Prevalence of Pseudomonas aeruginosa in Dental Unit Water-Lines

MATERIALS AND METHODS

A total of 82 dental unit water samples from 34 dental clinics of Amravati city were collected in the period of 6 months from June to December 2006. The water collected from 3 in 1 syringe 28, Air rotor 32 and Ultrasonic scaler 22 in sterilized plastic water sample collection bottle by flushing the water for 15 sec before collection. One milliliter dental water sample of each clinic from different sources was inoculated in 9 mL MacConkey broth and incubated at 37°C for 24 h. A loopful inoculum from MacConkey broth was subcultured on MacConkey agar and cetrimide agar plates (Hi-Media Laboratories, Mumbai). These plates were incubated at 37°C for 24-48 h. Plates were observed for growth and a Gram smear was performed from different types of colonies. Gram reaction, colony morphology, pigment formation, florescence, catalase, coagulase, urease and oxidase tests were performed and allocated to appropriate genera to the isolates. The cultural characteristics including lactose fermentation by enterobacteriaceae on MacConkey agar, pyocynin formation of Pseudomonas sp. on cetrimide agar were noted. Further identification to species level was carried out on the basis of various specialized tests (Collee et al., 1996).

All the confirmed P. aeruginosa strains were subsequently tested for antibiotic sensitivity patterns by Bauer et al. (1966) disk diffusion method using antibiotics discs obtained from Hi-Media Laboratories Pvt. Ltd Mumbai. The isolates were considered antibiotics resistant if the zone of inhibition was 10 mm or less. The antibiotic susceptibility pattern of P. aeruginosa strains was determined on the day of their isolation by the Bauer et al. (1966) disk diffusion method on Muller Hinton agar using the criteria of standard zone sizes of inhibition to define sensitivity or resistance to different antimicrobials (Table 1). Finally, the data were recorded and analyzed at the completion of the study as per recommendations of the Anonymous (2000). P. aeruginosa MTCC 424 was used as reference strain for the standardization of antibiotic susceptibility testing. The Multiple Antibiotic Resistant (MAR) index was determined by procedure described by Krumperman (1983).

RESULTS

A total of 82 DUWLs water samples from 34 dental clinics were collected and screened for isolation and identification P. aeruginosa. Out of these, 72 clinics used distilled water and 10 clinics overhead tank water by 19 postgraduate and 63 undergraduates qualified dentist’s clinics. On analysis, 59 DUWLs water samples showed presence of P. aeruginosa from 52 distilled water (40 U.G. qualified and 12 P.G. qualified dentist clinics and 7 overhead tank water supply (all U.G. qualified dentist clinics). Out of 59 P. aeruginosa isolates, 19 from 3 ways syringe, 22 from Air rotor and 18 from Ultrasonic scaler. Out of total 82 DUWLs water samples, 15 53% water samples from different hand-pieces of 8 U.G. qualified dentist clinics whereas 7 57% belonging to 4 P.G. qualified dentist clinics were found to be free from P. aeruginosa. The 59 isolates of P. aeruginosa were isolated from dental clinics where frequency of washing of water container was daily 17, twice a day 8, once in two days 22, once in three days 2, once in four days 3 and weekly 7 (Table 2).

Table 2:	Presence of P. aeruginosa in various DUWL water samples

DISCUSSION

Dental unit waterlines are an integral part of dental surgery equipment, supplying water as a coolant, primarily for air turbine and ultrasonic scaler. The patient and the attending dental staff may inhale a fine spray of this water, as it splashes off the surface of the patient’s mouth. The presence of biofilm in DUWLs is a universal problem and pathogens from patients and the dental clinic environment can be cultivated from biofilm removed from DUWLs. The waterlines of dental units remain a potential weakness in the control of infection in the dental practice, as they can easily become contaminated with both patient-derived and municipal water impurities (Franco et al., 2005).

A total of 82 DUWLs water samples from 34 dental clinics were screened for P. aeruginosa and 59 samples were contaminated. Out of these, the water sample from ultrasonic scaler showed maximum 82% contamination of P. aeruginosa then 3-in-1 syringe 68% and air rotor 69%. As the diameter and material of the tubing were same, the reason for this finding may be due to different water flow rates or by the fact that ultrasonic scaler is used more frequently than 3-in-1 syringe and air rotor in the clinics (Walker et al., 2000).

The dental clinics using distilled water 72 for flushing showed 72% (52) whereas clinics using overhead tank water showed 70% (7) P. aeruginosa contamination. The distilled water that was in use might be contaminated, addition of distilled water in residual water, improper as well as less frequency of cleaning the storage tank; all these factors might be contributed for the contamination or formation of biofilms containing of P. aeruginosa. Moreover it was observed that the dentist’s with post graduate qualified dentist keeps dental water less 63% P. aeruginosa contaminated or free from biofilm formation as compared to dentists with undergraduates qualified dentist clinic 75%. The 59 (72%) isolates of P. aeruginosa were isolated from dental clinics where frequency of washing of water container was daily 53%, twice a day 89%, once in two days 79%, once in three days 100%, once in four days 100% and weekly 88%.

Table 3:	MARI of P. aeruginosa isolated from DUWL water

The data indicated that the increase in frequency of washing of water container and water lines decreased the P. aeruginosa contamination and increase in the quality of DUWLs water (Table 2). Increase in washing frequency of water container and the flushing through of water lines between patient and at the beginning and end of the working day eliminates the bacterial contamination, which is a useful method to eliminate oral flora entering the waterline via suck-back.

The dental water lines’ P. aeruginosa showed 100% resistance to novobiocin and cefaclor followed by nitrofurantoin and erythromycin 98%, almost 100% sensitive to ciprofloxacin and amikacin and 75-78% sensitive to piperacillin and gatifloxacin. The MAR index of antibiotics against P. aeruginosa showed highest MAR index 0.125 against novobiocin and cefaclor followed by 0.122 for nitrofurantoin and erythromycin, respectively. Thus, these antibiotics should not be used against infection in dental clinics whereas amikacin (MARI 0.0021) and ciprofloxacin (MARI 0.004) can be drug of choice against P. aeruginosa in dental treatment (Table 3).

CONCLUSIONS

Since the origin of dental unit water contamination is now more clearly defined, dental manufacturers and the scientific community in approaches can make substantial progress to prevention and control. More contamination of P. aeruginosa was found in water samples due to the multiple ports of the entry to the DUW system for microbes, no single method or device will completely eliminate the potential for cross infection. The ultrasonic scalar showed highest contamination of P. aeruginosa than other two hand pieces (3-in-1 syringe and air rotor). Combinations of currently available procedures and equipment including anti-retraction devices, flushing, independent water supplies used in conjunction with biocide purges or fully autoclavable water line circuitry should provide water, which is of a higher standard than that of a drinking water. Sterile water or saline should be provided from a separate source, which cannot be contaminated by passage through the DUWLs. The dental clinics using distilled water as a source of water for treatment showed more P. aeruginosa contamination. Therefore chair side devices for monitoring microbial quality of the DUW need to be developed and are an essential component to satisfactory water quality. Existing recommendations for flushing through of water lines between patients and at the beginning and end of the day is a useful method to eliminate oral flora entering the waterlines via suck-back. All these systems required strict adherence to maintenance protocols to perform to their full potential.