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Objectives: The purpose of this research was to demonstrate the effectiveness of hydroperoxide
ion-phase transfer catalyst (HPI-PTC) cleaners and disinfectants for maintaining dental unit waterlines
free of planktonic organisms. Method and materials: Water samples were taken trom 117 sites, which
included a variety ot dental units and samples from the sink faucets ot most operatories. Samples were
plated on appropriate bactériologie media and incubated. The presence or absence oi biofilms was con-
firmed by scanning electron microscopy. Twenty-twc cf the dental units were retrctitted with independent
water systems; the cleaning procedure invclved an cvernight applicaticn otan HPI-PTC cleaner followed
by a 2-minute water rinse. Results: Water frcm both the air-water syringe and the high-speed handpiece
lines from all untreated units contained at least 6x10^ cclcny-forming units per milliliter of pianktonic or
froe-floating bacteria; the average was 1.4x10= CFU/mL. An initial 5% scluticn ot HPI-PTC successfully
cleared the lines of any apparent biofilm when applied for 3 ccnsecutive days. Thereafter, once weekly use
of the cleaner maintained the dental unit water supplies free cf significant numbers of planktonic organ-
isms. Conclusion: Routine weekly use of an HPI-PTC cleaner contrclled dental unit waterline biofilm and
reduced, with minimum effort, the microbial contamination level ct water used for patient treatment tc less
than 200 CFU/mL. fOti/n(essence int 2001:32:755-761)
Key words: biofilm, dental unit waterline, environmental bacteria, hydroperoxide ion cleaner, oral
streptccocci, phase transfer catalyst
increases in the biofílm mass are due primarily to bac- The level of toxicity for humans is low, as rep'^''''^°
terial replication. Growth of biofilm bacteria in in proprietary supporting documentation sul • ^°
DUWLs is slow because of lower (room) temperatures tbe US Food and Drtig Administration and i ^^
and nutrient limitation,' Environmental Protection Agency for app. '^te
Tbe organistns colonizing water supplies are gener- product registrations. Tbe present study évalua. • tbe
ally nonpatbogenic environmental bacteria,^ altbough clinical effectiveness of the HPI-PTC cleaner for both
patbogenic and/or opportunistic forms sucb as Le- the initial removal of biofilms from DUWLs and for
gionella^ Pseudomonas,^ and Mycobacterium^ species subsequent maintenance of the lines.
bave been isolated from waterlines in bealtb care facil-
ities. Tbe opportunistic organisms may pose a threat
to immunocompromised individuals. Viruses specifíc METHOD AND MATERIALS
for tbe buman bost are not found in biofilms, because
tbey require living bost cells to replicate. Sampiing sites
Tbe strengtb of attacbment of bacterial biofilms to
tbe interior surfaces of waterlines is sufficient to witb- Samples from DUWLs were collected from nine oper-
stand tbe sbear forces developed by water flow through atories within the University of Louisville Dental
the lines.'* Consequently, a water purge of DUWL lines School clinics and from 108 private practice operato-
bas little effect on ridding tbe lines of biofilm, altbougb ries located in both the Louisville metropolitan area
tbe number of planktonic bacteria may be reduced and other locations within the continental United
sligbtly. Tbe reduction in DUWL planktonic bacteria States. Tbe individual units all bad been in service for
does not approacb tbe levels needed to meet tbe ADA'S at least 1 year. Some bad been used continually for
goals."-^^ In addition, tbere is virtually no residual chlo- more than 20 years.
rine from tbe municipal water treatment in the DUWLs
to interfere with bacterial growtb.'^ Water coilection
Because Vess et aP demonstrated tbat bacteria
bound in biofílms attacbed to polyvinyl cbioride plas- Samples were collected in sterile specimen cups from
tic pipes were significantly more resistant to tbe effects tbe bigh-speed, air-water syringe, and, if present, tbe
of disinfectants, it was felt tbat removing the biofilm dental assistant's cart air-water syringe waterlines. Tbe
from DUWLs was preferable to killing tbe bacteria lines were purged for 2 minutes prior to sampling only
witbin tbe film. In addition, if the biofilm bactena if the unit had not been in service tbat day. Tap water
were killed but not removed, tbe matrix would remain samples were also taken from most locations. A mini-
and could provide a suitable structure for rapid recol- mum of 10 mL was obtained for eacb water sample.
onization of tbe DUWL. All samples were received in tbe laboratory witbin 24
Examination of the literature revealed a proprietary bours of collection, sent via surface or air courier,
product tbat is used in tbe food industry as an effec- depending on the location.
tive surface cleaner of biologic debris."*'' Tbe cleaner
is based on tbe powerful nucleopbilic bydroperoxide Microbioiogicai examination
ion (HPI) in conjunction witb pbase transfer catalysts
(PTC) and activators (Sterilex Ultra Dental Water Line Once received in the laboratory, samples were immedi-
Cleaner, Sterilex). Tbe enhanced nucleopbilic activity ately processed by plating either a 10"' or 10"^ dilution
is ascribed to an alpba effect tbat imparts a high den- on the following media: R2A agar for total het-
sity of negative charge on the terminal oxygen of the erotrophic CFU/mL,'* Mltis-Salivarius (M-S) agar for
peroxide ion.'^ The net effect is tbe hydrolysis of bio- selective enumeration of oral streptococci,'^ and
logic molecules containing ester linkages such as lipids DGVP Legionella Selective Agar for Legionella species
and the amide linkages of proteins. isolation (Becton-Dickinson Microbiology Systems).^"
A major reason most proposed cleaners and disin- The initial samples plated on R2A agar were incubated
fectants do not effectively remove biofilms is that both at either 23''C to 26°C or 37°C for a minimum of 7
the cleaner or disinfectant and the biofilm carry a net days. Those incubated at 23''C to 26°C produced about
negative charge, which results in repulsion or nonin- a tbird more growth from the same water samples;
teraction of the materials. The pbase transfer catalyst consequently, the ^I'C incubation set of R2A plates
in HPI-PTC eliminates this problem and is able to was discontinued. The selective plates for streptococci
penetrate the biofilm. Once inside the biofilm, the and Legionella were incubated at 37''C.
hydroperoxide ion is released, where it hydrolyzes and Unique colonies were selectively isolated from the
lifts tbe contaminating material away from the surface. R2A agar plates and were given preliminary identifica-
Tbe cleaner also has disinfecting properties.'''•'^•" tions using the RPI system of microbial characterization
DISCUSSION
TABLE 4 Recovery of planktonic bacteria
(CFU/mL) from University of Louisville dental unit Dental unit waterline biofilm is a mixture oí hving
wateriines before and after cleaning' bacteria, extracellular carbohydrates, and biologic
debris that adheres to the surfaces of plastic tubing.
Prior tc t D post- 7 D post-
HPI-PTC No. of units cleaning cleaning cieaning
The biofilm is conceptually similar to dental plaque on
the surfaces of teeth. The predominant planktonic
7.0% 0-tO 0-ao bacteria sloughed from the DUWL biofilms are typical
5.0% 0-tO 0-100
of the environmental bacteria encountered in normal
'Hydropercxide tor-phase transfer catalyst (HPI-PTC} Ireatment took municipal water systems.^ This was confirmed by com-
plaoe overnight and was followed by a 2 minute water flusli in the
morning. paring the general bacterial population obtained from
the DUWLs with those isolated from the tap water
samples. This strongly suggests that the DUWL biofilm
originates with the supply water.
The fact that oral streptococci were isolated from
Disassembly and scrubbing of the tap aeration devices 80% of the DUWLs indicates that contamination from
often reduced the bacterial count, and different taps patient-derived bacteria can occur from the functional
within the same office were not equally contaminated. end of the line also, Bagga et aP' presented data to
The first attempts to clean DUWLs were made in indicate that oral bacteria are sucked back into the
the institutional setting of the University of Louisville high-speed handpiece when there is no check valve in
Dental School, This allowed for close supervision of the water retraction device. The majority of the dental
the procedures. Table 4 presents before and after data units tested in this study were equipped with antire-
regarding treatment of the DUWLs with HPI-PTC, traction valves. The reason for the regression of oral
Scanning electron microscopic examination of sec- bacteria into these wateriines is not clear. It must be
tions cut from the wateriines revealed a dramatic dif- assumed that the oral streptococci were established in
ference foiiowing the cleaning procedures (Figs la and the biofilm, because fhey were never detected in the
TABLE 5 Recovery of planktonic bacteria (CFU/mL) from private practice dental unit
waterlines foilowing prescribed cleaning treatment*
Clinic
Test period 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Week 1 0 0 3000 6000 0 0 2900 32000 0 1700 0 0 17000 0
Week 2 0 0 0 0 0 0 0 0 0 0 200 350 14000 16000
Week 3 0 0 0 0 0 0 3000 0 0 0 0 0 15000t 300001
Week 4 0 0 0 0 0 0 2800' 15000t 0 4000t 0 0 0 0
Week 5 0 0 0 0 0 0 0 200 0 0 0 0 0 0
Week 6 0 0 0 0 0 0 —5 0 0
•All clinic personnel were instructed ¡n the use of the cleaner pnorto week 1, Those with detectable counts for the veek 1 s m pies were instructed to use the
cleaner for 3 consecutive days followed by 1 weekly treatment.
'Additional reinforcement of cleaning procedures was given to the clinic staff by University of Louisvllte personnel,
^Dashes indicafe waterlines were nof fested during that week.
lines after cleaning with HPi-i . .tamma- details. However, wben tbe cleaner was supr''' '"'^
tion was strictly transient, coming .'.'.¡m mi; previous private offices, the reliability of applicatio -,
patient, the oral bacteria would have been readily office staff came into question. The private ^]^^
detected regardless of the cleaning procedures. offices tbat did not meet the ADA's goals afte ^^""^^
No isolates of Legionelia were detected in this treatment were asked to repeat the treatme lor 3
study, either because tbey were not present or because consecutive days. One group was still not in compli-
tbe metbods used were not sufficiently sensitive to ance with the guidelines, and further questioning sug-
detect very iow levels.^-" The samples were not con- gested that the lines had yet to be cleaned. The lines
centrated, nor were molecular detection tecbniques were treated under University of Louisville staff super-
employed. An alternative explanation is that the vision and quickly came into compliance. Cooperation
Legionelia, if present, had formed somnicells, which of office personnel appears to be key factor in main-
are cells that are still viable but cannot be cultured on taining DUWLs.
routine media." Walker et aF' recently reported simi- An overriding question is wbether DUWL biofilms
lar results on the lack of Legioneila isolates from pose a bealtb risk to tbe dental patient.^' Tbere is
DUWLs. anecdotal evidence tbat seems to suggest tbat some ill-
Reduction of tbe planktonic bacteria in DUWLs to ness has been linked to tbe bacteria in DUWL.'
iess tban 200 CFU/mL by tbe year 2000 is a stated Dentists bave a significantly elevated antibody titer to
goal of the ADA.' Numerous approaches have been Legionella compared to the general population,^^'^"
suggested, including disinfection of tbe lines with and there is a strong probability that at least one den-
hypocblorite-containing products (bleacb)^^ or otber tist has contracted Legionella through his practice.' In
disinfectants, inciuding glutaraldebydes and iodo- addition, several opportunistic bacteria routinely iso-
pbores. None of these alternate disinfection lated from various dental water supplies can cause
approaches has formal approval for use in DUWLs. In serious disease in immunocompromised individuals. It
addition, most disinfectants have significant draw- is appropriate for healtb care facilities to reduce
backs, including damage to the equipment and the planktonic organisms in DUWLs to at least tbe levels
potential for residual toxicity to the patient. Further- proposed by tbe ADA and to use sterile water for sur-
more, disinfectants do not readily remove the biofilm gical procedures. Tbe data in tbis paper indicate tbat
matrix from plastic surfaces; consequently tbe matrix is tbe typical dental practice can meet tbe goal of less
available to trap and protect newly introduced bacter- tban 200 CFU/mL in all DUWLs with a minimum of
ial contamination. Antibacterial moutbrinses bave time expenditure by the dental staff.
been used to reduce tbe level of planktonic bacteria A commercial form of HPI-PTC (Sterilex Ultra
and bave been successful, aitbougb tbe time to reacb Dental Unit Water Line Cleaner, Sterilex) was issued a
satisfactory levels is a matter of weeks." Submicron fii- 510K approval by tbe FDA in 1999 for use as a
ters will adequately remove bacteria from DUWLs, but DUWL cleaner. On July 17, 2000, the ADA Council on
fail to remove bacterial by-products and can clog Scientific Affairs granted a Seal of Acceptance for
quickly wben placed in heavily contaminated lines. Sterilex Ultra "as being safe and effective for cleaning
It is our position that the best approacb to control- deposits and controlling aerobic, mesophilic, bet-
ling tbe release of planktonic bacteria is the removal erotropbic bacterial contamination in DUWL when
of tbe biofiim. Scanning electron micrographs of used as directed."
DUWLs treated witb HPI-PTC readily sbow tbat tbe
biofilm is removed from the plastic surfaces. Tbe
microbiologie data confirm the lack of significant CONCLUSION
numbers of planktonic bacteria in tbe treated DUWLs.
Independent studies by botb A-dec and tbe Sterilex 1. Essentially all dental unit waterlines harbor bacteria
Corp of tbe effects of the HPI-PTC on typical dental tbat develop into a biofilm, and significant numbers
unit parts demonstrated no significant deleterious of bacteria are continually released from the biofilm
effects on or loss of material from the parts. Parts were into the water flow passing througb the line.
made of different metals, plastics, and rubbers. Some 2. The majority of bacteria are typical environmental
discoioration of macbincd brass surfaces was noted. microbes, aitbougb, oral streptococci were isolated
The parts were exposed to a scries of different applica- from the majority of units tested.
tions of the HPI-PTC solutions for 29-day periods (E. 3. Dental unit waterlines can be effectively cleared of
deLaubenfels, personal communication, June, 1999). biofilms by application of bydroperoxide ions cou-
Application of the HPI-PTC to DUWL was pled to phase transfer catalysts. Weekly ap|:i[|ca-
straightforward in the defined setting of tbe dental tions are sufficient to keep tbe lines witbir he
scbool with laboratory personnel tending to tbe ADA'S goal of less tban 200 CFU/mL.
4. Tbe performance of tbe dental practice staff must 15. Kramer DN, Shaw PA. Methods of using a cleaner, sani-
be monitored to ensure that routine weekly clean- tizer, disinfectant, fungicide, sporicide, chemical sterilizer.
ing procedures are carried out to prevent reestab- US patent 5320805, 1994.
lishment of tbe biofilm. 16. Edwards JO. Peroxide Reaction Mechanisms. New York:
Interscience, 1960.
17. Shojaei M, Staat RH. Evaluation of hydroperoxide ions as
dental unit waterline disinfectants [abstract 282]. J Dent Res
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