Investigating contamination of dental-unit waterline systems and microbial biofilm ecology

Abstract

Introduction: Biofilms within dental-unit waterlines (DUWLs) are acknowledged sources of contamination in the dental clinical environment and affect the quality of clinical treatment water. As a standard for reducing exposure to potentially harmful microorganisms, the Department of Health (DoH), UK suggests that water discharged from DUWL should contain 100 to 200 CFU/mL. However, local audits suggest that the quality of clinical treatment water often fails to meet the standards required.
The aim: The aim was to be able to readily identify waterlines with higher levels of contamination via validation of a rapid existing “in-office” test and subsequently understand biofilm ecology.
Materials and Methods: Water samples from 31 DUWLs in general dental practices were taken during the working day and cultured using the PetrifilmTM AC plate test as per manufacturer’s instructions and for extended incubation periods under laboratory conditions. The samples were also cultured using the laboratory based benchmark R2A agar. Further culture methods were employed for investigating spread of human pathogens with aerosolization and splatter of DUWL water; retraction valve failure; waterborne biofilm ecology and environment within a simulated laboratory DUWL (sDUWL) and whether amoebae were harboring nosocomial bacteria.
Results: The bacterial concentration of the water samples cultured on R2A agar varied significantly (1 × 101 to 4.3 × 106); in surgeries (48%) which met DoH standards and those that failed (52%). A retest of water from surgeries which delivered safe and contaminated water revealed that approximately 55% of practices met the recommended threshold values whilst around 45% failed. The PetrifilmTM AC Plate method gave variable sensitivity values on different occasions with 100% specificity. Only the nosocomial clinical isolate of Serratia marcescens was recovered from one clinical water sample. The opportunistic yeast, Candida parapsilosis from 1 sample indicated possible retraction valve failure. The in-vitro sDUWL output water demonstrated a fully established biofilm community by day 2 consisting of bacteria, a fungus (Cladosporium cladosporioides), and one amoeba (Vermamoeba vermiformis) as the main organisms. When tested under laboratory culture conditions, V. vermiformis, appeared to feed on S. marcescens isolated from clinical water. Electron microscopy confirmed bacterial adherence characteristics for biofilm formation, and altered pattern of cell division in one Gram positive isolate from the in-vitro sDUWL. Despite the detection of a Legionella species, no metabolically active opportunistic human pathogens were observed within V. vermiformis in the sDUWL biofilm.
Conclusions: This study demonstrates the importance of regular monitoring of DUWL water because even clean DUWLs can quickly become contaminated. One aim of this study was to find an in-office testing method for dental needs but it appears that improving the sensitivity of in-office tests is a challenge that needs addressing in the first instance. A more positive outcome was that, on the whole, clinical output water was not harbouring opportunistic human pathogens at the time of testing and that clinical surfaces were clean. Also when dental units are used there was no evidence that contaminants were being drawn back into the DUWLs. Overall, achieving a low level of microbial contamination consistently in water to 100 - 200 CFU/mL appeared to be difficult. In the short-term, if water could be tested more often this would help to understand the related challenges associated with conforming to national standards of delivering clean treatment water. The laboratory sDUWL model showed defective cell division and altered phenotype of specific bacterial species, and that V. vermiformis appeared unlikely to be harboring the late coloniser L. pneumophila, as it was out-with the size-range of bacteria, amoebae choose to feed upon. As the laboratory sDUWL model closely mimicked the heterogeneous biofilm development including the type of main microorganisms as those of the clinical DUWL it can be used to accurately accesses commercial biocides in the control of the biofilm independently as literature continues to question the efficacy of commercial disinfections in waterline cleansing protocols that fail to meet the required standards.