THE MICROBIAL PROFILES OF DENTAL UNIT WATERLINES IN A
DENTAL SCHOOL CLINIC
Table 2. The bacterial distribution of the water samples according to the collecting points and isolated bacteria
Roof tank
Chairs connected
directly with water
pipe lines
Chairs receiving
distiled water
through attached
bottles
Hand piece/
scaler* (H/S)
MembraClean Plus
Disinfectant
Main water source*
(MWS)
Water line
tube* (WLT)
Water
pump
*
Distiled water
bottle (DWB)
Distilled water source*
(DWS)
Ground
reservoire
* = collecting point
Figure 1. The diagram of the RAKCODS Dental Units’ water supply system and the points of samples collection.
Plate Count Agar (HiMedia, India) was prepared
according to standard procedure and then cooled
at 44-46°C. Serial dilutions were prepared from the
water samples in addition to undiluted sample (1:1,
1:10, 1:100). One ml of each sample or dilution
was transferred to the properly labeled sterile
Petri dish. Approximately 15ml of the cooled agar
medium was then poured into each Petri dish. The
sample and agar were then mixed by rotating the
plate several times. After the media has solidified,
the plates were inverted and incubated at 35°C
for 48-72 hours. After incubation, the count of
colonies, mean and standard deviation were
calculated.
• Molecular identification of bacteria:
Pure cultures of the isolated bacteria were sent for
molecular identification in AccuVis Bio laboratories
in Abu Dhabi University Campus, Abu Dhabi,
UAE. Bacterial 16S rRNA gene sequencing was
performed according to the following protocol:
Bacterial DNA isolation AccuVis Bio’s Bacterial
Genomic DNA Isolation Kit (AV1003). PCR
amplification uses PCR Primers (Universal), forward:
27F - 5’- AGAGTTTGATCMTGGC TCAG - 3’, Reverse:
1492 R - 5’- TACGGYTACCTTGTTACGACTT - 3’.
DNA sequencing using BigDye® Terminator v1.1
Cycle Sequencing Kit, Sequencing reaction for
Forward (518F) and Reverse (800R), Data analysis -
Sequencing Analysis Software v5.2. Bioinformatics
tools used Fasta format conversion of both
sequences – NCBI, Pairwise sequence alignment
– LALIGN software, trimming of final sequence,
NCBI blast search, Similar sequence identification,
identification of bacteria.
3. Results
Table 1 shows the total count of bacteria (CFU/ml)
in water samples collected from the Main Water
Source, the Distilled Water Source and 12 dental
units of RAKCODS, counted according to ADA
guidelines (Table 1).
Based on the ADA guidelines, which state that
water used in dental treatment should contain
a bacterial level of ≤200 CFU/ml, the majority of
Table 1. Bacterial count in the water samples taken from different collecting points
Site of collection
Total number of
collected
samples Number (%) of
samples with
0–200 CFU*/ml Number (%) of
samples with
>200 CFU/m
Mean number of
CFU/ml±SD
Main Water Source 3 0 (0%) 3 (100%) 499±345
Distilled Water Source 3 0 (0%) 3 (100%) 1538±1165
Distilled Water Bottle 12 0 (0%) 12 (100%) 2397±1403
Water Line Tube 36 5 (14%) 31 (86%) 1867±1434
Handpiece/Ultrasonic
Scaler 36 2 (6%) 34 (94%) 2000±1535
Number (%) of samples in which the following bacteria were isolated
Ralstonia spp. Sphingomonas
paucimobilis Leifsonia spp. Brevundimonas
aurantiaca Main Water Source 3 1 (33.3%) 2 (66.7%) 3 (100%) 0 (0%) 0 (0%)
Distilled Water Source 3 3 (100%) 3 (100%) 2 (66.7%) 1 (33.3%) 0 (0%)
Distilled Water Bottle 12 12 (100%) 10 (83.3%) 10 (83.3%) 6 (50%) 3 (25%)
Handpiece/Ultrasonic Scaler 36 35 (97%) 33 (91.7%) 24 (66.7%) 14 (38.9%) 3 (8.3%)
Water Line Tube 36 36 (100%) 32 (88.8%) 27 (75%) 13 (36.1%) 3 (8.3%)
Total 90 87 (96%) 80 (88.8%) 66 (73.5%) 34 (37.8%) 9 (10%)
samples collected in our study showed CFU above
the standard.
The CDC recommended that non-surgical dental
water should have a heterotrophic plate count
(HPC) of ≤500 CFU/ml. The only samples that
fulfilled this criterion were the Main Water Source
samples (499 CFU/ml) which is equal to the levels
of HPC in drinkable water.
Since the dental units’ water supply systems were
of two types as shown in Fig. 1, it was found that
the bottled units contained significantly higher
numbers of CFU (2632±1231.783) compared
to the non-bottled units (1484.75±1395.093),
p<0.02.
RAKCODS had a prescheduled plan to replace
all of the distilled water bottled dental units with
new units receiving direct water connection. The
units were installed on time (September 2015)
and were allowed to work for 4 months. Random
water samples from 7 of the newly installed dental
units. Two samples from each water outlets (water/
air syringe and hand piece tubes) were collected
in the same manner as described earlier and
the bacterial colonies per ml were counted. The
average CFU/ml of these samples were compared
with the average counts of water line tubes of the
previous bottled units. The newly installed dental
unit counts showed remarkable reduction in the
number of CFU/ml (720, SD±969).
Table 2 shows the isolated bacteria and number
of water samples contaminated with each type
of bacteria out of the total number of samples
collected from the Main Water Source, the Distilled
Water Source and the 12 dental units (Table 2).
Ralstonia spp. was the most common bacteria
in the MWS, DWS and dental units’ WLT, as it
was found in 96% of the collected samples.
The other common isolated bacteria were
Sphingomonas paucimobilis 88.8%, Leifsonia spp.
(73.5%), Brevundimonas aurantiaca (37.8%) and
Pseudomonas aeruginosa (10%).
4. Discussion
The majority of the collected samples in this study
*CFU: colony forming units
128
Total number
of collected
samples
Source/site of sample
collection
Stoma Edu J. 2017;4(2): 126-132
http://www.stomaeduj.com
Stomatology Edu Journal
Pseudomonas
aeruginosa
showed CFU above the standards for drinking
water or water used for dental procedures
according to the CDC guidelines. The only samples
that fulfilled this criterion were the MWS samples
with an HPC of (499 CFU/ml), which is equal to the
levels of HPC in drinkable water. The fact that MWS
samples contained significantly lower CFU/ml of
bacteria compared t o the DWB, WLT or H/S clearly
indicates that the dental water pipelines provide
good environment for bacteria to thrive.
When bottled dental units were replaced with
new dental units, the average CFU/ml was
reduced dramatically. This result substantiates the
assumption that the DWB was the main source of
contamination.
In the examined water samples from the dental
units, bacteria of the Pseudomonadaceae family
were the most common. These obligate aerobic,
motile, gram negative bacilli are widely spread
and have the ability to survive and grow almost in
any environment. Their presence is associated with
the main water supply and failure of disinfection
methods to eradicate them totally or even
reduce their counts. The isolated bacteria tend
to categorize as non-fermenting gram-negative
bacilli (NFGNB) which are a group of organisms
that either do not utilize glucose as a source of
energy or utilize it oxidatively. 14
Pseudomonas aeruginosa, species of Pseudomonas
genus can be recovered from the oral cavity
of 4% of healthy individuals 4 and this indicates
the possibility of these microorganisms getting
aspirated into the DUWLs through a defective
check valve and colonized in the waterlines. This is
a drawback due to the fact that water after having
passed through DUWL, flows through hand pieces
during treatment and forms aerosol and splatter
therefore increasing the chances of cross infection
especially in immunocompromised patients.
Following is a list of bacteria tested for in our study,
in the order of their prevalence:
4.1. Ralstonia spp.
Ralstonia spp. was the most common type of
bacteria present in the MWS, DWS and dental
THE MICROBIAL PROFILES OF DENTAL UNIT WATERLINES IN A
DENTAL SCHOOL CLINIC
129