Biophysics of biofilm infection
Author: Philip S. Stewart
NOTICE: This is an Accepted Manuscript of an article published in Pathogens and Disease on 
January 2014, available online: http://www.tandfonline.com/10.1111/2049-632x.12118.
Stewart P, "Biophysics of biofilm infection," Pathogens and Disease 2014 70: 212–218.
Made available through Montana State University’s ScholarWorks 
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Biophysics of biofilm infection
Philip S. Stewart
Center for Biofilm Engineering, Montana State University, Bozeman, MT, USA
Abstract
This article examines a likely basis of the tenacity of biofilm infections that has received relatively little 
attention: the resistance of biofilms to mechanical clearance. One way that a biofilm infection persists is 
by withstanding the flow of fluid or other mechanical forces that work to wash or sweep microorganisms 
out of the body. The fundamental criterion for mechanical persistence is that the biofilm failure strength 
exceeds the external applied stress. Mechanical failure of the biofilm and release of planktonic microbial 
cells is also important in vivo because it can result in dissemination of infection. The fundamental 
criterion for detachment and dissemination is that the applied stress exceeds the biofilm failure strength. 
The apparent contradiction for a biofilm to both persist and disseminate is resolved by recognizing that 
biofilm material properties are inherently heteroge-neous. There are also mechanical aspects to the ways 
that infectious biofilms evade leukocyte phagocytosis. The possibility of alternative therapies for treating 
biofilm infections that work by reducing biofilm cohesion could (1) allow prevailing hydrodynamic shear 
to remove biofilm, (2) increase the efficacy of designed interventions for removing biofilms, (3) enable 
phagocytic engulfment of softened biofilm aggregates, and (4) improve phagocyte mobility and access to 
biofilm.
Keywords
cohesion
viscoelastic
failure
mechanical
neutrophil
detachment
Bacteria or fungi that aggregate in biofilms can cause persistent infections (Costerton et al., 1999). These 
infec-tions are typically localized, slow moving, recurrent, and poorly controlled by antibiotics or 
antiseptics (Parsek & Singh, 2003). The host immune response to the continued presence of 
microorganisms can give rise to collateral damage to neighboring healthy tissue, perpetuating a 
degradative, nonhealing state. A few well-known examples of biofilm-associated infections are cystic 
fibrosis pneumo-nia, catheter-associated urinary tract infection, prosthetic joint infection, periodontitis, and 
chronic dermal wounds.
One of the main reasons that biofilm infections are so persistent is the reduced susceptibility of 
microorganisms in biofilms to killing by antimicrobials including systemic antibiotics, topical antiseptics, 
and antimicrobial compo-nents of the host defense (Stewart & Costerton, 2001; Fux et al., 2005). Biofilm 
tolerance to antimicrobial agents has been extensively researched and discussed. The purpose of this article 
is to examine another likely basis of the tenacity of biofilm infections that has received much less attention: 
the resistance of biofilms to mechanical clearance. This topic requires engagement with structure, 
hydrodynamics, material properties, polymer gel physics, and adhesive and cohesive failure. The intent of 
this article is to outline the application of elementary biophysics concepts to the resil-ience of biofilm 
infections.
Structure
When thinking about mechanics, it is important to have a good grasp on the structure and geometry of the 
system. Much has been written about the three-dimensional archi-tecture of biofilms formed in in vitro 
reactors and environ-mental settings (Costerton et al., 1995; O’Toole et al., 2000). In vivo, however, 
biofilm structures are often quite different as has been recently articulated by Bjarnsholt et al.(2013a). The 
celebrated mushroom-shaped biofilm cluster observed in flow cells in the laboratory is not seen in vivo.
Introduction
qualitatively different aspects. G describes the reversible
stretching of the material under tension and can be thought
of as the stiffness of the material. Materials with larger
values of G are harder to deform. rB describes the breaking
of the material and can be thought of as its overall strength.
Materials with larger values of rB are harder to break. It is
clearly simplistic to think of these properties as single values
in the context of the systems begin discussed. The reality of
a viscoelastic material is that the measured failure strength
will depend on how quickly or slowly stress is applied. In
addition, we know that biofilms are heterogeneous with
regions that are stronger or weaker.
Biofilms resist mechanical clearance
One way that a biofilm infection persists is by withstanding
the flow of fluid or other mechanical forces that work to wash
or sweep microorganisms out of the body (Seymour et al.,
2004; Stewart, 2012). Here are four examples.
Consider the lumen of a central venous catheter, where
there are typically periods of no flow punctuated by intervals
of significant flow. For a biofilm to accumulate in these
locations, it is critical that the shear stress applied during
peak fluid flow is insufficient to remove the biofilm.
In the familiar example of dental plaque, a spectrum of
forces is exerted on the oral biofilm. These forces range
from mild shear associated with the flow of saliva (weak but
continuous), to contact with moving tongue, cheeks, or food
(intermediate and frequent), to water jets and toothbrushing
(occasional but intense.) Biofilm will only build up if it is
strong enough to resist disruption by the frequent forces or
is able to regrow in the intervals between the intense but
infrequent forces. Toothbrushing is effective precisely
because it is sufficiently powerful to remove much of the
biofilm. To be effective, it must be repeated regularly on a
cycle that is comparable to or shorter than the biofilm
regrowth time. Of course, there are locations in the dentition,
such as interproximal zones, that are sheltered from the
forces delivered by toothbrush bristles. These areas are
prone to greater plaque accumulation.
(a) (b) (c)
Fig. 1 Conceptual models of in vivo biofilm structures. (a) Small microbial aggregates (e.g. 5–50 lm in size) are distributed in a gel-like matrix which
may be composed of host extracellular matrix material, dead neutrophils and released neutrophil DNA, and microbial EPS. This model could apply to
biofilms in the cystic fibrosis lung or in chronic wounds. (b) Large aggregates of microorganisms comingle with precipitated mineral phases or
host-derived material such as platelets and fibrinogen. This model could apply to biofilms such as dental plaque, the infectious vegetation on a heart
valve, or the encrustation in a urinary catheter. In these examples, the biofilm/host material/mineral aggregation is attached to a surface. (c) Small
aggregates of surface-attached microorganisms are covered by a secondary layer of primarily host-derived material.
Instead, relatively small aggregates of biofilm cells are found 
intermixed or covered with extensive host-derived material 
(Gristina et al., 1985; Baltimore et al., 1989; Hall-Stoodley 
et al., 2006; James et al., 2008; Stoodley et al., 2008, 2010; 
Bjarnsholt et al., 2009). In other niches, thick biofilms form, 
but often contain substantial amounts of host or precipitated 
mineral particulates (Wright & Kirschner, 1979; Friskopp, 
1983; Marrie and Costerton, 1984; Marrie et al., 1987; 
Stickler et al., 1993; Tan et al., 2004). Conceptual models of 
in vivo biofilm structure are diagrammed in Fig. 1.
Biofilm composition and mechanical 
properties
As a material, a biofilm can be conceptualized as a 
dispersion of colloidal particles (microbial cells, mineral 
precipitates, host biological debris) in a hydrogel [microbial 
extracellular polymeric substances (EPS) and host extra-
cellular polymers such as mucus, collagen, or released 
DNA]. In the biofilm literature, the constituents of EPS have 
been identified as polysaccharides, proteins, and extracel-
lular DNA (Branda et al., 2005; Flemming & Wingender, 
2010). There has been less attention to understanding the 
composition of admixed host polymers and particulates, but 
these components will clearly be important in the mechanics 
of the in vivo biofilm.
Biofilms typically exhibit viscoelastic behavior when 
mechanically stressed (Klapper et al., 2002; Stoodley et al., 
2002; Wilking et al., 2011; B€ol et al., 2013). That is, they can 
deform in both an elastic, reversible manner and in a 
viscous, irreversible manner. Most biological materials, such 
as mucus or tissue, are also soft and viscoelastic (Levental 
et al., 2007). There are numerous parameters that can be 
appropriately used to characterize the mechanical behavior 
of these materials. In this article, I will mention only two: G, 
the elastic modulus, which can be thought of as a spring 
constant, and rB, the biofilm failure strength, which charac-
terizes the applied force per area that will cause the biofilm 
to break. Both of these parameters characterize the 
response of a material to an applied force, but they capture
The bacteria are thought to have travelled to the heart,
where they attached, following a transient bacteremia
associated with teeth cleaning. The biofilm on a central
venous catheter can be asymptomatic until enough micro-
organisms are released at once to trigger a bloodstream
infection. In cystic fibrosis pneumonia, acute exacerbations
may result from biofilm dispersion events.
There is an apparent contradiction in the requirement for
biofilms to be both strong enough to resist detachment and
weak enough to permit release of planktonic cells. This
contradiction is resolved by recognizing that biofilm mate-
rial properties, such as other chemical and biological
properties (Stewart & Franklin, 2008), are inherently
heterogeneous. Aggarwal et al. (2010) have reported
repeated microscale measurements of cohesive strength
of Staphylococcus epidermidis and Pseudomonas aerugin-
osa biofilms; they find these values are distributed over
more than two orders of magnitude (see Fig. 2). In other
words, there are parts of a biofilm that are strong enough
to remain attached even during high shear stress events
and there can also be parts of the same biofilm that are
weak enough to readily detach.
The fundamental criterion for detachment (dissemination)
is sF > rB, which says that for detachment to occur the
applied force must exceed biofilm cohesiveness.
Biofilms resist phagocytosis
Microorganisms interact with and evade phagocytic leuko-
cytes via a complex exchange of biochemical signals and
toxins (Nizet, 2007; Sarantis & Grinstein, 2012). Biofilms
may additionally thwart phagocytosis by simple mechanical
means. Here, I consider this possibility. The ability of a
phagocyte to engulf a biofilm aggregate likely depends on
both the size of the aggregate and on its material properties,
adhesive strength and cohesive strength in particular (M€oller
et al., 2013). For example, a neutrophil may be able to
engulf a sufficiently small biofilm aggregate even if it is
mechanically rigid and unbreakable. The critical threshold
size for engulfment of a biofilm cluster would logically be
similar to the size of the phagocyte itself. Neutrophils are
c. 10–12 lm in diameter. We can therefore speculate that
any biofilm aggregate less than about 10 lm in size may be
vulnerable to phagocytosis. Experimentally, neutrophils are
able to phagocytose 3- and 6-lm-diameter polystyrene
beads, but exhibit frustrated phagocytosis when presented
with 11-lm-diameter beads, engulfing only about 50% of the
bead circumference (Herant et al., 2006). If a biofilm
aggregate is larger than 10 lm, it may still be subject to
phagocytosis, but only if it is soft enough to be fragmented
by the forces applied by the engulfing cell. In other words,
there is likely a critical threshold value of biofilm cohesive
strength above which a biofilm is protected from phagocytic
engulfment. Using a mathematical model of the phagocyto-
sis process, Herant et al. (2006) suggest that the attractive
force between a neutrophil and its target body could be of
the order of magnitude of 103 Pa. Referring to Fig. 2, this
could be sufficient to allow phagocytosis of some, but not all
biofilms.
Fig. 2 Magnitude of calculated shear stresses in the human body and
measured biofilm failure strengths. Sources: Day (1997), Ohashi et al.
(1999), Bundy et al. (2001), Mareels et al. (2007), M€ohle et al. (2007),
Dasi et al. (2009), Aggarwal & Hozalski (2010), De Bartolo et al. (2011),
Clark et al. (2012), Vignaga et al. (2012), Rmaile et al. (2014). Sa,
Staphylococcus aureus; Se, Staphylococcus epidermidis; Pa, Pseudo-
monas aeruginosa.
Airborne particles, including microorganisms, are contin-
uously deposited in the lungs. In healthy individuals, these 
particles are swept out of the lung by the mucociliary 
escalator. Beating cilia steadily push the fluid mucus layer, 
along with entrapped particles, up and out of the lungs. In 
individuals with cystic fibrosis, the mucus is thickened by the 
disease and the motion of the escalator is much impaired. 
The residence time of bacteria in the mucus layer is long 
enough for them to grow into biofilm aggregates and exert 
pathogenic effects.
In endocarditis, the biofilm attached to a heart valve 
persists in a turbulent, high shear environment. In this case, 
the flow of fluid never ceases so the intrinsic mechanical 
strength of the biofilm/vegetation has to exceed the maxi-
mum stress developed by the blood flow.
One can begin to develop a quantitative basis for 
analyzing the interactions described above by comparing 
the shear stress applied to the biofilm with the biofilm 
cohesive strength (Fig. 2). Although measurements of 
biofilm cohesive strength range over orders of magnitude, 
this comparison allows us to appreciate that biofilms are 
strong enough to mechanically persist in many settings. The 
fundamental criterion for mechanical persistence is sF < rB 
where sF is the applied shear stress and rB is the biofilm 
failure strength. Put another way, when the force applied is 
not enough to overcome biofilm cohesiveness, the biofilm 
persists.
Dissemination of infection
Although the discussion thus far has focused on how 
biofilms stay stuck in place, we also know that biofilms can 
shed planktonic cells or aggregates of cells. This is 
important in the context of infection because planktonic 
release events can disseminate the infection to other parts 
of the body or spawn episodes of acute infection (Fux et al., 
2004; Hall-Stoodley & Stoodley, 2005). For example, oral 
streptococci are sometimes recovered from heart valves.
Treatments based on weakening biofilm
The discussion of issues above naturally leads to the
possibility of alternative therapies for treating biofilm infec-
tions based on weakening biofilm cohesion. Reducing
biofilm cohesive (or adhesive) strength could (1) allow
prevailing hydrodynamic shear to remove biofilm, (2)
increase the efficacy of designed interventions for removing
biofilms, (3) enable phagocytic engulfment of softened
biofilm aggregates, and (4) improve phagocyte mobility
and access to biofilm.
A wide variety of chemical, biochemical, and enzymatic
strategies can be envisioned for effecting biofilm weaken-
ing and dispersion (Chen & Stewart, 2000; Landini et al.,
2010; McDougald et al., 2011; Bjarnsholt et al., 2013b;
Kostakioti et al., 2013). I present a sampling of such
approaches here for sake of illustration; this listing is far
from comprehensive. The examples below focus on
targeting the biofilm extracellular matrix.
A direct chemical attack on the biofilm extracellular matrix
may be behind the relative success of halogens and other
oxidizing biocides as antibiofilm disinfectants. Free chlorine
caused erosion of a S. epidermidis biofilm that was not
observed with other antimicrobials (Davison et al., 2010).
The strong oxidant periodate is sometimes used to diagnose
the presence of polysaccharides in the biofilm matrix based
on its ability to oxidize and degrade these macromolecules
(Chaignon et al., 2007).
Enzymatic degradation of biofilm EPS holds promise as
a biofilm removal approach (Johansen et al., 1997;
Marcato-Romain et al., 2012). For example, an enzyme
discovered based on its involvement in the natural disper-
sion of aggregates of Actinobacillus actinomycetemcomi-
tans (Kaplan et al., 2003), Dispersin B, cleaves a linear
polymer of N-acetylglucosamine, a common biofilm extra-
cellular polysaccharide. Treatment with this enzyme can
remove biofilms of multiple bacterial species (Itoh et al.,
2005) and has been shown to alter the mechanical stability
of S. epidermidis biofilm under hydrodynamic challenge
(Brindle et al., 2011). The intriguing strategy of delivering
the coding potential for this same enzyme in the DNA of an
engineered bacteriophage has been demonstrated (Lu &
Collins, 2007).
Because biofilm EPS often contains proteins and extra-
cellular DNA, proteases and DNAases are also candidate
enzymes for breaking down biofilms (Whitchurch et al.,
2002; Chaignon et al., 2007; Boles & Horswill, 2008;
Hall-Stoodley et al., 2008; Lequette et al., 2010; Nijland
et al., 2010).
It should also be possible to alter biofilm matrix cohesion
by interfering with cross-linking interactions between matrix
polymers. Chelants that compete for multivalent cations
such as calcium or iron have been shown to remove biofilms
(Turakhia et al., 1983; Banin et al., 2006; Raad et al.,
2008). The efficacy of these agents may derive from
disrupting electrostatic interactions between bridging
cations and strands of negatively charged polymers (Chen
& Stewart, 2002). Concentrated urea, which disrupts
hydrogen bonding, has been shown to facilitate removal of
There is a second physical consideration in the phagocy-
tic attack on a biofilm. This has to do with the mobility of the 
phagocyte. Phagocytes respond to attractant molecules 
released by microorganisms and migrate toward them. Such 
migration requires a permissive matrix. It is possible that 
some biofilms affected milieus, for example the thickened 
mucus of the cystic fibrosis lung, are so viscous that they 
retard or prevent leukocyte chemotaxis. This could tip the 
balance in favor of the biofilm. In fact, Matsui et al. (2005) 
reported exactly this effect. Neutrophils migrated in mucus 
constituted at concentrations in the normal range (1.5–2.5%
dry weight) but exhibited little migration in any direction in 
thickened (6.5%) mucus representative of the airway mucus 
in the cystic fibrosis lung. Parkhurst and Saltzman (1994) 
measured leukocyte migration in cervical mucus and con-
cluded that neutrophils move effectively in normal mucus. 
These authors also measured human neutrophil motility in 
well-defined collagen gels and found that motility decreased 
with increasing collagen concentrations above 0.2 mg mL
1 
(Parkhurst & Saltzman, 1992). A conservative extrapolation 
of their data suggests that neutrophils would be completely 
immobile in a collagen gel of 1 mg mL
1 (0.1%).
The elementary biophysics of hydrogels suggests that 
modest increases in local polymer concentration, C, either 
biofilm EPS of host extracellular matrix materials, could lead 
to very large increases in the matrix strength. For example, 
the elastic modulus, G, of a gel increases approximately as 
G  C2.25 (De Gennes, 1979). In other words, a doubling of 
matrix polymer concentration could increase gel modulus by 
nearly a factor of five. Such increases in gel rigidity could 
obviously reduce phagocyte mobility.
Together, these observations suggest that the ability of 
leukocytes to effectively penetrate and police mucus layers 
can be strongly related to the extracellular polymer concen-
tration because the polymers determine the viscoelastic 
properties of the gelatinous matrix. The matrix polymers 
may derive from secreted mucus, necrotic tissue, DNA 
released from dead neutrophils or neutrophil extracellular 
traps, as well as from the EPS produced by microbial 
biofilm.
This argument, formulated here in terms of leukocyte 
migration in mucus, may extend to the limited penetration of 
leukocytes to the surface of an encapsulated implant. After 
implantation of a biomaterial, it is common for chronic 
inflammation and a foreign body reaction to ultimately 
conclude with fibrous encapsulation of the device (Anderson 
et al., 2008; Bryers et al., 2012). The capsule consists 
largely of collagen deposited by fibroblasts, and it effectively 
walls off the implant surface. If there is a biofilm on this 
surface, this process may effectively construct a fortress in 
which bacterial biofilm is shielded from host cellular 
defenses.
Of course, the EPS of a microbial biofilm itself may serve 
the function of excluding phagocytes. In the cystic fibrosis 
lung, there is a common selection over time for P. aerugin-
osa mutants that overproduce the polysaccharide alginate. 
Does the copious alginate gel limit physical access of 
neutrophils to bacterial cells? This function has been 
postulated (Mai et al., 1993; Bjarnsholt et al., 2009).
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bonding may contribute to matrix integrity.
One can also imagine a new class of drugs that inhibit the 
biosynthesis, export, or anchoring of EPS important for 
biofilm cohesion. These drugs, which are mostly hypothet-
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of such a strategy is a cocktail of chemistries reported to 
suppress exopolysaccharide synthesis in the cariogenic 
organism Streptococcus mutans (Falsetta et al., 2012).
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regulatory mechanisms that control elaboration of EPS. The 
leading example of this possibility involves interfering with a 
regulatory mechanism now understood to be common in 
bacteria centered on the secondary messenger molecule 
cyclic di-GMP (Hengge, 2009; R€omling et al., 2013). In 
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biofilm formation, whereas low levels of cyclic di-GMP lead 
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One caution with these approaches is that they must be 
performed in such a way so as to avoid dissemination of 
large numbers of planktonic microorganisms. Bacteria 
detached from a catheter would probably need to be 
deliberately withdrawn from the device and captured rather 
than allowed to escape into the bloodstream, for example. It 
may be necessary to simultaneously treat with antibiotics to 
control dispersed cells. It is also possible that in some 
instances, the immune defenses may be adequate to 
neutralize the released microorganisms on their own.
Conclusion
It should not be surprising that existing antimicrobials fail to 
remove or weaken biofilms: these agents have been 
discovered and developed based on their ability to kill 
germs without consideration or measurement of biofilm 
removal. Some biocides and antiseptics, such as glutaral-
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make it less prone to removal (Sim~oes et al., 2005; Brindle 
et al., 2011). Progress in developing new antibiofilm thera-
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mechanical structure and measurement of biofilm material 
properties and biofilm removal becomes more routine 
(Jones et al., 2011; Lieleg et al., 2011; B€ol et al., 2013). 
Mathematical and computer modeling of biofilm fluid–struc-
ture interactions also has an important role to play (Duddu 
et al., 2009; Taherzadeh et al., 2010; Vo et al., 2010; 
Lindley et al., 2012).
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PSS acknowledges support from NIH award R01GM109452 
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