3.1. Baseline Monitoring
Table 4 shows the results obtained at baseline, in relation to the disinfection treatment of the dental unit waterlines. At baseline all units were fed with water from the public water supply. As far as the HPCs at 37 and 22 °C are concerned, the tap water was found to satisfy the required standards for drinking water, but two out of nine samples were contaminated by
L. pneumophila SG1. Both samples had been taken from taps on the first floor, in operating spaces of the main building.
Overall, the output water from the dental units presented not compliant colony counts in 38.1% of samples, considering the Italian limit for HPC at 22 °C for drinking water, and in 66.7% of samples considering both the limits for HPCs at 22 and 37 °C.
P. aeruginosa and
Legionella spp. were detected respectively in 22.2% and 34.9% of the samples (
L. pneumophila SG1: 25.4%,
L. anisa: 9.5%), although the results varied depending on the type of disinfection treatment adopted (
Table 4).
The water from the dental units not subjected to any treatment showed microbial loads higher than the values recommended by the ADA and those set by the Italian regulations for drinking water (HPC at 22 °C) in 36.4% of samples. P. aeruginosa was detected in one sample out of 11 (9.1%); L. pneumophila SG1 was detected in two samples (18.2%), both taken from operating spaces on the first floor of the main building, and L. anisa was found in the output water of two dental units (18.2%) in the operating spaces situated on the ground floor of the secondary wing of the building.
The pattern of contamination was very similar in the output water of the units undergoing periodic treatment with Rely+OnTM Peracilyse, carried out only at the end of the day (untreated vs. periodically treated: p > 0.05 for both HPCs at 37 and 22 °C). Legionellae were detected from dental units in nine different operating spaces (24.3%), all on the first floor.
Table 4.
Microbial contamination values of water samples from supply water and dental unit water systems at baseline.
Table 4.
Microbial contamination values of water samples from supply water and dental unit water systems at baseline.
Parameters | Supply Water (Tap Water) | Dental Units |
---|
No Disinfection Treatment | Periodic Disinfection (Rely + OnTM Peracilyse) | Continuous Disinfection (ICX® 0.01%) | Continuous Disinfection (Calbenium® 2%) |
---|
n: 9 | n: 11 | n: 37 | n: 11 | n: 4 |
---|
Temperature | | | | | | |
mean (°C) | 17.9 | 24.9 | 23.3 | 24.5 | 26.3 |
SD (°C) | 2.1 | 2.2 | 3.3 | 1.6 | 2.6 |
HPC 37 °C | | | | | | |
not compliant samples (%) | 0 | 100 | 75.6 | 18.2 | 25.0 |
geometric mean (cfu/mL) | 5.3 | 519.3 | 202.3 | 7.1 | 9.9 |
range (cfu/mL) | (1–20) | (55–4800) | (1–8720) | (1–221) | (1–236) |
HPC 22 °C | | | | | | |
not compliant samples (%) | 0 | 36.4 | 45.9 | 18.2 | 25.0 |
geometric mean (cfu/mL) | 9.3 | 62.3 | 68.8 | 7.8 | 26.6 |
range (cfu/mL) | (2–98) | (14–634) | (1–5160) | (1–236) | (4–251) |
P. aeruginosa | | | | | | |
positive samples (%) | 0 | 9.1 | 27.0 | 0 | 75.0 |
range of positive samples (cfu/100 mL) | | (75) | (100–3700) | | (2–1020) |
L. pneumophila | | | | | | |
positive samples (%) | 22.2 | 18.2 | 13.5 | 63.6 | 50.0 |
range of positive samples (cfu/L) | (450–1250) | (200–300) | (350–3050) | (50–9000) | (250–1750) |
Other species of Legionella | | | | | |
positive samples (%) | 0 | 18.2 | 10.8 | 0 | 0 |
range of positive samples (cfu/L) | | (300–1100) | (50–250) | | |
The continuous disinfection systems, with both products (ICX
® and Calbenium
®), achieved the lowest levels of HPC at 37 and 22 °C, with 80.0% of samples conforming to the limits of the ADA as well as those of the Italian norms for drinking water. The differences were statistically significant for both the HPC at 22 °C (untreated
vs continuously treated
p < 0.05; periodically treated
vs continuously treated
p < 0.05) and the HPC at 37 °C (untreated
vs continuously treated
p < 0.001; periodically treated
vs continuously treated
p < 0.001). However, the continuous disinfection methods were not totally effective against
L. pneumophila, which was detected in 63.6% of samples treated with ICX
® and in 50% of sample treated with Calbenium
®, all collected from the first floor of the main building. Of those treated with Calbenium
®, three out of four were also contaminated by
P. aeruginosa with counts up to 10
3 cfu/100 mL, although much lower than the values considered to be the infective dose in healthy people (>1.5 × 10
6 cfu/mL) [
19].
These results show that the continuous introduction of disinfection products, necessarily used at low levels to minimize their potential toxic effect, was more effective in maintaining the heterotrophic bacterial counts within the recommended standards in output water of dental devices, while this treatment was not always able to control microorganisms such as
Legionella and
P. aeruginosa, which are very resistant to disinfectant treatments both on account of their intrinsic characteristics and because they are protected within the biofilm [
41].
L. pneumophila was isolated from 75% of the units in the Surgery Division, 57.1% of those in the Periodontics Division, 50.0% in the Endodontrics Division, 42.8% in the Prosthesis Division and from 10.0% units in the Dental School Area. However, it was not possible to relate the contamination to the type of work carried out in the different divisions since the dental units of each division came from different manufacturers and underwent different treatments. Instead, the contamination from Legionella spp. appears to be related to the location of divisions in the various parts of the building. Legionella spp. were never detected in the operating spaces situated on the ground floor of the main building; the first floor of the main building was found to be colonized by L. pneumophila SG1 (54.2% of positive dental units); the ground floor of the secondary wing by L. anisa (18.2%), and the first floor of the secondary wing by L. pneumophila SG1 (10.8%) and L. anisa (10.0%).
The dental clinic is supplied from a single water system with several branches that distribute water from the public network to the various parts of the building. Over time, the water system has undergone modifications that may have led to dead legs and points of stagnant water in certain parts of the network but not in others. This may explain the different patterns of contamination found in dental units in relation to their location. Water temperature may also have affected the level of contamination: the temperature of samples taken from dental units of positive divisions was on average about 2 °C higher than those taken from negative divisions (24.0 °C vs. 22.2 °C), with statistically significant differences (p < 0.001).
The highest concentrations of
Legionella spp. were detected on the first floor, in the dental units situated in the south-facing side of the main building (surgery and endodontrics), the same location as the positive samples of tap water. In these divisions 60% of the units were positive for
L. pneumophila SG1, at concentrations up to 9000 cfu/L of planktonic bacteria. According to the Italian guidelines for legionellosis prevention and control, concentrations above 10
3 cfu/L are considered to represent a health hazard in healthcare settings and a sign that corrective measures need to be implemented [
26].
Finally, no significant differences were found between the bacterial loads (HPCs at 37 and 22 °C) detected in samples positive or negative for L. pneumophila. The same was observed for P. aeruginosa.
3.2. Monitoring after the Adoption of the Risk Management Plan
To eliminate the contamination from
P. aeruginosa and
L. pneumophila, the units equipped with an independent supply system underwent shock treatment, by adding hydrogen peroxide 3% to the system. The shock procedure is described in
Table 3. This treatment allowed
P. aeruginosa and
Legionella spp. to be eliminated and the HPCs to be reduced to levels lower than those prescribed for drinking water in Italy. Since shock treatments are known to be ineffective in the long term, the protocol described in
Table 3 was adopted immediately afterwards, consulting the manufacturer about the products to be used in the different types of dental units. One of the units examined at baseline from the Castellini company and one from the Eurodent company had been replaced with two new units from A-dec. For technical reasons, periodic disinfection was not possible in the three remaining dental units from the Eurodent company, which were under continuous treatment with Calbenium
®. Moreover, all the units with an independent supply system used deionised water.
After about a year from the baseline monitoring, the post-intervention monitoring produced the results reported in
Table 5. The measures adopted allowed the contamination to be contained, with significant reductions in samples not conforming for HPCs. The results varied depending on the type of disinfection treatment implemented.
Table 5.
Microbial contamination values of water samples from dental unit water systems after implementation of the risk management plan.
Table 5.
Microbial contamination values of water samples from dental unit water systems after implementation of the risk management plan.
Parameters | Dental Units |
---|
No Disinfection Treatment | Disinfection Treatment (Supplied with Deionised Water) |
---|
Supplied with Tap Water | Continuous (H202 0.06%) + Periodic (Rely + OnTM Peracilyse) | Continuous (ICX® 0.01%) + Periodic (Sterilex Ultra) | Continuous (Calbenium® 2%) |
---|
n: 10 | n: 37 | n: 13 | n: 3 |
---|
Temperature | | | | | |
mean (°C) | 24.8 | 22.6 | 23.6 | 22.3 |
SD (°C) | 2.2 | 2.1 | 1.0 | 1.1 |
HPC 37 °C | | | | | |
not compliant samples (%) | 100 | 35.1 | 38.5 | 33.3 |
geometric mean (cfu/mL) | 874.9 | 17.1 | 9.4 | 13.0 |
range (cfu/mL) | (225–1980) | (1–121) | (2–100) | (3–21) |
HPC 22 °C | | | | | |
not compliant samples (%) | 90.0 | 13.5 | 7.7 | 33.3 |
geometric mean (cfu/mL) | 456.2 | 53.6 | 24.6 | 36.4 |
range (cfu/mL) | (77–1720) | (2–242) | (3–282) | (20–115) |
P. aeruginosa | | | | | |
positive samples (%) | 20.0 | 0 | 0 | 0 |
range of positive samples (cfu/100 mL) | (240–300) | | | |
L. pneumophila | | | | | |
positive samples (%) | 10.0 | 0 | 0 | 0 |
range of positive samples (cfu/L) | (250) | | | |
Other species of Legionella | | | | |
positive samples (%) | 10.0 | 0 | 0 | 0 |
range of positive samples (cfu/L) | (1850) | | | |
The dental units which, for technical reasons, were not treated with the combined continuous and periodic disinfection protocol, showed an increase of HPCs (HPC 37 °C,
p < 0.05; HPC 22 °C,
p < 0.05). On the contrary, the HPCs decreased significantly in the output water from dental units treated with the combined H
2O
2 + periodic Peracilyse system (HPC 37 °C,
p < 0.001; HPC 22 °C,
p < 0.05) and remained at low concentrations (not significant differences pre-post) in dental units treated with continuous disinfection at baseline (ICX
® and Calbenium
®) (
Table 6).
L. pneumophila and
P. aeruginosa were detected respectively in 10% (
vs 18.2% at baseline) and 20% (
vs. 18.2% at baseline) of the samples from untreated dental unit waterlines. The implementation of the combined continuous and periodic disinfection allowed
L. pneumophila and
P. aeruginosa to be completely removed from dental unit waterlines treated with H
2O
2 + Peracilyse and ICX + Sterilex ultra systems. The complete removal of these bacteria was also obtained in the dental units which continued to be treated only with Calbenium
® (
Table 5). This result could be attributable to the implementation of management measures other than the disinfection methods, and suggests that all the combined interventions foreseen in the water safety plan contribute to the control of contamination and none of them should be overlooked.
Table 6.
Comparison of the bacterial contamination of output water from dental units before and after implementation of the risk management plan.
Table 6.
Comparison of the bacterial contamination of output water from dental units before and after implementation of the risk management plan.
Parameters | Dental Units Grouped for Disinfection Treatment | |
---|
No Disinfection Treatment | Continuous (H202 0.06%) + periodic (Rely+OnTM Peracilyse) | Continuous (ICX® 0.01%) + Periodic (Sterilex Ultra) | Continuous (Calbenium® 2%) | |
---|
n: 10 | n: 35 | n: 11 | n: 4 |
---|
before | after | before | after | before | after | before | after |
---|
HPC 37 °C | | | | | |
geometric mean (cfu/mL) | 519.3 | 874.9 | 202.3 | 17.1 | 7.1 | 9.4 | 9.9 | 13.0 |
pre-post comparison (paired t test) | p < 0.05 | p < 0.001 | ns | ns | |
HPC 22 °C | | | | | |
geometric mean (cfu/mL) | 62.3 | 456.2 | 68.8 | 53.6 | 7.8 | 24.6 | 26.6 | 36.4 |
pre-post comparison (paired t test) | p < 0.01 | p < 0.05 | ns | ns |
The combination of a continuous introduction of low levels of a minimally toxic agent associated with a periodic treatment using a more concentrated active product, has shown itself to be a potential method for the control of contamination. In addition, the periodic treatment may be useful in preventing the adaptive resistance of bacteria that could be induced by continuous exposure to low concentrations of biocides [
42]. The combined procedure is easy to perform and therefore favours staff compliance to the protocol. Under our working conditions, we used various active products for dental units made by different manufacturers and obtained similar reductions in the microbial contamination, thus confirming that the efficacy is not so much linked to the product used, but rather to the type of treatment protocol applied and, above all, to the compliance of personnel to the management plan [
3]. The literature reports that non-compliance and technical errors are the most probable causes of failure to properly disinfect dental unit waterlines [
3,
5].
This study was carried out in a complex clinical dental setting where various confounding factors may have affected the results, such as the age and technical characteristics of the dental units, their different activity and frequency of use, the various disinfection systems, and the location of the operating spaces. Despite this limitation, the results highlight the importance of identifying and implementing a management protocol for Legionella control, including microbiological monitoring focused on Legionella spp.