Pseudomonas aeruginosa outcompetes other bacteria in the manifestation and maintenance of a biofilm in polyvinylchloride tubing as used in dental devices

In a PVC tube as a model system for dental devices, Pseudomonas aeruginosa outcompetes Staphylococcus aureus and Klebsiella pneumoniae for the biofilm formation. P. aeruginosa has advantage over the other strains due to higher tolerance for low-nutrient situations or direct killing by the production of soluble factors like pyocyanin.


Findings
Polyvinylchloride (PVC) tubes are widely used in medical and dental devices. These tubes can easily come in contact with human skin and mucosa during odontology procedures and can be contaminated with the following bacteria that play a role in human medicine.
Pseudomonas aeruginosa is a Gram-negative rod-shaped bacterium found in moist to wet habitats. These include human mucosal surfaces, e.g., the nasopharynx (Fothergill et al. 2014), and surfaces in tap water lines (Rozej et al. 2015 count of the formed biofilms for 5-8 weeks. To imitate a dental device system, we cultivated the biofilm at room temperature and provided flow of water by a peristaltic pump. We used scanning electron microscopy to visualize the biofilms on the tube lumen and collect data on their spatial distribution at the end of the incubation period. After fixation and dehydration, samples were investigated in a scanning electron microscope (Jeol 6010, Eching, Germany) at 5 kV acceleration voltage using a spot size of 40 or 50, respectively. We used the REF detector setting in which data are collected through the secondary electron detector without using the suction current to attract secondary electrons. We found that biofilms are clustered in small groups of high bacterial counts instead of forming a single low-density biofilm spread over the entire tube lumen (Fig. 1a, b). Similar results have been observed with confocal microscopy using ground water and no specific bacterial input (Martiny et al. 2003).
The single biofilms showed a thick ECM spreading over the biofilm core, while single bacteria could still be visualized at the edges of the individual biofilms. A representative image is shown in Fig. 1b. At weekly intervals, a sample of the PVC tube was cut and stained with 0.5 % crystal violet for 5 min for macroscopic evaluation of the biofilm growth. We observed a gradual covering of the lumen along the time beginning with isolated clumps. Additionally, we weekly cut a 10-cm piece of tubing. 3 cm were cut from the non-pump side, washed extensively, and placed in 6 ml of MH medium. The biofilm was then detached from the tube by ultrasound (41 kHz, Bandelin Bactosonic, Berlin, Germany) for 1 min. Bacterial counts were determined after transferring 100 µl of the solution to a MH agar plate leading to a limit of detection of 10 cfu/ml. At end, we calculated how many bacteria were attached to the tube lumen per square centimeter. Figure 2 shows the determined values for both experimental settings combined.
Pseudomonas aeruginosa proved to be the predominant bacterial strain overgrowing both S. aureus and K. pneumoniae. All strains were initially detected at a similar level of approximately 10 5 cfu/cm 2 . After only 2 weeks, S. aureus had vanished from the system, while P. aeruginosa showed still increasing colony counts. K. pneumoniae, after slightly increasing from 5.01 to 5.83-5.89 log 10 cfu/cm 2 during the initial 2 weeks, decreased rapidly in the third week and was undetectable at any time point in the following weeks. The overgrowth by P. aeruginosa may either be an indirect effect of this species being better adapted to low-nutrient situations, or it may reflect a direct killing of the partners in co-culture by secreted toxins like pyocyanin (Voggu et al. 2006). In contrast to the vanishing of S. aureus or K. pneumoniae, the cfu counts of P. aeruginosa plateaued rather stably at approximately 200 cfu/cm 2 of tube lumen from week six on. This is in agreement with early reports showing that in dental unit water lines shedding of 10 2 to 10 6 cfu/ml can occur (Gross et al. 1976;Furuhashi and Miyamae 1985).
Our data show that biofilms can be formed and maintained in tap water on the lumen of PVC tubes using the Fig. 1 Scanning electron microscopy image of bacterial biofilms spread over the PVC tube lumen. a Small areas of biofilms spread on the PVC tube lumen; b Staphylococcus aureus biofilm on PVC lumen; c PVC lumen populated by Pseudomonas aeruginosa biofilm method developed here. Pseudomonas outcompeted the respective partners in co-cultures and established long-lived biofilms in water according to our experiments. The longtime incubation of P. aeruginosa with water has recently been shown to alter the phenotype significantly (Mendis et al. 2014). Our future experiments will focus on the genotypic and resulting phenotypic changes in long-term dental unit biofilms as well as the sterilization of biofilms with novel agents.