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I, ...v..... .. khan...“ .i xll \...I..c Kiri»... 1.1.9:..- n...\1b..l ......fl.s\. 911. .. a"... rah... r $ . 3.. .-: a» . in. . - x). - 2.6.5.103. a}! 2521...} 3. .t 3.3., . . .(......-1:\1- .. . .. .1. 9., .1.-. x3...“ .r...r._._1.;.. C . .flwruwwyih. 1s ITYUBRAR Illlllllllllllllllll\Illlllslilll 3 1293 01044 so .—. This is to certify that the thesis entitled MICROBIAL CONTAMINATION OF DENTAL UNIT WATER LINES presented by JOSE IVAN SANTIAGO SANTIAGO has been accepted towards fulfillment of the requirements for MAS I ERS degree in SC IENCE Major professor November 18, 1994 MS U i: an Aflimative Action/Equal Opportunity Institution LIBRARY Mlchlgan State Unlverslty PLACE IN RETURN BOX to man»... mum your record. TO AVOID FINES Mum on or baton «to duo. DATE DUE DATE DUE DATE DUE ' ; _| ll:l_—J [:14 ES [:1 J MSU IoMMmm-MMMOWIW W MICROBIAL CONTAMINATION OP DENTAL UNIT WATER LINES BY José Ivan Santiago Santiago A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology 1994 ABSTRACT MICROBIAL CONTAMINATION OF DENTAL UNIT WATER LINES BY José Ivan Santiago Santiago Specimens of dental unit water line (DUW) samples were collected from different dental instruments and their microbiological quality assessed, Extensive contamination of DUW was found and comparisons with other potable water sources emphasize the relatively high concentrations of microorganisms in DUW. Evidence of the presence of bacteria, amoebae, and nematodes in DUW points up the need for further studies of these components of DUWL biofiLm, as well as health risks posed to personnel, patients, and immunocompromised individuals. The data confirmed the short term value of two minute flushes, but these findings were offset by first, occasional increases in bacterial concentrations, rather than decreases, followed.flushing, and.second, reductions.in bacterial numbers were often trivial. Microbial contamination was frequently restored to pre-flush levels or higher after brief stasis or use. .Additional prophylactic measures are needed.to limit DUW microbial contamination. Le dedico este trabajo cientifico a mi familia, pero en especial a mis abuelos que no presenciaron esta parte de mi vida, en memoria de José Santiago Rivera y Ana Santiago. iii ACKNOWLEDGEMENTS I am deeply grateful to my mentor, Dr. Jeffrey F. Williams. You taught me a lot about science and guided me when I needed it; I will always run controls in experiments. Thanks for providing me with a second chance when others would not; I hope I did not waste it, MUCHAS GRACIAS. To Dr. Charles D. Mackenzie thanks for his insight and support. Special thanks go to the "Gang" of the Pathobiology Lab, who made research enjoyable. And finally thanks to all dental health care professionals involved in this research endeavor without whom this investigation would not have been possible. iv TABLE OF CONTENTS OF TABLES OF FIGURES OF APPENDICES Appendix A - Appendix B - OF ABBREVIATIONS LITERATURE REVIEW Introduction . . . . . . . . . . . Infectious disease risks and infection control in the dental office . . . . . . . . ADA/CDC infection control guidelines HIV transmission in a dental setting Other infection control problems in dental clinics . . . . . . . . . . . Dental instruments . . . . . New 1993 CDC guidelines . . . Dental unit water microbial contamination Dental unit water microbiota Potential health risks in dentistry Dental aerosols . . . . Other infection routes . Biofilms: Their nature and the extent of in medical care . . . . Biofilm formation . . . . . . Biofilms in biomedical devices Dental unit water line biofilms Preventive measures for the control of water contamination . . . . . . . dental unit Page viii ix xii 114 127 xiii 10 12 13 14 19 19 24 26 27 29 31 33 TABLE OF CONTENTS (continued) Biocide flushes of dental unit water lines . Sterile water reservoirs . . . . . Flushing of dental water lines . . In line filters . . . . . . . . . . In line checkvalves . . . . . . . . conc lus ion 0 C O O O O O O O O O O O O O O O O O 0 List of References . . . . . . . . . . . . . . . . Page 33 35 35 37 38 39 40 Article # 1 - MICROBIAL CONTAMINATION OF DENTAL UNIT WATER LINES:SHORT AND LONG TERM EFFECTS OF FLUSHING Introduction . . . . . . . . . . . . . . . . . . . Materials and Methods Specimen collection . . . . . . . . . . . . . Electron microscopy of DUWL biofilm . . . . . Evaluation of water quality . . . . . . . . . Identification of DUWL microbiota . . . . . . Results . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . Summary and Conclusion . . . . . . . . . . . . . . List of References . . . . . . . . . . . . . . . . Appendix A - EVALUATION OF DENTAL UNIT WATER BACTERIAL CONTAMINATION CONTROL METHODS Introduction . . . . . . . . . . . . . . . . . . . Materials and Methods a) Extended water flushes of DUWL . . . . . . b) Clean water systems and line disinfection c) Copper/silver ionization of building water supply . . . . . . . . . . . . . . . . . . Results a) Extended water flushes of DUWL . . . . . . b) Clean water systems and line disinfection c) Copper/silver ionization of building water supp 1y 0 O O O I O O O O O O O O O O O O 0 vi 55 58 6O 61 61 62 89 100 103 114 115 115 115 116 116 116 TABLE OF CONTENTS (continued) Page Discussion . . . . . . . . . . . . . . . . . . . . 123 Summary and Conclusion . . . . . . . . . . . . . . 124 List of References . . . . . . . . . . . . . . . . 125 Appendix B - INSTITUTIONAL PROFILES OF DENTAL UNIT WATER BACTERIAL CONTAMINATION Introduction . . . . . . . . . . . . . . . . . . . 127 Materials and Methods . . . . . . . . . . . . . . 128 Results . . . . . . . . . . . . . . . . . . . . . 128 Discussion . . . . . . . . . . . . . . . . . . . . 145 Summary and Conclusion . . . . . . . . . . . . . . 148 List of References . . . . . . . . . . . . . . . . 149 vii Table Bl B2 LIST OF TABLES Page Article # 1 Concentrations of heterotrophic bacteria in colony forming units per milliliter in water samples from dental unit water lines and domestic and environmental water sources . . 67 Appendix B Heterotrophic bacterial contamination of dental unit water in colony forming units per milliliter delivered by dental instrument lines and faucets at different institutional sites throughout the United States . . . . . . . . 132 Average heterotrophic bacterial contamination of dental unit water, in colony forming units per milliliter, delivered by dental instruments at institutional sites throughout the United States . . . . . . . . . . . . . . . . . . . 144 viii Figure LIST OF FIGURES Page Article # 1 Scatter plot showing heterotrophic plate counts in water samples from dental unit water lines and domestic and environmental sources . . . . . . . . . . . . . . . . . . . 65 Scatter plot showing a comparison of heterotrophic plate counts of bacterial contamination of dental unit water samples collected from water lines after overnight stasis and during the working day . . . . . . 69 Scanning electron micrograph of microbial biofilm on the inner aspect of a dental unit water line from dental clinic # 1 . . . . . . 73 Scanning electron micrograph of the inner aspect of dental unit water line # 2 . . . . 73 Transmission electron micrograph of microbial biofilm from a dental unit water line, showing an amoebic cyst . . . . . . . . 75 Transmission electron micrograph of microbial biofilm from a dental unit water line, illustrating a cross section of a nematode . . . . . . . . . . . . . . . . . . 75 Histogram showing heterotrophic plate counts of bacterial contamination of dental unit water samples collected from 20 air/water syringe lines in dental operatories, pre-flush of syringe line and post 2 minute water flush . . . . . . . . . . . . . . . . . 78 Histogram showing heterotrophic plate counts of bacterial contamination of dental unit water samples collected from 14 high speed handpiece lines pre-flush of the handpiece line and post 2 minute water flush . . . . . 80 ix Figure 10 11 12 13 A1 A2 LIST OF FIGURES (continued) Page Histogram showing heterotrophic plate counts of bacterial contamination of dental unit water samples collected pre-flush from dental water lines and 30 minutes after the lines had the 2 minute water flush . . . . . 82 Histogram showing heterotrophic plate counts of bacterial contamination of dental unit water samples collected post 2 minute flush of dental water lines and 30 minutes after flush . . . . . . . . . . . . . . . . . . . . 84 Histogram showing heterotrophic plate counts of bacterial contamination of dental unit water samples collected pre-flush from dental water lines and immediately after completion of a routine procedure . . . . . . 86 Histogram showing heterotrophic plate counts of bacterial contamination of dental unit water collected post 2 minute flush from dental water lines and immediately after completion of a routine procedure . . . . . . 88 Diagrammatic representation of laminar flow of water in a dental unit line . . . . . . . 99 Appendix A Scatter plot showing a comparison of heterotrophic bacterial plate counts in water from dental unit water lines after flushing for 30 seconds, 60 seconds, 2 minutes, 20 minutes, or 1 hour . . . . . . . . . . . . . 118 Scatter plot showing a comparison of heterotrophic bacterial plate counts in dental water from Clean Water Systems using reservoirs filled with sterile water and Clean Water Systems using sterile water reservoirs and which have the lines treated with a 1:6 dilution of household bleach over the weekend and are flushed extensively Monday morning . . . . . . . . . . . . . . . 120 Appendix A LIST OF APPENDICES Page Evaluation of dental unit water bacterial contamination control methods . . . . . . . . 114 Institutional profiles of dental unit water bacterial contamination . . . . . . . . . . . 127 xii 6 a» AUX AWS CDC cfu CWS DHCP DU DUW - DUWL= EPA = EPS HBV HIV HSH min PPm SBA TSA 3 Ctifi Gigi? LIST OF ABBREVIATIONS American Dental Association air water syringe - auxiliary lines air water syringe The Centers for Disease Control colony forming units Clean Water Systems dental health care professionals dental unit dental unit water dental unit water line Environmental Protection Agency extracellular polymeric substance hepatitis B virus human immunodeficiency virus high speed handpiece minute milliliter nanometer parts per million sheep blood agar trypticase soy agar microliter micrometer ultrasonic scaler xiii LITERATURE REVIEW It may appear from current accounts of the controversy in the press that the rigorous evaluation of dental office infection control practices was brought about entirely by the rapid increase of human immunodeficiency virus (HIV)-infected individuals in the population. Much emphasis has been given to the possible transmission of HIV by an HIV-positive dentist to six of his patients, none of whom was considered to be at high risk of exposure (CDC, 1990a, 1991). However, better infection control procedures in the dental office have been developing for years in parallel with a greater understanding about communicable diseases in general, and their possible transmission using dental instruments (Stevens, 1963; Belting et a1., 1964; Hausler and Madden, 1964; ADA, 1978, 1984, 1986, 1988a, 1988b; Holbrook et a1., 1978; Bagga et a1., 1984; Miller' and IPalenik, 1985; CDC, 1986, 1993; Crawford. and Broderius, 1988, 1990; Christensen, 1991; Cottone and Molinari, 1991; Anonymous - Lancet, 1992; Epstein et a1., 1992; Lewis et a1., 1992; Lewis and Boe, 1992; Faecher et a1., 1993; Mandel, 1993; Miller, 1993; Mills et a1., 1993; Pankhurst and Philpott-Howard, 1993; and Watson and Whitehouse, 1993). The need to develop firm guidelines on infection control 1 2 has become apparent because dental professionals and their patients are clearly at risk of exposure to a variety of microorganisms. The Centers for Disease Control (CDC) states that dental patients and dental health care professionals can be exposed to: ". . cytomegalovirus, hepatitis B virus (HBV) , HIV, herpes simplex virus type 1 and 2, Mycobacterium tuberculosis, staphylococci, streptococci, and other viruses and bacteria- specifically, those that infect the upper respiratory tract. Infections may be transmitted in the dental operatory through several routes, including direct contact with blood, oral fluids, or other secretions; indirect contact with contaminated instruments, operatory equipment, or environmental surfaces; or contact with airborne contaminants present in either droplet spatter or aerosols of oral and respiratory fluids." (CDC, 1993) . There is also an emerging recognition of additional serious contributors to the infective hazards present in the dental unit: the formation of a stable biofilms in cooling and irrigation lines (Kelstrup et a1., 1977; Oppenheim et a1., 1987; Mayo et a1. , 1990; Whitehouse et a1. , 1991; and Williams et a1., 1993) and the unusually high number of pathogenic and opportunistic bacteria present in the water delivered by the instruments to patients and to dental health care professionals with the production of contaminated aerosols by the instruments (Williams et a1. , 1993) . The need for better infection control procedures in the dental clinic is stressed by the more than 200,000,000 dental procedures performed annually (ADA, 1992). In the following review an account is provided of infectious disease hazards and contemporary regulatory practices for infection control in the dental office. A history is documented of those studies which have characterized dental water microbiota, their origins, and potential significance in infectious disease transmission in dental operatories. Introduction: Infection control practices in dentistry have come under great scrutiny in recent years. This thesis concerns microbiological studies of the much-neglected area of dental unit water contamination, a potential contributor to infection transmission in the dental practice. It also examines several important features of the origins of these contaminants and the typical contamination levels of microbes in dental water. The literature review covers the background to the current regulatory environment on dental infection control procedures. Infection control practices that have been brought to the forefront by recent episodes of disease transmission in dental offices are reviewed, with special reference to the significance of microbial contamination of water. Early work, reviewed here, on infection control in the dental office was concerned with infection risks to the dentists and dental staff, and focused on aerosolization of potential pathogens present in the mouths of patients (Stevens, 1963; Belting et a1., 1964; and Holbrook et a1., 1978). More recent developments have centered upon blood- borne pathogens such as human immunodeficiency virus (HIV), 4 hepatitis B virus (HBV), and others. The realization that extensive biofilms form within the coolant and irrigant water lines of hand-held dental instruments (high speed drills, ultrasonic scalers, and air-water syringes) has led to the analysis of the microbiota involved in their production. Investigators in the United States and Europe have identified a variety of pathogens, some of them opportunistic, some of them primary, that reside in the dental unit water lines, and are commonly dispensed in high numbers into patients' mouths, including onto exposed lesions and surgical sites. The literature reviewed suggests that some of these microorganisms are derived from the very low level of contaminants in municipal water supplies, while others contaminate the lines from water retraction, or "suck back", of oral fluids from the patient's oral cavity which may harbor microorganisms (Bagga et a1., 1984; Miller and Palenik, 1985; Crawford and Broderius, 1988, 1990; Lewis et a1., 1992; Lewis and Boe, 1992; and Mills et a1., 1993). Health risks associated with contamination of dental unit water is emphasized by reports in the literature of unusual nasal microbiota of dentists, characterized by the presence of frequent dental water contaminants like Pseudomonas and Proteus (Clark, 1974). Colonization of the oro/nasal mucosa by aquatic bacteria is apparently not limited to dentists. After the report of infection and production of abscesses by Pseudomonas in two immunocompromised patients following dental treatment, a unique study subsequently demonstrated that 5 Pseudomonas from dental water frequently colonizes the mouths of recipient patients and dentists working with tainted dental units (Martin, 1987). Also, common water contaminants have been determined to be among the primary contributors to severe adult periodontitis (Slots et a1., 1988). Another cause for concern is the mounting evidence of widespread and extensive contamination of dental water lines with Legionella species (Rheinthaler and Mascher, 1986; Oppenheim et a1. , 1987; Michel and Borneff, 1989; Pankhurst et a1., 1990; Lflck et a1., 1993; and Williams et a1., 1993). There are indications that dental personnel are at increased risk of exposure to Legionella (Fotos et a1., 1985; Rheinthaler et a1., 1987,1988; and.Lfick.et a1., 1993), and one fatality of a dentist with legionellosis is circumstantially linked to this pathogen (J.F. Williams et al., submitted for publication). Furthermore, reports on respiratory ailments and upper respiratory tract infections have indicated that dental health care professionals and dental students have a greater number of respiratory ailments than other health care professionals (Carter and Seal, 1953; Burton and Miller, 1963; and Mandel, 1993). However, direct evidence of the extent of the health hazard of dental water organisms has been hard to come by. The potential health risk created by water microorganisms cannot be ignored, given the proven ability of water contaminants to 'use aerosols as infection ‘vehicles (Macfarlane, 1983; Hambleton et a1., 1983; Zuravleff et a1., 6 1983; Muder et a1., 1986; Midulla et a1., 1987; CDC, 1990b; and Faecher et a1., 1993) , the large number of dental and hygiene treatments are performed.annually, and the increase in the number of immunocompromised persons in the population. One widely used "so called" infection control practice for water borne disease agents, recommended by the American Dental Association (ADA) and the Centers for Disease Control (CDC), consists of the flushing of water lines each.morning to eliminate microbial contamination of water, and again between each patient (CDC, 1993). The procedure is examined thoroughly in the work reported in this thesis, and found ineffective. Literature concerning flushing and other possible preventive approaches is also reviewed, and some preliminary observations on the utility of several measures are presented in the appendices. The dental profession will inevitably be faced with the adoption of preventive practices for water contamination in the coming years, and a spectrum of chemical and physical solutions seems likely to appear for this purpose. The work reported in this thesis may contribute to defining the needs, and establishing the urgency of attending to this problem in dentistry in the United States and elsewhere. Infectious disease risks and infection control in the dental office: The need to establish better infection control procedures in dentistry was emphasized in the late 1970's and 1980's by 7 sporadic outbreaks of hepatitis B and viral gingivostomatitis in dental practices (Levin et a1., 1974; Hadler et a1., 1981; Reingold et a1. , 1982; Manzella et a1. , 1984; and Shaw et a1. , 1986). Viral infections, which on one occasion resulted in the death of 2 patients with acute hepatitis (Shaw et a1., 1986) , were caused by dental professionals who had become viral carriers and had unknowingly exposed and transmitted HBV and herpes simplex virus to patients during dental procedures. Dental health care professionals did not routinely wear gloves during the procedures and it was suspected that repeated and vigorous hand washing between patients, to assure infection prevention, caused breaks in the skin which released infective viruses to patients (Levin et a1., 1974; Hadler et a1., 1981; Reingold et a1. , 1982; Manzella et a1. , 1984; and Shaw et a1. , 1986). The transmission of infectious diseases by dental health care providers to patients was not limited to viruses; in two dental clinics fifteen patients were unknowingly infected with tuberculosis by a carrier dentist (Roderick Smith et a1. , 1982) . At the time there was no recommendation in effect that a protective mask should be worn during dental procedures, and infection of the patients appeared to have started by the colonization of the tooth socket by Mycobacterium tuberculosis. ADA/CDC infection control guidelines: As early as 1978, the ADA began making recommendations to decrease the risks of infection transmission during dental 8 procedures (ADA, 1978). This work culminated in the development of guidelines meant to decrease the possibility of transmission of microorganisms from dental professionals to patients (ADA, 1986) . Recommendations were made on the prevention of the transmission of infectious diseases, and on infection control practices in the dental office. These included the need to obtain detailed medical histories of the patients, the use of protective barriers, such as the use of gloves (to be changed after each patient), protective masks, and clothing by dental care providers during all dental procedures, and the use of rubber dams in the patients. It was also recommended that environmental surfaces should be kept clean and, disinfected, and instruments ‘were to be sterilized or disinfected after use. Handpieces, irrigation syringes, and ultrasonic scalers were to be flushed 20-30 seconds between patients in order to eliminate potentially infective materials from the inside of the instruments. Also any waste should be treated as a potential health hazard. In 1988, the rapid increase in the population of HIV- infected individuals, coupled with the number of HBV-infected individuals (CDC, 1985), required modifications to be made in the recommended infection control procedures for dental practices (ADA, 1988a). To insure the safety of patients and dental health care providers, dental professionals were advised to obtain vaccination against HBV, to use disposable instruments ‘when. possible, and. to follow' the guidelines established in 1986. 9 HIV transmission in a dental setting: The possible transmission of HIV from an infected dentist in Florida to a patient raised doubts about the extent of adoption of infection control procedures recommended by the ADA/CDC in 1988 in dental clinics (CDC, 1990a). An investigation of the infected dentist's former patients has identified five more HIV infected individuals to date (1994). DNA analysis of the dentist's and patients' HIV strains revealed that it was highly likely that the dentist was the infection focus of the virus due to the similarities between the viruses, and dissimilarities to other HIV strains present in the area. Upon review, the mode of transmission appeared to be the use of contaminated dental instruments. Staff of the office indicated that instruments were not routinely sterilized, instruments were only wiped with alcohol after use, and ADA/CDC guidelines were not followed (CDC, 1991). Other infection control problems in dental clinics: The HIV outbreak in Florida, due to an apparent lack of infection control protocols, seems to accentuate the inadequacy of preventive measures in dentistry. A survey on the sterilization of dental instruments in 1989 indicated that less than 50% of dentists who answered the survey sterilized their instruments daily, and only 25% of those sterilized the instruments between patients (Dental Products Report, 1993). A report in 1991 indicated that 80% of dentists continue to 10 surface-disinfect handpieces, and air-water syringes were virtually never sterilized (Christensen, 1991). The lack of implementation of suitable measures in the dental office, and the fear of HIV transmission through invasive dental procedures, made necessary continuing reevaluation and modification of ADA/CDC guidelines (CDC, 1993). The Florida case emphasized the need to establish regulated infection control practices in dentistry, and brought home to national public health officials the need to change the classification of dental instruments to invasive medical instruments. Dental instruments: Some dental instruments are very much like surgical instruments, in that they become contaminated with the patient's blood and secretions which may contain microorganisms, making these instruments possible vectors in patient to patient transmission of infectious diseases (Bagga et a1., 1984; Anonymous - Lancet, 1992; Lewis et a1., 1992; Lewis and Boe, 1992; CDC, 1993, and Mills et a1., 1993). The practice of external disinfection and cold sterilization of medical instruments is only effective in cleaning instruments which are not internally contaminated with patients' material (Anonymous - Lancet, 1992; Lewis et a1., 1992; Lewis and Boe, 1992; Mills et a1., 1993; and Epstein.et a1., 1993). However, in dentistry, the use of water retraction in irrigation syringes and high speed cutting drills of dental units (used 11 to prevent water from dripping on the patients) raised the possibility of influx of oral fluids and blood into dental water lines. An ADA report revealed that some dental units can retract fluids up to ten inches into the dental instrument water line (ADA, 1988b). Patient-derived materials may include tooth particles, blood and oral secretions, tissue fragments, and microorganisms present in the oral cavity, and all of these could enter the water line (Bagga et a1., 1984; Miller and Palenik, 1985; ADA, 1988b; Crawford.and Broderius, 1988, 1990; Lewis, 1991; Lewis et a1., 1992; Lewis and Boe, 1992; and Miller, 1993) . This influx of patients' materials contaminates internally both the dental instruments in use, and the water line attached to the instrument (Bagga et a1., 1984; Miller and Palenik, 1985; Crawford and Broderius, 1988, 1990; Lewis, 1991; and Lewis and Boe, 1992), and creates possible means of patient to patient cross contamination. Although dental instruments have been known to be contaminated internally with patient debris since 1978 (ADA, 1978), and the recommendation to sterilize dates from 1986 (CDC, 1986), external disinfection of handpieces and irrigation syringes remains common (Christensen, 1991; Anonymous - Lancet, 1992; and Dental Products Report, 1993). The problem of aspiration of patients' material into the instrument and the attached water line is compounded by the presence of biofilm in the dental water line. This could affect the elimination of patient material by flushing of the 12 water line, by permitting adhesion of patient materials to the biofilm. In these circumstances, external disinfection of instruments and flushing would be ineffective modes of decontamination of dental instruments. The need to establish better disinfection techniques has recently been brought to the limelight by Lewis et a1. (1992). This research group demonstrated that infective virus particles can contaminate the inside of dental instruments and could potentially be transmitted to the next patient, revealing a manner by which any blood borne pathogen, including HIV, could also be transmitted. New 1993 CDC guidelines: CDC has recommended the between-patient sterilization of instruments for infection control (CDC, 1993). Dental instrument use is similar to medical devices associated with hepatitis B outbreaks.in.clinical settings (Kent.et a1., 1988; and Polish et a1., 1992). The issue of sterilization or disinfection of instruments between patients was addressed by determining how instruments are used; any instrument considered. to be used in invasive procedures should be sterilized between patients by heat, and any other instruments should be disinfected by using high level disinfection (CDC, 1993) . This CDC document suggests that contamination of dental units by retraction of patients' materials could be eliminated with the sterilization of hand held instruments between patients, and the installation of anti-retraction, 13 one-way check valves (Bagga et a1., 1984; Miller and Palenik, 1985; and Crawford and Broderius, 1988, 1990). .Although these recommendations have been in place since May 1993, the practice of sterilization of dental instruments between patients is not always followed (Dental Products Report, 1993). Also, irrigant syringes are, on the whole, not autoclavable and the vast majority are not sterilized at all, and certainly not between patients (Christensen, 1991). Dental unit water microbial contamination: The firm position taken by national authorities about the need to sterilize high speed drills and irrigant syringes is at odds with the known significance of dental unit water as a possible source of infective microorganisms (CDC, 1993). Dental unit water is not examined routinely for the presence of microorganisms, and contamination of the water line could be critical because few dental procedures are performed without water. The development of high speed handpieces created a need for a coolant substance in order to prevent damage to the dental pulp. Water is that coolant. Today, dental unit water is still used to prevent damage of the dental pulp by the heat produced by high speed handpieces, but it is also used in the irrigation of dental sites, cleaning the area and rinsing out debris“ Thus, any‘ contamination. of ‘water violates the sterility of the procedure and contaminates all instruments through which the water flows. 14 Dental unit water microbiota: At first glance it would seem that water transmitted by dental instruments should have only low levels of bacterial contamination, because dental units are supplied with potable water from municipal water sources. Municipal water, due to national and state health regulations, must have low levels of microbial contaminants (EPA, 1989). The US Army defines potable water as any treated water with less than 200 colony forming units (cfu) per milliliter (mL), or raw water with less than 500 cfu/mL (Simmons and Gentzkow, 1955). EPA regulations prescribe limits on the contamination of potable water by coliforms and stipulate zero tolerance for the presence of Legionella, Giardia lamblia, and viruses in water (1989) . The presence of heterotrophic bacteria is also limited to 500 cfu/mL in order to prevent interference by these bacteria in coliform tests (Geldreich, 1986; and EPA, 1989). The numbers of bacteria present in dental unit water vary from one dental unit to another, and within dental units during the working day (Abel et a1., 1971; Tippett et a1., 1988; and Williams et a1., 1994). A recent report indicated that 72% of the water samples taken at different times during a working day and analyzed for the presence of heterotrophic bacteria could not be considered suitable for drinking by EPA standards (Williams et a1., 1993). The problem of microbial contamination of dental water is not a new one. The presence of bacteria in dental water has 15 been known for at least 30 years (Blake, 1963). Blake isolated bacteria of the genera PBeudomonas and Klebsiella from the water reservoir of a dental unit. Characterization of the composition of dental unit water microbiota over the last 30 years has extended Blake's observations to include aquatic bacteria and inhabitants of the skin and oral cavity of humans. Bacteria identified in dental unit water include Gram negative and Gram positive genera, among them: Flavobacterium, Bacteroides, Pausterella, Acinetobacter, Staphylococcus,.Klebsiella, Neisseria, Moraxella, Klebsiella, Streptococcus, and Legionella (Larato et a1., 1966; Abel et a1., 1971; Clark, 1974; Kelstrup et a1., 1977; Holbrook et a1., 1978; Scheid et a1., 1982; Fitzgibbon et a1., 1984; Oppenheim et a1., Mayo et a1., 1990; Pankhurst et a1., 1990; Whitehouse et a1., 1991; and Williams et a1., 1993). Contaminants of dental unit water (DUW) are not limited to prokaryotic organisms, but there are also eukaryotes. Filamentous fungi, free-living amoebas, and nematodes have been isolated from dental water (Kelstrup et a1. , 1977; Michel and Borneff, 1989; and Williams et a1., 1993). The extent of the problem of tainted dental water has been recently reviewed by Williams et a1. (1993). In their study, water specimens from 150 dental operatories in the states of Washington, Oregon, & California were analyzed for the composition of the aerobic, microaerophilic, and facultative anaerobic microbiota in the samples, and the concentrations of these bacteria in each sample were 16 determined. The investigation revealed the presence of great numbers of bacteria in many of the samples, which contained at least 20 different species of bacteria and 4 different genera of fungi at contamination levels exceeding potable water standards. For many years dental procedures have been associated with the development of endocarditis in dental patients (Bayliss et a1., 1983) . In controlled experiments oral manipulations of rabbits are known to produce endocarditis in some animals (McGowan and Hardie, 1974) . Also, common microbial contaminants of dental water are among the most common isolates in adult severe periodontitis (Slots et a1., 1988) . Contamination of dental water could be potentially hazardous to patients and staff in a dental office because of the presence of pathogens and opportunistic microorganisms (Blake, 1963; Kelstrup et a1, 1977; Holbrook et a1., 1988; Scheid et a1., 1982; Fitzgibbon et a1., 1984; Martin, 1987; Oppenheim et a1., 1987; Mayo et a1., 1990; Pankhurst et a1., 1990; Whitehouse et a1., 1991; and Williams et a1., 1993). A review of the literature from 1970-1993 revealed the occurrence of high bacterial contamination levels in water delivered by dental operatories using an in-line water system connected to a municipal supply (Abel et a1. , 1971; McEntegart and Clark, 1973; Clark, 1974; Gross and Devine, 1976; Gross et a1., 1976; Dayoub et a1., 1978; Scheid et a1., 1982; Mills et a1. , 1986; Tippett et a1. , 1988; Mayo et a1. , 1990; Whitehouse et a1. , 1991; J.F. Williams et a1., 1993; and H.M. Williams et 17 a1., 1994). Contamination in water delivered by ultrasonic scalers was determined to range from 48,100 cfu/mL to 2,600,000 cfu/mL (Gross and Devine, 1976; Gross et a1., 1976; Dayoub et a1., 1978; and Williams et a1., 1993). Bacterial concentrations in water delivered by air/water syringes was from 250 cfu/mL to 1,200,000 cfu/mL (Gross and Devine, 1976; Gross et a1., 1976; Mayo et a1., 1990; and Williams et a1., 1993). High speed handpieces delivered contaminated water that contained from 5,700 cfu/mL to 3,600,000 cfu/mL (Abel et a1., 1971; Clark, 1974; Gross and Devine, 1976; Gross et a1., 1976; Dayoub et a1., 1978; Scheid et a1., 1982; Fitzgibbon et a1., 1984; Mills et a1., 1986; and Williams et a1., 1993). Reports in 1993 issues of the Journal of the American Dental Association indicates that the delivery of dental unit water with high bacterial contamination through dental instruments is an accurate representation of typical dental water contamination in clinics today (Mills et a1., 1993; and Williams.et a1., 1993). The numbers of bacterial contaminants in dental unit water in the report by Williams et a1. were similar to contamination levels previously reported in the literature. They reported that heterotrophic plate counts of bacteria from water delivered by air/water syringes, from different operatories, ranged from less than 30 cfu/mL to 1,200,000 cfu/mL, while contamination of water delivered by high speed handpieces ranged from less than 30 cfu/mL to 550,000 cfu/mL. The degree of contamination found in DUW is remarkable, given that similar bacterial concentrations have 18 been found only in dilute sewage or in heavily contaminated bodies of water near sewage treatment.plants (Gainey and Lord, 1950; and Rheinheimer, 1991). Dental unit water bacterial counts were much higher than the bacterial presence found in unpolluted lakes and streams (LeChevallier et a1., 1990; and Rheinheimer, 1991). The ability of dental unit water contaminants to cause medical problems could be the result of either aerosolization of pathogens or direct injection of pathogens. Aerosolization of Chlamydia trachomatis, during a dental procedure of a Chlamydia infected patient, is suspected to be the cause of the (development. of’ purulent conjunctivitis by' a dentist (Midulla.et a1., 1987). The ability by DUW’bacteria to infect humans is demonstrated by the development of dental abscesses, caused by Pseudomonas, in the oral cavity of immunocompromised patients immediately after dental treatments (Martin, 1987). Subsequent investigations revealed that Pseudomonas was a contaminant of the dental water, and that healthy individuals who received treatment in the same‘dental units had their oral cavity colonized by the same microorganisms. The contamination of DUW is also a cause of concern due to a recent report which indicates that consumption of water with high numbers of heterotrophic bacteria is associated with gastrointestinal maladies (Payment et a1., 1991). Contamination of dental unit water by eukaryotes could also increase the health risks. Certain free living amoeba species have been identified a serious water borne agents of 19 disease (Ma et a1., 1990; and Martinez and Vivesvara, 1991) and isolates of DUW belong to this classification group (Michel and Borneff, 1989). Amoebae also serve as hosts for pathogenic organisms, like Legionella and coliforms, and are suspected to be the source of sensitizing allergens in Pontiac Fever (Newsome et al. , 1985; Barbaree et al. , 1986; Rowbotham, 1986; King et al., 1988; and Fields et al., 1993). Potential health risks in dentistry: Dental aerosols: A contributor to the health risks associated with dental water contamination is the production of aerosols during dental procedures. High speed handpieces, specifically, produce aerosols contaminated with bacteria present in the water (Kazantzis, 1961; Madden and Hausler, 1963; Stevens, 1963; Belting et al., 1964; Hausler and Madden, 1964; Larato et al., 1966; Abel et al., 1971; and Earnest and Laesche, 1991) . Aerosols produced by dental instruments are made up of particles of an average size of 50 um or less, which form a colloid (Kazantzis, 1961; Belting et al., 1964; Hausler and Madden, 1966; Micik et al., 1969; and Abel et al., 1971) . The colloid permits aerosol particles to be suspended in air for long periods by Brownian motion and to be carried by air currents, such as those produced by air conditioners. The formation of aerosols also prevents desiccation of contaminating microorganisms, prolongs the infectivity of pathogenic organisms like Legionella pneumophila (Hambleton et 20 al., 1983), and exposes all areas of the dental office and all personnel to dental water (Belting et al., 1964; Hausler and Madden, 1966; and Micik et al., 1969). The danger of contaminated aerosols is that particles of 5 um or less in diameter can be inhaled and trapped in the alveoli of the lungs during respiration. Greater than 95% of the droplets produced by handpieces are less than 5 um in size (Micik et a1., 1969), and these could serve as a means of transmission of DUW contaminants known to be stable in aerosol particles (Hambleton et al., 1983; Macfarlane, 1983; Zuravleff et al., 1983; Muder et al., 1986; and CDC, 1990b). The production of contaminated aerosols by dental instruments was first demonstrated in 1963 by Kazantzis with the isolation of human oral microbiota from aerosols. Madden and Hauser (1964, 1966) also reported that aerosols produced during dental procedures could be contaminated by microorganisms present in the oral cavity of humans. These findings uncovered a potential health risk to dental professionals. The problem was highlighted further by the isolation from the air of Mycobacterium tuberculosis during dental treatment of patients suffering from tuberculosis, who had mycobacteria in their sputum (Belting et al., 1964). The investigators determined that mycobacteria could be isolated from the air within a 4 foot semicircle in front of the patient, with the greater concentration of aerosol particles present in the dental health care workers' close working area. This obviously exposes dental professionals to the 21 majority of the aerosol particles produced by the instruments. Characterization of the composition of the contaminated aerosols produced by handpieces, air water syringes, and ultrasonic scalers identified the presence of bacteria like pneumococci, alpha and beta hemolytic streptococci, Pseudomonas aeruginosa, and various Staphylococcus species, with contaminating bacteria originating from the oral cavity of patients and contaminants of DUW (Kazantzis, 1961; Belting et al., 1964; Hausler and Madden, 1964; Larato et al., 1966; Holbrook et a1., 1978; and Earnest and Laesche, 1991). Contaminated aerosols could be associated with the propensity of dental personnel to have respiratory problems (Carter and Seal, 1953; Burton and Miller, 1963; and Mandel, 1993). A survey of dentists in the US has determined that respiratory maladies are the leading afflictions suffered by dentists today (Mandel, 1993). Reports by Carter and Seal (1953) and Burton and Miller (1963) , have indicated that dental personnel and dental students have a higher incidence of colds and upper respiratory tract infections than their counterparts in other'health.science fields. IFurther proof of the potential danger of contaminated aerosols is the altered nasal microbiota of dentists (Clark, 1974). Dentists working in units contaminated with bacteria of the genera Pseudomonas and Proteus, common contaminants of dental unit water and not normal members of nasal microbiota, have their nasal cavity colonized by them. Also, contaminated aerosols were suspected to be the infective vector for Chlamydia trachomatis in a 22 dentist that developed purulent conjunctivitis (Midulla et al., 1987). Further cause for' concern. about the association. of aerosols and disease transmission is the finding that Legionella pneumophila is a common contaminant of dental unit water (Rheinthaller and Mascher, 1986; Oppenheim et al., 1987; Pankhurst et al., 1990; and Lfick et al., 1993). The etiological agent of Legionnaires' Disease and Pontiac Fever is transmitted.by aerosols similar'to those produced by dental instruments (Macfarlane, 1983; Zuravleff et al., 1983; Muder et al., 1986; and CDC, 1990), and remains viable for up to 2 hours in a mist colloid suspension (Hambleton et al., 1983) . Studies of the exposure of dental personnel to Legionella in Austria and Czechoslovakia (Liick) , Germany (Rheinthaler, 1987, 1988) , and the United States (Fotos) , have revealed the presence of increased anti-Legionella antibodies in dental professionals' sera when compared to other health professionals and members of the population (Fotos et al., 1985; Rheinthaler et al., 1987, 1988; and Lfick et a1., 1993). Increased antibody titers to Legionella pneumophila in dental personnel appear to be associated to their work experience (Fotos et al., 1985; and Rheinthaler et al., 1987 and 1988) . Dentists and dental staff with greater work, experience appear to have a greater exposure to Legionella and have higher antibody titers (Fotos et al., 1985; and Rheinthaler et al., 1987, 1988)). The recent death of a dentist in California as a result of Legionella pneumonia 23 raises questions about the presence of this bacteria in‘dental water and its potential as a serious health issue. An investigation of the dentist's death revealed that he most likely acquired his Legionella infection from his dental operatory (J.F. Williams et al., submitted for publication). Another health risk associated with the presence of Legionella in dental water and aerosols could be the presence of free living amoebae in dental water (Michel and Borneff, 1989). Legionella is known to infect free living amoebae, to survive in these organisms through exposure to environmental hazards, and to multiply in them (Newsome et al., 1985; Barbaree et al., 1986; Rowbotham, 1986; Fields et al., 1993; and Kuchta et al., 1993). The ability of Legionella to infect and multiply within protozoa, makes amoebae the perfect disease carrier for inoculation of high numbers of Legionella into the lungs by aspiration of aerosol particles (O'Brien and Bhopal, 1993). Another problem with the bacterial contamination of dental water and the production of contaminated aerosols is the recent increase of tuberculosis cases in the general population (Faecher et al., 1993) . Mycobacterium has the ability to infect the oral mucosa of humans and to infect the tooth socket after extractions (Roderick Smith et al., 1982; and Penderson and Reibel, 1989). The disease can produce ulcers with infective microorganisms in the oral cavity and tongue in the early stages of the disease and prior to the development of systemic infection, and in sputum. in an 24 infection of the lungs (Prabhu et al., 1978; Rauch et al., 1978; and Dimitrakopoulos et al., 1991). Thus, the presence of mycobacteria in the oral cavity can produce contaminated aerosols, as demonstrated in sputum positive individuals by Belting et al., and this may expose dental professionals and patients to the bacteria. Other infection routes: Another caused for concern is the potential for cross contamination of patients by water retraction into the dental unit and the saliva ejector (Bagga et al., 1984; Miller and Palenik, 1986; ADA, 1988b; Crawford and Broderius, 1988, 1990; Anonymous - Lancet, 1992; Lewis and Boe, 1992; Lewis et al., 1992; and Watson and Whitehouse, 1993). Retraction of water during dental procedures contaminates the inside of dental instruments and the attached water line with microorganisms, bacteria and viruses, present in the oral cavity, blood, and debris (Bagga et al., 1984; Miller and Palenik, 1986; ADA, 1988b; Crawford and.Broderius, 1988, 1990; Lewis et al., 1992; and Lewis and Boe, 1992). Thus, this could provide material for the production of contaminated aerosols and.transfer it to another patient directly during a dental procedure. The possibility of cross contamination caused the ADA/CDC to recommend the use of anti-retraction check valves and the sterilization of dental instruments and flushing of the water lines between patients (CDC, 1993). Although this recommendation has been in place for some time, the internal 25 contamination of dental instruments and water lines remains a problem. Part of the problem is that anti-retraction check valves are ineffective after prolonged use, and these devices are not monitored once installed. Also, dental instruments are not always sterilized between patients (Christensen, 1991; Anonymous - Lancet, 1992; and Dental Products Report, 1993). A recent publication indicated that the common practice of creating a seal around the saliva ejector with the mouth, creates negative pressure which could also release retracted fluids derived from the mouth of a previous patient (Watson and Whitehouse, 1993). Thus any microorganisms present in dental instruments and the saliva ejector could be released into the oral cavity, injected into surgical sites, or aerosolized during dental procedures, exposing dental professionals and patients to potentially pathogenic bacteria. Aerosol contamination is not the only health risk associated with the use of contaminated water during dental procedures. Microorganisms in dental water could cause bacteremias in patients, as a result of the injection of microorganisms directly into the bloodstream during invasive dental treatments and surgical procedures. Pathogens like Legionella pneumophila, Mycobacterium, and Pseudomonas, have the ability to infect a human host also through open wounds, making a dental procedure an excellent mode of infection by the direct inoculation route (Roderick Smith et al., 1982; Brabender et al., 1983; Martin, 1987; Lowry et al., 1991; and Pendersen and Reibel, 1991). 26 A recent scientific investigation by Ely et al. associated contaminated air used during dental procedures with the development of pneumomediastinum, fatal descending necrotizing mediastinitis, and Lemierre's syndrome in four dental patients. However, the authors did not consider that the water lines could equally well have been the source of the infections they observed; they attributed the source of the infection to the air, even though there is little evidence that air delivered by the dental unit is contaminated. BIOFILM8:Their nature and the extent of the problem in medical care. The design of the dental unit, with its long and narrow tubes, and the stasis of water inside the unit, create ideal conditions for the colonization of the inner surface of dental unit water lines and subsequent biofilm formation by aquatic bacteria and human contaminants retracted inside the water lines. Studies have revealed that bacteria prefer to live in association; in alpine streams 1 out of every 1000 bacteria are found in a planktonic (motile) stage, and the others associate to produce a slimy biofilm on rock surfaces (Costerton et al. , 1978) . This association appears to produce a selective advantage to the adherent bacteria (Costerton et al., 1981, 1987; LeChevallier et a1., 1988). Microorganisms present in.‘water' distribution. systems .have an increased resistance to chlorine just by surface attachment 27 (LeChevallier et al. , 1988) . Control experiments with a fastidious laboratory organism, like Legionella, have revealed that this organism. is 'more resistant to biocides, like chlorine, when grown in association with other bacteria, than when grown in pure cultures (Cargill et al., 1992). Biofilm formation: Biofilm development on any surface begins with the change of bacteria from a jplanktonic stage to a sessile one. Microorganisms adsorb to surfaces by hydrophobic forces in a reversible manner. With adsorption certain bacteria begin to secrete extracellular components, an "extracellular polymeric substance" (EPS) necessary for a more permanent adhesion by polar bonds. This begins the formation of a glycocalyx matrix and serves as the foundation of the biofilm. Bacterial adhesion by polar bonds causes attached bacteria totdivide and to secrete more EPS. The increased production of EPS provides optimal conditions for the attachment of planktonic bacteria to the biofilm and the formation of heterogeneous microcolonies. This glycocalyx acts like an anion exchange resin which can trap nutrients according to ionic charge. "Maturation" of the biofilm creates a new ecosystem formed by a mucopolysaccharide glycocalyx matrix of varying layers and widths, with channels, embedded with microcolonies of heterogenous bacteria, filamentous fungi, and other protozoa. These organisms act as interacting communities with different survival requirements that are met within the biofilm. 28 Biofilm formation also creates the development of oxygen and nutrient gradients within the structures. Depending in their position in the biofilm or on the microcolonies, microorganisms could live in an aerobic environment located closer to the flow of water, in.a microaerophilic environment found in the middle layers of the biofilm or inside of the microcolonies, or in a strictly anaerobic one, found in the inner layers of the biofilm or inside the microcolonies. Nutrients present in the water are readily used by organisms present in the aerobic and.microaerophilic layers. Anaerobic organisms use byproducts of the other organisms as nourishment and process these nutrients by fermentation. Also, they respire by using electron acceptors other than oxygen. For a review of biofilms and their formation refer to Costerton et al. (1981, 1987), Characklis and Cooksey (1983), Marshall (1992) or Mayette (1992). Maturation of the biofilm also provides its inhabitants with protection from the action of antibiotics and biocides. The formation of biofilms inhibits the access of antibiotics and biocides to constituent microorganisms, thus increasing their resistance to these compounds. Only microorganisms in contact with fluids are affected (Costerton et al., 1981, 1987; LeChevallier et al., 1988; Fackelmann, 1990; van der Wende and Characklis, 1990; Anwar and Costerton, 1992; Marshall, 1992; and Mayette, 1992). The ability of bacteria to produce biofilms is expressed in biotic and abiotic conditions. Biofilm formation occurs in 29 industrial settings and is associated with corrosion, the blockage of pipelines and filters, fouling of products, and the production of harmful metabolites, like H28 (Characklis and Cooksey, 1983; and Costerton, 1984). The formation of biofilms is not always harmful and it is exploited in the treatment of water and waste water, using the microorganisms' abilities to break down pollutants (Charaklis and Cooksey, 1983). In animals, one of the best described biofilms in nature is the formation of dental plaque on teeth, a potentially important biofilm in humans (Costerton et al., 1987). Biofilms are also formed in the gastrointestinal tract and genitourinary tract of mammals, protecting the organisms against pathogenic microorganisms (Costerton et al. , 1981, 1987; and Anonymous - ASM News, 1993). Any disturbances of these biofilms could result in the development of infections. Biofilm formation in humans is also the cause medical problems. Osteomyelitis results from the development of biofilm in the bones, and its treatment requires the elimination of biofilm structures. Prolonged treatment is needed with antibiotics at higher than normal concentrations due to the drugs' limited access to microorganisms in the biofilm (Anwar and Costerton, 1992; and Marshall, 1992). Biofilms in biomedical devices: The production of biofilms in biomedical devices is also associated with prolonged use of implanted catheters, and may 30 cause urinary tract infections, endocarditis, and catheter- related sepsis in patients with implants (Peters et al. , 1981; Kluge, 1982; Marrie et al., 1982; Costerton et al., 1987; Russell et al., 1987; Nickel et al., 1992) . Biofilms found in implanted medical devices are produced by microbiota of the skin, Staphylococcus aureus and Staphylococcus epidermidis, and gram negative organisms, like Pseudomonas aeruginosa and Alcaligenes calcoaceticus (Peters et al., 1981; Kluge, 1982; Marrie et al., 1982; Gristina et al., 1988; Anwar and Costerton, 1992; Marshall, 1992; Nickel et al., 1992; and Passerini et al., 1992). These bacteria can form biofilms along the catheters at a rate of 2-3 cm per hour, even against the flow of antibiotic containing fluids, by using fibronectin to attach to surfaces with polar bonds, and immediately beginning development of a glycocalyx matrix after the implant of the devices (Russell et al., 1987; and Nickel et al., 1992). The development of endocarditis and septicemia in patients with implanted pacemakers has been determined to be caused by biofilm formation in pacemaker leads and appears to depend on the contamination of the device prior to surgery (Peters et al., 1981; and Marrie et al., 1982). Similar medical problems are observed in patients with implanted arterial grafts and prosthetic cardiac valves (Kluge, 1982). The implantation of prosthetic hip joints and artificial hearts leads to the formation of biofilms in the devices, necrosis of tissue surrounding the implanted device, and 31 finally' rejection. and. removal. of implants (Gristina and Costerton, 1984; Gristina et al., 1988; and Marshall, 1992). Dental unit water line biofilms: The contamination of water in water distribution systems with high numbers of bacteria and fungi result of the formation of biofilms in the pipes of the water distribution system (Rigway and Olson, 1981; Rosenzweig et al., 1986; and LeChevallier et al., 1987). Something similar occurs in dental units where the production of biofilm is the result of the creation of optimal conditions for the formation of biofilms. The long and narrow tubes with static water are combined with known biofilm producers like Staphylococcus, Pseudomonas, Legionella, and fungi, all of which are common contaminants of DUW (Kelstrup et al., 1977; Peters et al., 1981; Kluge, 1982; Marrie et al., 1982; Oppenheim et al., 1987; Russell et al., 1987; Gristina et al., 1988; Mayo et al., 1990; Pankhurst et al., 1991; Anwar and Costerton, 1992; Marshall, 1992; Nickel et al., 1992; Passerini et al., 1992; Williams et al., 1993; and Wireman et al., 1993). The microscopic examination of discolored, badly tasting, foul smelling water with flakes, by Kelstrup et al. (1977) revealed the presence of aggregated bacteria and fungi in the floccules present in DUW. Upon inspection of the dental water line by phase contrast and electron microscopy a similar arrangement was found in the inner surface of dental water lines. The inner walls of the dental unit water lines were 32 covered with different bacteria and fungal hyphae embedded in a‘matrixu This report established the presence of biofilms in dental unit water lines. Examination of biofilms formed in dental unit water lines has revealed the presence of complex microbial communities (Williams et al., 1993). Microscopic examination of dental water line biofilms in vivo and in situ with the use of Nomarski optics and electron microscopy uncovered the presence of not only bacterial and fungi in the biofilm structures, but also the presence of amoebae and nematodes, which feed on the bacteria of the biofilm (Williams et al., 1993). Mayo et al. and Whitehouse et al. associated bacterial contamination found in dental water with the formation of biofilms in dental water lines, providing a'~-r:-;!:‘.1:‘.t.-._.. 41.2.“;- .':..-. 5. .. .. ~9~~u-.. 1“ 7;»; .z-l :' .' 1 :1 2.11.5. -- J -c— .e- ... 2500000 —‘ 2 3 4 5 6 7 8 910111213 1 Dental Operatories t Ien Post pat FIGURE 11 87 FIGURE 12 Histogram showing heterotrophic plate counts of bacterial contamination of dental unit water samples collected post 2 minute flush from dental water lines and immediately after completion of a routine procedure. Samples 1-7 were from air/water syringe lines and samples 8-13 from high speed handpiece lines. Bacterial presence in the samples of 30 cfu/mL or less are expressed as 30 cfu/mL. aria”: ‘ ,, .. , ! ',-:.Lo~—-4- - .... .. ; “J 34.01:; 1133.3 :73: ___,.;.;_1; ..'-_ ' — ,' '~-'.°‘.."‘:'l'.-L";r;g- n'u 1: 73.5-- "Eu-"Ji- .... 11.2... . "'1‘ ' "" "-‘»' ---|--~""“T'." 1“" HEN—"- ‘ 7:”;"1‘15‘1’ 7?" ".h‘i:*‘~."-'1-"-'E.I "-3133: I ' ‘ . ' I ' ‘ . 4 I. ,, t J .-~' .-v ‘ ..1- ..~ . ‘ . ’ ..: ; ~ 1313127511.; ; . 3;... . L.,.T- ;._ 5,. ,. .. L.-.._. 1' u . . .7 "1.) I” --‘ a... )- - ' - . ', .. ..:.-.. 9’ . m his): ‘m: “: .-..r..-x.::-:~‘-Iii-£3.34"..,. 11';',‘_,'.3".“' :.:.x':-:.::r: a ‘c-an‘l' ’- - a , . 2 .v . . :2 ... .. .. n... .-u .-::o. '51. .m , - .z' in.- '_ ' ‘ ‘ . - - . . . » — fl .: i‘n.“J'--7¢" ' .3 L 7 :- '-r ‘ . . 7 V q . . ‘.-.-_ , ' 0 .0 . 2500000 “ 250000 __ 25000 — 25 “ 2 3 4 5 6 7 8 910111213 1 Dental Operatories 1 Ian Post pat FIGURE 12 89 DISCUSSION: The extent of bacterial contamination of dental unit water lines encountered in this study can not be considered unusual; instead, based on a series of recent reports (Mayo et al., 1990; Whitehouse et al., 1991; Pankhurst and Philpott- Howard, 1993; J.F. Williams et.al., 1993; and.H.M.‘Williams et al. , 1994) , it appears to be the normal profile to emerge from similar sample analyses. The literature reveals that contamination levels of dental unit water vary, but generally range from less than 30 cfu/mL to 10,000,000 cfu/mL (Able et al., 1971; McEntegart and Clark, 1973; Clark, 1974; Dayoub et al., 1978; Gross and Devine, 1976; Gross et al., 1976; Scheid et al., 1982; Tippett et al., 1988; Mayo et al. , 1990; Whitehouse et al., 1991; J.F. Williams et al., 1993; and H.M. ‘Williams et al., 1994). Analysis of readily available domestic and environmental samples served to establish a basis for comparison and to validate dental water analysis methods. Bacterial levels in domestic water samples were consistent with the standards of the United States Safe Drinking Water Act Of 1989 (EPA). Heterotrophic bacterial counts in environmental samples varied widely among specimens, but were within the range for natural bodies of fresh water reported.historically by Gainey and Lord (1950) and recently by Armstrong et al. (1982), LeChevallier et al. (1990) and Rheinheimer (1991) . The (contrast between contamination levels in dental unit water, Eind domestic and environmental samples is attributable to the 9O extraordinary productivity of microbial biofilms in dental unit water lines and the static nature of the of water in DUWL (Kelstrup et al., 1977; Mayo et a1., 1990; Whitehouse et al., 1991; and Williams et al., 1993). The biofilm produced in a dental unit is composed of microcolonies embedded in an anionic glycopeptide-glycocalyx matrix which stabilizes microbial communities, helps in the gathering of nutrients acting as a charged net, and protects participants from the effects of biocides and antibacterials by limiting their access to microorganisms embedded in the biofilm (Costerton et al., 1981, 1987; LeChevallier et al., 1988; Fackelman, 1990; van der Wende and Characklis, 1990; Anwar and Costerton, 1992; Cargill et al., 1992; Marshall, 1992; and Mayette, 1992). The production of biofilm within DUWL is not surprising due to the presence of common dental unit. water’ contaminants.‘which are Iknown biofilm "slime" formers, and include opportunistic pathogens like Pseudomonas sup. (Peters et al., 1981; Fackelman, 1990; van der Wende and Characklis, 1990; and Nickel et al., 1992) and.Staphylococcus sup. (Peters et al., 1981; Kluge, 1982; Marrie, et al., 1982; Russell.et al., 1987; Anwar and Costerton, 1992; and.Marshall, 1992) and pathogens like Legionella pneumophila (Cargill et al., 1992; Marshall, 1992; and Wireman et al., 1993). The presence of pathogenic and opportunistic bacteria in