DUWL water at the end of the working day, at 500 CFU/ml (Kohn et al., 2004). On the other hand, the average bacteri-al count for each DUWL water sample at beginning of the working day was as follows:1) 6,090 CFU/ml,2) 6,325 CFU/ml,3) 3,925 CFU/ml,4) 4,915 CFU/ml,5) 5,122 CFU/ml,6) 795 CFU/ml,7) 2,810 CFU/ml,8) 6,345 CFU/ml.SUMMARyBacterial contamination in dental unit waterlines (DUWLs) was evaluated by molecular techniques in addition to the conventional culture method. Water samples (n=8) from DUWLs were investigated for heterotrophic bacteria by culture method using R2A agar. The selected bacterial antibiotic-resistance genes and Legionella species-specific 16SrDNA were identified by PCR. The profiles of bacterial contamination in DUWLs were further identified by PCR-DGGE. In this study, no antibiotic-resistant or Legionella genes were detected. Polycyclic aromatic hydrocarbon-degrading bacterium, Novosphingobium sp. was the most prevalent in DUWLs. Conventional PCR and PCR-DGGE were shown to be potentially useful for monitoring of bacterial contamination in DUWLs.Received September 19, 2015 Accepted March 18, 2016Key words:Bacterial contamination, Dental unit water line, Molecular analysis, PCR-DGGE.A. Watanabe, N.Tamaki, M. Matsuyama, S. Kokeguchi144Additionally, after 3 and a half days of not being in use, the bacterial count in each DUWL was highly increased. Thus, the quality of water from the DUWLs sampled did not fulfilling the water standards criteria set by the CDC. Colonies formed were used for 16SrDNA analysis. As a result, Novosphingobium sp. and Sphingomonadaceae sp. were identified as predominant contaminants in DUWL samples based on 16SrDNA sequencing of isolates on R2A agar.Several previous reports have indicated that flushing with water for several minutes at the beginning of the work-ing day effectively reduces the bacterial load in DUWLs (Pankhurst et al., 1998; Cobb et al., 2002; Watanabe et al., 2008). However, biofilm formation within the DUWLs presents a challenge for complete elimination of bacteria by flushing with water (O’Donnell et al., 2011). O’Donnell et al. concluded that chemical DUWL cleaning, dental chair unit design, and development of automated DUWL clean-ing control systems provide effective long-term solutions to DUWL maintenance. Water from DUWLs and hospital wastewaters is not sterile and contains high numbers of bacteria. Several studies have reported the presence of op-portunistic pathogens and antibiotic-resistant bacteria in DUWLs (Petti et al., 2004; Al-Hiyasat et al., 2007; Máayeh et al., 2008; Arvand et al., 2013) and hospital wastewaters (Schwartz et al., 2003; yomoda et al., 2003; Thompson et al., 2013).In this study, contamination by antibiotic-resistant patho-gens and Legionella species in DUWLs was further exam-ined by conventional polymerase chain reaction (PCR) method. Each 50-mL water sample collected at the begin-ning of the working day was centrifuged at 10,000×g for 30 min at 4°C. The supernatant was discarded and the pellet was suspended in 200 μL InstaGene Matrix (Bio-Rad Lab-oratories, Hercules, CA, USA). Bacterial DNA was extract-ed according to the manufacturer’s instructions and used for PCR. Antibiotic-resistant pathogens, namely methicil-lin-resistant S. aureus (MRSA), multi-drug resistant Pseu-domonas, extended-spectrum beta-lactamase (ESBL) producing bacteria, and vancomycin-resistant Enterococ-ci were identified by PCR, by amplification of respective bacterial genes, mecA, blaIMP, blaVIM, blaTEM, vanA, and vanB, as previously described (Murakami et al., 1991; Kariyama et al., 2000; Heneriques et al. 2006). Contami-nation by Legionella sp. was also investigated using PCR for specific 16SrDNA (yamamoto et al., 1993). Neither bacterial antibiotic-resistance genes nor Legionella-specif-ic 16SrDNA were detected in any of the DUWL samples analyzed by PCR.Moreover, the profiles of bacterial contamination in DU-WLs were evaluated by PCR-denaturing gradient gel elec-trophoresis (DGGE) in this study. The PCR-DGGE method has become one of the new analytical tools for microflo-ra (Fujimoto et al. 2003). A forward primer with a 40-bp GC-rich sequence added to the 5ʹ-end (5ʹ-CGCCCGCCG-CGCCCCGCGCCCGTCCCGCCGCCCCCGCCCG-CCTAC-GGGAGGCAGCAG-3ʹ) and a reverse primer (5ʹ-CCGT-CAATTCCTTTRAGTTT-3ʹ) were used to amplify by PCR, approximately 585-bp 16SrDNA fragments corresponding to nucleotide positions from 341 to 926 in the genome sequence of Escherichia coli. PCR was performed using EX-Taq® kit (TaKaRa Shuzo Ltd., Otsu, Japan) as previ-ously described (Fujimoto et al., 2003). DCode Universal Mutation Detection System (Bio-Rad Lab.) was used for sequence-specific separation of the PCR products on 6% (w/v) polyacrylamide gel containing a linear gradient de-naturant, ranging from 20% to 50%. The gel was run for 4 h at 200 V in 0.5X Tris-acetate-EDTA (TAE) buffer (1X TAE buffer contains 0.04 mol/l Tris base, 0.02 mol/l acetic acid, and 1.0 mmol/l EDTA, pH 8.5) at a constant tempera-ture of 60°C. After staining with ethidium bromide, the gel was viewed under UV transillumination. The dominant 16SrDNA bands on the denaturing gradient gel were ex-cised, purified, and re-amplified using PCR as previously described (Fujimoto et al., 2003). The re-amplified DNA fragments were cloned into a vector (TOPO TA Cloning kit®, Invitrogen, Carlsbad, CA, USA), and the DNA inserts were sequenced using a kit (ABI Prism BigDye® termina-tor v1.1 cycle sequencing ready reaction kit, Perkin Elmer, Foster City, CA, USA) and an automated DNA sequencer (ABI PRISM 3100, Perkin Elmer). The sequence data were subjected to the BLAST sequence homology search pro-gram at GenBank and analyzed. Bacterial species were identified at similarity values above 99%.PCR-DGGE analysis revealed that polycyclic aromatic hydrocarbon-degrading bacterium, Novosphingobium sp., were the most prevalent and dominant contaminat-ing bacteria in all DUWL samples tested. Dechloromonas sp., Blastobacter sp. and Sphingomonadaceae sp. were also identified as other, minor contaminants (Figure 1, Table 1). Novosphingobium sp. and Sphingomonadaceae sp. were identified in all DUWL water samples, by both, culture method and PCR-DGGE analysis. Additionally, PCR-DGGE analysis revealed the profiles of bacterial con-tamination in the DUWLs. Moreover, polycyclic aromatic hydrocarbon-degrading bacterium, Novosphingobium sp. was the most prevalent and dominant, whereas, Dechloro-monas sp., Blastobacter sp., and Sphingomonadaceae sp. Figure 1 - DGGE profiles of amplified 16SrDNA from 8 DUWL water samples. Lanes 1 to 8 correspond to the sample number of DUWLs described in the text. The 4 intense bands (A-D) obtained were cloned and sequenced. The bacterial species identified by sequencing are shown in Table 1.Table 1 - Bacterial species identified from DGGE major bands.Bands in Fig. 1 Identified species (the number of clones isolated)A Novosphingobium sp. (8)B Sphingomonadaceae sp. (1)C Blastobacter sp. (1)D Dechloromonas sp. (1)ABCD1 2 3 4 5 6 7 8Molecular analysis for bacterial contamination 145were identified as minor contaminating bacteria in the DUWL samples tested in this study. The pathogenicity of Novosphingobium sp., Dechloromonas sp., Blastobacter sp. and Sphingomonadaceae sp. in healthy and immunocom-promised subjects is still unknown (Rutebemberwa et al., 2014). These bacteria might be useful for one of indicators of water quality in DUWL.Hospitalized patients with infectious diseases and the el-derly requiring nursing-care are immunocompromised and more likely to visit dental clinics for dental care and treatment, where both patients and clinic staff could be exposed to microorganisms in DUWL output water and contaminated aerosols. Although the case of infection resulting from exposure to water from the contaminated DUWL is limited (Ricci et al., 2012), it is necessary to mon-itor the level of bacterial contamination in DUWLs and maintain the water quality.In conclusion, PCR-DGGE was shown to be a potentially useful molecular analysis tool to monitor DUWL bacteri-al contamination. Conventional PCR for antibiotic-resis-tance genes and Legionella sp. 16SrDNA could also be used for rapid monitoring and tracing of the source of nosoco-mial infections via DUWLs.AcknowledgementsThis work was supported by Grant-in-Aid for Scientific Re-search from the Japan Society for the Promotion of Science (JSPS) (KAKENHI Grant Number 26463163). The authors declare no conflicts of interest in the publication of this study.ReferencesAl-Hiyasat A.S., Màayeh S.y., Hindiyeh M.y., Khader, y.S. (2007). The pres-ence of Pseudomonas aeruginosa in the dental unit waterline systems of teaching clinics. Int. J. Dent. Hyg. 5, 36-44.Arvand M., Hack A. (2013). Microbial contamination of dental unit water-lines in dental practices in Hesse, Germany: A cross-sectional study. Eur. J. Microbiol. Immunol. (Bp) 3, 49-52.Cobb C.M., Martel C.R., McKnight S.A. 3rd, Pasley-Mowry C., Ferguson B.L., Williams K. (2002). How does time-dependent dental unit wa-terline flushing affect planktonic bacteria levels? J. Dent. Educ. 66, 549-555.Depaola L.G., Mangan D., Mills S.E., Costerton W., Barbeau J., Shearer B., Bartlett J. (2002). A review of the science regarding dental unit water-lines. J. Am. Dent. Assoc. 133, 1199-1206.Fujimoto C., Maeda H., Kokeguchi S., Takashiba S., Nishimura F., Arai H., et al. (2003). Application of denaturing gradient gel electrophore-sis (DGGE) to the analysis of microbial communities of subgingival plaque. J. Periodont. Res. 38, 440-445.Güngör N.D., Kadaifçiler D.G., Peker O.Ö. (2014). Investigation of the bac-terial load and antibiotic susceptibility of dental units. Environ. Monit. Assess. 186, 1847-1853.Henriques I.S., Fonseca F., Alves A., Saavedra M.J., Correia A. (2006). Oc-currence and diversity of integrons and beta-lactamase genes among ampicillin-resistant isolates from estuarine waters. Res. Microbiol. 157, 938-947.Kariyama R., Mitsuhata R., Chow J.W., Clewell D.B., Kumon H. (2000). Simple and reliable multiplex PCR assay for surveillance isolates of vancomycin-resistant enterococci. J. Clin. Microbiol. 38, 3092-3095.Kohn W.G., Harte J.A., Malvitz D.M., Collins A.S., Cleveland J.L., Eklund K.J. (2004). Centers for Disease Control and Prevention. Guidelines for Infection Control in Dental Health-Care Settings-2003. J. Am. Dent. Assoc. 135, 33-47.Lancellotti M., Oliveira M.P., Ávila F.A. (2007). Research on staphylococcus spp in biofilm formation in water pipes and sensibility to antibiotics. Braz. J. Oral Sci. 6, 1283-1288.Ma’ayeh S.y., Al-Hiyasat A.S., Hindiyeh M.y., Khader y.S. (2008). Legionel-la pneumophila contamination of a dental unit water line system in a dental teaching centre. Int. J. Dent. Hyg. 6, 48-55.Murakami K., Minamide W., Wada K., Nakamura E., Teraoka H., Watanabe S. (1991). Identification of methicillin-resistant strains of staphylococ-ci by polymerase chain reaction. J. Clin. Microbiol. 29, 2240-2244.O’Donnell M.J., Boyle M.A., Russell R.J., Coleman D.C. (2011). Manage-ment of dental unit waterline biofilms in the 21st century. Future Mi-crobiol. 6, 1209-1226.Oliver J.D. (2005). The viable but nonculturable state in bacteria. J. Micro-biol. 43, 93-100.Pankhurst C.L., Johnson N.W., Woods R.G. (1998). Microbial contamina-tion of dental unit waterlines: the scientific argument. Int. Dent. J. 48, 359-368.Petti S., Iannazzo S., Tarsitani G. (2004). Allogenic succession between Pseudmonas and Legionella in the water distribution system of a dental hospital. Annals of Microbiology. 54, 25-30.Ricci M.L., Fontana S., Pinci F., Fiumana E., Pedna M.F., Farolfi P., et al. (2012). Pneumonia associated with a dental unit waterline. Lancet. 379, 684.Rutebemberwa A., Stevens M.J., Perez M.J,, Smith L.P., Sanders L., Cos-grove G., et al. (2014). Novosphingobium and Its Potential Role in Chronic Obstructive Pulmonary Diseases: Insights from Microbiome Studies. PLoS One. 9, e111150.Schwartz T., Kohnen W., Jansen B., Obst U. (2003). Detection of antibiot-ic-resistant bacteria and their resistance genes in wastewater, surface water, and drinking water biofilms. FEMS Microbiol. Ecol. 43, 325-335.Su X., Chen X., Hu J., Shen C., Ding L. (2013). Exploring the potential environmental functions of viable but non-culturable bacteria. World J. Microbiol. Biotechnol. 29, 2213-2218.Thompson J.M., Gündoğdu A., Stratton H.M., Katouli M. (2013). Antibiotic resistant Staphylococcus aureus in hospital wastewaters and sewage treatment plants with special reference to methicillin-resistant Staphy-lococcus aureus (MRSA). J. Appl. Microbiol. 114, 44-54.Watanabe E., Agostinho A.M., Matsumoto W., Ito I. (2008). Dental unit wa-ter: bacterial decontamination of old and new dental units by flushing water. Int. J. Dent. Hyg. 6, 56-62.yamamoto H., Hashimoto y., Ezaki T. (1993). Comparison of detection methods for Legionella species in environmental water by colony iso-lation, fluorescent antibody staining, and polymerase chain reaction. Microbiol. Immunol. 37, 617-622.yomoda S., Okubo T., Takahashi A., Murakami M., Iyobe S. (2003). Pres-ence of Pseudomonas putida strains harboring plasmids bearing the metallo-beta-lactamase gene bla(IMP) in a hospital in Japan. J. Clin. Microbiol. 41, 4246-4251.

Molecular analysis for bacterial contamination

https://repo.lib.tokushima-u.ac.jp/files/public/11/115825/20210317101812442053/nm_39_2_143.pdf

Molecular analysis for bacterial contamination

Abstract

Similar works

Full text

Available Versions

Tokushima University Institutional Repository