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I ‘ .Vto.vlbl..| «I‘ln'bCI‘lfi‘. in!!! 4/ .o p.t('i}1l|l)f iii}- lint-i ‘ 091.3: .I v l 1.} ‘.l I t‘hluf 19’..1‘u|| .: 1 ~ .I‘qw . .‘tonrn-.l MICHIGAN STATE UN II II IIII I III IIIIII IIIIIIIIIIIIIIIIII 01571 1546 ”I This is to certify that the dissertation entitled A STUDY OF WATER-BORNE BACTERIAL PATHOGENS IN THE CONTEXT OF THE DENTAL OPERATORY presented by MARK KENNETH HUNTINGTON has been accepted towards fulfillment of the requirements for Doctor Philosophy degree in Major professor ’t/oL/éé MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 .- Jaw- 4» «gr- LIBRARY Michigan State Unlverslty PLACE N RETURN BOX to romovo this chookout from your rocord. TO AVOID FINES rotum on or baton dot. duo. DATE DUE DATE DUE DATE DUE MSU lo An Affirmative MIONEQJII Opponunlly Inotltwon WM! A STUDY OF WATER-BORNE BACTERIAL PATHOGENS IN THE CONTEXT OF THE DENTAL OPERATORY by Mark Kenneth Huntington A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology 1 996 ABSTRACT A STUDY OF WATER-BORNE BACTERIAL PATHOGENS IN THE CONTEXT OF THE DENTAL OPERATORY by Mark Kenneth Huntington Reports of microbiologically contaminated dental water come from all corners of the globe. As early as 1963, significant pathogens were isolated from dental water systems. In light of increased attention to infection control in recent years, my studies reported herein were undertaken to evaluate and characterize the microbiological quality of water in the modern dental operatory. Culture of dental unit water revealed enormous contamination by bacteria, including a variety of well known pathogens and NV 1. saprophytic organisms, identified by their biochemical profiles. Bacterial concentrations in time dental. water samples were exponentially greater than those of potable water systems. The proximate source of this contamination is the biofilms lining the small caliber instrument cooling and irrigation water lines. Gram negative bacteria were the most prevalent organisms in the dental water samples. Correspondingly high concentrations of endotoxin were measured in the water using the Limulus amoebocyte lysate assay. Endotoxin concentration correlated. positively' to the bacteria load present. Aerosolization (n3 endotoxin chnfljm; dental procedures was detected using an aerosol-sampling impinger. Also prominent among the contaminants was Legionella. Surveys utilizing the Ekndixmmmfm polymerase chain reaction (PCR) detection system found Legionella present in two- thirds of time dental turn: water samples. This prevalence rate was similar to that found in potable water samples collected over tflue same period” However, semiquantitation of the levels revealed a vastly greater intensity of contamination in time dental samples. The' feasibility ea patient's mouth with the potential for ingestion or inoculation into the oral mucosa may initially seem the most serious hazard, an unrecognized and unexplored risk is represented by the aerosols. Dental procedures, especially' those involving 15 high-speed handpieces, produce aerosols [Abel et al 1971, Belting et al 1964, Earnest et al 1991, Hausler et al 1964, Kazantzis et al 1961, Larato et al 1966, Madden et a1 1963, Stevens et al 1963]. Stable dental aerosols are composed of a colloid of droplets which may be suspended many hours and carried to every corner of the dental office [Abel et a1 1971, Belting et a1 1964, Hausler et a1 1964, Kazantzis et al 1961, Micik et a1 1969]. Pathogenic organisms such as Legionella and Mycobacterium within aerosolized droplets may avoid dessication and remain infective for several hours, allowing the inhalation of aerosols to serve as a means of transmission [Belting et al 1964, Hambleton et a1 1983, Macfarlane et al 1983, Muder et al 1986, Zuravleff et al 1983]. Aerosols are aspirated into the air-intake port of the dental air line apparatus. Once aerosolized organisms are within the air lines, they colonize them [Costerton et al 1987]. The air line biofilm has the potential to amplify the contaminants, and shed them into the lumen where they are blown into the dental office air and the patient's mouth. Very little research has been done to investigate the medical significance of air line organisms in the dental office, though they have recently been implicated in two infectious intrathoracic complications of routine dental 16 care [Ely et al 1993]. While the authors attributed these infections to bacteria aerosolized by the dental air line, others have suggested the waterline as an alternative source [Ely et a1 1995, Mackenzie et a1 1995]. Vacuum lines receive microbial contamination directly from the patients' mouth. These organisms also generate biofilms. Backflow of microorganisms into the disposable saliva ejector and ultimately the next patientls mouth has been reported [Watson et al 1993]. When the airflow through the lines suddenly decreases (as occurs when a patient seals his lips around the saliva ejector), the likelihood of this "suck—back" phenomenon occurring increases. CKXTTAEDHWKTICRIlfiONEEKIL REH¥§UFEK3 In addressing person—to-person contagion and instrument- borne cross-infection, time dental profession and (XXI recommend diligent attention to sterilization of dental handpieces, disinfection of operatory surfaces, and the use of gloves, goggles, and masks by dental professionals [Centers for Disease Control and Prevention 1993]. As these measures are adopted transmission of pathogens between patients and dental professionals will be reduced, though not eliminated [Crawford et al 1985, Merchant et al 1992]. 17 Unfortunately, current practices do not always reflect current (xx: recommendations. Zhi 1989, fewer tflmni 50% of dentists responding to a survey sterilized their instruments on a daily basis, and only 12.5% sterilized them between patients [Dental Products Report 1993]. In a 1991 report, 80% of dentists claimed to surface-disinfect their handpieces rather than autoclave/gas-sterilize them, and only rarely was any form of disinfection or sterilization applied to air—water syringes [Christensen et a1 1991]. The latter is still true today. CDC recommends flushing of dental waterlines as a means to control waterline microbial contamination [Centers for Disease Control and Prevention 1993]. However, this measure has minimal effect on the quality of the water delivered to the patient. Flushing may sometimes increase the intensity of' bacterial contamination, with day-to-day ‘variation in contamination levels exhibiting' as great. a magnitude as preflush-to-postflush variation [Gross et al 1976, Santiago et a1 1994]. There are physical reasons for these findings. The physics of landnar flow (ME fluids through cylindrical structures results in maximum velocity of water occurring at the lumenal center of the waterline, with the laminae juxtaposed. to the biofilm—coated ‘wall remaining stagnant 18 [Cutnell 1989]. Flushing 'merely rinses out ‘planktonic organisms 111 the lumen, leaving tjua mature biofilm essentially undisturbed. Multiplication of organisms in the biofilni releases rmnv flora into tin; lumen. The large surface area-to-volume ratio of the lines leads to generation of high concentrations of bacteria in a relatively short time. Ultraviolet irradiation of the incoming operatory water supply is of limited value. A recent study of the effects of ultraviolet irradiation on the growth of Legionella and other heterotrophic bacteria in water in a circulating cooling system failed to show a reduction in bacterial numbers. This failure was attributed by the authors to the protective effects of the biofilm in which the organisms reside [Kusnetsov et a1 1994]. Approaches tx> the problem (ME waterline contamination that are more effective than flushing or irradiation include the addition of chemical disinfectants, the use of bottled water reservoirs, bacteriological membrane filters, autoclavable operatory components (i.e. waterlines in addition to instruments), and various combinations of these. Each has potential to reduce significantly the levels of bacterial contamination ill the dental water system, run: all require 19 diligent, conscientious maintenance in order to be effective [Williams et al 1996]. Presently, however, few dental offices employ any of these methods. The medical community has taken the approach of installing in-line bacteriological filters between the patient and the infusion solution reservoir, with all infusion lines and reservoirs employing a single-use, disposable design. Such an approach may well become standard dental practice, as well. SIGNIFICANCE OF HETEROTROPHIC BACTERIA Heterotrophic bacteria have frequently been considered primarily saprophytic, with little innate pathogenic potential [Matsen et al 1975]. The only medical significance attributed txi them was iii the case of immunodeficient or otherwise compromised patients. 'They have always been important in water quality assessment because overgrowth of water heterotrophs will obscure the presence of "marker" pathogens (coliforms) [Enviromental Protection Agency 1987, Environmental- Protection Agency 1989, Matsen et a1 1975, Payment et al 1994]. An extensive study of these pigmented water bacteria in the context of hospital infections was undertaken by' Matsen 20 [Matsen et a1 1975]. From it, they concluded that "pigmented water-associated bacteria, with the exception of Pseudomonas aeruginosa, are infrequent hospital isolates of low—grade pathogenicity." 'In 1987, Martin diagnosed Pseudomonas aeruginosa infections in two chemotherapy patients [Martin et al 1987]. Upon obtaining a thorough history, he discovered that they had both recently received care at the same dental clinic. Cultures of water samples obtained from the operatories in the clinic grew out organisms which were identical to those found in the lesions. Through serial sampling of the oral flora (If 78 subsequent patients visiting' the clinic, he found colonization by the same strain of organism for up to 10 weeks following exposure. In all the prospective cases, this colonization was asymptomatic. Recent surveys of dental clinics have revealed that dental unit water contamination by pseudomonads of a variety of species is a ubiquitous problem [Mayo et al 1990, Williams et al 1993]. In recent years there has been a growing conviction that not only P. aeruginosa, but virtually all heterotrophic bacteria are opportunistic pathogens whose delivery in potable water should be controlled. In a study by Payment, 57% of tap water samples grew out cytolytic colonies, and 6% contained 21 bacteria having three or‘ more of the virulence factors assessed [Payment et a1 1994]. Similar findings were reported by Lye, who found that 1% of all bacteria isolated from drinking water samples produced cytotoxic factors [Lye et al 1991]. While most of these organisms were of genera that are not traditionally associated with significant public health problems, on occasion they have been implicated in clinically significant infections. The presence of multiple virulence factors increases their disease-causing potential [Payment et a1 1994]. During a prospective epidemiological study of gastrointestinal diseases associated with drinking water, a statistically significant univariate correlatbmn was found between rate and duration of reported symptoms and counts of heterotrophic organisms grown from the water source [Payment et al 1991a, Payment et al 1991b]. This correlation observed in the absence of known enteropathogenic organisms is strong evidence of the clinically and statistically significant pathogenic potential of heterotrophic water microbes in healthy, immunocompetent individuals., Such hazards are accentuated for the inmmnocompromised patient, and the numbers of immunocompromised individuals in the general population are increasing with the rise in organ transplant recipients and AIDS patients. The counts of 22 heterotrophic bacteria observed in dental water are much higher than those found in unpolluted lakes and streams [LeChevallier et al 1987, Reinheimer et al 1991, Santiago et al 1994], being instead in the range found in dilute sewage [Gainey et al 1950, Reinheimer et al 1991]. They are well in excess of the number seen in Payment et al's studies. The literature also contains a report of endocarditis caused by' nonstreptococcal dental water“ organisms ' generally considered saprophytes [Zinman et al 1991]. This indicates cardiovascular as twitl as gastrointestinal pathologies may be caused by these organisms. SIGNIFICANCE OF LEGIONELLA Legionnaire's disease, first described in 1976 following an outbreak of fulminant pneumonia in 221 individuals attending an American Legion convention, is caused by bacteria of the genus Legionellaceae. Infection by these organisms is responsible for an acute fibrinopurulent pneumonia histologically characterized tn/.a hypoxia-inducing exudate of neutrophils, macrophages, and fibrin in the alveoli secondary to capillary damage. This presents clinically with a variable severity, ranging from mild pneumonia to 23 adult respiratory distress syndrome (ARDS) with major systemic manifestations. Following an incubation period of 2 to 10 days, high fever, nonproductive cough, chills, cephalgia, and myalgia abruptly appear. Hyponatremia, diarrhea and neurological deficits are also present in 50, 36, and 25 percent of patients, respectively [Fang et al 1989, Reingold 1988, Winn et al 1981]. This may progress to empyema, pneumothorax, and respiratory failure with an untreated mortality of 10 to 20 percent. In 1985 alone there were 75,000 cases of clinical legionellosis in the U.S., with over 11,000 resulting in death [Schleicher et al 1995]. Legionella pneumonia.:may' affect both humans and nonhuman mammals [Fabbi et al 1993]. The route of infection has long been believed to be via inhalation of aerosols generated from. contaminated ‘water sources such as air conditioners, showers, decorative fountains, humidifiers, supermarket produce Sprayers, etc. [Bolin et al 1985, Dondero et a1 1980, LaMaine et al 1990, McDade et al 1977, Stout et a1 1993, Winn et al 1988]; however, more recently it has been shown that ingestion, aspiration, and direct inoculation (including via irrigation of surgical wounds) play significant roles, as well [Blatt et al 1993, Lowry et a1 1991, Lowry et al 1993]. 24 Following invasion, Legionella are phagocytized by neutrophils and macrophages [Nash et al 1988]. Once inside they nmltiply within the cells. While antibodies enhance phagocytosis, they ck) not decrease the intracellular survival cm? the organisms. .Activated necrophages ck) kill the organism suggesting a role for cell-mediated immunity in recovery from and prevention of the disease [Nash et a1 1988]. Intracellular multiplication of Legionella results in cell death and lysis followed by release of host and bacterial enzymes. At least two cytotoxins, an endotoxin- like substance, and numerous extracellular enzymes ‘which have been described, but not yet fully characterized, are believed to be responsible for the lung tissue damage [Fang et al 1989, Reingold 1988, Winn et al 1981]. In addition to Legionnaire's disease and Pontiac Fever, Legionella has been implicated 1J1 endocarditis, myocarditis, pericarditis, pyelonephritis, pancreatitis, sinusitis, post- pericardiotomy-like syndrome, and abscess formation [Bernstein et al 1991, Camara-Gonzalez et al 1993, Kaye et a1 1991, Tompkins et al 1988]. Legionellaceae have been shown to invade and multiply intracellularly' within. a :number' of free-living’ protozoa, including' .Acanthamoeba, Tetrahymena, .Hartmannella, and 25 Naegleria, with the amoebae being the most significant natural hosts [Anand et al 1983, Breiman et a1 1990, Fields et al 1984, Fields et al 1990, Harf et al 1987, Henke et a1 1986, Newsome et al 1985, Rowbotham et al 1986, Tyndall et al 1982, Wadowsky et 5L1 1988]. Legionella survive and multiply within these protozoa; in fact, their growth in the environment in the absence of protozoan hosts has not been documented, suggesting that such intracellular sequestration is a prerequisite for survival of the genus [Fields et a1 1989, Fields et a1 1993, Wadowsky et al 1988]. Legionella's role as an intracellular symbiote/pathogen of both protozoan and mammalian hosts is representative of an ecological interaction only recently being appreciated and investigated by the scientific community. Barry Fields postulated "some intracellular pathogens of higher vertebrates may have acquired the ability to infect these organisms by first adapting tx> intracellular life 1J1 protozoa. Predation tn? free-living protozoa appears to be an ideal selective pressure for the evolution of these intracellular bacteria. In: is tempting t1) imagine tflmn: they subsequently acquired mechanisms for infecting higher eukaryotic cells." He concedes, however, that this "contradicts a central doctrine of evolution, that more highly evolved organisms tend to become more specialized...studies of the evolution of 26 Legionellae may have implications concerning the origins of intracellular bacteria" [Fields et a1 1993]. The epidemiological efforts to track down the sources of Legionella infections have employed a variety of different methods. While culture on specialized media remains the gold standard for detection of the organisms, other more rapid techniques are rmnxe often employed. Direct fluorescent antibody (DFA) screen of both clinical and environmental specimens is widely used [Broome et a1 1979]. The literature reports on the sensitivity and specificity of this technique are mixed. Reports of 30—80% sensitivity and 30-95% specificity suggest that reliability of results may be largely dependent on the skill of the individual technician [Tompkins et a1 1993]. More recently, a variety of molecular biology techniques have been employed. Nucleic acid hybridization based on cDNA probes complementary to specific regions of 168 RNA sequences has been. used, ‘with reported sensitivity' of 56-74% and 100% specificity [Edelstein et al 1987, Tompkins et a1 1993]. A head-to-head comparison with DFA resulted in 7 of 11 positive clinical specimens being detected by probe, while only 4 of 11 were DFA positive [Pfaller et al 1988, Tompkins 27 et al 1993]. These probes have not been used to evaluate contamination of water samples. Polymerase chain reaction (PCR) has also been employed, using primers based CH1 Legionella specific SS enmi 16S RNA and L. pneumophila specific mip and pro sequences. Sensitivity and specificity of 100% are attainable using this technique, including detection of viable-nonculturable specimens [Atlas et al 1995, Loutit et al 1990, Miller et a1 1993, Tompkins et al 1993, Yamamoto et al 1993]. The procedure has been applied to both enviromental and clinical specimens [Tompkins et a1 1993]. A similar technique, based on the ligase-chain reaction (LCR) is also being developed [Tompkins et al 1993]. Once a Legionella-positive source of contamination is identified, confirmation of the presence of the strain responsible for the infection is imperative. There are over 30 Legionella species and more than 40 distinct 'L. pneumophila subtypes described 1J1 the literature. Given the ubiquitous nature of these organisms in water and moist environments, it is common to isolate several different Legionella species/strains from a single plate of primary culture [Barbaree et a1 1993]. Subtyping procedures are necessary to make the link between the environmental and 28 clinical isolates. .Methods used include polyclonal and monoclonal serological techniques [Barbaree et al 1993, Cherry et a1 1978, Joly et a1 1986], plasmid analyses [Aye et al 1981, Ehxnni et al 1985], electrophoretic alloenzyme typing [Barbaree et £11 1993], ribotyping [Grimont et .al 1989, Mao et al 1989], restriction length polymorphism (RFLP) analysis [Pang et £11 1990, Saunders et £11 1990], nucleotide probes of DNA digests [Stout et al 1988], pulsed-field gel electrophoresis of DNA digests [Barbaree et al 1993, Ott efi:.al 1991], 16SrRNA-based speciation [Hookey et a1 1995], and repetitive element PCR (repPCR)[Georghiou et al 1994, vanBelkum et al 1993]. High levels of exposure to Iegionella among dental professionals are well documented. Serological studies from Europe and the United States show seropositive rates among dental clinic staff are substantially higher than that of the general population [Fotos et a1 1985, Luck et a1 1993, Reinthaler et al 1988]. Eufferences among the staff have been related to their level of exposure to aerosols generated in the operatory [Reinthaler et al 1988]. While most of these exposures apparently result in asympomatic seroconversions or undiagnosed Pontiac Fever manifestations (largely indistinguishable from other "flu-like" symptoms), a fatal case of Legionnaire's disease in a dentist was 29 linked to his dental operatory water [Mackenzie et a1 1994]. Surveys of the dental operatory water demonstrate that Legionella -are present at high intensities [Luck et a1 1993]. Given the high intensity of Legionella contamination of dental water, and the fact that no source of infection is established in the vast majority of all cases of Legionnaire's disease [Stout 6%: al 1992], :U: seems reasonable that exposure in the dental operatory is the source of some of these cases. However, prior to the studies reported in this dissertation, a direct comparison of Legionella prevalence and intensity in dental water as opposed to other potable water sources had not been made. Thus, while levels of Legionella contamination were known to be high, no clear conclusions regarding their significance as a possible source of exposure could be reached. SIGNIFICANCE OF ENDOTOXIN Since dental unit water commonly contains high concentrations of gram negative organisms, it is correspondingly likely to be a rich source of endotoxin. Endotoxin, a heat-stable toxin associated with gram negative bacterial cells, is composed of lipopolysaccharides (LPS). 30 These amphilic molecules consist of three regions, including O-specific polysaccharide (the somatic O antigen), core polysaccharide, and lipid A. The lipid A region is associated with the toxic effects induced and the other two regions confer the serological specificity, which is widely variable. The mammalian gastrointestinal tract is heavily colonized by a variety of gram-negative organisms existing symbiotically with their host as normal gut microflora. They produce endotoxin which is absorbed across the mucosa into portal venous tfltxxi following lHichaelis-Menton kinetics reaching levels of 1 EU/mL [Jacob et al 1977, Nolan et al 1977, Tokyay et al 1993]. The endotoxin then travels to the liver where it is removed from circulation via a biphasic clearance consisting (fl? uptake tn! the reticuloendothelial system (RES) and modification of the endotoxin to a lower density form [Mathison et al 1979, Premaratne et a1 1995]. This continuous uptake of endotoxin appears to serve a vital physiological function in priming and maintaining optimal phagocytic activity in the RES [Van Leeuwen et al 1994]. Hence, endotoxin is ea normal constituent (ME portal blood; however, it is only found systemically in disease states [Jacob et a1 1977]. 31 Pathological endotoxin exposure nay kxe seen ix) conditions with decreased RES function such as liver disease, or conditions with increased absorption that overwhelms the RES such as bmrns, trauma, radiation, gastrointestinal disease [Van Leewen et al 1994]. However, endotoxemia. is most significantly! associated vnifli gram—negative infection. and sepsis. The presence of these organisms in an abscess or as 51 bacteremia results 1J1 circulation cu? endotoxin 1J1 the systemic blood. Other modes of exposure such as inhalation [Castellan et a1 1987, Teeuw et al 1994] have only recently been appreciated. Little is known about the significance of aspiration, ingestion, inucosal (n: dermal exposure. Gram negative organisms in the biofilms of plumbing and climate— control ductwork have the potential to contribute medically significant quantities (ME endotoxin tx> their surroundings [Costerton et a1 1987, Hugenholtz et al 1992, Teeuw et al 1994]. Many of the pathogenic effects of gram negative bacteria are mediated by the endotoxin [Berczi et a1 1993, Natanson et al 1994] to the extent that the clinical syndrome present during gram negative sepsis may occur in the absence of bacteremia, and is referred to as endotoxemia or endotoxic shock [Danner et a1 1991, Graham et al 1994]. This 32 dependence (N1 the toxin rather than time organism explains why septic shock may occur even following successful antibiotic treatment of the active infection. This distinction may be hard to insist upon, given the cell-wall associated nature of the endotoxin. Nevertheless, endotoxin Imay be present even in the absence of viable, intact bacteria. A 3- to 20-fold increase in endotoxin concentration is observed following antibiotic treatment both in vitro and in vivo [Hurley 1992, Shenep et al 1988]. This is seen with both beta-lactam and nonbeta-lactam antibiotics, and occurs after an unexplained delay following antibiotic exposure. The mechanism of this increase is unknown, but both lytic and non-lytic mechanisms were suggested by the authors. Much of the pathology seen in endotoxemia is primarily due to the host response to the molecule rather than direct injury by the endotoxin itself (though endotoxin has been shown to directly damage pulmonary endothelium [Martin et al 1992]). Endotoxin stumulates the production of interleukin 1, 6 and 8 (1L1, 1L6, 1L8) [Levi et al 1993, vanDeventer et al 1990], tumor necrosis factor alpha (TNF) [Beutler et al 1987], and the activation of the alternative complement pathway and extrinsic coagulation cascade [Grahani et a1 1994, Shoemaker et al 1987]. Interleukin 1 directly causes a 33 pyrogenic reaction and. muscle proteolysis [Graham et .al 1994, Suffredin et al 1989]. The cytokines, especially TNF, damage the endothelium of the lung parenchyma and vasculature, yielding an increase in vascular permeability [Graham et a1 1994, Herbert et a1 1992, Horgan et a1 1993, Mannel et al 1987]. The resulting' pulmonary edema ‘may progress to adult respiratory distress_ syndrome (ARDS) followed by multisystem organ failure (MSOF), which is lethal in 60-90% of cases [Graham et a1 1994, Herbert et a1 1992, Horgan et a1 1993, Martin et al 1992 ]. Experimental injection of endotoxin into healthy volunteers elicits all the symptoms of endotoxemia [Herbert et a1 1992, Suffredin et al 1989, vanDeventer et al 1990]. Interestingly, while TNF appears to be the molecule principally responsible for the ‘pathogenesis (n? endotoxin-induced. tissue injury, administering TNF prior to administration of a lethal dose of endotoxin to laboratory animals actually protected them from death or injury [Alexander et al 1991]. The reason for this apparent paradox is unclear. Inhaled endotoxin significantly lowers spirometric values in otherwise healthy subjects [Castellan 6M: al 1987]. The likely mechanism is the same as that described for endotoxemia. Obviously, breathing endotoxin-tainted air has serious implication for exacerbation of chronic obstructive 34 pulmonary disease and asthma in individuals with pre- existing conditions. The clinical significance in healthy individuals is not known. However, recent studies linking airborne endotoxin to "sick building syndrome" [Teeuw et al 1994] would suggest that even uncompromised individuals are at risk for the development of medical conditions due to inhaled endotoxin. In the study correlating airborne endotoxin .to "sick building syndrome", 19 mechanically ventilated buildings were (divided into "sick" and "healthy” groups, based (N1 symptom prevalence (>15% or <15%, respectively). Airborne endotoxin levels were 6 to 7 times higher in sick buildings than in healthy ones (254 vs 46 ng/m2), both of which were higher than naturally ventilated buildings (35 ng/mz). These findings suggested ea significant contribution (n3 airborne endotoxin to the etiology of sick building syndrome [Teeuw et a1 1994]. Biofihn has been mend ix) air conditioning systems, and may represent the source of this endotoxin [Hugenholtz et al 1992]. The finding is important to the work reported here: dental waterlines have limfli biofilms within them [Mayo et a1 1990], and tremendous aerosols are generated during the course of routine dental procedures [Abel et al 1971, Belting et a1 1964, Earnest et al 1991, Hausler et al 1964, Kazantzis et a1 1961, Larato et al 1966, 35 Madden et a1 1993, Stevens et al 1963]. If dental water contains endotoxin, these aerosols could be contributing to the development or exacerbation of numerous medical conditions, run: the least of'vfiUtxi is periodontitis [Trope et al 1995, Yoshinuma et al 1994]. SUMDflUKY Infectious complications of dental care may involve nearly any site Cfll the patient, both anatomically and temporally distant from the dental care. A majority of these conditions may be attributed to the dental water as the source (n? exposure, either through ingestion, inoculation into the operative site, or inhalation of the aerosols generated by dental procedures. The full extent of the public health and occupational health implications of these complications is not yet appreciated. Within this dissertation is presented a characterization of what may be three of the most prevalent, and significant, microbial contaminants encountered in a trip to the dentist: heterotrophic bacteria, Legionella, and endotoxin. Chapter 1 Microbial contamination of dental unit waterlines: prevalence, intensity, and microbiological characteristics INTRODUCTION Microbiological contamination (n? dental water systems has been recognized for nearly a third of a century [Abel et a1 1971, Blake 1963, Clark et al 1974, Fitzgibbon et al 1984, Martin et a1 1987, Micik et a1 1969, Miller et a1 1991]. Initially, studies focused on oral microbiota and the significance of their aerosolization [Holbrook et al 1978, Micik et a1 1969, Miller et al 1971]. Later, at a time when dental water microbes were widely regarded as harmless nonpathogenic saprophytes, time emphasis shifted tx> concern over the aesthetic aspects of delivering large numbers of bacteria of any sort to patients [Abel et a1 1971, Clark et al 1974, Fitzgibbon et :11 1984, McEntegart et all 1973]. Contemporary thought is that these saprophytic organisms do have pathogenic potential and are, in fact, significant in human diseases, and modern dental microbiology reports 36 37 reflect this concept [Pankhurst et al 1993, Mayo et a1 1993, Williams et a1 1993]. Since the first recognition that dental water microbes may represent ea health. concern, numerous changes 1J1 hygienic practices and instrument design have occurred. This chapter reports the profiling of bacterial contamination of dental unit water from operatories in the western United States to evaluate what effect, if any, these changes in the practice of dentistry might have had on microbiological profiles of the water in dental lines. The scope of the contamination and the characteristics of the macrobiological populations involved suggest that there is much room for improvement. MATERIALS AND METHODS In 1992, dental unit waterline (DUWL) samples were collected :fmmn 116 three-way syringe lines, Efll high-speed handpieces and 12 scaler lines, from 150 operatories at 54 sites in Washington, Oregon and California. The sites of sampling were selected with a conscious effort to obtain representative samples from a variety of dental practice types, including private practices, university-based clinics, new practices, and well established. practices. 38 Collections were made by collaborating dentists and by colleagues at the University of Washington. Samples were taken at various times during the working day, but sampling was avoided in the early morning when stasis of water after the weekend or overnight .can lead to artificially high concentrations of bacteria. No attempt was made to flush lines immediately before sampling as the aim was to collect water representative of that issuing from instruments during typical procedures. However, in some instances specified here, tests were conducted before and after a defined flushing period. Office faucet water samples were also collected from 11 sites for comparison with the corresponding DUWL. Seven additional operatories were included whose water was supplied by distilled water reservoirs. Water samples were handled and processed according to standard practices for water quality evaluation using heterotrophic bacterial plate counts [American Public Health Association 1985]. Samples (2 to 13 Kid were collected aseptically in sterile 12 )( 75 polystyrene tubes, avoiding any contact between the instrument parts and the collection tube during the collection. Samples were shipped overnight to the laboratory, usually but not always, with a cold pack. 39 Cultures were generally established within 36 hours. Some late-arriving samples were refrigerated overnight before culture. Delays longer tjuui 36 hours are luxnni to lead to artifactual declines or increases in bacterial counts depending on the flora, although these changes are severely limited by refrigeration. Log dilutions were made in sterile water for culture on TSA plates and incubated at room temperature (~20C) for 96 hours before counting. Scores below 25 colony—forming units (cfu) on undiluted samples were recorded for statistical reasons as too few to count (TFC). Microbiological typing of colonies selected from tjme bacterial growth vans conducted according to the procedures described by the American Society of Microbiology, Clinical Microbiology Manual [American Society for Microbiology 1989]. These involve the classical microbiological techniques of recording microscopic and colony characteristics, gram stain reactivity, growth on selective media and use of biochemical typing profile reagents. The latter involved testing for nitrate reduction, tryptophanase activity, glucose fermentation, arginine dihyrolase activity, urease activity, esculin hydrolysis, gelatinase activity, beta-galactosidase activity, and assimilation of D—glucose, L-arabinose, D- mannose, D-mannitol, N-acetyl-D-glucosamine, maltose, D— 4o gluconate, caprate, adipate, L-malate, citrate, and phenylacetate by way of inoculation and incubation of convenient, commercially available Hmlti—cupule typing strips (API kits, Sherwood Medical, Plainview, New York). IREEHDUTS Seventy-two percent of DUWL samples contained bacterial populations that would qualify them as "unfit.for human consumption" (>500 colony-forming units per milliliter [cfu/mL]), according to U.S. Army standards [Abel et a1 1971] (Figure 1). Mean heterotrophic colony-forming unit counts were 49,700 (a = 156,200), with a maximum of 1,200,000 cfu/mL for the three-way syringe samples, and 72,500 (0 = 140,300), with a maximum of 550,000 cfu/mL for high-speed handpiece lines. Only 28 DUWL samples yielded plate counts classified as TFC, whereas nine of 11 office faucet sample counts were TFC. Only one faucet sample qualified as unfit for consumption (Figure 2). Most faucet samples showed zero growth, even after prolonged incubation. Samples from 12 ultrasonic scalers showed a similar pattern of severe microbial contamination (mean 19,800 cfu/mL, o = 37,300). No trends emerged with regard to 41 ill [ill] 50 OPERATORIES OPERATORIES Figure 1. Histograms showing heterotrophic bacterial plate counts (vertical bars) in cfu/mL in dental unit water samples from (a) 116 individual high-speed handpieces and (b) 54 individual three-way syringes. Note the wide range of concentrations (on logarithmic scale) and the small proportion of samples with bacteria levels in the “potable" range of 500 cfu/mL or below. Reprinted by permission of ADA Publishing Co., Inc. [Williams et al 1993]. 42 types or models of dental units and the degree of contamination of water samples, nor were there noticeable differences between geographic sites, or influences of collection and shipping. Although systematic quantitative Hucrobial typing profiles were not done on all samples, colony form characteristics were used to select and speciate as many distinct bacterial organisms as was practical from the DUWL samples (Table 1). Pseudomonas spp. predominated in most samples, but the most usual finding was a variety of identifiable genera on TSA cultures. Pseudomonas, Staphylococcus and Mi crococcus were cultured from samples collected at operatories using closed distilledwater delivery systems (Figure 3). Counts in these samples were comparable to those from units receiving water derived -from inunicipal water supplies (mean 28,300, 0' = 35,800 cfu/mL); only one sample was in the TFC category. Flushing of handpieces continuously for two minutes lowered cfu/mL, reducing counts in most cases to a third or less of the pre-flush concentrations. However, they were not reduced to TFC in any instance. 43 -' toe-1".“ - mmuunu'rwAm .. DENTAL OFFICE .lxvcls equal In or trio“ 50 CFI'lml are uprrunl a\ fill ('Fl ImL In [his figure Figure 2. Histogram showing heterotrophic bacterial plate counts in paired faucet and dental unit water samples. Log scale shows the wide range of bacterial concentrations in the dental water with only one in the “potable” range of 500 cfu/mL or below. All except one of the faucet samples had levels in the “potable" range. Reprinted by permission of ADA Publishing Co., Inc. [Williams et al 1993]. Table 1. Organisms identified in dental water samples Bacteria: Pseudomonas aeruginosa Pseudomonas cepacia Pseudomonas fluorescens Pseudomonas vesiculan's Pseudomonas paucimobilis Pseudomonas pickettii Pseudomonas acidovorans Pseudomonas testosteroni Pseudomonas stutzeri Xanthomonas maltophilia Pasteurella haemolytica Pasteurella spp. Achromobacter xyloxidans Micrococcus Iuteus Klebsiella pneumoniae Bacillus spp. Streptococcus spp. Flavobacterium indologenes Staphylococcus saprophyticus Staphylococcus capitus Staphylococcus warnen' Staphylococcus spp. Legionella pneumophila LegioneI/a spp. Ochromobacterum anthropi Alcaligenes denitrificans Acinetobacter spp. CDC Group ch—2 Fungh Alternan'a spp. Penicillium spp. Scopulan'osis spp. Cladosporium spp. 44 m0- MD WON-”I'D twuuo- "RING- moi m Figure 3. Histogram showing heterotrophic bacterial plate counts on samples from instruments receiving water from distilled water supplies. Log scale depicts wide range of bacterial concentrations in the dental unit water. Only one sample's bacterial level falls within “potable" range. Reprinted by permission of ADA Publishing Co., Inc. [Williams et a/ 1993]. DISCUSSION These findings are consistent with previous reports and show that theree has been IN) demonstrable inmrovement in water quality in the dental operatory in spite of significant changes in the practice of dentistry and infection control in recent years [Clark et a1 1974, Fitzgibbon et a1 1984, Martin et al 1987]. 45 The difference between the microbial quality of water from office faucets and operatory samples.(at least 3 orders of magnitude) suggests that the waterlines themselves are the proximate source of the contamination. This is due to the formation of biofilm on the walls of the fine bore tubing of the operatory through vflfltfli the water flows. .As reviewed above (Literature Review, page 10), these biofilms form On surfaces exposed to aqueous flow and consist of complex, dynamic, interdependent microbial populations of bacteria, protozoa, fungi, and even occasionally nematodes [Costerton et al 1987]. Adherent organisms have their origins in both the incoming municipal water supplies as well as in material sucked back into the lines from the patients. Biofilms trap nutrients, offer varied oxygenation Hdcroenvironments, and limit influx of biocides, making them ideal sites for the persistence of resident microbes and the production and release of planktonic forms to pioneer adherence at surfaces downstream. . Some (If the variation 1J1 levels of contamination between water samples seen in this study may be attributed to "clumps" of organisms being dislodged during manipulation of the DUWL in some samples and not others. The majority of heterotrophic water bacteria do not grow on tryptic soy agar, requiring specialized media or even being entirely unculturable in vitro [American Society for Microbiology 1989]. As a result, the levels of 46 microbial contamination presented in this chapter represent underestimations of time extent. of contamination actually present. Prior to this study few attempts at speciation had been reported [Clark et al 1974, Fitzgibbon et al 1984, Martin et a1 1987]. Nowhere in the literature is there reported so extensive a characterization of dental unit water flora as is presented If) this chapter. The majority (Mi organisms identified. were gram. negative pseudomonads. These "heterotrophic bacteria" or "pigmented water associated bacteria" were historically considered to be saprophytic and of little medical consequence, except to the already severely debilitated patient [Matsen et al 1975]. In recent years, however, there has txxnu a growing appreciation that many of these bacteria are actually opportunistic pathogens, and their growth in potable water supplies can have significant disease consequences even in consumers with no immunological impairment [Payment et a1 1994]. HeterotrOphic organisms have been implicated in endocarditis and gastrointestinal maladies [Payment et al 1991a, Payment et al 1991b, Zinman et al 1991] and produce numerous virulence factors, antibiotic resistance genes, and cytotoxins [Lye et al 1991, Payment et al 1994]. In addition to the medical significance of the high levels of 47 microbial fouling demonstrated in this study, the putrid odor and bad taste and texture associated with dental operatory water is a consequence of the types and quantities of organisms seen here. Some organisms identified are widely recognized as pathogens. Pseudomonas and Klebsiella have dental significance [American Public Health Association 1985, American Society for Microbiology 1989, Costerton et al 1987, Martin et al 1987, Miller et a1 1991, Slots et al 1988], and Staphlylococcus, Acinetobacter, Nocardia, Pasteurella, Serratia, Pseudomonas, Klebsiella, Legionella, and others are associated with important medical conditions as a result of infection and production of allergens and toxins, including endotoxin [American Society for Microbiology' 1989, Clark et all 1974, Martin et all 1987, Miller et al 1991]. Legionella, one of the more significant in terms of dental water contamination, will be discussed in greater detail in subsequent chapters. Dispensimg either primary (n: opportunistbc pathogens into dental patients' mouths may be responsible for post-dental care complications, including many which have no oral component. Administration of water with these characteristics is clearly undesirable, and has legal 48 consequences [Zinman 1991]. In addition to exposing dental patients to water-borne pathogens, dental professionals also receive occupational exposure [Clark et al 1974, Fotos et a1 1985, Mackenzie et a1 1994, Reinthaler et al 1987]. None of the solutions to the problem of dental water contamination discussed in the literature review have come into general use [Centers for Disease Control and Prevention 1993, Clark et al 1974, Kusnetsov et al 1994, Martin et a1 1987, Miller et al 1991, Santiago et al 1994, Williams et al 1996]. One measure growing in popularity is to use commercially available bottled water (H: saline reservoirs. Our finding of contamination on the order of that seen with municipally supplied operatories suggests that even waterlines attached to such reservoirs become rapidly colonized by a biofilm of patient-derived organisms or organisms due to a failure to naintain the system aseptically. This negates the initially perceived benefit of the sterile water supply. Contamination of the lines often extends to the reservoir, probably as a result of poor maintenance and improper handling and filling procedures. In the absence of residual antimicrobials in the water, the extent of contamination may become massive in a short time [Williams et al 1993, Williams et al 1994]. 49 CONCLUS ION Microbial contamination of dental water continues to be widespread and extensive. Organisms identified included both opportunistic and primary pathogens. The presence in dental waterlines of high numbers of these organisms, some of which are patient-derived, needs to be addressed in the context of current concerns over handpiece sterilization and infection control practices in the dental office. Until the issue its addressed satisfactorily, infection control practices based on more widespread and frequent sterilization of handpieces will remain flawed. The value of this practice in reducing risks of patient—to-patient transmission (n? pathogens :hs unquestionable. (hm: results suggest, however, that sterile instruments may become heavily contaminated with bacteria as soon as the handpieces are attached to the DUWL. Chapter 2 Legionella Contamination of Dental-Unit Water DNTRODUCTION The literature contains reports that dentists, dental students, and dental staff have higher rates of respiratory tract infections, common colds, and other respiratory ailments than the general public [Burton et al 1963, Cuthbertson et al 1954, Carter et al 1953, Mandel et al 1993, Scheid et al 1982]. Both viruses and bacteria, including Legionella, have been proposed as possible agents involved. in the pathogenesis of respiratory symptoms in dental personnel [Paszko-Kolva et al 1991]. Higher rates of seropositivity for Legionella antibodies have been found among dental personnel than among the general public [Fotos et al 1985, Paszko-Kolva et al 1993, Reinthaler et al 1988]. The aerosols generated in dental operatories during the course of routine dental care are a likely source of exposure to .Legionella spp. Water-cooled, high-speed 50 51 handpieces generate stable aerosols [Abel et al 1971] that may contain Legionella spp. As discussed in the literature review and the preceeding chapter, the complex design of the dental operatory with its long runs of tubing and intermittent water flows results in the stagnation of water within the water lines, where bacteria, including Legionella spp., can. proliferate withiri a biofilm [Pankhurst et' al 1993]. Legionella pneumophila and other Legionella spp. have been isolated from dental water [Pankhurst et a1 1990, Reinthaler et al 1986]. These organisms are often difficult to isolate because of overgrowth by other microorganisms and because Legionella spp. often are sequestered within amoebae [Anand et al 1983, Breiman et al 1990, Fields et al 1990, Fields et al 1993, Fields et al 1984, Harf et al 1987, Henke et al 1986, Newsome et al 1985, Rowbotham et al 1986, Tyndall et al 1982, Wadowsky et al 1988]. As a result, detection of Legionella spp. by viable-culture methods frequently gives variable results even from. sources believed to tme responsible for disease outbreaks [Miller et al .1993]. Historically, culture lmus been time first step 1J1 studying any’ bacteria. Only recently has there been a growing recognition of the importance of viable-nonculturable forms of many if not all environmental organisms [Atlas et al 52 1988, Byrd et al 1991, Chmielewski et al 1995, Davies et al 1995, England et al 1995, Gribbon et al 1995, Jacob et al 1993, Oliver et al 1995, Rahman et al 1996, Servis et a1 1995, ]. The viable-nonculturable form of Legionella may be clinically significant: to date there has never been a clinical isolate of Legionella obtained from ea case of Pontiac Fever, suggesting that the disease may result from an. allergic reaction ti) bacterial components rather‘ than active infection. Because of time difficulty (Hi culturing these organisms, direct fluorescent-antibody tests anmi PCR detection of Legionella spp. have been recommended for epidemiological investigations [Broome et al 1979, Miller et al 1993, Tomkins et al 1993]. These approaches have proven valuable when standard culture techniques were unsuccessful in tracing the source of Legionella. One such example was a 1992 outbreak of Pontiac fever in the United States which was linked to a resort hot tub by using PCR techniques [Miller et al 1993]. Currently, the vast majority of cases of community-acquired pulmonary legionellosis occur sporadically rather than epidemically, and the sources are never identified [Stout et al 1992]. The presence of Legionella in dental water may represent a previously unrecognized but important element of the medical history of a proportion of clinical legionellosis cases. In this chapter, the prevalence and intensity of L. pneumophila-and other Legionella spp. in dental-unit water samples was compared with that in potable water samples using time PCR-gene probe detection procedure described by Mahbubani et al. [Mahbubani et al 1990]. The hypothesis was that the prevalence and/or intensity of Legionella contamination of dental water would be higher than that of randomly collected potable water samples. FUrthermore, it was considered likely that. a molecular probe-based test woubd reveal higher rates (NE contamination than imxi been detected in previous studies that employed traditional culturing techniques. EHTEERJJULS AWHDIMEIEKNDS Samples In 1993, 119 dental-unit water samples were collected from 28 dental facilities located in time United. States (California, Massachusetts, Michigan, Minnesota, Oregon, and Washington), China, and ‘Venezuela. Included. in ‘the .study were both institutional clinics and private practices. Samples included water from high-speed drill handpiece lines, dental—syringe lines, and scaler lines. For 54 comparison, 76 potable water specimens were also collected through convenience sampling of domestic and institutional facilities and water fountains in Michigan and California. Water samples of 50 to lfifllxnl were collected 1J1 sterile containers and shipped on ice to the laboratory. PCR detection of Legionella spp. The PCR method detects all species of the genus Legionella, including viable nonculturable organisms that are net detected by conventional selective cultivation. It also identifies the presence of L. pneumophila and permits a semiquantitative evaluation of the intensity of contamination. Each water sample was filtered through a Durapore filter to trap bacterial cells, and the trapped bacteria were lysed by treatment with EnviroAmp lysing reagent (Perkin Elmer-Roche Molecular Systems, Nutley, N.J.). An aliquot of the sample was transferred ti) a reaction vessel for ammflification of the diagnostic gene sequences. The EnviroAmp detection kit was used for PCR amplification and gene probe detection. This kit includes PCR buffer, Taq DNA. polymerase, and biotinylated primers for amplification of a genus-specific region of the Legi onella SS rRNA gene (5’-poly-dT- GCGCCAATGATAGTGTG—3’ 'and 5’-po|y-dT-GCGCCGATGATAGTGTG-3’) and of 55 the L. pneumophila mip gene (5’-GCATTGGTGCCGATTTGG—3’ and 5’- GCT'ITGCCATCAAATCTTTCTGAA-3'/5’-Gl l | IGCCATCAAATCI | | | lGAA-3’) . A Quarterbath (Inotech Biosystems, Inc., Lansing, Mich.) thermal cycler was used for ammflification with 23 30-cyc1e program of 0.5 min at 95C for denaturation and 1 min at 63C for primer annealing and DNA extension. The PCR-amplified 5S rRNA and mip DNA sequences were detected by reverse dot blot strip analysis with an immobilized probe. Specific probes complementary to internal sequences of the amplified regions are immobilized on nylon membrane strips in the detection kit. Biotinylated PCR products generated by amplification with biotinylated primers are specifically hybridized to the immobilized probes. After stringent washing of the strips, the presence of hybridized biotinylated PCR products are detected. by incubating the strips with 23 streptavidin-horseradish peroxidase conjugate, washing, and adding the substrate for horseradish peroxidase. A blue dot appearing on the nylon membrane indicates the presence of bound PCR product. An internal positive control (IPC) is included as a means for detecting poor amplification or hybridization. The IPC is a synthetic DNA. sequence that is coamplified by ‘the primers for the mip gene, and its template is included in 56 the EnviroAmp PCR mixture. When the IPC fails to yield the positive blue dot, it indicates poor amplification such as may be caused by the presence of inhibitors of PCR in the environmental specimen. In an effort to overcome this inhibition, aliquots of the samples were diluted 1:10 and ‘processed 1J1 parallel vniii the undiluted .aliquots. The positive control also provides a semiquantitative basis for estimating the number of Legionella cells in the sample. The IPC corresponds to 1,000 copies of the mip gene sequence prior to amplification, and thus the intensity of the sample hybridization signal can be graded on a scale to determine the relative number of Legionella cells. An internal negative control is also included in the kit as a means (n5 ensuring appropriate hybridization stringency. The positive control probe is perfectly complementary to a sequence in the IPC amplification product. The negative control probe has a 1-bp mismatch with this sequence. When the hybridization reactions are done correctly, the PCR product generated from amplification of the IPC will hybridize with the positive control probe but not with the negative control probe. The hybridization conditions have been optimized in this system to be stringent enough to allow detection of a 1-bp mismatch between the probe and a PCR product. An agarose gel demonstrating the PCR products generated using this system is shown in Figure 1. The 108, 168, and 135 base pair products represent the amplified 58 rRNA, mip, and IPC sequences, respectively. The use of stringent L LOp. -.+i (a) Lane:1 2 3 4 5 6 7 8 Figure 1. Schematic diagrams of an agarose gel (left) and reverse dot-blots (right) of EnviroAmp” PCR products. Lane 1 is the molecular marker, 2 and 8 are blank, 3 is positive for L. pneumophila (the corresponding dot-blot is (a)). 4 and 6 are negative for Legionella (the corresponding dot-blot is (c)). and 5 and 7 are positive for non-pneumophila Legionella (the corresponding dot-blot is (b)). The 108, 135, and 168 base pair bands represent the amplified SS rRNA, IPC, and mip sequences. respectively. hybridization during the detection of these sequences allows nonspecifically amplified products to be disregarded as they are not visualized on the reverse dot blot. REKNILTS Legionella spp. were detected by time PCR amplification—DNA gene probe method in 89% of the dental—unit water samples(Figure 2), and L. pneumophila was detected in 11%. Of the 119 dental—unit water samples, 63% contained concentrations of Legionella spp. of 1,000 to 10,000 organisms per ml, whereas of 76 domestic potable water samples, only' 19% contained concentrations of Legionella spp. of 1,000 to 10,000 organisms per ml (Table 1). Fifty- three percent of domestic potable water samples had detectable levels of Legionella spp. (Figure 2), but <2% of domestic potable water samples tested positive for L. pneumophila. L. pneumophila, when present, was in the range of the lowest detectable concentrations. None of the domestic potable water samples contained concentrations of Legionella spp. of >10,000 organisms per ml; however, 12% of the dental—unit waters examined were in the category of >10,000 organisms per ml. None of the domestic potable water 59 and <2% of the dental-office water samples had concentrations of L. pneumophila of >1,000 organisms per ml. The percentage of samples positive for Legionella spp. at different dental sites was highly variable (Table 2). For example, of 10 locations around Seattle, WA, and Portland, OR, 8 provided Legionella-positive dental—unit water samples, with 4 of these sites showing evidence of L. pneumophila. The proportion of Legionella-positive samples 60 [a Dental um Water a 50 - . .9 [I Potable Water a. E 40 * a a) _ I. 30 i [If “ . a g 20 i t; ., “a .\° 1°“ x“ 0 ‘ ‘ . . " I 91.351795 <10 10-1000 100040.000 >10,000 OrganismsImL Figure 2. PCR-DNA probe test results for Legionella spp. in dental-unit water samples and potable water samples. This histogram shows that of the samples testing positive for Legionella, over 90% of potable water samples had low intensities of contamination while 37% of dental water samples had high contamination intensities of greater than 103 organismsImL. Table 1. Percent of samples positive for organisms. Dental Office Water Domestic Potable water OrganismslmL Legionella L. pneumophila Legionella L. pneumophila 399- 399- <1 1 1 89 47 99 1-100 26 9 33 1 1 00-1000 24 1 1 5 0 100040.000 28 0 5 0 >1 0,000 1 2 1 0 0 Table 2. Legionella spp. and L. pneumophila in dental water‘ Site it of samples # positive for # positive for tested Legionella L. pneumophila 9 2 28 19 12 OceanximanwN—s oorooo-soom-i A mammmmm A msmwomao —l 'Samples colected from 10 locations in Portland, OR. and Seattle. WA 61 at each site ranged from 0 to 100%. None of the samples from two large teaching institutions was positive for L. pneumophila, whereas 42% from another school were positive, several at high levels (>1,000 organisms per ml). Approximately two—thirds of all the specimens collected came from dental air-water syringe lines, with most »of the remainder collected from high—speed handpiece lines. Thirty samples from scalers were processed. The samples from lines to high-speed dental handpieces were positive less frequently than those from lines to syringes, though not significantly so. Pdthough fewer scaler line samples were evaluated, the prevalence of contamination (85%) was higher than. that seen in samples from lines supplying other instruments, though the small sample size precluded a proper comparison. DISCUSSION Our findings indicate that high levels of Legionella contamination of water may be encountered in dental operatories. The actual concentration of Legionella spp., however, varied from one dental operatory to another, consistent with the literature [Pankhurst et al 1990]. This 62 suggests that the microbial ecological conditions in dental lines may fluctuate and affect Legionella populations. Compared with contamination of domestic potable waters, the intensity (n? the Legionella contamination (Hf dental—unit water samples was at least an order of magnitude higher. The levels seen in this study in both the dental water and the potable water samples were higher than have been recorded for each previously; this may be a reflection of the higher performance characteristics of the PCR-DNA detection system relative to those of conventional cultivation procedures [Mahbubani efi:.al 1990, Stout 6N: al 1992]. Given this greater sensitivity of the PCR—DNA probe detection method [Mahbubani et al 1990], it appears that low counts (in the <10,000/ml range) may prove clinically significant only for individuals whose exposure to the aerosol occurs over prolonged periods (for example, for dental office personnel), or immunocompromised individuals [Winn et al 1988]. The growth of Legionella within amoebae [Anand et al 1983, Breiman et al 1990, Fields et al 1990, Fields et al 1993, Fields et al 1984, Harf et al 1987, Henke et a1 1986, Newsome et al 1985, Rowbotham et al 1986, Tyndall et al 11982, Wadowsky' et al 1988] is one of ‘the reasons that 63 detection of the bacteria by culture is extremely difficult. While DFA allows visualization of the intraprotozoan Legionella, the effects (n5 this sequestratmmi on PCR—gene probe detection are not known. That both PCR and DFA give similar. prevalence and intensity results suggests that effects on PCR are minimal [Atlas et al 1995]. The higher concentrations of Legionella detected by PCR than by viable-plate-count methods also raise the question as to whether nonviable Legionella species that have rm) clinical significance are detected. by the PCR. method. Relatively short amplicons, as in the EnviroAmp detection system, can be used to detect nonviable Legionella spp. [McCarty et al 1993]. Detection of Legionella spp. by direct fluorescent- antibody detection gave results similar to those by PCR detection; both methods gave results that are higher than those obtained by viable-culture methods, and both could detect nonviable rather than exclusively viable Legionella [Atlas et al 1995]. The viable-culture methods 'for Legionella detection, however, often fail, and the Centers for Disease Control and Prevention has turned to PCR for epidemiological investigations of Legionnaires' disease and Pontiac fever [Miller et a1 1993]. Furthermore, viable nonculturable Legionella spp. have been shown to be capable (bf causing pulmonary legionellosis [Reingold 6H: al 1984]; 64 exposure to high concentrations of viable nonculturable Legionella spp. may also be an important cause of Pontiac fever [Miller et al 1993]. Legionella spp., the causative agents of Legionnaires' disease, Pontiac Fever, and a variety of local infections, are ubiquitous in aquatic environments, [Dondero et al 1980, Fliermans et al 1981, Stout et al 1992, McDade et al 1977, Winn et al 1988]. Infection is most often acquired through the inhalation of aerosols containing high levels of Legionella spp. by susceptible individuals; though ingestion, direct inoculation, and irrigation of surgical wounds by contaminated water have also been implicated [Dondero et al 1980, Fliermans et a1 1981, Stout et al 1992]. Exposure to low numbers of Legionella organisms is generally not viewed as a health risk for immunocompetent individuals [Winn et al 1988]. While air-conditioning cooling towers have been considered the most likely source of heavy exposure, potable water supplies, hospital showerheads, and even vegetable moisturizers in produce markets have been implicated in past outbreaks of Legionella infections [Bolin et al 1985, Dondero et al 1980, LaMaine et a1 1990, Stout et al 1992, Winn et al 1988]. 65 Dental-unit water, especially when aerosolized, is a potential source of exposure to Legionella species [Abel et a1 1971].-Previous studies utilizing traditional culturing approaches have found that Legionella species could only be cultured sporadically, with between 9% and 40% of the dental-unit and potable water samples examined yielded culturable Legionella spp. [Borneff et a1 1986, Reinthaler et al 1986]. In. spite (Hf this sporadic: culture (detection, serological surveillance of a dental staff and a control group revealed elevated seropositivity among dental personnel. In Dresden, Germany, an elevated level of anti-L. pneumophila 8G6 antibodies was found among dentists [Luck et al 1993], particularly among those working in dental operatories where l“ [pneumophila serogroup 6 “KN” was isolated. Similar results were obtained in a study of dentists, dental assistants, and dental technicians in Austria [Reinthaler et al 1988]. In the latter study, there was a strong positive correlation between seropositive individuals and the degree of exposure to aerosols from high-speed drills and dental syringes. Thirty-four percent of the sample group was seropositive for L. pneumophila, compared with only 5% testing positive in a control group of nonmedical workers. Of the 36 positive serum samples, 13 (36%) reacted with SG6, 66 12 (33%) with SGl, 12 (33%) with SGS, and 3 (8%) with 864; 8 samples were positive for antibodies to other Legionella species. Among the sample population, dentists had the highest prevalence (50%) of L. pneumophila antibodies, followed by dental assistants (38%) and technicians (20%). In another study, 20% of the students and employees at a dental clinic in West Virginia were seropositive for Legionella antibodies [Fotos 6%: al 1985]. These results suggest that dental personnel are at an increased risk of Legionella exposure. These findings of high-prevalence/low-intensity contamination of Legionella in potable water supplies was somewhat unexpected. The concentrations seen enme higher than those ‘permitted try EKVX regulation [Hansen 1988] or those seen in previous studies based on the viable culture technique [Marrie et a1 1994, Ta et al 1995]. This finding reflects the increased sensitivity of the PCR technique [Mahbubani et al 1990, Stout et al 1992]. The low intensity seen in the majority of these samples (in the <100/ml range) is probably clinically insignificant [Atlas et al 1995, Winn et a1 1988], although more ckfima on environmental sources, their degree of contamination, and links to disease occurrences now need to be calculated for the new, more sensitive technique. It would seem appropriate for 67 regulatory agencies to reevaluate the acceptable limits for Legionella contamination of potable water, basing their rules on clinically significant intensities, taking into account the method of detection employed. lime high intensity cm? contamination vdiil Legionella, well above the 10,000/mL level, in dental-unit water samples as compared to potable water samples may be a reflection of the rich microbial biofilms commonly present along the runs of the fine-bore dental water tubing [Williams et al 1993]. In previous studies, Legionella organisms appeared to be growing within biofilm in the dental—unit water supply, often within protozoa [Pankhurst et a1 1990, Michel et al 1989]. Amoebae lmnme been SKKHI frequently 1J1 dental-unit biofilms [Michel et al 1989, Williams et al 1993]. In light of the results reported here, it is surprising that no definitive clinical associations between dentistry and legionellosis have thus far emerged. There were no related cases of human infection detected in two studies at dental institutions in Britain where Legionella spp. were isolated from dental-unit water [Oppenheim et al 1987, Pankhurst et a1 1990]. There may be several reasons for the lack of association (Hf dental-unit water anmi occurrences of Legionnaires' disease. Nonpneumonic legionellosis of the Pontiac fever type may occur in dental personnel or their patients and cause seroconversion but may be indistinguishable clinically from other flu—like episodes . experienced. by the general ’population. Legionella spp. vary with regard to their virulence. Most Legionella spp. detected in this study were not L. pneumophila; thus they would be expected to be less virulent than L. pneumophila [Winn et al 1988] because they lack the macrophage infectivity potentiator gene and other factors of L. pneumophila which are involved in cell invasion and subsequent intracellular growth. On the basis of guinea pig infectivity studies, it has been suggested that even time L. pneumophila strains commonly present in dental-unit water may have limited invasive capacity [Luck et al 1993]. While L. pneumophila was more prevalent in dental water than in potable water supplies, concentrations of L. pneumophila generally were much lower than those of the other Legionella species, and L. pneumophila was detected at levels below those normally considered to present a health risk for immunocompetent individuals [Winn et al 1988]. Interestingly, the sources of most cases of community- acquired pulmonary legionellosis are never identified [Stout 69 et al 1992]. This is because the majority occur as sporadic cases rather than as 'outbreaks, and no epidemiological investigation into the source is attempted. In order to make a definitive connection between a source and clinical disease, specific comparisons of isolates must be made between the organisms cultured from the water and the clinical isolates (when they are obtained). One method Of making this comparison employing a modern molecular biology technique is reported in a subsequent chapter of this dissertation. Unfortunately, cultures are not generally obtained from clinical specimens CM? sporadic cases. The) diagnosis of legionellosis is typically made when a patient presents with an atypical pneumonia eumi organiams are seen (x1 indirect fluorescent antibody staining of sputum, positive urine antigen test, or a rise in titer is seen on serial serology [Henkel et al .1995]. When a high index of clinical suspicion. of legionellosis surrounds ea case even. in “the absence of demonstrable organisms, empiric antibiotic treatment. is ibegun following initial serology. In. this author's limited clinical experience, when the patient recovers, follow-up titers are (fifixfll not obtained. .As a result, the total number of cases of Legionnaire's disease may be significantly under-reported. Given the findings of 70 Legionella contamination of dental water, the potential implication of dental exposure may represent a previously unrecognized but important element of the medical history of a proportion of the cases for which a source has not been found. Regardless (H? the) lack (n5 specific: clinical association, exploration of possible preventive measures ‘against Legionella spp. and other opportunistic waterborne pathogens [Williams et ail 1993] in time dental health. care setting would be prudent. Preventive strategies increasingly adopted by hospitals faced with nosocomial legionellosis outbreaks have ranged from chemical disinfection to steam sterilization to rid water lines of contaminating biofilm. Hyperchlorination and charcoal filtration have been shown to be ineffective in controlling Legionella spp. contamination of dental lines [Pankhurst et al 1990], but the application of other measures such as those based on microfiltration, germicidal flushes, or fully autoclavable fluid reservoirs and connecting tubings would clearly be appropriate. Chapter 3 Contamination of Institutional Dental Unit Water by Heterotrophic Bacteria and Legionella INTRODUCTION Microbiological profiles of dental unit water (DUW) in institutional facilities at university dental schools and in the dental clinics of large hospitals have been featured in a number of reports from North America and Europe [Abel et a1 1971, Fitzgibbon et al 1984, Fotos et a1 1985, Reinthaler et al 1986]. Contamination with Legionella species and the resulting occupational exposure of personnel have been identified as potential problems as discussed in chapter 2 [Atlas et al 1995]. The quality of water in these institutional settings may be affected by the deterioration that often characterizes jplumbing systemm; in large .aging buildings [Bezanson et a1 1992, Hart et al 1991, Memish et a1 1992]. Declining water quality has been shown to result in chronically contaminated water supplies in many medical facilities, and there is correspondingly a high frequency of 71 72 nosocomially acquired water-borne infections in hospitalized patients, including legionellosis (Legionnaires' Disease) and .Mycobacterium avium infections [Hart et al 1991, Slosarek et al 1994, Sniadack et al 1993]. Legionella, especially L. pneumophila, the species responsible for the original outbreak of acute pneumonia amongst conventioneering legionnaires that gave the disease its name [Fraser et a1 1977] is also recognized as the fourth most common cause of community-acquired (i.e. non— nosocomial) pneumonia [Mandel 1995, Ostergaard et a1 1993]. The intensity? of Legionella contamination in [NWT samples drawn predominantly from private dental offices has been shown to be much higher than in domestic potable water sources [Atlas et al 1995], although the relationship between these degrees of contamination and the occurrence of clinical legionellosis remains unknown. This chapter reports the results of 23 study which encompassed several important aspects of Legionella contamination of dental units. It is based primarily upon assembled data on the extent of microbial contamination, including Legionella, in dental unit water samples from fourteen institutions: nine in the USA and five overseas. 73 First, the presence of Legionella was detected and semi- quantitatively assessed using a PCR amplification procedure which employs genus and species specific molecular probes [Mahbubani et al 1990]. Second, primary isolation and identification of Legionella was undertaken on some samples, employing the direct fluorescent antibody (DFA) immunostaining and repetitive-element PCR-amplification (rep-PCR) product electrophoresis to identify organisms. This part of the study provided an opportunity to compare the sensitivity and utility of these approaches to the evaluation of Legionella in water samples. Third, the study permitted EH1 exploration (Hf certain critical factors that influence the outcome of the widely used Legionella-PCR environmental detection kit which is commercially available. Finally, the molecular detection method was used to investigate the potential for detection of Legionella in aerosols created around dental operatories during the use of powered dental instruments that use coolant water. The above techniques and the experiences gained in their use were subsequently applied to a series of water samples from the offices of a practicing dentist who had suffered an episode of subacute pneumonia believed to be legionellosis, 74 possibly associated with time high numbers of cmganisms in the water throughout his dental equipment. The results are discussed in terms of their significance to the emerging picture of Legionella contamination in dental ‘facilities and. the potential health importance of these findings. The advantages and shortcomings of molecular biological approaches to detection of environmental pathogens like Legionella are also discussed. hflflnEFUJULS ADHDIMEEHKNDS ‘Water samples A total of 241 dental unit water samples were processed in the course of this study. Samples in this study, as opposed to those of the studies reported in the preceding chapters, came exclusively from institutional dental clinics, i.e. those affiliated with a university or major medical center. Such clinics, in our experience, are more likely to follow strict infection-control and disinfection protocols than are private practices. As such, one might expect contamination levels to be lower than those seen in the samples from mixed populations of clinics already discussed. Conversely, these institutional clinics are more likely to be in older buildings, and use equipment that has been in service for a 75 longer period of time; these features may result in higher contamination levels than those reported in the earlier chapters. Institutional samples were furnished through the cooperation of personnel at the following sites; University of Minnesota Dental School, Minneapolis MN, lhdversity of Oregon, Dental School, Portland, (ML Forsyth School of Dental Hygiene, Northeastern University, Boston, MA, Pierce College School of Dental Hygiene, Tacoma , WA, Lansing Community College School of Dental Hygiene, Lansing, MI, Sacramento Community College, School of Dental Hygiene, Sacramento, CA, Harborview Hospital Dental Clinic, Seattle, WA, Children's Hospital Foundation Pediatric Dentistry Clinic, Cincinnati, OH, and Jackson State Prison Dental Clinic, Jackson, MI. Overseas samples were collected at the Dental Clinic of the University of Zulia, Venezuela, and at the dental clinics associated with several of the major hospitals in Beijing, China, including the Beijing Hospital, the Xian Hospital and the China-Japan Friendship Hospital. Samples collected by cooperators in the U.S. were handled as described previously [Williams et al 1993] and shipped via overnight carrier using ice-packs to keep the water cool in transit. Samples from overseas sites were hand-carried by colleagues (Dr. Neuro Guanipa, University of Zulia, and Dr. Ziao Tan Qiao , University of Beijing) on ice in coolers 76 and processed for heterotrophic plate counts as soon as possible on arrival, typically within 12-24 hours. Samples from the local site in Lansing were hand carried to the MSU laboratory on ice and processed the same day. Dental unit water from the offices of the private practitioner in San Francisco were collected by office staff approximately four months after' the practitioner' had been hospitalized for subacute pneumonia associated with El high titer of antibodies against L. pneumophila. These were-handled as specified above for the institutional samples and shipped overnight to MSU for processing. Water from a wide variety of dental hand instruments water lines was studied, including several connected to so-called “self-contained” water systems which utilize a refillable bottle as the source of water rather than the municipal tap water. Some samples came from.300 or <30 colony forming units (CFU) were considered too many, or too few to count, respectively, for statistical reasons [American Public Health Association 1985]. Every effort was made to avoid undue delays between collection and HPC determinations, since ideally these should tme carried out as siiil as possible after sample collection ti) avoid artifactual changes in bacterial numbers. Samples were routinely chilled, although this is known to alter the culturability of certain water organisms. Preliminary studies in our laboratory showed that changes in HPC numbers were not significant over the first week of storage at 4C, but that storage tinr longer periods leads ti) unacceptable declines. Mblecular Detection of Legionella Each water sample was filtered through a Durapore filter to trap bacterial cells, and the trapped bacteria were lysed by treatment with EnviroAmp lysing reagent (Perkin Elmer-Roche Molecular Systems, Nutley, N.J.). An aliquot of the sample was transferred to a reaction vessel for amplification of the diagnostic gene sequences. The EnviroAmp detection kit 79 was used for PCR amplification and gene probe detection. This kit includes PCR buffer, Taq DNA pOlymerase, and biotinylated primers for amplificatbmi of a genus-specific region of the Legionella 58 rRNA. gene HipdydT- GCGCCAATGATAGTGTG—B’ and 5’-poly-dT-GCGCCGATGATAGTGTGB’) and of the L. pneumophila mip gene (5’-GCATTGGTGCCGATTTGG-3’ and 5’- GCl'lTGCCATCAAATCTl'TCTGAA-3’/5’-G| I | IGCCATCAAATCI | l | IGAA-3') . A Quarterbath (Inotech Biosystems, Inc., Lansing, Mich.) thermal cycler was used fOr emmflification with El 30-cyc1e program of 0.5 min at 95C for denaturation and 1 min at 63C for primer annealing and DNA extension. The PCR—amplified 58 rRNA and mip DNA sequences were detected by reverse dot blot strip analysis with an immobilized probe. Specific probes complementary to internal sequences of the amplified regions are immobilized on nylon membrane strips in the detection kit. Biotinylated PCR products generated tn; amplification idiil biotinylated primers are specifically hybridized to the immobilized probes. After stringent washing of the strips, the presence of hybridized biotinylated PCR products are detected by incubating time strips with ea streptavidin-horseradish peroxidase conjugate, washing, and adding the substrate for horseradish peroxidase. 21 blue (kn: appearing til the nylon , membrane indicates the presence of bound PCR product. An internal positive control (IPC) is included as a means for detecting poor amplification or hybridization. The IPC is a synthetic DNA sequence that is coamplified by the primers for the mip gene, and its template is included in lthe EnviroAmp PCR mixture. When the IPC fails to yield the positive blue dot, it indicates poor amplification such as may be caused by the presence of inhibitors of PCR in the environmental specimen. In an effort to overcome this inhibition, aliquots of the samples were diluted 1:10 and processed in parallel with the undiluted aliquots. As a result of the pattern of inhibition experienced in processing these samples, duplicates were processed independently for comparative purposes by Dr. Claudia Thio at Perkin Elmer Cetus PCR Laboratories in Palo Alto, CA, and by Dr. C. Paszko—Kolva at Advanced Technology Laboratories in Alta Loma, CA. The positive control also provides a semiquantitative basis for estimating the number of Legionella cells in the sample. The IPC corresponds to 1,000 copies of the mip gene sequence prior to amplification, and thus the intensity of the sample hybridization signal can be graded on a scale to determine the relative number of Legionella cells. 81 An internal negative control is also included in the kit as a means (n3 ensuring appropriate hybridization stringency. The positive control probe is perfectly complementary to a sequence in the IPC amplification product. 'The negative control probe has a 1-bp mismatch with this sequence. When the hybridization reactions are done correctly, the PCR product generated from amplification of the IPC will hybridize with the positive control probe but not with the negative control probe. The tummidization conditions have been optimized in this system to be stringent enough to allow detection of a 1—bp mismatch between the probe and a PCR product. In this study as well as that reported in chapter 2, we found frequent, significant inhibition of the PCR amplification by unknown substances within the dental unit waters; thus, it was necessary to process an aliquot of such samples both directly and as a 1:10 dilution in' an effort to overcome the inhibition. This modification of the protocol, as recommended by the kit manufacturer, was modestly effective as will be discussed later in this chapter. 82 Direct Fluorescent Antibody staining The direct fluorescent antibody (DFA) procedure was employed to identify Legionella in sediments of certain samples to determine the comparability of DFA- and PCR-based techniques for the detection of these organisms in dental unit water. DFA was also used to specific identify isolates obtained using time primary isolation and selection procedure described above. Sediments or suspensions of organisms from picked colonies were dispensed (approximately 108 cells/mL) in 10 microliter aliquots onto acid washed microscope slides and allowed to dry before acetone fixation tin? 10 mdnutes. Slides were stained using pooled FITC-conjugated polyclonal rabbit antisera provided by the Michigan Department of Public Health. Following staining at room temparature for 30 minutes, slides were rinsed in PBS and distilled water, air dried, and examined by fluorescence microscopy. The above antisera have been prepared to discriminate based on two pools of antigenic characteristics (pool A: L. pneumophila serotypes, and pool B: non—pneumophila Legionella species). Once positive results were obtained with 53 pool reagent, antibody preparations (n3 more restricted specificities were applied (pool A1, A2, A3 83 etc.). These pools consisted of pool A1 (specific for L. pneumophila 8G2, 8G3, 8G4, 8G12); pool 1&2 (L. pneumophila 8G5, SG6,- 8G8, SG10, L. lansingensis); pool A3 (L. pneumophila 8G7, 8G9, SGll, SG13, 8G14); Pool B1 (L. dumoffii, 1L gormanii, 1L. longbeachae 8G1, 1L longbeachae 8G2, .L. jordanis, 1L anisa, I” tucsonensis); pool IE2 (L. bozemanii 8G1, L. bozemanii 8G2, L. feeleii 8G1, L. feeleii 8G2, L. micdadeii), pool BB (L. birminghamensis, L. cincinnatiensis, .L. lansingensis, 1L maceachernii, I” sainthelensi 8G1, .L. sainthelensi 8G2); gum) pool PM (L. hackeliae 8G1, L. hackeliae 8G2, L. wadsworthii, L. oakridgensis). Speciation beyond that obtained using time above pools was not attempted. In this study identification as to specific serotypes ‘within species yum; not considered. necessary' in order to accomplish a comparison of sensitivity and to a degree, specificity, between methods. Control tests with known reference serotypes of Legionella spp were provided by the Michigan Public Health Laboratory, and conjugated preimmunization. sera. fromi the rabbits offered ea negative control for the extent of non-specific binding with these reagents with known standards and with test samples. Legionella cultivation Primary isolation of Legionella was undertaken from certain sample collections following the procedures described by Yu et al [1987]. Water samples were centrifuged at 5000G for 30 minutes and time supernatant discarded. Sediments were resuspended in 200 uL KCl-HCl buffer, pH 2.0, and incubated at room temperature (25C) for 30 mdnutes after which they were neutralized with 0.1N NaOH. A one hundred microliter aliquot of each sample was plated on DGVP supplemented BCYE agar plates and incubated at 35C for up to 14 days, following established protocols [Centers for Disease Control and Prevention 1992]. Individual colonies that grew on the DGVP-BCYE were selected and suspended in 0.5mL sterile water, and 100 uL was plated on unsupplemented BCYE and on sheep blood agar (SBA). Those colonies which grew on BCYE but not SBA were used in these studies. Isolates of L. pneumophila 8G1 provided by the Michigan Department of Public Health were used as controls. Isolates were maintained on BCYE slants under the same culture conditions. rep-PCR fingerprinting of Legionella Repetitive PCR fingerprinting of Legionella employed an adaptation of the methods used Verslovic and others 85 [Georghiou et a1 1994, vanBelkum et al 1993, Verslovic et al 1994, Woods et al 1993]. Briefly, colonies were scraped from the BCYE plate (described above) and washed in 1M NaCl, then suspended in sterile ultrapure water. A 25 uL aliquot of this suspension was removed and subjected to repeated freeze—thaw cycles to lyse a portion of the cells. Twenty-five microliters was then placed in a) 0.5 mL thin— walled reaction tube with 50pmol each of two opposing primers. These included either ERIC2 (5'-AAGTAAGTGACT GGGGTGAGCGB’) and BG2 (5'-TACATTCGAGGACCCCTAAGTG-3’) [vanBelkum et al 1993] or REPlR-Dt (3'-CGGNCTACNGCNGCCNIIl-5’) and REP2-Dt (3'-CATCCGGNCTATTCNGCN-5’) [Georghiou et a1 1994] . In addition, 1.25mM of dATP, dTTP, dGTP, and dCTP, 2U of Taq polymerase, and 10% DMSO were present in each tube. Total reaction volume was 25uL in a PCR buffer (5x buffer included 83mM sodium acetate, 335mM TRIS-HCl, 33.5mM NaClg, 33.5uM EDTA, 150mM B—mercaptoethanol, 850ug/mL bovine serum albumin; pH 8.8). Amplification proceded with an initial 7 minute denaturation at 95C followed by 30 cycles of 30 seconds at 90C, 1 minute at 52C, and 8 Iminutes at 65C in a Perkin Elmer Cetus thermocycler. Following amplification, products were stored at -20C until they were electrophoresed on a 1% agarose gel. RESULTS Heterotrophic bacteria The results of microbiological analysis of dental unit water samples from the U.S. institutions are shown in Figure 1. Heterotrophic plate counts for samples from institutions in Venezuela and China are shown in Figures 2 and 3, respectively. The~ range (n? bacterial concentrations ‘was from <30 CFU/mL to 2.65 x 107 CFU/mL. PCR Legionella detection Prevalence and the semi—quantitative intensity assessment of Legionella contamination are illustrated. in Table 1 for samples from the institutions evaluated. Legionella pneumophila was ‘clearly not the preponderant organism detected in most samples, but in one case almost half of those samples testing positive for the Legionella probe were also positive for L. pneumophila. 87 100000000 D 10000000 .- 1000000 B D i n E a h c D a E B _. E a a E 100000 «~ 5 n g E '3 n 5 n n o I "- 10000 .. a D U E B E U D B n D 1000 . a E 0 n E 100 ~» 0 B D U U U D 10 1 2 3 4 5 6 7 8 9 Institution Figure 1. Scattergram depicting heterotrophic bacterial plate counts in USA institutional dental clinics. All samples from institution #8 are from waterlines connected to "self-contained’bottled water reservoirs where the lines are subject to routines of exposure to 5% bleach solutions usually on a weekly basis. Gross bacterial contamination is evident in the majority of samples from every institution. 100000000 CFUImL Waterline Figure 2. Histogram representing heterotrophic bacterial plate counts of dental unit water samples collected from an institutional dental clinic in Maracaibo, Venezuela. Each vertical bar represents the bacterial concentration in a sample from an individual dental unit waterline within the clinic. Gross contamination is evident in nearly every sample. 100000000 10000000 . 1000000 ' 100000~ CFUImL 10000 15‘ 1000 Waterline Figure 3. Histogram representing heterotrophic bacterial plate counts of dental unit water samples collected from four institutional dental clinics in Beijing, China. Each vertical bar represents the bacterial concentration in a sample from an individual dental unit waterline within the clinic. Gross contamination is evident in every sample. 89 Table 1. Legionella and L. pneumophila contamination of U.S. and international institutions. Source identification of U.S. samples reflects the identification numbers used in Figure 1. Institutions 5, 9, and two of the Chinese institutions were not assayed for Legionella contamination. Source # of samples with it of samples with # of samples with of the n 104 0’ Legionella >103 Legionella L. pneumophila sample oganisms misms detectable U81 6 3 3 0 U82 5 1 4 0 U83 28 15 2 0 U84 1 9 3 1 5 8 U86 1 3 6 7 1 U87 20 15 0 2 U88 12 0 0 0 China 2 5 2 0 0 China 4 5 4 1 0 Venezuela 15 1 14 O 90 Relationship between heterotrophs and Legionella A comparison of the heterotrophic bacteria concentrations for individual lines from.cmme ‘ L.P -.+ L' p - Figure 5. Schematic representation of EnviroAmp“ reverse dot-blots demonstrating the PCR inhibition observed in dental water samples. A dot at “L” indicates a positive signal for Legionella, at “p” indicates a positive signal for L. pneumophila, and “-“ and “+” represent the negative and positive controls, respectively. The first row demonstrates inhibition of the PCR reaction, as evidenced by lack of amplification in the positive control in the neat preparation, overcome in the 1:10 dilution. Row 2 shows the results of dilution in the absence of inhibition. Rows 3-4 show partial inhibition, where the positive control amplifies in the neat, but the Legionella and L. pneumophila signals are seen only in the dilution. The final row shows that very weak signals drop below the limit of sensitivity of the test when the sample is diluted, precluding processing all samples exclusively at the dilution without also looking at the neat preparation. See text for further discussion. Recognizing the constraints of the labeled probe technique revealed by these comparisons, all DNA probe results were recorded only after each sample had been examined both neat and at 1:10 dilutions in the test. .Aliquots of the samples used. to (generate the series of result shown in Figure 5 were sent, blinded, to two collaborators who were expert in the use of EnviroAmptm and other PCR modalities. Their results, shown in Table 2, suggested that variables produced by inhibitory factors confounded the interlab comparisons and. were not easily overcome by either dilution or protocol modifications introduced by the manufacturer. At Perkin Elmer Cetus, the samples were processed both neat and at 1:10 dilution, and in each case the samples were processed in both the standard way, and using a modification to remove nonspecific inhibitors. This consisted cm? the addition (n3 3% bovine serum albumin (BSA) to the preparation immediately prior to amplification. This modification permitted detection of a L. pneumophila signal in one neat sample that had not been evident under standard sample preparation conditions. Cme other sample gave interpretable results (nO- nonspecific inhibition)on1y using the 1:10 dilution with 3% BSA, despite 95 having been interpretable at MSU at the 1:10 dilution using the standard preparation. Perkin Elmer Cetus results using either the modified or unmodified protocols failed to detect seven L. pneumophila-positive signals detected by both Advance Technology Laboratory and PERL Overall, agreement Ibetween MSU and Advance Technology was 70%, between MSU and Perkin Elmer Cetus was 40%, and between Advance Technology and Perkin Elmer Cetus was 60%. A comparison of the sensitivity of the DFA and PCR techniques in detecting the presence of Legionella is shown in Table 3, using samples from Institution #4. It is clear that PCR tests positive more frequently than DFA, but there was a) troublesome inconsistency III the extent ti) which L” pneumophila could be found. Samples were seen that were L. pneumophila positive iii DFA but 1mm: in PCR/DNA tests, and vice versa. Concordance of results was high when these procedures were applied to isolates. The ability of EnviroAmptm to contribute to studies of heterogeneity of Legionella populations in samples is limited ti) differentiating .L. pneumophila :finmn non— pneumophila species. DFA, on the other hand, is well suited for this purpose. In Table 4 the results are shown of the application of this procedure to the exploration of ea set 96 of 12 non-pneumophila isolates (i.e., negative by both PCR/DNA and DFA reagents for pneumophila specificity) Table 2. Comparison of PCR-based Legionella detection in three laboratories. Fifteen water samples were processed by the three separate laboratories which were blinded to the results obtained at the other laboratories. Results noted as “inhibited” indicate that neither positive nor negative controls were amplified in any preparation. lnterlaboratory agreement ranged from 7 to 40%. - Sample MSU Perkin Elmer Cetus Advanced Technology 1 L+p+ L+p— L+p+ 2 L+p+ L+p- L+p+ 4 L+p+ L+p— L+p- 7 inhibited l_+p- L+p- 9 L+p+ L+p- L+p+ 10 L+p+ ' L+p— L+p- 12 L+p+ inhibited inhibited 13 L+p- L+p- L+p- 14 L+p+ L+p- L+p+ 15 L+p+ (spilled) L+p+ 97 Table 3. Comparison of DFA and PCR/DNA probe methods for detecting Legionella and L. pneumophila in sediment of samples from dental unit waterlines (n=19). Although the overall prevalence of L. pneumophila contamination was similar with each technique, there was not close agreement between the techniques for the individual samples. Legionella L pneumophila DFA 1 1 7* PCR 19 8“ 'Five samples positive for L. pneumophila by DFA were negative by PCR. ** Six samples positive for L. pneumophila by PCR were negative by DFA. derived from dental unit water samples collected at institution #6. Reactivities with the distinct pools of antisera to control cultures of non-pneumophila species (all of which had been isolated from clinical cases in Michigan) showed that the the group was heterogenous, with the preponderance of reactivities being to antigens in pool B2. Legionella [pneumophila was detected in the sediments of several of the samples from institution #6 with the PCR/DNA probe procedure but not the viable culture method. PCR tests of aerosol samples :fnmn operatories in this ciinic were positive for the L. pneumophila. Altogether, three of eight aerosols sampled were Legionella positive by PCR, one from an operatory where an ultrasonic scaler was used (the 98 water samples from this line had been consistently positive for L. pmeumophila by PCR) and two from operatories where highspeed handpieces were being used (water samples from both the highspeed handpiece lines had been positive for Legionella by PCR). Semiquantitiation of the DNA probe signals indicated that there were in excess of 100 Legionella organisms per cubic meter of aerosol collected in' one case. In another institutional setting (#4) vnmnme.L. pneumophila appears to have a substantial presence in the dental waterlines via EKHL DEA serogroup-specific antibodies were used to type organisms seen in sediments. The results are shown in Table 5. No reactions were seen with antisera to 861, the most common clinical isolate in the U.S. There were, however, reactions with 8G6 and SG10, the next most important subtypes of L. pmeumophila. Reactions with other serotypes were common, with some samples testing strongly positive with a variety of serotype-specific antibody conjugates. Fluorescent-stained organisms in sediments were occasionally seen iii clusters, suggesting intracytoplasmic localization within the amoebae which were commonly present in these samples. Table 4. Heterogeneity of DUW non-pneumophila Legionella isolates. DFA testing of dental unit water isolates of Legionella (non-pneumophila) from three instrument lines from institution #6. In 42% of the samples, multiple species of Legionella are detectable, demonstrating the complex heterogeneity of the DUWL Legionella populations. Reactivities of isolates are shown with pools of antisera directed against Legionella species groups. See text for details. Reactivity is recorded from - to ++++. SAMPLE SOURCE B1 32 B3 B4 ‘ Airwater syringe #11 - ++ - - Airwater syringe #11 - +++ - - Airwater syringe #11 + ++ - - Airwater syringe #1 1 - +++ - - Airwater syringe #11 + ++++ + - Airwater syringe #1 1 +/- +++ - - Airwater syringe #11 » - ++ - - Airwater syringe #11 + + - - Ainivater syringe #11 - + - - AinNater syringe #13 + - - _ Highspeed handpiece #11 + + - - Highspeed handpiece #11 + - - - 100 Table 5. Heterogeneity of L pneumophila in dental unit waterlines. DFA testing of dental unit water sediments from instruments at instituition #4. Each sample contains multiple serotypes of L. pneumophila, indicating complex, heterogeneous populations of Legionella within the dental waterlines. Reactivities of isolates are shown with L. pneumophila serotype-specific reagents. Intensities of contamination are recorded as - to ++++. WATER SAMPLE NUMBERS SEROTYPE 8 ‘I 5 16 1 7 18 1 - - - - - 2 - ++ +++ +++ ++ 3 - ++++ +++ ++++ ++ 4 - - - - - .5 ++ - - - - 6 ++ - - - - 7 +++ - ++++ - - 8 ++++ ++++ +++ ++ +++‘ 9 ++ - ++++ - - 10 +++ ++++ - - ++++' 11 +++ - - - +++* 12 - - - ‘ +++ - 13 ++++ - - - - 14 +++ - ++++ +++ - LN3 ++ ++++ +++ ++++ ++++ 'Fluorescing organisms arranged in clumps, possibly within protozoan cells within the sediment 101 rep-PCR fingerprinting of Legionella Repetitive PCR fingerprinting revealed four distinct clonal subpopulations (Figure 6). For our purposes, nomenclature of the strains detected by repPCR consisted of arbitrarily assigning the strain the identification of the colony in .which the specific pattern was identified, i.e. the pattern seen in colony #1 of water sample #1 was named "1-1", even though it is also the pattern of colony #2 of sample #3. Clone 1-1 was the most prevalent population isolated, representing 44% of the colonies isolated and present in over half of the samples from which organisms were isolated. In two of the samples, multiple distinct clonal subpopulations were identified. As these samples were also evaluated via DFA, a direct comparison of the DFA and rep- PCR results is possible, and shown in Table 6. Application of methods to a clinical case When the opportunity arose to apply Legionella detection techniques to water samples from the offices of a dentist inm) had. experienced. a «clinical episode attributed. to .L. pneumophila, I was eager to apply the above techniques to an “in vivo” situation. It 60 year old dentist in San Fiancisco experienced marked respiratory distress and was diagnosed with an acute 102 ladder MDPH 1~1 1-1 3—1 3~1 3-1 7—1 1-1ladder1-1 1-1 1-1 SampleOrigin: 1 244477 333 1-1 3-1 1-1 3-1 1-1 3—1 1-11—1 ladder 3 3 3 3 3 3 3 3 Figure 6. (a) photograph of, and (b) schematic representation of an agarose gel demonstrating the fingerprinting patterns observed between individual colonies within a single water sample, and between colonies from separate water samples. The top line of text under the schematic diagram denotes the strain identification (MDPH being the control Legionella culture provided by the Michigan Department of Public Health), and the bottom line of text indicates the water sample from which the colony was isolated. Nomenclature of the strains detected by repPCR consists of arbitrarily assigning it the identification of the colony in which the specific pattern was identified, i.e. the pattern seen in colony #1 of water sample #1 was named "1-1", even though it is also the pattern of colony #2 of sample #3. Clone 1—1 was the most prevalent population isolated, representing 44% of the colonies isolated and present in over half of the samples from which organisms were isolated. In two of the samples (3 and 7), multiple distinct clonal subpopulations were identified. 103 Table 6. Comparison of rep—PCR and DFA typing of Legionella. All samples contained multiple Legionella species, including multiple L. pneumophila serotypes, indicating a heterogeneous population of Legionella within DUWL as seen in previous tables. Despite the variety of Legionella detected by DFA, only a limited number of strains were culturable, and thus analysed by rep-PCR. Sample EnviroAmp Number of repPCR DFA antisera Number PCR results‘ colonies patterns pools reactive (organismlmL) cultured present to sample 1 >1 OOOImL 1 1-1 A1 , A2, 83 2 >1 OOOImL 1 1-1 A2, A3, 83, 84 3 >1000/mL 11 1-1, 3-1 A1, A2, A3, 81, 82Jfit84 4 >1 OOOImL 3 3-1 A1 , A2, A3, 82, '83EM 5 >1 OOOImL 0 NA A1 , A2, A3, 82 6 >1000ImL 0 NA A1, A2, A3 7 >1 OOOImL 2 1-1, 7-1 A1 , A2, A3, 83 8 >1000ImL 0 NA A1, A2, A3, 82, 3384 'Concentrations refer to non-pneumophila species, aI samples were negative for L pneumophila by PCR. community-acquired pneumonia. Ike was empirically treated with rifampin and erythromycin and made a complete recovery. Serology <1rawn (N1 presentation revealed ea L” .pneumophila titer of 1:512. No effort was made to isolate the etiological agent from his sputum, and no convalescent serum was obtained. Sediments of water samples from his office were examined using the PCR/DNA probe approach and the results are shown in Table 7. High HPC counts were detected, as usual. 104 Table 7. HPC and PCR characterization of water samples from dental unit lines in the office of a dentist who experienced a probable Legionella infection. Each of the 16 samples had high levels of heterotrophic bacteria, and 100% were positive for Legionella, the majority at high levels. Over half were also contaminated by L. pneumophila. SAMPLE NUMBER CFUImL Legionella L. pneumophila LegionellalmL 1 6300 + - >103 2 71,000 + + <103 3 620,000 + + <103 4 97,000 + - >103 5 18,400 + - >103 6 148,000 + - >103 7 79,000 + - >103 8 124,000 + - >103 9 133,000 + - >103 10 181,000 + - >103 11 50,000 + - >103 12 98,000 + - >103 13 131,000 + - >103 14 300,000 + - >103 15 320,000 + - >103 16 38,000 + - >103 'Forty isolates of Legionella were obtained from these samples. L. pneumophila was present in samples 2, 4, 9, 10, 11, 12, 13, 14, and 15, based on PCR characterization of the isolates. 105 However, all of the 16 water samples from the dental lines in the office were Legionella positive, two with L. pneumophila. The majority of the samples contained well in excess (n? the 103 organisms per ndlliliter. Approximately forty Legionella isolates were obtained from primary culture of the samples. L. pneumophila were identified via PCR/DNA probe analysis in isolates from nine of the water samples. Unfortunately, ill that rm) clinical isolate inns obtained, comparison of the rep-PCR fingerprints of the clinical and water sample isolates was not possible, precluding confirmation of the dental water as the proximate source of the infection. DISCUSSION These results permit a number of important conclusions to be drawn regarding the microbial contamination of dental unit waterlines and the issues surrounding the presence of Legionella in these lines in institutions. Overall, the data on HPC substantiate earlier findings from our laboratory on the extent of microbial contamination of coolant and irrigant water in dental equipment. 106 The data from samples collected in hospitals in Venezuela and China appear to be the first from these geographic areas to have been examined in this way, and they confirm the occurrence of heavy contamination in dental equipment of a variety of designs and origins. Institutions, which might be considered more likely to have commitments to infection control practices (such as periodic hyperchlorination episodes) than would be likely in private offices, do not appear any less likely to be delivering microbially contaminated water to patients. On the contrary, some of the bacterial concentrations were extraordinarily high, although overall the HPC profiles from large clinics and.private dental offices are very similar. The high concentrations seen in samples from dental Operatories that were equipped with “self—contained” water reservoirs routinely subjected to disinfectant flushes, were especially remarkable in tin; samples studied here. These results are a tribute to a) the readiness with which bacteria are able to get access to these supposedly “cleaner" self-contained systems, that are theoretically not subject to contamination by regrowth of municipal water organisms in the water supply, and b) the tenacity of the adherent microbes and their ability to maintain viable 107 biofilms ill the face (Hf periodic exposure tx> high concentrations of disinfectant chemicals. This finding has been reported recently by Williams and colleagues (H. Williams et al 1994] in a study of the use of self-contained water reservoirs by private dental practitioners. They found (water samples issued from dental instruments in some of these offices t1) contain more than 53 x 106 organisms per milliliter , even though the dental practitioners believed that by using the reservoirs they had eliminated contamination. Indeed, most practitioners referred to their bottled. water systems as “sterile water systems”. In a survey of the practitioners [H. Williams et al 1994], a mere 15% followed protocols necessary to maintain acceptable contamination levels in the water in their equipment lines. The high numbers of bacteria are due to the presence of well established and prolific biofilm layers inside the tubing, and their prodigious resistance to chemical dislodgment and disinfectant treatments are by now well known as a result of the published reports [Costerton et al 1987]. Biofilm organisms produce a glycocalyx slime matrix (known as extracellular polymeric substance, or EPS) which serves both as an anchor and as an anion exchange resin, capable of trapping nutrients based on their ionic charge, and preventing many biocides from reaching the resident 108 organisms. Biofilms form at many fluid—solid interfaces and are associated. with pacemakers, prosthetic joints, other medical devices, anui prolonged.tn%3 of implanted catheters. Because of their resistance to antimicrobial agents, biofilm contamination of the surface of prostheses 'necessitates their removal and replacement [Costerton et al 1987, Gristina et al 1984, Gristina et al 1988, Kluge et a1 1982, Marrie et al 1982, Nickel et al 1992, Peters et al 1981, Russell et al 1987]. It is certainly possible that the plumbing systems in some of the older educational institutions that furnished samples are contaminated with Legionella that were detected in the dental units [Bezanson et al 1992, Hart et al 1991, Memish et al 1992]. However, the amplification which leads to such high numbers of the organisms in dental unit water is most probably taking place within the long fine-bore hoses in the coolant and irrigant delivery systems. Conditions in these hoses, where prolonged stagnation is a characteristic of the use jpattern favorable to .Legionella growth, lead. to 'the heterogeneous biofilms, especially Iflxfii in amoeba content, that can result in extremely high contamination in the output water. This has been observed in previous studies of the Legionella content of water in dental clinics associated with hospital facilities, especially in Europe [Michel et al 109 1984, Michel et al 1989, Williams et al 1993]. Seroconversion of staff and students to Legionella antigens have been reported under these circumstances, suggesting a degree of occupational exposure to these environmental organisms that is unique, there being no other published reports of occupational risks for exposure to Legionella to date. Apart from the issue of Legionella and its detection and significance, the numbers of heterotrophic organisms to which dental staff are exposed is rather high. At one of the sites used in this study, the concentration of microorganisms, largely Gram—negative pigmented colony formers in all probability [Williams et al 1993], averaged more than lOG/mL. Some of these samples, collected as patients were being worked on, the samples had a mucoid appearance to the naked eye, probably because of the large amounts of carbohydrate substances.produced.iJi'the biofilm and being continually sloughed off into the water during use. This process may be aggravated by the pulling and stretchimg of the tubing during dental instrument use for procedures [Santiago et al 1995]. Although no speciation of heterotrophs was attempted in this study, it is a' reasonable that in addition to the biofilm 110 residents of an innocuous nature, there would also be opportunistic pathogens present and even some primary pathogens. They do not appear to bear any obvious relation to the presence or type of Legionella contamination, judging from the results summarized in Figure 4. Legionella were detected to similar degrees in both heavily contaminated and lightly contaminated samples, whether from high speed' handpiece lines, scalers or air/water syringe lines. This is consistent with pmevious observations 1J1 the literature, although the recently published suggestion that certain equipment brands are more subject to Legionella proliferation than others is an interesting and potentially important development if confirmed [Challacombe et a1 1995]. It suggests that specific features of equipment design may be contributory factors, and that by paying attention to them, the extent of contamination may be reduced. There was also no obvious relationship between HPC characteristics and the presence and numbers of Legionella “pneumophila, as opposed ‘to tflue genus Legionella only. The PCR/DNA. probe results and the DFA analysis of the enormous heterogeneity suggest that, while the numbers of Legionella are exceedingly high, these organisms are usually mixtures of a number of species, and tjmn: L. pneumophila, when present, may be represented as a number of serotype variants. The absence of serogroup 1 type L. pneumophila in the limited 111 numbers (ME organisms specifically typed fill one sample set here is reassuring since this is the agent associated with approximately 70% of clinical cases of legionellosis in the USA [Marston et a1 1994]. Nevertheless, other serotypes and species are then left as the causes of almost a third of pneumonic legionellosis in the U.S.; these organisms are plentiful ill the dental setting. Sui at least (flue fatal legionellosis case in a dentist, the causative organism, L. dumoffi, was obviously not in the LpSGl category [Atlas et al 1995]. These observations about the occurrence (n3 Legionella 1J1 dental unit water need now to be considered in the light of the findings reported here of the variables that affect the outcome (ME PCR/DNA detection techniques. In time past, the gold standard ikn: Legionella detection 1J1 the environment had been primary isolation. This approach is at times cumbersome and unsuccessful in identifying sources of the organism [Atlas et al 1995, Mahbubani et al 1990, Stout et al 1992, Yamamoto et al 1993]. A recent investigation into recovery of organisms from water seeded with a known concentration of organisms using viable culture detection revealed that centrifugation concentration of organisms yielded recoveries of only 4 to 32%, while use of filter concentration exhibited a 50% recovery [Boulanger et al 112 1995]. For this reason tests such as DFA and the newly available [flux amplification procedures appear ideal. Interpretation can however be problematic, and although the procedures are finding more and more favor in the investigation of clinical episodes [Miller et a1 1993], the results of this study make it clear that care must be taken to avoid implicating a water source as the source of clinical infection merely because Legionella are present. With the increased sensitivity of the modern techniques it is possible to detect clinically insignificant contamination. The utility of the PCR/DNA test system is clear, and its superior sensitivity in detecting the presence of Legionella contamination over primary culture and DFA are becoming well known. The better detection rate of PCR over DFA. when applied to dental unit samples seen here (Table 3) was not surprising, but the poor correlation with L. pneumophila detection between the two techniques was disappointing. Obviously, from the proven influence of unknown inhibitors on the outcome of PCR tests, there remain to be identified variables that are by no means insignificant. Whether or not there are interfering factors in the test outcome that are attributable to the presence of multiple .species and serotypes of Legionella in one sample mixture is unclear, 113 but the findings with the samples from the dentist's office in San Francisco suggest such is the case: the L. pneumophila PCR detection rate on water sediments was low, but the primary culture results indicate that pneumophila organisms were present and isolatable from the majority of the samples. The inconsistency of outcomes in PCR/DNA probe applications is very important for the adoption of PCR technology for environmental sampling. The inconsistencies that surfaced when identical samples were processed by three different laboratories suggest that the issue of inhibitors and their practical significance if; still cloudy aumi needs further study. PCR as a means of environmental evaluation is certainly rapid in comparison to culture and isolation, but it is expensive and if samples need to be routinely examined at two dilutions, and perhaps by both standard and modified (BSA addition) protocols if] order tx> get optimally interpretable results (i.e. minimal false negatives) the cost will be prohibitive. However, cost is not the most important factor. This kit is designed to detect Legionella in environmental water samples, samples likely tx> contain PCR inhibitors similar to those in dental water. Given the EPA's “zero tolerance” level for Legionella in potable water, the potential failure to detect lower levels of 114 contamination in the presence of inhibition of the PCR is unacceptable. It raises serious .questions about the suitability of EnviroAmp Legionella or similar kits for use in other ‘than investigational applications. 'There is .a trend towards use of the same PCR primers in clinical sample processing and to date the problems of inhibition have not appeared [Matsiota-Bernard et al 1994]. The possibility that it might needs to be cmmsidered. It seems likely that the combination of culture/isolation and PCR-based characterization of Legionella isolates from different environments will be extremely useful in clarifying the epidemiology of Legionella, and in the process, modifications of the PCR protocol may be developed that will overcome the difficulties. Certainly the convenience of the PCR approach is attractive and permits collection of data in circumstances that would otherwise not be likely to yield results. In the analysis of aerosols, for example, PCR techniques are now beginning to find acceptance [Alvarez et al 1995, Palmer et al 1995, Sawyer et al 1994], although the experience with Legionella to date appears to be very limited. The observation recorded here that Legionella, including L. pneumophila, was detectable in aerosols to which staff are occupationally exposed. in aa dental setting, has important implications. 115 Some of these relate to the practicalities of infection control in the idental office, and. others revolve around legal and regulatory aspects of on-the-job exposure as discussed in chapter 2. Of course the central issue of the clinical significance, if any, of the high levels of Legionella in the samples processed fill this study MHJJ. remain controversial, ill the absence of prospective analysis of exposure rates and risk assessment. There were no related cases of human infection detected ix) two studies an: dental institutions ix) Britain where Legionella spp. were isolated from dental-unit water [Oppenheim et al 1987, Pankhurst et al 1990]. There may be several reasons for the lack of association of dental-unit water auxi occurrences (Hf Legionnaires' disease, including nonpneumonic or other unrecognized legionelloses, low virulence of the dental-water-derived organisms, or other situations as discussed in chapter 2. Interestingly, the sources of most cases of community- acquired pulmonary legionellosis are never identified [Stout et al 1992]. This is because the majority occur as Sporadic cases rather than as outbreaks, and no epidemiological investigation into the source is attempted. In order to make a definitive connection between a source and clinical 116 disease, specific comparisons of isolates must be made between the organisms cultured from the water and the clinical isolates. One method of making this comparison is by employing a modern molecular biology fingerprinting technique such as rep—PCR. This technique exploits tflua naturally—occurring repetitive palindromic sequences interspersed throughout the genome of many prokaryotes. Using PCR primers based on these sequences, one may amplify numerous segments of the genome, with the fragments varying in number and size even between closely related strains [Verslovic et al 1994]. The results of the present study demonstrate that it is possible to obtain a molecular fingerprint of the organisms in dental water utilizing repetitiwe PCR technology. The application (n? this technique tx> compare operatory-derived and patient-derived isolates could prove useful in firmly establishing or disproving a clonal relationship between the isolates, though the natural fluctuations in Legionella populations and the existence of nonculturable strains will preclude its utility in all instances. Such an approach may kxe a useful tool 1J1 epidemiological studies cm? the relationship between dental operatory Legionella contamination and clinical legionellosis. 117 Unfortunately, cultures are not generally obtained from clinical specimens (ME sporadic cases. The diagnosis of legionellosis is typically made when a patient presents with an atypical pneumonia and organisms are seen on indirect fluorescent antibody staining of sputum, positive urine antigen test, or a rise in titer is seen on serial serology [Henkel et al 1995). When a high index of clinical suspicion of legionellosis surrounds a case even in the absence of demonstrable organisms, empirical antibiotic treatment is txxnni following initial serology. In this author's limited clinical experience, when the patient recovers, follow-up titers are cflfixni not obtained. .As a result, cases of Legionnaire's disease may be significantly under-reported. Given time findings (If Legionella contamination of dental water, the potential implication of dental exposure may represent a previously unrecognized but important element of the medical history of the cases for which a source has not been found. Over 30 Legionella species and more than 40 distinct L. pneumophila subtypes are described in the literature. Given the ubiquitous nature of these organisms in water and moist environments, i1: is common ix) isolate several different Legionella species/strains from a single plate of primary 118 culture [Barbaree et al 1993]. In spite of this, a previous study of dental clinic waters [Luck et al 19931 found that a stable, clonal population of L. pneumophila SG6 was present longitudinally, which suggested that Barbaree's observation might not hold true for dental water samples. Our findings are more consistent with the former report. DFA revealed multiple serogroups in all samples, and multiple species in all but one sample. Of these, we were able to culture and rep-PCR fingerprint four distinct clonal populations. Eight colonies each were identified as clones 1-1 and 3-1, and in both cases these colonies were isolated from more than one sample. While these clones were the most prevalent strains among those isolated via culture, they' may not represent the most prevalent subpopulations actually present in the original samples. There may be many other, more prevalent, strains present which. are run: as amenable to isolation via culture as are clones 1-1, 3-1, or even 7-1. Viable nonculturable Legionella spp., shown to be capable of causing pulmonary legionellosis [Reingold 6%: al 1984] and Pontiac fever [Miller et a1 1993], are detected by the PCR- gene probe method but not the viable culture method [Negron- Alvira et a1 1989, Paszko-Kolva et al 1991, Stater et al 1987, Yamamoto et al 1993]. Our inability to culture, and thus fingerprint, the viable-nonculturable organisms 119 represents an important limitation to this study. the are unable to include them in any cfiscussion of the genotypic clonality or heterogeneity of Legionella populations based on the current study. ‘The presence ci'tn«> of the clones in nmltiple operatories within a single practice suggests the source for the organisms within the cmeratory lines originates upstream, possibly derived from the building plumbing or municipal water supply. Clones such as 7-1, which were only isolated from a single operatory, may represent strains of the practice-wide water system which have been selected by the ecosystem within the individual operatory, or patient- derived origins (M5 the contamination“ .Although person-to- person transmission of Legionella has never been demonstrated, the organisms are present in sputum and could easily be introduced into the dental line via the suck-back phenomencmu However, given the close similarity' of the banding patterns stains isolated 1J1 this study, especially compared to the divergence seen from the control strain, the former option is most likely the case in the practice on which this study is based. A comparison of subpopulations isolated longitudinally from these operatories to investigate the progress of genetic divergence of Legionella 120 clones within the individual ecosystems of each operatory is a topic worthy of further study. The presence (Hf L. pneumophiLa in dental water should not overshadow time potential contribution CHE other waterborne opportunists and pathogens to infectious complications of dental procedures as discussed in previous chapters. Ely et al [1993] has raised the question of how frequently unrecognized instances cm? infectbmn might fellow invasive dental surgery, where this feature of the clinical history was not elicited. Whatever the true extent of the problem, the steady accumulation of evidence of widespread and serious contamination of dental water can no longer be ignored in the context of responsible infection control in the dental office. Chapter 4 Endotoxin contamination of dental water IlflERCEflflETICEI Endotoxin, a heat-stable toxin associated with gram negative bacterial. cells, 113 composed (n3 lipopolysaccharides (LPS) derived from the bacterial cell wall. Most of the pathogenic effects seen in gram-negative bacterial infections are mediated by endotoxin [Berczi et al 1993, Natanson et al 1994]; the associated clinical syndrome may even occur in the absence of bacteremia [Danner et al 1991, Graham et a1 1994]. Endotoxin has been implicated in the pathogenesis of hepatotoxicity, hepatorenal failure, hepatic encephalopathy [Lang et al 1993, Odeh et al 1994, Shibayama et al 1992], periodontitis [Trope et al 1995, Yoshinuma et al 1994], mastitis [Tyler efizial 1994], adult respiratory distress syndrome [Graham et a1 1994, Herbert et a1 1992, Horgan et al 1993], disseminated intravascular coagulation [Graham et al 1994], humidifier fever [Flaherty et al 1984, Mamolen et al 1993], and sick building syndrome [Teeuw et al 1994]. 121 122 Dental unit waterlines are lined by a thriving biofilm which is composed. predominantly' of gram. negative heterotrophic bacteria [Mayo et al 1990, Oppenheim et al 1987, Pankhurst et al 1990, Pankhurst et al 1993, Williams et al 1993, Williams et al 1994]. These organisms are released in high numbers into the dental unit coolant and irrigant water and' delivered through the cdistal outlet of the (dental instrument. With them is delivered the potential for endotoxin exposure. In medicine, endotoxin concentrations in fluids have to be carefully controlled and United States Pharmacopoeia (U.S.P.) standards for irrigation and parenteral fluids must be observed [U.S. Food and Drug Administration 1987]. Despite the fact that there are numerous reports of gram- negative bacteria in dental water, there are no published reports (x1 endotoxin, though there is (Hue abstract of 23 presentation on the topic [Bourassa et al 1995]. The Centers for Disease Control and Prevention currently recommends that U.S.P. sterile water be used for all dental surgical procedures, enui this stipulation requires use (Hf “pyrogen—free” irrigant [Centers for Disease Control and Prevention 1993, United States Food and Drug Administration 1987]. 123 Three questions were asked in this study: (1) Are there detectable levels of endotoxin in dental water, (2) Is there any correlation between the number of bacteria and the level of endotoxin present in dental water, and (3) Is endotoxin aerosolized in detectable levels by routine dental procedures? EH¥EERJJULS AQHDIME15KNDS 'water and aerosol samples Water samples were collected from the dental lines of institutions #6 described in the previous chapter and handled anmi processed according tx> standand practices for water quality evaluation using heterotrophic bacterial plate counts [American Public Health Association 1985] as described in Chapter 1. Samples (2 to 3 mL) were collected in sterile, pyrogen-free 12 X 75 polystyrene tubes, avoiding any contact between the instrument parts and the tube during collection. Samples were shipped overnight to the laboratory with a cold pack. Aerosols were cmdlected using 5M1 Anderson sampling vacuum pump (Anderson Co., Atlanta, GA) and AGI-3O glass impingers (Ace Glass Co., Vineland, NJ) containing sterile water, as 124 described by Trudeau et al [1994]. In this procedure, a calibrated vacuum generator draws a measured volume of air containing the aerosol into glass vessels (impingers) set up a specific distance from the aerosol source. Over the course of a known time interval, aerosolized particles in the sample strike the surface (n? the sterile collection fluid within. the impinger" and. are retained. therein. In this study, impingers were set up at a distance of 60 cm from the area of dental work in a patient's mouth and a total of 0.33 cubic meters of air were sampled for each of eight tested operatories: four from operatories using ultrasonic scaler lines and four from. high speed handpiece lines. Control collections done in the early morning prior to the generation of any dental aerosols were collected for comparison. Samples were shipped on ice via overnight courier and the aerosol particles retained in the collection fluid were analyzed for endotoxin. In view of the natural day-to-day fluctuations of bacterial contamination in dental water [Santiago et al 1994], both water and aerosol samples were obtained on nmltiple occasions, ultimatelyr providing 4Y7 separate ‘water-aerosol pairs for our analyses. 125 Bacterial contamination Heterotrophic bacterial contamination of time water samples was assessed by plating lOO uL aliquots of serial dilutions of each sample onto R2A plates and incubating as previously described [Santiago et al 1994, Williams et a1 1993]. Plates .with >300 or <30 colony forming units (CFU) were considered too many, or too few to count, respectively, for statistical reasons . At each of the 8 aerosol collection sites (four from scalers, four from highspeed handpiece lines), additional collections were made using Anderson plate chambers. In this procedure, air is drawn over the surface of agar plates through steel plates which deflect the particles onto the surface of the R2A agar for subsequent incubation and colony formation. In this way, the number of bacteria may be roughly determined and the resultant colonies permit standard microbiological isolation and identification methods to be applied. Endotoxin Assay The presence of endotoxin in the samples was assessed using the Limulus amoebocyte lysate (LAL) test [U.S. Pharmacopoeia 1993], commercially available as the PyrotellTM kit (Associates of Cape Cod, Inc.). The LAL assay is H5 exquisitely sensitive to endotoxin and mandates devoted adherence to the use of endotoxin-free glassware and plasticware (including tubes and. pipette tips), diluent, etc. The assay was performed in accordance with the specified protocol. Briefly, water samples were serially diluted in pyrogen—free water and a 0.1 mL aliquot of each dilution was added to 0.1 mL of LAL, vortexed, and incubated at 37C for 1 hour. A positive test result consists of the formation of a gel (as the lysate coagulates in response to the endotoxin) that is stable on inversion of the tube. This corresponds to the presence of 20.03 endotoxin units (EU) per nulliliter. The level (Hf endotoxin if} the cmiginal sample is calculated by multiplying this value by the dilution. Commercially available endotoxin standards were used as controls. IflBSEHIPS Consistent with all studies to date, the extent of bacterial contamination in the dental waters sampled for this investigation far surpassed the levels associated with potable water, with counts in excess of 2.0 x IIW CFU/mL in some samples. Correspondingly, high concentrations of endotoxin (up to 15,000 EU/mL) were present. 127 The relationship between bacterial concentrations and the results of the endotoxin assay are shown in Figure l. A positive correlation of 0.33 between EU/mL and CFU/ml. is demonstrated, which is significant at p <0.05. Collection of aerosols from the institutional clinic dental lines resulted in the accumulation of a varied mixture of bacterial organisms which were rapidly overgrown by abundant growth of a number of fungal varieties. This precluded quantification of bacterial numbers in aerosols. Nevertheless, a variety of bacterial forms were isolated, including Gram positive cocci, Gram positive coccobacilli, Gram negative cocci, and Gram negative bacilli. Not all of the isolates were identifiable using standard microbiological and biochemical profiling techniques, but of those that could be definitively speciated, the following were found: Staphylococcus epidermidis, Staphylococcus aureus, Staphylococcus haemolytica, .Micrococcus spp., .Micrococcus varians roseus, Pseudomonas vesicularis, Sphingomonas paucimobilis, Acinetobacter spp., and Flavomonas spp. Other distinct organisms in the gram- negative pigmented colony forming categories were plentiful but did not provide satisfactory profiles in any system for identification. 128 100000 y = 0.0214xo-695 R2 = 0.3266 I. 100001 1000 ~- 100 -- EU/mL 10 0.1 4r 0.01 § f i 1 100 1000 10000 100000 1000000 10000000 CFUImL Figure 1. Correlation between bacterial contamination and endotoxin levels in dental waterline samples. A positive correlation of 0.33 between bacterial load and endotoxin level, signifimnt at p<0.05, is evident. 129 All of the aerosol samples collected in impingers contained detectable endotoxin, though the concentration did not exceed 2.7 EU per cubic meter in any instance. No endotoxin was detectable in control air samples. DISCUSSION Water obtained from properly functioning distillation, reverse osmosis, and ultrafiltration systems generally has undetectable levels of endotoxin [Associates of Cape Cod 1990]. Clearly, the concentrations seen in the dental water samples far exceed any acceptable standard for water intended tin: medical purposes. The ‘usuallgr acknowledged range of 0.06 EU/mL to 0.5 EU/mL for medical devices (including liquid devices), depending on application, applies to irrigation fluids and is regulated by the federal government [U.S. Food and Drug Administration 1987]. The presence of endotoxin in dental water has potential clinical significance, both medically and dentally. Endotoxin stimulates time production (ME numerous cytokines which result in tissue injury [Graham et al 1994]. These may 130 inhibit healing following dental or periodontal treatment. The significance of endotoxin in the pathogenesis of periodontitis is well documented [Trope et al 1995, Yoshinuma et al 1994], and irrigation of highly vascular mucosal lesions with endotoxin-laden water during treatment for this condition is, at the very least, inappropriate. Irrigation of the site of any dental intervention with endotoxin-laden water has the potential for introduction of the endotoxin into the patient's bloodstream. Experimental injection of endotoxin into the systemic circulation of healthy volunteers elicits the signs and symptoms of endotoxemia, including fever, elevated white blood cell count, elevated blood concentrations of stress hormones, and decreased blood oxygenation [Herbert et al 1992, Suffredini et al 1989, vanDeventer et al 1990]. Endotoxemia is typically associated with gram-negative infection and sepsis, but significant exposure via inhalation [Castellan et all 1987, Teeuw’ at 511 1994] has recently been reported. Little is known about the significance of aspiration, ingestion, mucosal or dermal exposure. 131 Gram-negative organisms in the biofilms of plumbing and climate-control ductwork may contribute medically significant. quantities CH? endotoxin ti) their‘ surroundings [Costerton et al 1987, Hugenholtz et al 1992, Teeuw et al 1994]. Inhaleml endotoxin significantly' lowers spirometric values in otherwise healthy subjects [Castellan et al 1987]. Breathing endotoxin-tainted air has serious implications for exacerbation of chronic obstructive pulmonary disease and asthma in individuals with pre-existing conditions [Michel et al 1991]. The clinical significance in healthy individuals is not known. However, recent studies linking airborne endotoxin to "sick building syndrome" [Teeuw et al 1994] suggest that even uncompromised individuals are at risk for the development of medical conditions due to inhaled endotoxin. While there is no specific concentration of airborne endotoxin above which is defined as hazardous, experimental animals show clear respiratory dysfunction at 0.3 ug/m3. In the above study correlating airborne endotoxin to "sick building syndrome", 19 mechanically ventilated buildings were divided into "sick" and "healthy" groups, based on symptom prevalence (>15% or <15%, respectively). Airborne endotoxin levels were 6 to 7 times higher in sick buildings than in healthy ones (254 vs 46 ng/m3), both of which were 132 higher than naturally ventilated buildings (35 ng/m3). These findings suggested 53 significant contribution (n5 airborne endotoxin to the etiology of sick building syndrome [Teeuw et al 1994]. Biofilm found in air conditioning systems may represent time source (n? this endotoxin [Hugenholtz 6%: al 1992]. That finding is important to the work reported here: dental waterlines have lush biofilms within them [Mayo et al 1990], and tremendous aerosols are generated during the course of routine dental procedures [Abel et al 1971, Belting et al 1964, Earnest et al 1991, Hausler et al 1964, Kazantzis et al 1961, Larato et al 1966, Madden et al 1993, Stevens et al 1963]. The endotoxin-laden aerosolized dental water could be contributing to the development or exacerbation of numerous medical conditions, especially in occupationally exposed staff. In this study, I have demonstrated that operatory air contains detectable levels of endotoxin in addition to a variety of airborne microorganisms. Further study will be needed to determine whether these aerosols represents contamination from time waterlines, time airlines [Ely et al 1993], or a combination. In light of the significant correlation demonstrated in Figure l, the proximate source of the dental water endotoxin HE contamination is likely the gram-negative organisms resident in the dental waterline biofilms [Mayo et al 1990, Oppenheim et al 1987, Pankhurst et al 1990, Pankhurst et al 1993, Williams et al 1993, H.N. Williams et al 1994]. The correlation. between. bacterial and.iendotoxin contamination levels observed in this study is at odds with recently reported findings from another laboratory [Bourassa et al 1995]. In that study, a correlation between the levels was sought as a “simple test for monitoring .bacterial contamination”. They found no significant relationship existed between CFU and endotoxin concentrations even when CFU's varied by up to three orders of magnitude. They proposed that this lack of relationship may be due to bacterially produced inhibitors of the Limulus chromogenic test employed in their investigation. The LAL assay may, in fact, be less prone than the chromogenic test to such inhibitors. Other explanations could relate to sample size, variation iii aliquot handling’ protocol and 'technique, or contamination of diluents or labware by exogenous endotoxins. Whatever the cause of the differences, while it is not an adequate basis on which to compute bacteria per milliliter, the correlation between endotoxin and CFU/mL is clear in this study. 134 In addition to time clinical implications (n5 dental water endotoxin, there are legal ramifications. On top of the obvious malpractice liability incurred by exposing patients to massive amounts of endotoxin, there is legal risk from an employee occupational health perspective. The Indoor Clean Air Act Ammendments of 1990 mandate that within buildings, exposure to airborne pollutants cannot exceed levels of exposure in the local outside air [Hodson et al 1994]: dental office airborne endotoxin exposure may cross that threshohd. Blaming an exacerbation of chronic obstructive pulmonary disease or asthma on workplace exposures could lead to both civil and criminal penalties. The endotoxin literature contains an interesting report on the inhibition of Legionella growth within endotoxin-treated macrophages [Egawa et al 1992]. In this study, macrophages fitmi a murine strain permissive for Legionella growth became highly resistant to growth of the organisni when pretreated with endotoxin. This enhanced cytolyic activity occurs at some point subsequent to the initial bacteria- macrophage interaction. in: is tempting ti) speculate that the high levels of endotoxin delivered in dental water might exert some sort (ME protective effect, contributing ti) the infrequency of severe legionellosis in those exposed [Fotos et al 1985, Reinthaler et al 1988, Luck et al 1993] to the 135 high levels of Legionella present in dental water [Atlas et al 1995]. In conclusion, this study has demonstrated that dental unit water contains high concentrations of endotoxin and that there is as statistically significant positive correlation between endotoxin and time bacterial load. present. This water is readily aerosolized during routine dental work. Exposure to either the endotoxin-laden water or the aerosolized endotoxin represents a potential health threat. Further epidemiological and cflinical studies of the consequences of dental endotoxin exposure as well as evaluation of means by which the exposure may be prevented are warranted. APPENDIX APPENDIX, PART 1: Bombesin-like Neuropeptides in Nematodes: Literature Review Filarial parasites are responsible for debilitating diseases that affect several hundred million people world-wide. Although a variety of treatments can be used to decrease the severity of symptoms, there is no effective, safe means of curing patients of filarial infection. Not enough is known about the parasites' physiology to predict which systems or pathways may provide suitable targets for pharmacological intervention. This study was undertaken ti) begin characterization of 53 previously undescribed physiological system within the adult nematode: bombesin-like peptides and their binding sites. This system is a potential target for therapeutic interventions. By way of background, a brief review has been assembled of the major features of the filariae, the 'ways these confound current therapeutic approaches, and the anatomical and 136 137 physiological peculiarities that may be exploitable as targets for therapeutic interventions. REVIEW OF FILARIAL DISEASE At least eight species of filarial worm infect a total of over half a billion people in the world today [Wharton et al 1986]. The manifestations of these diseases vary widely, from the subcutaneous "Calabar" swellings caused by Loa loa to the grotesque elephantiasis (Figure 1) induced by Brugia and Wuchereria. These infections exact a high toll in human suffering and lost productivity from those who can least afford it: the masses living in tropical developing nations. The life cycle of these parasites (Figure 2) is simple to comprehend, but difficult to interrupt. Nficrofilariae (Ll larvae) are taken up in blood meals by a biting vector (several species of nmminnii) for lymphatic filariae, black flies for Onchocerca and deerflies for Loa). In .the vectors, they undergo two moults to the infective L3 stage which is transmitted to another human host during the next meal. The adult worms develop in the tissues, breed, and produce Inicrofilariae. 'These Inicrofilariae then. migrate through the skin (in the case of Onchocerca) or blood Figure 1. Sudanese elephantiasis patient (photo by MKH). 139 In Human Host In Insect Vector L2 Figure 2. Filarial life cycle (lymphatic filariae) and guie eventually 'taken in) by’ the insect vector. Onchocerca volvulus causes perhaps the most dramatic filarial disease. Following transmission by the bite of an infected blackfly, the worm develops to the adult stage over the course of approximately one year; A. granulomatous reaction consisting (n? mononuclear‘ cells enmi eosinophils forms the characteristic onchocercal nodule around the adult females. Within these nodules, the parasite may live for decades [Plaisier et al 1991, Remme et al 1990]. The male worms appear to migrate between nodules, fertilizing the um females dwelling therein [Collins 6%: al 1982, Duke 6%: al 1990]. The gravid female produces large numbers of microfilariae which then migrate through the skin and subcutaneous tissues. During the (Simmnims or so that these nucrofilariae remain alive, a ‘variably effective immune response is directed against them. This appears to be due to active immunosuppression (Hf local reactivity in; the ndcrofilariae [Akuffo et al 1996, Elkhalifa et al 1991, Soboseay et al 1994, Williams et al 1986]. However, upon their death a localized inflammatory reaction occurs. .Although directed against the "foreign body" of the worm, the eosinophils, leukocytes, and interleukins called into action also cause damage to the host tissue. This small reaction, multiplied by the huge number of dead microfilariae per square centimeter of skin, can result in a very severe rash known as onchodermatitis (Figure 3). Just as inflammation results in the skin rash, reaction to microfilariae dying in the cornea leads to punctate keratitis and scar formation terminating in sclerosing keratitis and permanent blindness at EN) early age (Figure 3). This expression of disease is known as River Blindness, 141 because infection occurs only along fast moving rivers in which the insect vector can reproduce. In spite of the morbidity induced by O. volvulus, there are no safe, effective means of eliminating the infection. As is the case with most helminthic infections, the immediate goal of intervention is reducing the clinical «disease, whether or rmn: this includes elimination (n? the parasite. This is a legitimate and realistic goal in one sense, for symptoms in helminthiases are often proportional to the number of worms present: reduction of the worm burden can eliminate the disease state. There is "no direct evidence of protective immunity in man against reinfectitui with filaria." [UNDP 1989]. Thus, despite the advent of molecular biology and the ability to clone and mass-produce select antigens, there is no prospect of an effective vaccine on the horizon. Therefore, the most promising approach to intervention is pharmacological. However, anthelmintics that are effective on luminal- dwelling nematodes are generally not effective against filariae. Often, their mode of action targets the worms' motility apparatus, resulting in paralysis and expulsion from the intestine by the host's peristalsis. Such a strategy is not effective in the tissue-dwelling worms. For Figure 3. Sudanese onchocerciasis patients demonstrating (a) depigmentation onchodermatitis, (b) Sowda onchodermatitis - note the increased pigmentation of the right leg, and (c) river blindness (photos by MKH). 143 this reason, other targets have been exploited, with varying degrees of success. As can be seen in Table 1, both microfilariae and adults (macrofilariae) have been the focus of drug actions. Microfilariae are the stage most often hit by medications, and such an approach to therapy has the potential of reducing symptoms of infection as well as transmission of the parasite. Their side effects result mainly from the host's reactions to the sudden death of the larvae. This acceleration of the disease process is known as the "Mazzotti" reaction. Unfortunately, agents which exert no effect on the adult worm leave the patient open to problems caused by the next batch of microfilariae shed from the intact adults. As a result, even the best microfilaricide in use today (ivermectin) must be administered twice annually1 for' decades ti) prevent overt disease. Such a requirement presents logistical problems that, in many areas where the disease is endemic, are insurmountable. Macrofilaricides have greater potential for affecting worm populations. By eliminating the adult worm, production of microfilariae is halted. Used in conjunction with a microfilaricide to clear the patient of existing microfilariae, mass treatments with an adulticidal drug w 32955.82 32955.82 50m 50m 32283922 32955282 32235992 accesses: $532 990m 99mm 29822 29322 2880.2 938 2:2 28822 .363: 82.852: 2.832350 300 F m_m_coE_E< Em__onm~oE 9.25920 .E:__o£_co _ swoop .mczmQE .Em__on£oE 29350950 m_mo_comc< 290m. ocsEE_ sec: 89.839: 30.0 F 283528 .5386 Lm_:om:EoSoz 282.93 33: ommsEm .ommp»%3_oac-.m .383 me. o mo 0.0 555 gap YN .99: a Sweep conqmomfiw‘mtnm resonam $252.3 a 8355258. 9.82, m . 3.32995. mm_onp_E_~com 82.3525 .2525 .mommci 5205 9.3; o .EmgonfloE 232 can Barefoot—so £695 9909 256:: mmoow “mo: 829m Lm_:om:EoSoz 5250:: £83.82 29.08 mcaEg “mo: .Em__oo£oE 28.2 new 293598 68:3 .230 966 my -230 .8206 BiomanSoz .Emtooc: 6on oc_~mEm€mo_>£o_o E3568» :0. o o o . cow to 39:8 .u <._ rm u < $.on Bahamas—352m Bob coccmpmv «.3:qu .mEotmE do 38.5.20 .H 035. 145 could eliminate the parasite from a given region in one fell swoop. Although immigration of infected humans and vectors could reintroduce infection, such an approach would be logistically more feasible than any of the other options previously discussed. Unfortunately, current macrofilaricidal agents are extremely toxic. This precludes mass treatments. Still, the idea Ci :miii an approach is. appealing: what is needed is a better adulticidal drug. To get there we need exploration of filarial physiological peculiarities, and the function of the hypodermis is a good starting point. REVIEW OF HYPODERMAL PHYSIOLOGY Filariae, as ch) all nematodes, imnme a remarkable structure within their body wall known as the hypodermis. This subcuticular syncytiunl participates in a number of physiological processes, encompasses the nerve cords and excretory ducts, and is in contact with the body wall musculature. In many ways, this pluripotent structure could be considered the "brains" of the worm. Developing filariae undergo four moults. During each of these, the cuticle is at least partially replaced. The regulation of this process, which was one of the subjects of a recent comprehenSive review of nematode surfaces [Geary et al um 1995], appears to be largely under the control of the hypodermis. Some of the enzymes necessary for cuticle protein synthesis anme present iii the hypodermis. Other enzymes, required for the detachment of the previous cuticle fnmn the hypodermis prior to time new cuticle's formation, are also hypodermal in origin. During the growth between moults, the proteins needed for cuticular growth again form in the hypodermis and migrate out to their proper location, though the exact mechanism of this translocation is uncertain. What triggers moulting is also unclear. All nematodes maintain an internal turgor pressure, and are capable of osmoregulation when placed in hypotonic or hypertonic solutions. The site at which this water balance is maintained is primarily the body wall, likely the hypodermis, with only minor contributions from the gun: of the wornu The mechanism of osmoregulation requires an intact metabolism to function, and it has been. suggested that it may be hormonally controlled [Fuse et al 1993]. The cuticle is permeable to both organic and inorganic ions, with the relative permeabilities of: BC>Naf=ClT>acetate‘ >gluconate' in Ascaris suum. The underlying hypodermis has a membrane potential of -74.9mV (Eo)/-47.6mV (Ei) which can be depolarized by. increasing the [W] or decreasing the 147 [acetate'] on the muscle side but not by such ionic changes on the cuticular side [Pax et al 1995]. This indicates that the hypodermal faces are differentially permeable to ions. The hypodermal electrochemical gradient may be the result of active ion transport and/or diffusion of metabolic organic ion by-products. Indeed, it has been shown that both occur. The hypodermis can directly transport Cl". In nematode muscle, it is this ion that is principally involved in establishing and neintaining the nematode myocyte membrane potential [Geary 6%: al 1995]. In contrast, mammalian or cestode myocytes maintain potentials that are dependent on potassium flux. Thus, it: appears that time hypodermis is involved in regulation of the membrane potential of the body wall musculature and nerves through the control of the flow of key ions. Filarial parasites obtain their energy from a number of different substrates via homolactate fermentation glycolysis, malate dismutation, and to some degree an electron transport system [Bryant et al 1989]. Intermediary metabolism of these compounds produces lactate, succinate, acetate, and formate [MacKenzie et al 1989]. These metabolically produced organic ions diffuse into the cuticle from the hypodermis, setting Lm)ee pH gradient [Sims et al 148 1992]. The gradient could influence the ionization states of molecules within the cuticle/hypodermis microenvironment, affecting the permeability of the body wall. The means by which filariae take up nutrients from their environment is controversial. Although they have a [functional gut, evidence is mounting that they are more dependent on transcuticular nutrient uptake rather than oral feeding [Bryant et al 1989, Howells et al 1980, Howells et al 1981, Howells et al 1983]. Eflndlarly, filariae have a less well developed excretory system than do the intestinal- dwelling worms and it lmns been suggested that the cuticle/hypodermis of the former is more permeable than that of the latter to compensate for this fact [Howells et al 1981]. Thus, both nutrient absorption and waste excretion occur via the transcuticular route, mediated by the hypodermis Hk) et al 1992]. Naturally, there are limitations to transcuticular feeding and. excretion. Molecules over 3000 Da in size do not traverse the cuticle [Thompson et al 1993]. Additionally, while the above data clearly demonstrate transcuticular absorption and excretion by filariae, there is little evidence to suggest that the orthodox view of the intestinal nematode cuticle's impermeablity to most small polar molecules is in need of revision [Masood et al 1983]. 149 The hypodermis seems to be a key site in the tissue-dwelling nematode. It. is involved in tuitrient uptake) and utilization, developmental moulting, osmoregulation, and maintenance of the internal ionic milieu. Indeed, it would seem to be the main control center of the corpus filariae. How these myriad of functions of the tissue are regulated is unknown. Suggestions include neuronal, hormonal, or neuroendocrine systems. REGULATORY SYSTEMS OF NEMATODES The nervous system of nematodes, while anatomically simple, is chemically very complex. Containing relatively few neuronal cell bodies, the basic "wiring diagram" appears to be conserved between all nematodes thus far examined, both free-living and. parasitic [Geary' et al 1992]. Unfortunately, 1'very little it) yet known about the functioning of the helminthic nervous system. The nematode nervous system differs significantly from that of their vertebrate hosts. Anatomically, the difference is profound: nematode inuscles send CNN: processes ‘which communicate with the nerve cord; in vertebrates, it is the nerve that sends out the axon. In contrast to vertebrate 150 nervous transmission, nematodes exhibit a graded rather than all—or-nothing quantal response to stimulation. The action potential so well characterized in vertebrates is absent in the worm. immetode nerve membrane potential is primarily dependent CH1 Cl‘ flux, with the pminciple anions involved being CaH'rether than Na+ and K1 [Stretton et al 1992, Geary et al 1992]. A number of neuromodulators have been found in the nervous system <1f nematodes. There its evidence of acetylcholinergic, GABAergic, serotoninergic, glutamatergic, and peptidergic components [Geary et al 1992]. If little is known about nematode neurobiology, even less is known about their endocrinology. Moulting is presumed to be controlled tux three Ibiochemically' distinct categories of hormone. These are the neuropeptides which stimulate secretion (n5 ecdysteroids, juvenile hormones which have 21 role in maintenance of the larval form during development, and the ecdysteroids which control the moulting process itself [Howells et al 1987]. Hormones are also involved in neuromuscular modulation, nutrient absorption, and osmoregulation as well [Fuse et al 1993, Stretton et al 1992]. 151 An area of contemporary research in helminthology is peptidergic regulatory systems. Nematode nerve membrane potentials exhibit rather regular oscillations which occur in bouts, with muscle contractions occurring during the interbout intervals. This suggested time possibility that the nervous activity stimulated the release of some hormonal factor. Indeed, evidence for the presence of a large number of neurohormonal peptides in nematodes has been found [Brownlee et al 1993, Stretton et al 1992]. While helminth peptides research is a new field, this ground has proven fertibe and produced some very interesting findings. One such finding was by our laboratory, demonstrating that antibombesin antiserum reacted very strongly with material at the hypodermocuticular junction zone of a filarial worm [Huntington et al 1993]. This discovery provided the springboard for the studies presented in this appendix. REVIEW OF BOMBESIN-LIKE PEPTIDES In 1970, Nakajima and co-workers isolated a peptide isolated from frogs of the genus Rana which they named ranatensin due to its actions on blood pressure [Nakajima et al 1970]. Later that year, Vittorio Erspamer and colleagues isolated a 152 similar peptide, bombesin, from the skin of the European frog, Bombina bombina. Bombesin had a number of pharmacological actions, including "hypertensive action in the dog; stimulation of the rat uterus, rat and guinea-pig colon, and the cat ileum; stimulation of gastric secretion in chicken and dog; hyperglycemic action in the rat and dog; increased insulin levels in the dog; and stimulant action on the active transport (n5 Cl' ions from.time serosal ti) the mucosal side of the isolated gastric mucosa of amphibians" [Anastiasi et al 1970]. Other' bombesin-like peptides have since been identified, including gastrin releasing peptides (GRP), neuromedins (NM), litorins, and phyllolitorins. Bombesin-like peptides have a wide distribution across the animal kingdom, from helminths [Gustafsson ei:.al 1985, Gustafsson 6%: al 1986, Halton et al 1990, Huntington et al 1993] and insects [Penzlin et al 1989] to mammals [Sunday at al 1987]. Tissue distribution studies have shown that these peptides may be iIt the» central. and. peripheral nervous systems (including sensory centers), the gut and related organs [Sunday et al 1987], and are developmentally expressed in the lung [Spindel et al 1993]. 153 The sequences of some bombesin-like peptides are shown below. Bombesin: pEQRLGNQWAVGHLMa Litorin: pENWAVGHFMa Ranatensin: pEVPNWAVGHFMa Neuromedin 8: GNLWATGHFMa Human Gastrin-Releasing Peptide: VPLPAGGGTVLTKMYPRGNHWAVGHLMa As a general rule, the C-terminal 7 to 9 residues are required tin: specific: association. ‘with. the receptor [Campbell et al 1990]. It has further been elucidated that the C-terminal residue (methionine) is vital for initiating the biologic response but is not an essential determinant of receptor affinity [Wang et al 1990]. Upon binding its receptor, the bombesin—like peptide triggers a series of intracellular responses. The throtein complex activates phospholipase C with the resulting phospholipid metabolites mobilizing calcium and activating a protein kinase. The subsequent flurry of activity is best summarized in Figure 4. Nonhydrolysible GTP analogs can block the ligand's actions, confirming time role