ANTIBIOTIC SENSITIVITY OF TRUE AND OPPORTUNISTIC PATHOGENS IN DENTAL UNIT WATER LINES

A total of 38 water samples were obtained from the dental unit water lines in three(3) hospital/clinics within Zaria, Nigeria viz: The Kaduna State Dental Clinic, Tudun Wada, University Health Services (Sick Bay), A.B.U. Zaria, Ahmadu Bello University Teaching Hospital, Shika, Nigeria and assessed for true and opportunistic pathogens. Sixteen (16) bacterial isolates consisting of five isolates of Pseudomonas aeruginosa and 11 isolates of Klebsiella pneumoniae were recovered. The antibiotic susceptibility profile of K. pneumoniae showed that it was susceptible to gentamicin and nalidixic acid but resistant to tetracycline, streptomycin ampicillin, cotrimoxazole, colistin and nitrofuratoin. Ps.aeruginosa was susceptible to gentamicin and colistin and resistant to streptomycin, ampicillin, nalidixic acid, cotrimoxazole and nitrofuratoin. These true and opportunistic pathogens may pose a health risk to the immunocomprised patients visiting the dental clinic for surgery and other dental procedures.


INTRODUCTION
Dental unit wate r lines (DUWLs) may be heavily contaminated with microorganisms.These organisms may be environmental organisms such as Alcaligenes denitrificans, Moraxella sp and Caulobacter sp or opportunistic and true human pathogens such as Pseudomonas aeruginosa, Klebsiella pneumoniae, Mycobacterium sp (Williams et al., 1996;Pankhurst et al., 1998;Walker et al., 2000;Franco et al., 2006).Opportunistic pathogens may account for more than 30% of the total bacterial population in water distribution systems (Barbeau et al., 1996).Certain types of Gramnegative bacteria can persist in water and multiply actively in aqueous environments associated with some medical equipment such as the dental hand pieces.This can lead to accumulation of large numbers of Gramnegative bacteria which can directly or indirectly affect patients by causing septicaemia, abscesses and endotoxemia (Favero et al., 1992;Lonnemann et al., 1992).
The laminar flow of DUWLs encourages maximum flow at the centre and minimal flow at the periphery.This results in formation of biofilms by many bacteria on the inner surface of the tubing (Douglas and van Nourt, 1993;Fayle and Pullard, 1996).
Organisms in biofilms are derived from incoming mains water supply.Bacteria can be released from these biofilms slowly into DUWLs.During dental procedures, these organisms are sprayed in aerosols arising from the low or high hand speed device attached to the dentist's chair.Many tasks performed in dentistry can produce aerosols.Many organisms particularly bacteria can produce fatal pneumonias in immunocompromised individuals (Franco et al., 2005).
Against this background information, this study was undertaken to ascertain the possible health risks to which the unknowing public may be exposed to.Moreover, no records of such studies in Northern Nigeria are known to us.

Sample collection
Thirty-eight dental unit water samples were collected in sterile bottles that were dechlorinated with sodium thiosulphite (Na 2 SO 3 ).These water samples were obtained from three different hospitals within Zaria, Nigeria; The Kaduna State Dental Clinic, Tudun Wada; Ahmadu Bello University (A.B.U) Teaching Hospital, Shika, Nigeria; and University Health Services (Sick Bay), A.B.U.Zaria, Nigeria.

ISOLATION OF MICROORGANISMS
Pre-enrichment of the water samples was done by introducing 1ml of water sample into 5ml of Mueller-Hinton broth and the tubes were incubated at 37 0 C for 18 h.A sterile pipette was used to deliver 0.25mls of the overnight broth culture into blood agar and cetrimide agar.The inoculum was spread with a sterile bent glass rod.All inoculated plates were incubated at 37 0 C for 24 h.Cultures were purified, and thereafter stored on nutrient agar slants.

BIOCHEMICAL IDENTIFICATION OF BACTERIAL ISOLATES
Specific biochemical tests for the true and opportunistic pathogens were done according to Barrow and Feltham (1974).

ANTIBACTERIAL SUSCEPTIBILITY TEST
The Kirby-Bauer method (Clinical Laboratory Institute Standards.(CLIS), ( 2005) was used.Mueller-Hinton Agar (MHA) was prepared according to manufacturer's instructions.Sterile swab sticks were used to spread-inoculate agar plates with the standardized suspension of the isolates (0.5 MacFarland).Using a sterile pair of forceps, antibiotic discs were evenly placed on the inoculated plates.The plates were incubated at 37 o C for 18-24 h after which zones of growth inhibition were observed and their diameters measured with a metric rule.The results were classified as resistant, intermediate and susceptible according to the interpretative chart of the National Committee for Clinical Laboratory Institute Standards (CLIS, 2005).

RESULTS
Table 1 shows the bacterial isolates recovered from the three dental unit water lines and the biochemical identification.Out of the 16 isolates, five were identified as Pseudomonas aeruginosa and 11 as K. pneumoniae.There were six (6) K. pneumoniae isolates and no P. aeruginosa detected in the water samples obtained from the dental unit water lines in Sick bay,UHS, ABU, Zaria, Nigeria.The antibiotic susceptibility profiles of the isolates are presented in Table 2. K. pneumoniae was susceptible to gentamicin and colistin while P.aeruginosa was susceptible to gentamicin and nalidixic acid only.

DISCUSSION
Pseudomonas aeruginosa and Klebsiella pneumoniae were the opportunistic pathogens isolated from the water supply in the dental units of hospitals used in this study.
Sixteen bacterial isolates consisting of 11 isolates of K.pneumoniae and five isolates of Ps.aeruginosa were recovered.These two organisms are likely to cause pneumonia and/or bacteriemia in immunocompromised subjects.Water delivered through dental handpieces does not meet potable water standards because it has much higher counts as seen in this study.The reason is the high area to volume of small-bore water -lines which offers a lot of surface area on which microorganisms can settle and form biofilms (Franco et al., 2005).The American Dental Association (ADA) states that water used in dental units should be less or equal to 200 cfu/ml while the European Union has drinking water standard set at 100cfu/ml.These values are equivalent to WHO standards for dental unit water and drinking water respectively.
The water samples analysed had colonies Too Numerous To Count (TNTC/ml) which exceeded the ADA and EU standards for dental unit water.Fulford et al (2004) found counts that exceeded the ADA limit of 200cfu/ml.
The slow and quiescent nature of water in DUWLs encourages the formation of biofilms.These biofilms may adhere to the inner lining of the tubes of the dental unit lines.Tuttlebee et al (2002) showed that biofilms can be shed on a continuous basis into DUWLs and these organisms may be sprayed into the patient's mouth during dental surgery.P. aeruginosa is a known nosocomial pathogen and can colonize wounds and establish infections such as oral abscesses dental caries and wound infections (Anaissie, 2002;Rossolii and Mantengoli, 2005).
Predisposing factors such as Diabetes mellitus, immunoglobulin deficiency states, steroid therapy and surgery may be responsible for nosocomial infections by P.aeruginosa.Sterile water supplies have been recommended for surgical procedures (Parkhurst and Johnson, 1998).Ps.aeruginosa derived from Dental Unit Water Lines (DUWLs) is the only opportunistic Gramnegative pathogen that has been implicated in causing infection and is a marker of the presence of opportunistic pathogens (Walker et al., 2000).P.aeruginosa strains were resistant to many antimicrobial agents including the older quinolones like nalidixic acid, tetracycline,ampicillin, among others.All the strains were multiresistant with a Multiple Antibiotic Resistance (MAR) index greater than 0.2.This means that the organisms must have been derived from an environment where antibiotics are used without prescription (Paul et al., 1997).
The presence of a number of active multidrug efflux systems are known to contribute to the intrinsic resistance or reduced susceptibility of this species to several antimicrobial agents (Poole, 2004).Resistance to ampicillin could be due to the presence of AmpC chromosomal β-lactamases (Livermore, 1995).
The most important anti-pseudomonal agents include some aminoglycosides( of which gentamicin is an example) and all the isolates of P. aeruginosa were susceptible.They were susceptible to colistin-an effective but toxic anti-pseudomonal drug (Giamarellou, 2002) There are various approaches to risk reduction to both the patient and the dental surgeons.These include the use of anti-retraction valves and retrograde aspiration of oral fluids (Beierle, 1998), filtration (Parkhurst andJohnson, 1998), flushing, use of biocides, chlorination, autoclavable systems, use of peroxide, ozone and ultra violet light (Parkhurst and Johnson,1998).

Table 2 :
Susceptibility profiles of klebsiella pneumoniae and pseudomonas aeruginosa