Oral Biofilms. Группа авторов

7]. Potential pathogens grow rapidly (within 2 days) on ordinary solid bacteriological media in 36 °C. Samples could be incubated at room temperature (22 °C) but grow slower [8]. A longer incubation time (1 week) will increase the number of CFU due to the slow-growing nature of most heterotrophic bacteria for which the water lines are their natural habitat [4]. High numbers of slow-growing bacteria therefore indicate poor water quality, the presence of a biofilm, and a rich inner life of the DUWL.

A special concern relating to biofilms and the microbiota in water lines such as in DUWLs is the presence of protozoa/amoebae [13]. There are various examples, with the most recognized being Acanthamoeba species. Protozoans are of special interest in Legionella pathogenicity (see below).

      Specific Pathogens in DUWLs

      Although numerous microorganisms have been found in the water of DUWLs, some opportunistic pathogens are of special concern for patients and personnel, for example aerobic Gram-negative bacilli such as Pseudomonas spp. and Legionella spp. [4, 13].

Pseudomonas spp. are regularly present in water, soil, and moist environments, and should be considered an indicator of poor water quality. Pseudomonas should not be present in drinking water and therefore not in the water of DUWLs. Pseudomonas spp. typically cause opportunistic infections, and respiratory tract infections in particular. Patients with respiratory diseases such as COPD and cystic fibrosis are a particular risk for Pseudomonas infections [14–17], with P. aeruginosa being the most common species.

Legionella infections are typically spread through aerosols from water lines containing various Legionella spp. [3, 18, 19]. L. pneumophila is responsible for most Legionella infections and can be serologically classified, which makes it possible to trace the origin [11]. Some outbreaks can be endemic, when many compromised patients can be exposed at the same time, for example in hotels, hospitals, and public baths. An aggravating circumstance of Legionella in water lines is their tendency to hide intracellularly in protozoa/amoebae residing in the biofilms [20, 21]. This makes the action of some disinfectants, especially chlorine, less efficient [19].

      Methods to Reduce Bacterial Counts in DUWLs

A number of solutions have been introduced on the market, but it is beyond the purpose of this paper to review the various products that are available. Principally, the methods are of two approaches, those that disinfect/reduce bacteria from the output water, and those that attack and eliminate/prevent the biofilm in the DUWLs [4, 22]. The first is attractive since it is based on a continuous delivery of the disinfectant, needs less manipulation and care, but has the disadvantage that it may expose patients to antimicrobials that are still present in the water when it is used. A continuous delivery, such as using hydrogen peroxide, may also efficiently prevent biofilm formation in the DUWLs once they have been eliminated [23]. Another disadvantage is that disinfection is only performed at use – and bacterial growth and biofilm formation may take place in units that are only sporadically used or over the weekend and during vacations. The principle of non-continuous delivery is based on the disinfection being active for longer periods of time, such as overnight or at weekends, which allows for a more efficient elimination of the biofilm using chlorine products. An established biofilm is much more difficult to eradicate. It is sometimes necessary to expose the entire DUWL system to a strong disinfectant (shock treatment) in order to remove the biofilm. Sodium hydroxide 0.1 M (0.8%) or sodium hypochlorite 0.5% has been suggested [3, 24, 25]. However, it should only be used when necessary due to the risk of damage to metal pieces (e.g., valves and connections) within the DUWL by corrosion if used frequently and for longer periods of time. Importantly, these strong solutions should be stained (e.g., methylene blue) to make it possible to check that the chemicals have been washed off. In severe cases, when there is a total blockage in the water lines, the radical solution is to exchange the lines with new ones, although this is expensive [3].

The threshold for a sufficient water-cleaning system within the EU has been recommended to be <200 CFU/mL and the water in a DUWL should be checked yearly. The American Dental Association (ADA) has recommended the same, while the recommendation of the Center for Disease Control (CDC) for drinking water is 500 CFU/mL [4]. The Swedish recommendations, which were set in 2006 [26] and before the EU recommendation was decided, used the threshold of 100 CFU/mL and is still in use [27, 28]. In practice it means that we accept, although with attention, up to 500 CFU/mL for heterofermentative fast-growing (<2 days) bacteria at room temperature (22 °C). In order to get a better picture of the “internal life” and presence of a biofilm, in our lab we practice incubation for 7 days for slow-growing bacteria, which should not exceed 5,000 CFU/mL according to the drinking water standard for tap water in Sweden [3]. Such high levels indicate the presence of biofilms in a DUWL and should be shock treated. However, when regular antimicrobial treatment of each DUWL in a clinic is performed, such high numbers of bacteria are rare.

      Reasons for Failure

      While most DUWLs with functioning water-cleaning systems show few or no bacteria, others still harbor bacteria to an unacceptable level. A 4-year follow-up study [28] showed that the installation of water-cleaning systems (added systems or inbuilt systems) leads to a significant improvement in output water quality. For 1,200 DUWLs at 140 clinics followed between 2013 and 2016, a 95% acceptance of the water quality was attained. The Alpro/Bilpron system was used in the majority of DUWLs (81.6%), but also Oxygenal (2.0%), Unit-Clean (5.2%), Sterilox (0.6%), as well as inbuilt systems (4.8%) were included in 2016. This study did not show any significant differences between the systems. In the remaining 5% of DUWLs that did not reach an acceptable level, the failures could be divided into problems related to the unit or to the clinic. Even if the unit is equipped with a water-cleaning system there is no guarantee of successful results. The most common reason for failure is neglect to run the water-cleaning system regularly. If the bacterial level in the output water is not acceptable, additional cleaning steps have to be taken. It is of outmost importance that the personnel are sufficiently informed and instructed and that necessary precautions are taken for servicing each unit.

      Clinic-related