Pseudomonas aeruginosa outcompetes other bacteria in the manifestation and maintenance of a biofilm in polyvinylchloride tubing as used in dental devices
Arch Microbiol
Pseudomonas aeruginosa outcompetes other bacteria in the manifestation and maintenance of a biofilm in polyvinylchloride tubing as used in dental devices
Christoph Gert Ammann 0 1
Markus Nagl 0 1
Michael Nogler 0 1
Débora Cristina Coraça‑Huber 0 1
0 Division of Hygiene and Medical Microbiology, Department of Hygiene, Microbiology and Social Medicine, Medical University of Innsbruck , Schöpfstrasse. 41, 6020 Innsbruck , Austria
1 Experimental Orthopaedics, Medical University of Innsbruck , Innrain 36 - 1. Floor, 6020 Innsbruck , Austria
2 Débora Cristina Coraça-Huber
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. Communicated by Erko Stackebrandt.
Bacterial biofilm; PVC tubing; Dental device; Co-culture
-
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)
. Klebsiella pneumoniae is a Gram-negative
rod-shaped bacterium that can colonize mouth mucosal
tissue
(Bagley 1985)
and cause pneumonia
(Podschun and
Ullmann 1998)
. Staphylococcus aureus is a Gram-positive
bacterium that typically colonizes skin. Approximately
20 % of all human beings are long-term carriers of S.
aureus in nose mucosa
(Kluytmans et al. 1997)
.
Once in contact with the moist surface of a tube lumen,
bacteria can adhere to the material and start biofilm
formation. Biofilms are formed in several stages during
propagation of bacteria after adherence
(Stoodley et al. 2002)
.
Once a sufficient number of bacteria are reached, the
biofilm matures, ultimately establishing an extracellular
matrix (ECM). The ECM contains water, polyglycans,
proteins, and nucleotides
(Branda et al. 2005)
. Bacteria in the
biofilm use these stored materials in times of malnutrition
from the exterior.
We here investigate the growth of three bacterial strains
which are typical components of human flora. These can
transfer from human to a dental device and subsequently
grow in the lumen of the PVC tubing. We sought to
determine the growth pattern and dynamics of S. aureus and K.
pneumoniae in co-culture with P. aeruginosa, a well-known
contaminant of water systems.
Pseudomonas aeruginosa ATCC 27853, S. aureus ATCC
25923, and K. pneumoniae (clinical isolate) single colonies
from Mueller–Hinton (MH) agar were grown separately
overnight at 37 °C in MH broth to 2–5 × 109
colony-forming units (cfu)/mL. Subsequently, P. aeruginosa was mixed
with S. aureus or K. pneumoniae and diluted in MH broth
to approximately 1 × 105 cfu/ml for each strain. One tube
system each was filled with P. aeruginosa plus S. aureus or
P. aeruginosa plus K. pneumoniae. We here sought to speed
up the process of initial biofilm formation by providing
ample nutrients (MH broth) for 72 h. After this period, we
provided tap water for the system and followed the bacterial
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-c (...truncated)