Biofilms and Device-Associated Infections

Special Issue
Microbial biofilms develop when microorganisms irreversibly
adhere to a submerged surface and produce
extracellular polymers that facilitate adhesion and provide a
structural matrix. This surface may be inert, nonliving
material or living tissue. Biofilm-associated microorganisms
behave differently from planktonic (freely suspended)
organisms with respect to growth rates and ability to resist
antimicrobial treatments and therefore pose a public health
problem. This article describes the microbial biofilms that
develop on or within indwelling medical devices (e.g., contact
lenses, central venous catheters and needleless connectors,
endotracheal tubes, intrauterine devices, mechanical heart
valves, pacemakers, peritoneal dialysis catheters, prosthetic
joints, tympanostomy tubes, urinary catheters, and voice
Characteristics of Biofilms on Indwelling Medical
Biofilms on indwelling medical devices may be composed
of gram-positive or gram-negative bacteria or yeasts. Bacteria
commonly isolated from these devices include the grampositive
Enterococcus faecalis, Staphylococcus aureus,
Staphylococcus epidermidis, and Streptococcus viridans; and
the gram-negative Escherichia coli, Klebsiella pneumoniae,
Proteus mirabilis, and Pseudomonas aeruginosa. These
organisms may originate from the skin of patients or healthcare
workers, tap water to which entry ports are exposed, or
other sources in the environment. Biofilms may be composed
of a single species or multiple species, depending on the device
and its duration of use in the patient. Urinary catheter
biofilms may initially be composed of single species, but
longer exposures inevitably lead to multispecies biofilms (1).
A distinguishing characteristic of biofilms is the presence of
extracellular polymeric substances, primarily polysaccharides,
surrounding and encasing the cells. These polysaccharides,
which have been visualized by scanning electron
microscopy (Figure 1), appear either as thin strands
connecting the cells to the surface and one another or as
sheets of amorphous material on a surface. Most biofilm
volume is actually composed of this extracellular polymeric
substance rather than cells, a fact that has been confirmed by
ruthenium red staining and transmission electron microscopy
(2). This biofilm matrix may act as a filter, entrapping
minerals (1) or host-produced serum components (3). Biofilms
are both tenacious and highly resistant to antimicrobial
treatment; Anwar et al. (4) showed that treatment with levels
of tobramycin far in excess of the MIC reduced biofilm cell
counts for P. aeruginosa by approximately 2 logs, while the
same dosage provided a >8-log decrease in planktonic cells of
this organism.
Factors Influencing Rate and
Extent of Biofilm Formation
When an indwelling medical device is contaminated with
microorganisms, several variables determine whether a
biofilm develops. First the microorganisms must adhere to
the exposed surfaces of the device long enough to become
irreversibly attached. The rate of cell attachment depends on
the number and types of cells in the liquid to which the device
is exposed, the flow rate of liquid through the device, and the
physicochemical characteristics of the surface. Components
in the liquid may alter the surface properties and also affect
rate of attachment. Once these cells irreversibly attach and
Biofilms and Device-Associated Infections
Rodney M. Donlan
Centers for Disease Control and Prevention Atlanta, Georgia, USA
Address for correspondence: Rodney M. Donlan, National Center for
Infectious Diseases, Hospital Infections Program, Centers for Disease
Control and Prevention, 1600 Clifton Road, Mailstop C16, Atlanta, GA
30333, USA; fax: 404-639-2322; e-mail: vog.cdc|8dlr#vog.cdc|8dlr
Microorganisms commonly attach to living and nonliving surfaces, including those of indwelling medical
devices, and form biofilms made up of extracellular polymers. In this state, microorganisms are highly
resistant to antimicrobial treatment and are tenaciously bound to the surface. To better understand and
control biofilms on indwelling medical devices, researchers should develop reliable sampling and
measurement techniques, investigate the role of biofilms in antimicrobial drug resistance, and establish the
link between biofilm contamination and patient infection.
Figure 1. Scanning electron micrograph of a Staphylococcus biofilm
on the inner surface of a needleless connector.
Photograph by Janice Carr, Centers for Disease Control and Prevention,
Atlanta, GA USA.
Emerging Infectious Diseases 278 Vol. 7, No. 2, March–April 2001
Special Issue
produce extracellular polysaccharides to develop a biofilm,
rate of growth is influenced by flow rate, nutrient composition
of the medium, antimicrobial-drug concentration, and
ambient temperature. These factors can be illustrated by
examining what is known about biofilms on three types of
indwelling medical devices: central venous catheters,
mechanical heart valves, and urinary (Foley) catheters.
Central Venous Catheter Biofilms
Scanning and transmission electron microscopy has
shown that virtually all indwelling central venous catheters
are colonized by microorganisms embedded in a biofilm
matrix (5). The organisms most commonly isolated from
catheter biofilms are Staphylococcus epidermidis, S. aureus,
Candida albicans, P. aeruginosa, K. pneumoniae, and
Enterococcus faecalis (6,7).
These organisms originate from patient’s skin microflora,
exogenous microflora from health-care personnel, or
contaminated infusates. They gain access to the catheter by
migration externally from the skin along the exterior catheter
surface or internally from the catheter hub or port (8).
Colonization of these devices can occur rapidly (within 24
hours) and may be a function of host-produced conditioning
films (platelets, plasma, and tissue proteins) (8). Raad et al.
(9) found that biofilm formation on central venous catheters
was universal, but the extent and location of biofilm
formation depended on the duration of catheterization: shortterm
(<10 days) catheters had greater biofilm formation on
the external surface; long-term catheters ( 30 days) had more
biofilm formation on the catheter inner lumen. The nature of
the fluid administered through central venous catheters may
affect microbial growth: gram-positive organisms
(S. epidermidis, S. aureus) did not grow well in intravenous
fluids, whereas the gram-negative aquatic organisms (e.g.,
P. aeruginosa, Klebsiella spp., Enterobacter spp., Serratia
spp., and Pantoea sp.) sustained growth (10-14). Because
many of these solutions have limited nutrients, bacterial
growth rarely produces turbidity, meaning that numbers are
<107 organisms per milliliter. The number of organisms on
the catheter tip is related to occurrence of bloodstream
infection in the patient (7,15-17), supporting the concept of a
critical level of biofilm development above which substantial
cell detachment and embolism occur.
Several studies have examined the effect of various types
of antimicrobial treatment in controlling biofilm formation on
these devices. Freeman and Gould (18) found that addition of
sodium metabisulfite to the dextrose-heparin flush of the left
atrial catheter eliminated microbial colonization of these
catheters. Darouiche et al. (19) found that catheters
impregnated with minocycline and rifampin were less likely
to be colonized than those impregnated with chlorhexidine
and silver sulfadiazine. In a study by Kamal et al. (20),
catheters coated with a cationic surfactant
(tridodecylmethylammonium chloride), which was in turn
used to bond cephalosporin to the surface, were less likely to
become contaminated and develop biofilms than were
untreated catheters. Flowers et al. (21) found that an
attachable subcutaneous cuff containing silver ions inserted
after local application of polyantibiotic ointment conferred a
protective effect on catheters, resulting in lower rates of
contamination. Maki (8) suggested several ways to control
biofilms on central venous catheters, including using aseptic
technique during implantation, using topical antibiotics,
minimizing the duration of catheterization, using an in-line
filter for intravenous fluids, creating a mechanical barrier to
prevent influx of organisms by attaching the catheter to a
surgically implanted cuff, coating the inner lumen of the
catheter with an antimicrobial agent, and removing the
contaminated device.
Mechanical Heart Valve Biofilms
Microorganisms may attach and develop biofilms on
components of mechanical heart valves and surrounding
tissues of the heart, leading to a condition known as prosthetic
valve endocarditis. The primary organisms responsible for
this condition are S. epidermidis, S. aureus, Streptococcus
spp., gram-negative bacilli, diphtheroids, enterococci, and
Candida spp. These organisms may originate from the skin,
other indwelling devices such as central venous catheters, or
dental work (3). The identity of the causative microorganism
is related to its source: whether the contaminating organism
originated at the time of surgery (early endocarditis, usually
caused by S. epidermidis), from an invasive procedure such as
dental work (Streptococcus spp.), or from an indwelling device
(a variety of organisms). Implantation of the mechanical
heart valve causes tissue damage, and circulating platelets
and fibrin tend to accumulate where the valve has been
attached. Microorganisms also have a greater tendency to
colonize these locations (3). The resulting biofilms more
commonly develop on the tissue surrounding the prosthesis or
the sewing cuff fabric used to attach the device to the tissue
(22,23) than on the valve itself (24). Antimicrobial agents are
usually administered during valve replacement and
whenever the patient has dental work to prevent initial
attachment by killing all microorganisms introduced into the
bloodstream. As with biofilms on other indwelling devices,
relatively few patients can be cured of a biofilm infection by
antibiotic therapy alone (25). Illingworth et al. (22) found that
a silver-coated sewing cuff on a St. Jude mechanical heart
valve (St. Jude Medical Inc., St. Paul, MN) implanted into a
guinea pig artificially infected with S. epidermidis produced
less inflammation than did uncoated fabric. Although the
number of attached organisms was not determined, the
authors concluded that the degree of inflammation was
proportional to the number of viable organisms. Carrel et al.
(23) also found this approach was effective in in vitro studies
with different organisms.
Urinary Catheter Biofilms
Urinary catheters are tubular latex or silicone devices,
which when inserted may readily acquire biofilms on the
inner or outer surfaces. The organisms commonly contaminating
these devices and developing biofilms are S.
epidermidis, Enterococcus faecalis, E. coli, Proteus mirabilis,
P. aeruginosa, K. pneumoniae, and other gram-negative
organisms (1). The longer the urinary catheter remains in
place, the greater the tendency of these organisms to develop
biofilms and result in urinary tract infections. For example,
10% to 50% of patients undergoing short-term urinary
catheterization ( 7 days) but virtually all patients undergoing
long-term catheterization (>28 days) become infected (1).
Brisset et al. (26) found that adhesion to catheter materials
was dependent on the hydrophobicity of both the organisms
and the surfaces; catheters displaying both hydrophobic and
hydrophilic regions allowed colonization of the widest variety
of organisms. Divalent cations (calcium and magnesium) and
Vol. 7, No. 2, March–April 2001 279 Emerging Infectious Diseases
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increase in urinary pH and ionic strength all resulted in an
increase in bacterial attachment. Tunney et al. (27) stated
that no single material is more effective in preventing
colonization, including silicone, polyurethane, composite
biomaterials, or hydrogel-coated materials. Certain component
organisms of these biofilms produce urease, which
hydrolyzes the urea in the patient’s urine to ammonium
hydroxide. The elevated pH that results at the biofilm-urine
interface results in precipitation of minerals such as struvite
and hydroxyapatite. These mineral-containing biofilms form
encrustations that may completely block the inner lumen of
the catheter (27). Bacteria may ascend the inner lumen into
the patient’s bladder in 1 to 3 days (28); this rate may be
influenced by the presence of swarming organisms such as
Proteus spp. (D. Stickler, pers. comm.). Several strategies
have been attempted to control urinary catheter biofilms:
antimicrobial ointments and lubricants, bladder instillation
or irrigation, antimicrobial agents in collection bags,
impregnation of the catheter with antimicrobial agents such
as silver oxide, or use of systemic antibiotics (29). Most such
strategies have been ineffective, although silver-impregnated
catheters delayed onset of bacteriuria for up to 4 days. In a
rabbit model, biofilms on Foley catheter surfaces were highly
resistant to high levels of amdinocillin, a beta-lactam
antibiotic (30). However, Stickler et al. (31) found that
treatment of a patient with a polymicrobial biofilm-infected
catheter with ciprofloxacin allowed the catheter to clear and
provide uninterrupted drainage for 10 weeks. Morris et al.
(32) found that time to blockage of catheters in a laboratory
model system was shortest for hydrogel- or silver-coated latex
catheters and longest for an Eschmann Folatex S All Silicone
catheter (Portex Ltd., Hythe, Kent, England). Biofilms of
several gram-negative organisms were reduced by exposure
to mandelic acid plus lactic acid (33). In a study in which
ciprofloxacin-containing liposomes were coated onto a
hydrogel-containing Foley catheter and exposed in a rabbit
model, the time to development of bacteriuria was double that
with untreated catheters, although infection ultimately
occurred in the rabbits with treated catheters (34).
Directions for Future Research
To better understand and control biofilms on indwelling
medical devices, research must progress in several key areas.
More reliable techniques for collecting and measuring
biofilms should be developed. For central venous catheters,
the reference method for quantification of biofilms on catheter
tips is the roll-plate technique, in which the tip of the catheter
is removed and rolled over the surface of a nonselective
medium. Quantification of the biofilm depends on the number
of organisms recovered by contact with the agar surface.
Biofilm-associated cells on the inner lumen of the device are
not detected with this method, which has low diagnostic
sensitivity and low predictive value for catheter-related
bacteremia (7). In addition, this method cannot detect more
than 1,000 colony-forming units (CFU) per tip. A method that
used sonication plus vortexing as a means of quantifying
biofilms on catheter tips showed that a level of 104 CFU per tip
is predictive of catheter-related septicemia. Although this
method is an improvement over the semi-quantitative rollplate
technique, the recovery efficiency of the method needs to
be determined (i.e., the percentage of cells that are not
recovered and quantified). Zufferey et al. (35) described a
method for rapidly detecting biofilm cells on catheters by
direct staining of the catheter with acridine orange. Although
they found good agreement with culture techniques and noted
that this technique provided more rapid results, they did not
quantify cells; instead, they recorded a simple positive or
negative result. Techniques that allow counting of biofilm
cells directly on the catheter surface would be an
improvement over established methods.
Model systems should be developed and used to study
biofilm processes on various indwelling medical devices.
These systems should closely simulate the in vivo or in situ
conditions for each device, while at the same time providing
reproducible, accurate results. To investigate biofilm
formation on needleless connectors, Donlan et al. (14) used a
biofilm disk reactor system (Figure 2) that incorporated a
medium (intravenous fluid), a material (teflon coupons or
needleless connectors), an organism (Enterobacter cloacae),
and a flow rate (1 mL/min) that closely simulated conditions
of use for these devices. Results were both reproducible and
precise, and the system was capable of developing a steady
state biofilm (Figure 3). This system design could be used to
investigate and compare various biofilm control treatments,
device design modifications, or different media formulations.
By performing a similar experiment in an animal model
system, biofilm processes in vivo could be predicted.
Another area of great importance from a public health
perspective is the role of biofilms in antimicrobial-drug
resistance. Bacteria within biofilms are intrinsically more
Figure 2. Biofilm disk reactor system.
Figure 3. Enterobacter cloacae biofilm formation on needleless
Emerging Infectious Diseases 280 Vol. 7, No. 2, March–April 2001
Special Issue
resistant to antimicrobial agents than planktonic cells
because of the diminished rates of mass transport of
antimicrobial molecules to the biofilm associated cells (36) or
because biofilm cells differ physiologically from planktonic
cells (37). Antimicrobial concentrations sufficient to
inactivate planktonic organisms are generally inadequate to
inactivate biofilm organisms, especially those deep within the
biofilm, potentially selecting for resistant subpopulations.
This selection may have implications for treatments that use
controlled release of antimicrobial agents to prevent biofilm
growth on indwelling devices. Bacteria can transfer
extachromosomal genetic elements within biofilms; Roberts
et al. (38) demonstrated transfer of a conjugative transposon
in a model oral biofilm. Hausner and Wuertz (39)
demonstrated conjugation in a lab-grown biofilm with rates
one to three orders of magnitude higher than those obtained
by classic plating techniques. Resistance-plasmids could also
be transferred within biofilms on indwelling medical devices.
The link between biofilm contamination of an indwelling
device and patient infection is often unclear. Raad et al. (9)
noted that biofilm formation was universal on vascular
catheters collected from patients, yet observed that this
universal colonization rarely resulted in bloodstream
infection. A better understanding of the factors that control
cell detachment may help answer the questions: Is there a
critical biofilm density threshold above which detachment
occurs? What is the role of the exopolymers in this process?
Davies et al. (40) demonstrated the role of acyl homoserine
lactones (HSL) in biofilms of P. aeruginosa and showed that
HSL-knockouts were deficient in biofilm architecture and
much more readily detached than wild-type organisms.
Stickler et al. (41) detected these quorum-sensing molecules
in biofilms on urethral catheters. A greater understanding of
cell-to-cell communication within biofilms may lead to better
predictability of biofilm processes such as detachment, as well
as more effective control strategies.
Microbial biofilms may pose a public health problem for
persons requiring indwelling medical devices. The microorganisms
in biofilms are difficult or impossible to treat with
antimicrobial agents; detachment from the device may result
in infection. Although medical devices may differ widely in
design and use characteristics, specific factors determine
susceptibility of a device to microbial contamination and
biofilm formation. For example, duration of use, number and
type of organisms to which the device is exposed, flow rate and
composition of the medium in or on the device, device material
construction, and conditioning films on the device all may
influence biofilm formation. More effective biofilm control
strategies should result as researchers develop more reliable
techniques for measuring biofilms and better model systems
for evaluating control strategies. A clearer picture of the
importance of biofilms in public health should also result as
the role of biofilms in antimicrobial-drug resistance is
investigated and the link is established between biofilm
contamination and patient infection.
Dr. Donlan is team leader for the Division of Healthcare Quality
Promotion Biofilm Laboratory, National Center for Infectious Diseases,
CDC. His research interests focus on biofilms on indwelling medical
devices, the role of biofilms in antimicrobial-drug resistance, and survival
and treatment of pathogenic organisms in potable water system
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