Reviews
Microbial biofilms
Bacterial adhesion at the
single-​cell level
Cecile Berne
, Courtney K. Ellison
1
, Adrien Ducret2 and Yves V. Brun
1
*
1
Abstract | The formation of multicellular microbial communities, called biofilms, starts from the
adhesion of a few planktonic cells to the surface. The transition from a free-​living planktonic
lifestyle to a sessile, attached state is a multifactorial process that is determined by biological,
chemical and physical properties of the environment, the surface and the bacterial cell. The initial
weak, reversible interactions between a bacterium and a surface strengthen to yield irreversible
adhesion. In this Review, we summarize our understanding of the mechanisms governing
bacterial adhesion at the single-​cell level, including the physical forces experienced by a cell
before reaching the surface, the first contact with a surface and the transition from reversible
to permanent adhesion.
Shear
The fluctuation developed by
particles in movement relative
to each other in a liquid
environment, reflecting the
motion between two adjacent
layers of a liquid with flow.
Extracellular appendages
Filamentous structures that are
present on the surface of
bacteria, including flagella, pili
and curli.
1
Department of Biology,
Indiana University,
Bloomington, IN, USA.
2
Molecular Microbiology and
Structural Biochemistry,
CNRS UMR 5086, Lyon,
France.
*e-​mail: ybrun@indiana.edu
https://doi.org/10.1038/
s41579-018-0057-5
Microorganisms have the ability to form biofilms, which
are complex, heterogeneous, multicellular communities
that adhere to surfaces. This attached lifestyle presents
numerous advantages compared with that of free-​
living planktonic microorganisms, including increased
nutrient availability and protection from predators and
xenobiotic stresses1. Microbial biofilms are ubiquitous
and are present on biotic and abiotic surfaces exposed to
bulk liquid environments in natural, industrial and
medical settings. Indeed, biofilms have been observed
on surfaces as diverse as river rocks, deep-​sea vents and
plant roots in the rhizosphere in nature, water pipes
and food-​processing surfaces in industrial settings and
indwelling medical devices in the human body. Biofilms
can be detrimental to both human health and industry,
as they have a role in many infections and diseases, biofouling and corrosion. By contrast, biofilms can also be
useful for various biotechnological processes, including
bioremediation, biological fuel cells or the production
of biofertilizers. Hence, it is important to manage
microbial biofilms both to enhance their formation to
improve industrial applications and to disrupt their
development to reduce infections, biofouling and
contamination. Biofilm-​targeting strategies include the
regulation of established biofilms or the modulation
of single-​cell attachment, the step that initiates biofilm
formation. For the latter, it is crucial to have a better
understanding of the first steps in bacterial adhesion
and how single cells switch from planktonic growth to
surface attachment.
Surface colonization involves a transition from a free-​
living planktonic lifestyle in the bulk liquid to a sessile,
surface-​attached state. This transition begins with the
reversible adhesion of a few single cells to a surface, with
two different outcomes: weakly attached cells can return
616 | OCTOBER 2018 | volume 16
to the planktonic bulk medium, or the initial interactions
between the cells and the surface strengthen to yield
irreversible adhesion. When conditions are favourable
and suitable for growth, irreversibly attached cells on
the surface divide to form multicellular microcolonies,
which then grow and develop into a mature biofilm. As
the biofilm matures, factors that will prevent sustainable
growth, such as limited nutrient availability or decreased
oxygen levels, can trigger dispersion, which is the release
of cells from the biofilm to the bulk liquid environment.
Dispersed single cells can then attach to a new hospitable
surface to initiate colonization2.
For single-​cell adhesion, three main factors must be
considered: the liquid environment, the solid surface
and the bacterium itself. In this Review, we discuss the
stages of attachment, beginning with a free-​swimming
bacterium and its approach to the surface, followed by
the initial reversible attachment and the transition to
irreversible adhesion. For a comprehensive review on the
processes that occur after single-​cell attachment occurs,
see Ref.3. We relate how the properties of each factor
are interconnected and consider how physical and/or
chemical changes in one factor can have an impact
on the others. Flow and shear as well as pH and ionic
strength are major factors that can alter the ability of a
bacterium to reach and adhere to the surface. Nutrient
availability in the liquid environment can either promote or impair bacterial adhesion1,4. For example, for
Agrobacterium tumefaciens, iron and manganese limitations independently impair cell adhesion5, whereas
phosphate limitation triggers cell attachment6. Moreover,
bacteria often harbour extracellular appendages that are
crucial during the adhesion process: flagella power the
swimming motility that enables the cell to approach
the solid surface, but they can also be involved in
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Flagella
Filamentous extracellular
appendages that are
responsible for the active
movement of cells in a liquid
environment. The bacterial
flagellum is composed of three
main substructures: the motor
that uses proton motive force
to generate the torque, the
basal body and hook that
anchor the flagellum filament
to the cell membrane and
transmit the motor torque and
the flagellar filament, which is
composed of flagellin proteins
that are arranged in a long, thin
filament and functions as a
propeller.
Pili
Thin, extracellular protein
fibres that are involved in
various bacterial behaviours,
including attachment, twitching
motility, horizontal gene
transfer and virulence.
Brownian motion
The continuous movement of
micrometre-​scale particles that
are suspended in liquid as a
result of random collisions with
each other.
Chemotaxis
A sensing mechanism that
enables bacteria to modify
their swimming behaviour in
the presence of a chemical
gradient. Bacteria can sense
and swim towards attractants
or away from repellents.
Box 1 | Methods to study single-​cell adhesion
Live imaging via optical microscopy is the most widely used method to visualize bacterial cell behaviour. In the past
decade, powerful new techniques have been developed that help researchers visualize, track and analyse single cells in
real time. The use of microfluidic devices in microscopy analysis of bacterial behaviour has increased recently because
they enable the precise manipulation of environmental conditions. Bacteria are typically seeded in microchannels within
microfluidic devices and can then be subjected to changes in both flow rates and nutritional conditions103. The most
common microfluidic devices are designed using soft lithography104 on inexpensive, biocompatible materials, such as
polydimethylsiloxane (PDMS), that enable the microchannel size and shape to be easily customized105. Microfluidic
technology has been successfully used to study adhesion at the single-​cell level35,42,82,99, as it provides a well-​defined
environment where different parameters relevant to bacterial adhesion, such as flow rate, surface chemistry and
topography, or composition of the environment can be modified106.
Recent advances in the use of microfluidic devices coupled with microbial tracking software have enhanced our
understanding of bacterial attachment23,102,107,108. The development of cell-​tracking programmes has bypassed the
previous limitations of quantifying bacterial attachment at the single-​cell level and has enabled high-​throughput analysis
of surface colonization at both the single-​cell level and population level.
In addition to microscopy and cell-​tracking techniques, methods focused on understanding biophysical properties of
adhesion have provided new insights into bacterial attachment. New atomic force microscopy (AFM) techniques were
developed to understand how single cells interact with a surface and characterize the physicochemical properties of
different adhesins present on the surface of bacteria33,100. Single-​cell force spectroscopy (SCFS), whereby a single
bacterial cell is irreversibly immobilized on an AFM tip, has enabled mechanistic studies of type IV pili in Pseudomonas
aeruginosa109 and the characterization of adhesin proteins in Staphylococcus aureus110–112 and Staphylococcus
epidermidis113. Another AFM-​based approach, single-​molecule force spectroscopy (SMFS), has been successfully
developed to unravel the material properties of purified bioadhesives in cell-​free systems114. For example, SMFS
experiments were crucial to understanding the physicochemical properties of the LapA surface adhesin of Pseudomonas
fluorescens61 and the holdfast polysaccharide adhesin of Caulobacter crescentus56. More recently, SMFS helped to uncover
the presence of a polymer brush layer on the holdfast of C. crescentus that is mainly composed of DNA and peptide
residues89; these components are important for mediating initial surface–holdfast interactions, a finding that expands our
understanding of adhesin structure, composition and function. In addition, the recently developed fluidic force
microscopy (FluidFM) combines AFM with microfluidics, whereby a single cell is immobilized or released by applying
underpressure or overpressure on a hollow cantilever with pressure control115. This technique has been successfully used
to probe adhesion of single cells of Escherichia coli, Streptococcus pyogenes and C. crescentus116,117.
reversible adhesion and the regulation of irreversible
adhesion. Thin fibres called pili are another class of
extracellular appendages that have a role in adhesion
in many different Gram-​positive and Gram-​negative
bacteria7.
During the past decade, the development of new,
more sensitive and more rapid microscopy techniques,
as well as the advent of robust computer-​assisted analysis
tools, have enabled the dissection of single-​cell adhesion
with unprecedented spatial and temporal resolution
(Box 1). We highlight some of the most relevant studies
that contribute to a better understanding of the signals
and factors involved in bacterial adhesion at the single-​
cell level as well as the regulation of this complex process.
These studies use combinations of genetic approaches
with live imaging or atomic force microscopy (AFM) to
dissect the first events of a bacterium reaching a surface
and when this interaction with the surface transitions
from reversible to permanent adhesion.
Reaching the surface
Bacteria need to successfully achieve proximity to a
surface to initiate attachment. In liquid environments,
bacteria in the planktonic medium, or bulk liquid,
approach the surface by passive and/or active movement.
Some bacteria exhibit swimming motility (see below)
to generate a self-​propelled active movement, but even
nonmotile bacteria are subject to physical forces that
bring them close to the surface in a passive manner.
Although passive transport by flow in the bulk liquid,
Brownian motion and gravitational forces can bring cells
NATuRE REvIEwS | MICRoBIology
close to the surface8,9, active motility is the most efficient way to reach it. Swimming motility is driven by
flagella; flagellar rotation generates a propulsive force
that provides enough energy to reach the surface.
Indeed, flagellar motility has a crucial role in initial bacterial attachment, as nonmotile mutants are impaired
in the first steps of adhesion in various bacteria such
as Escherichia coli 10, Listeria monocytogenes 11 and
Caulobacter crescentus12. Flagellated bacteria can use
flagellar-​based motility to swim directionally towards
a surface in response to environmental cues, such as
chemical signals, light, temperature, magnetic fields
and oxygen13. One example of a directional movement
towards the surface in response to chemical signalling
via chemotaxis involves Vibrionaceae in marine environments: Vibrio spp. can attach to and metabolize chitin,
the major constituent of the cuticles and exoskeletons of
crustaceans, molluscs and arthropods. The degradation
of chitin by soluble enzymes secreted by the bacteria
releases chitin hydrolysis products in the liquid environment, generating a chemical gradient that bacteria use to
modulate swimming direction. Driven by the presence
of the gradient, motile cells swim towards and attach
to cuticles and exoskeletons. This positive feedback
mechanism leads to a rapid and effective colonization
of these chitin surfaces14. Using microfluidics technology and live imaging, it was shown that single motile
Bacillus subtilis cells can also be attracted to surfaces by
other bacteria, of the same or different species, within
a surface-​associated biofilm via electrical signalling:
the release of potassium ions from the biofilm directs
volume 16 | OCTOBER 2018 | 617
Reviews
A
High flow
Low flow
Flow
Flow
Bulk liquid
Bulk liquid
Hydrodynamic
boundary layer
Hydrodynamic
boundary layer
Surface
B
Surface
C
Ca
No flow
Ba
Subcritical flow
Bb
50 μm
Bc
50 μm
Cb
Cc
20 μm
20 μm
Supercritical flow
Fig. 1 | The impact of flow and solid surfaces on bacterial swimming behaviours.
A | Schematics of the different flow patterns observed in aqueous environments.
The liquid environment can be separated into three regions: the bulk liquid
environment, where cells move freely ; the hydrodynamic boundary layer, which is
closest to the surface and where moving cells are subjected to strong hydrodynamic
effects; and the solid surface, where cells can adhere and adopt a sessile lifestyle.
When the flow rate of the bulk liquid increases, the size of the boundary layer
decreases, which generates more shear forces for bacteria that are near the solid
surface. B | Escherichia coli K12 swimming trajectories near the surface under
different flow conditions. In the absence of flow (part Ba), cells exhibit a circular
swimming motion (indicated by the yellow arrows). Cells swim towards the flow
(positive rheotaxis) up to a flow rate of 6.4 s−1 (subcritical flow , indicated by the white
arrows (part Bb)), and they are pulled away with the flow at higher shear rates (35 s−1
shown; supercritical flow (part Bc)). Time-​lapse movies of cells swimming in a
microfluidic channel subjected to various shear flows were recorded using an
inverted microscope. Images are contrast-​enhanced overlays of every fifth sequential
frame of the movies (original frame rates: 30 frames s−1 for no flow and subcritical
flow and 240 frames s−1 for supercritical flow). C | Bacteria swim in a circular motion
when approaching a solid surface. Cells exhibit more circular trajectories close to
the surface but straighter trajectories farther away from the surface. E. coli K12
swimming behaviour in the bulk liquid, far from the surface (part Ca). Trajectories
of smooth-​swimming E. coli (the surface is pictured as a red plane, the white
line indicates the Z-​plane (part Ca)), Caulobacter crescentus (part Cb; the arrows
indicate the swimming direction) and Vibrio cholerae (part Cc; different tracks are
represented by different colours) cells swimming near a solid surface. E. coli
trajectories (part Ca) were generated by computer visualization of the x-​y-z
coordinates over time (12 frames s−1) from the trace of a single swimming cell
captured by a 3D microscope; yellow and magenta spheres represent data points
collected during runs or tumbles respectively. C. crescentus trajectory (part Cb)
was generated as an overlay of consecutive frames (10 frames s−1) of a single cell
imaged by dark-​field microscopy with a ×10 objective. V. cholerae trajectories
(part Cc) were extracted from high-​speed movies of 100 s at 5 ms resolution during
the first 5 min after inoculation. Part B adapted with permission from Ref.17, Elsevier.
Part Ca adapted with permission from Ref.21, Proceedings of the National Academy
of Sciences. Part Cb adapted with permission from Ref.22, Proceedings of the National
Academy of Sciences. Part Cc adapted from Ref.23, Macmillan Publishers Limited.
618 | OCTOBER 2018 | volume 16
the motility of distant cells towards the biofilm by
affecting their membrane potential15.
It may seem obvious to state that the initial step of
attachment is to go from the bulk liquid environment to
the surface, but this step is not trivial. Aqueous environments can be divided into three regions: the bulk liquid,
where cells can move freely; the liquid–surface interface,
called the hydrodynamic boundary layer, where moving cells are subjected to strong hydrodynamic effects on
the surface; and the surface, where cells can adhere and
adopt a sessile lifestyle (Fig. 1A). Bacteria can live in low-​
flow environments, such as ponds or the human oral
cavity, but they experience high flow in environments
such as moving ship hulls and the urinary tract. Flow
dynamics in the bulk liquid environment are affected
by the surface, with the flow rate being negligible at
the hydrodynamic boundary layer close to the surface8
(Fig. 1A). The size of the boundary layer depends on the
flow rate in the bulk liquid: the higher the flow rate,
the thinner the boundary layer. Therefore, at a high flow
rate in the bulk liquid, the boundary layer decreases, and
bacteria near the surface are subjected to higher shear.
Flow and shear rates dramatically affect the efficiency of
bacteria approaching the surface. If flow velocity is too
high, bacteria are transported with the flow without having a chance to approach the surface16. For example, high
flow and high shear affect swimming behaviour of single
E. coli cells over a surface, as observed by live microscopy
of swimming cells in microfluidic chambers where different shear rates are applied (Fig. 1B). This bacterium
exhibits random trajectories under low flow conditions,
and whereas E. coli swims towards the flow under moderate shear rates (~6 s−1), it fails to swim upstream at
extremely high shear rates (>30 s−1) and is pulled away
with the flow17. The enhanced upstream motility against
the direction of flow, termed rheotaxis, observed under
moderate flow contributes to the efficient propagation
of E. coli cells in environments such as medical catheters,
water pipes or river streams17.
Bacteria that swim close to the surface are subject
to different mechanical constraints than those bacteria
that swim in the bulk liquid environment. Swimming is
randomly altered by Brownian motion in the bulk liquid environment18, but this phenomenon is amplified at
the near-​surface boundary, yielding more circular trajectories close to the surface but straighter trajectories
farther away from the surface, as observed by single-​
cell tracking19 (Fig. 1C). These random circular motions
enable bacteria to spend more time in close proximity
to the surface than cells that swim in straight trajectories, and this provides a greater probability for contact
between the bacterium and the surface20. Furthermore,
drag forces on the bacterial cell are stronger near the
surface, yielding a slower swimming velocity at the surface boundary than in the bulk liquid environment21,22.
In some cases, extracellular appendages present on
the bacterial cell envelope can also help to increase the
time the cell spends near the surface. In Vibrio cholerae,
single-​cell tracking showed that mannose-​s ensitive
haemagglutinin pili affect near-​surface swimming by
generating periodic mechanical contacts with the surface. This brief interaction between pili and the surface
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Hydrodynamic effects
Different forces that are caused
by a liquid in motion.
modifies the swimming behaviour by generating shorter,
near-​circular trajectories and increasing the time spent
near the surface23.
Owing to the complex dynamics between the bulk
liquid, the hydrodynamic boundary layer and the surface, bacteria use several strategies to achieve close
proximity to the surface, including active motility, chemotaxis and cell appendages. Once sufficiently close to the
surface, the major challenge for efficient attachment is
initiating direct contact with that surface.
Factors affecting initial attachment
Forces involved in bacterial adhesion. When bacteria
approach the surface, the fate of adhesion is dictated by
the sum of attractive and repulsive forces24 between the
cell and the surface. If attractive forces outweigh repulsive forces, the contact between the cell and the surface
proceeds. The first physicochemical forces that bacteria
experience when moving from the liquid environment to
the solid surface are: van der Waals interactions, which
are generally attractive; electrostatic interactions, which are
modulated by ionic strength and pH of the liquid environment; and acid–base hydrophobic interactions,
which are attractive or repulsive depending on the
environment, bacterium and surface chemistries24 (see
below). Those forces act at long range, tens of nanometres away from the solid surface. Although van der
Waals interactions act at the longest ranges compared
with the other forces, their magnitude decreases sharply
as distance from the surface increases, with repulsive
forces preventing bacterial adhesion if the bacterium is
not close to the surface. However, close to the solid surface, the magnitude of van der Waals force is the strongest and leads to adherence of the bacterium. Steric forces
are another type of force that determine the success of
adhesion. Some bacteria, such as Pseudomonas aeruginosa, Pseudomonas putida and E. coli, harbour on their
surface a network of long polysaccharide chains and
other biopolymers, called the polymeric brush layer. The
composition and charges of this layer differ depending
on the bacterial species, and they generate long-​range
steric forces that are involved in the interaction with the
surface25,26. AFM experiments that measured the interaction forces between a single bacterium and the tip of
the AFM cantilever showed that steric interactions can
be more important than van der Waals and electrostatic
forces for bacterial adhesion25.
Physical and chemical properties of the surface.
Bacteria encounter surfaces that are extremely diverse,
and they can colonize any natural or man-​m ade
material16,27. However, physical and chemical properties
of the surface dictate the efficiency of adhesion. Surface
topography at the micrometre and nanometre scale is an
important determinant of bacterial attachment24. This
patterning of the surface can modify its hydrophobic
properties, a crucial parameter for bacterial adhesion
(see below). In addition, any microscopic structure or
irregularity on the surface tends to increase the overall
surface area and therefore promotes bacterial adhesion28.
Besides maximizing the contact area between the bacterial cell and the surface, patterning at the micrometre
NATuRE REvIEwS | MICRoBIology
scale (micropatterning) also reduces the shear experienced by attached cells29. It has been shown that, in
model organisms such as P. aeruginosa30, Shewanella
oneidensis31, Staphylococcus epidermidis and E. coli32,
bacterial adhesion is favoured on recessed portions of
micropatterned surfaces (Fig. 2A), and bacteria tend to
attach preferentially to patterns in the micrometre range
rather than to smooth surfaces. By contrast, the outcome
of patterning at the nanometre scale (nanopatterning) of
surfaces is still up for debate. Different studies report
conflicting results about the relationship between
nanopatterning and bacterial adhesion33,34. For example, the pathogens E. coli, P. aeruginosa, S. aureus and
S. epidermidis experience greatly impaired adhesion
when the patterning on the surface is smaller than the size
of the bacterium32,34,35 (Fig. 2A). However, earlier studies
showed that nanopatterning of surfaces prevents initial
adhesion by the Staphylococci, whereas adhesion by
P. aeruginosa or E. coli was unaffected36,37. The difference
might be due to the cell shape (spherical Staphylococci
versus rod-​ s haped P. aeruginosa and E. coli) 37
and the composition of the cell envelope (Gram-​positive
Staphylococci versus Gram-​negative P. aeruginosa and
E. coli)36. As the number of studies on diverse bacterial
systems increases, a clearer consensus on the effect of
nanoscale patterned surfaces on adhesion may emerge.
Bacteria can attach to a wide range of surfaces with
diverse chemical properties16. Charge and hydrophobicity are two major components that influence the
bacterium–surface interaction. The chemical properties
of the bacterial cell envelope can vary (see below), but as
a general rule, as bacteria are often negatively charged,
surface materials with positive to neutral charges are
more readily colonized than those that display negative
charges38. Similarly, the effect of surface hydrophobicity
on bacterial adhesion depends on the hydrophobicity of
the bacterial cell. Bacteria with a more hydrophobic cell
surface preferentially colonize hydrophobic materials and
vice versa38. Inspired by lotus leaves, dragonfly wings
and shark skin39, recent work has begun to investigate
bacterial colonization of superhydrophobic surfaces.
These novel surfaces combine material with nanopatterned structures and very low affinity for water that are
extremely resistant to bacterial colonization by diverse
bacterial species, including S. aureus, L. monocytogenes,
E. coli and P. aeruginosa34,40.
In aqueous environments, various organic and
inorganic compounds adsorb on the surface, forming
deposits described as conditioning films. Conditioning
films modify physicochemical surface properties by
altering charge, potential and surface tension41. These
irregular changes to surface attributes result in varying local adhesion efficiency (Fig. 2B). The composition
of conditioning films varies greatly, as any molecule
present in the bulk liquid environment can settle on
the surface and become part of the conditioning film.
Molecules deposited on the surface come from the
environment and from substances excreted by bacteria that are present in the liquid environment and/or
already attached to the surface, producing numerous conditioning films with varying composition41.
Adsorbed molecules may attach to binding sites on
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A
B
Micropatterned surfaces
Aa
Ab
Bacterial
cell
1 μm
Cell surface
hydrophobic
components
–
–
– +–
Ac
Charge
Flagellum
Adhesin
Interfacial
water
++––+
Curli
Pilus
Conditioning film
Surface
500 μm
500 μm
2 μm
C
Pilus
Flagellum
Nanopatterned surfaces
Ad
Ae
Non-fimbrial
adhesins
Curli
<50 nm
1 μm
1 μm
Fig. 2 | Interactions between the surface and the bacterium. A | Impact
of surface topography on single-​cell adhesion. Bacterial adhesion is
favoured on recessed portions of micropatterned surface (parts Aa–Ac),
and bacteria tend to attach preferentially to patterns in the micrometre
range rather than to smooth surfaces. On micropatterned surfaces, cells are
arranged to maximize the contact area with the surface. Shown is
Shewanella oneidensis, attached to a silicon nanowire pole (Aa) (scanning
electron microscope (SEM) image showing a single cell, false coloured in
yellow). The inset shows the merged images of the same cell (stained
in green), imaged by fluorescence microscopy, and the silicon nanowire (red
arrow), imaged by dark-​f ield microscopy. Shown are Pseudomonas
aeruginosa attached to a periodically structured epoxy surface (part Ab)
(SEM image showing single cells, false coloured in green) and Pseudomonas
fluorescens attached to the grooves of a gold micropatterned structure
(part Ac) (atomic force microscopy (AFM) image showing isolated bacteria;
the white arrow indicates the flagella). In contrast to micropatterned
surfaces, on nanopatterned surfaces (parts Ad,Ae), cells adhere to the tip
of the nanostructured pillars and cannot maximize the contact area with
the surface; therefore, cells are more vulnerable to shear forces, and
adhesion is less efficient. Shown are Staphylococcus aureus (part Ad)
and Escherichia coli (part Ae) cells attached to nanopillared aluminium
surfaces (field emission-​SEM image showing single cells, false coloured in
green for S. aureus and red for E. coli, respectively) (both images from Ref.34).
B | First interactions between the surface and the bacterium. Once the
bacterium is close to the surface, the heterogeneity of both the surface and
the bacterium at the microscale level affects the adhesion process.
Although the bacterial cell is usually negatively charged, the cell surface is
highly heterogeneous, exhibiting different charges around the cell body. In
addition, the presence of the conditioning film (orange) can modify the
physicochemical properties of the solid surface (by altering charge,
potential and surface tension) and thus affect local adhesion. At the
Micrometre range
nanoscale level, the thin layer of water (interfacial water, light blue) present
on the surface can potentially be a barrier to cell adhesion. Hydrophobic
components on the cell surface (dark blue), such as proteins, the polymeric
brush layer and extracellular polysaccharides, can displace interfacial water
between the bacterium and the surface and enhance hydrophobic
interactions, thereby promoting close contact between the bacterium and
the surface. Once the bacterium is sufficiently close to the surface (<1 nm),
adhesins (red) and bacterial cell appendages (such as flagella (brown), pili
(blue) and curli (purple) can interact with the solid surface and have direct
or indirect roles in adhesion. C | The bacterial cell surface is highly
heterogeneous. Several types of molecules and structures are involved in
the first interaction between the bacterium and the solid surface, including
long fimbriae adhesins (such as pili and curli, depicted in blue and purple,
respectively) and flagella (depicted in brown) and non-​fimbrial adhesins
(such as Ag43 and LapA proteins, depicted in red). Whereas non-​fimbrial
adhesins are more compact and usually protrude tens of nanometres
(<50 nm) from the cell envelope, longer fimbrial adhesins can extend at the
micrometre scale. Part Aa adapted with permission from Jeong, H. E., Kim, I.,
Karam, P., Choi, H. J. & Yang, P. Bacterial recognition of silicon nanowire
arrays. Nano letters 13, 2864–2869 (2013). Copyright © 2013 American
Chemical Society. Part Ab adapted with permission from Hochbaum, A. I. &
Aizenberg, J. Bacteria pattern spontaneously on periodic nanostructure
arrays. Nano letters 10, 3717–3721 (2010). Copyright © 2010 American
Chemical Society. Part Ac adapted with permission from Díaz, C.,
Schilardi, P. L., Salvarezza, R . C. & Fernández Lorenzo de Mele, M.
Nano/Microscale Order Affects the Early Stages of Biofilm Formation on
Metal Surfaces. Langmuir 23, 11206–11210 (2007). Copyright © 2007
American Chemical Society. Parts Ad and Ae adapted with permission from
Hizal, F. et al. Nanoengineered superhydrophobic surfaces of aluminum with
extremely low bacterial adhesivity. ACS Appl Mat Interfaces 9, 12118–12129
(2017). Copyright © 2017 American Chemical Society.
the cell surface, thus reducing the ability of bacteria
to attach; by contrast, such compounds may support
the formation of adhesive bonds, thereby increasing
adhesion27,41,42. Adhesion of the plant pathogen Xylella
fastidiosa to abiotic surfaces is more efficient when
620 | OCTOBER 2018 | volume 16
a conditioning film is produced on the surface by
molecules in the culture medium, probably owing
to stronger interactions between the bacterium and
specific phosphate groups present in the conditioning film43. Single P. aeruginosa cells deposit a trail of
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Psl exopolysaccharide when they first encounter a clean
surface. Subsequent attaching cells preferentially bind
to the Psl on the surface, creating nucleation points
for the formation of microcolonies44. In other cases,
molecules secreted by bacteria can, when deposited
on the surface, inhibit bacterial attachment, exemplified by the secreted group II capsule in E. coli45 or
biosurfactants in P. aeruginosa46.
Psl exopolysaccharide
(Named after the
polysaccharide locus). A
polysaccharide that is
composed of mannose,
glucose, rhamnose and
possibly galactose residues
and is found on the surface of
Pseudomonas aeruginosa
cells. It is involved in cell–cell
and cell–surface interactions,
and it is a scaffolding molecule
in the biofilm matrix.
cyclic di-​GMP
(ci-​di-GMP). A second
messenger signalling molecule
involved in the regulation of
various bacterial behaviours,
including motility, adhesion,
cell cycle progression and
virulence.
The bacterial surface and appendages. The bacterial
cell envelope is highly heterogeneous and contains
various exposed proteins, lipids and exopolysaccharides, as well as fimbrial and non-​fimbrial structures7
(Fig. 2C). The conformation of these various biopolymers is dynamic and depends on environmental pH
and ionic strength. Thus, the bacterial cell envelope
exhibits different charges and hydrophobicity around
the cell body dependent on growth conditions47 (Fig. 2B).
Heterogeneity of the bacterial cell envelope can exist
both between bacterial species and among individual
cells within a clonal population48, which highlights the
importance of using single-​cell techniques to dissect
the specific behaviour of individual cells within a
population (Box 1).
Many bacterial cells harbour various proteinaceous
extracellular appendages that have a direct or indirect
role in adhesion (Fig. 2B, C). For example, in addition to
actively propelling bacteria to the surface, the flagellum has an important role in adhesion by providing a
physical contact with the surface and functioning as an
adhesin27,28. Upon contact with the surface, bacteria can
use their flagella to explore the local surface topography or access microenvironments inaccessible to the
relatively large cell body as a way to increase attachment efficiency. Adhesion of E. coli harbouring flagella
is improved on surfaces with rough microtopography
compared with flat surfaces, probably because flagella
can access crevices and initiate adhesion42.
Besides flagella, other thin filamentous protein extensions from the cell surface, including fimbriae, curli
and pili, are involved in nonspecific initial adhesion to
abiotic surfaces7 (Fig. 2C). In various bacterial species,
different types of pili are involved in the first steps of
adhesion, for example, E. coli type I pili10, C. crescentus
and Agrobacterium tumefaciens type IVc tight adherence (Tad) pili49,50 and P. aeruginosa type IVa pili51.
Although some pili, including the type I pili in E. coli,
have specific receptors that bind to specific substrates,
the majority of pili can bind wide ranges of nonspecific substrates through an unidentified mechanism.
Dynamic pilus activity has been shown to be extremely
important for attachment, as reflected by recent work in
both V. cholerae52 and C. crescentus50. In V. cholerae, the
levels of cyclic di-​GMP (c-​di-​GMP) control pilus biogenesis and near-​surface behaviours that influence whether
cells attach efficiently. By tracking labelled Tad pili of
single C. crescentus cells by live microscopy, it was shown
that these pili are dynamic, undergoing multiple cycles
of extension and retraction upon surface contact. This
dynamic activity ceases concomitantly with permanent
adhesion, and obstruction of pilus activity stimulates
adhesin synthesis in the absence of surface contact,
NATuRE REvIEwS | MICRoBIology
demonstrating that pili have a key role in this process
(see below).
The possession of many surface appendages that
independently mediate adhesion does not necessarily
translate to more efficient adhesion: in some instances,
longer adhesins can sterically prevent the interaction
between a shorter molecule and the surface in a process
often referred to as surface shielding. For example, E.coli
harbours short cell surface adhesin proteins such as
Ag43 and AIDA-​I that are masked by longer components
also present on the cell surface (Fig. 2C). These masking
components include capsular polysaccharides, lipopolysaccharides and fimbriae, which prevent interaction of
small adhesin proteins with the surface53–55.
Strengthening the initial attachment
Once bacteria overcome the repulsive forces near the
surface and initiate surface contact, they must secure
and strengthen the adhesion to generate a more permanent type of attachment. This is achieved by the
maturation of some interactions between the cell and
the surface through the repositioning of the cell body
and surface structures and by the production of adhesin
molecules (Figs 3,4), which often involves small molecule
signalling (Box 2). Once irreversible adhesion of single
cells is achieved, the colonization of the surface and the
establishment of a multicellular biofilm can initiate.
Maturation of interactions and cell repositioning. The
first step in achieving permanent adhesion is dictated
by short-​range interactions mediated by cell appendages and hydrogen bonds on the surface and by the
maturation or strengthening of these interactions over
time. Bond strengthening after initial contact is mostly
governed by rearrangements of both bacterial and
surface components9. For example, the C. crescentus
adhesin holdfast, which mediates permanent adhesion,
is thought to experience an intramolecular rearrangement upon contact with the surface, accompanied by
the diffusion of molecular adhesins within the bulk
of the holdfast 56. This rearrangement over time is
believed to be responsible for the increase in the number
of bonds with the surface and the strengthening of the
adhesion force between the holdfast and the surface. The
displacement of interfacial water between the bacterium
and the surface enhances hydrophobic interactions and
thereby promotes close contact between the bacterium
and the surface (Fig. 2B). Then, additional rearrangements, such as unfolding of cell surface structures (for
example, curli or pilus proteins), may occur to create
additional strong contacts with the surface57 (Fig. 2B, C).
The transition from reversible to irreversible attachment is usually, but not always, due to repositioning of
the cell body from a polar to a longitudinal attachment.
Sometimes cells initially attach along the body of the cell,
where they are reversibly adhered, and the transition to
irreversible adherence can still occur. In some bacteria,
such as E. coli and P. aeruginosa, initial surface contact
is mediated by flagella and pili, which leads to polar
adhesion (see below). These bacteria transition from
reversible to irreversible adhesion by repositioning the
cell body to a longitudinal position4. This repositioning
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Box 2 | Small molecules and sRNAs in the regulation of adhesion
Once the bacterial cell is in contact with the surface, its physiology is severely modified at both the transcriptional level
and the post-​translational level8: motility genes, such as flagellar genes, are downregulated, and genes that promote the
sessile lifestyle, such as those involved in the production of adhesins, are upregulated. Small intracellular molecules have
an important role in regulating motility and adhesion genes. The second messenger cyclic-​di GMP (c-​di-GMP) is a key
signal that controls the switch between a free-​living and surface-​bound lifestyle in many species (recently reviewed in
Ref.118). Briefly, the intracellular level of c-​di-GMP is regulated by proteins of two antagonistic enzymatic functions:
diguanylate cyclases synthesize c-​di-GMP, and phosphodiesterases degrade it. High levels of c-​di-GMP trigger the
synthesis of molecules favouring adhesion and/or suppressing motility. For example, in Pseudomonas aeruginosa, at least
four diguanylate cyclases have been implicated in single-​cell adhesion119 by upregulating the expression of
polysaccharide adhesins120 and downregulating flagellar motility121. Unipolar polysaccharide adhesins, such as
Agrobacterium tumefaciens UPP or the Caulobacter crescentus holdfast, are also regulated by the intracellular
c-​di-GMP pool83,92,122.
Other small molecules regulate motility and adhesion in different bacteria. In P. aeruginosa PAO1, accumulation of cyclic
AMP (cAMP) inhibits irreversible adhesion123. cAMP is involved in surface sensing and initial adhesion of this bacterium,
and mechanosensory pili modulate cAMP and c-​di-GMP signalling84,124. cAMP also regulates adhesion in Vibrio cholerae125,
Escherichia coli126 and Serratia marcescens127. Finally, cyclic-​di AMP (c-​di-AMP) has a role in the production of
polysaccharide128 and the regulation of biofilm formation129 in Streptococcus mutans and Bacillus subtilis130. The production
of guanosine tetraphosphate and pentaphosphate ((p)ppGpp), the master regulator of the stringent response131, regulates
adhesion in E. coli132 and Enterococcus faecium133 by inducing the synthesis of type 1 fimbriae or other extracellular
filamentous structures. The stringent response also has a role in biofilm formation in other diverse bacteria, such as
V. cholerae134, Streptococcus mutans135, Bordetella pertussis136, Actinobacillus pleuropneumoniae137 and P. putida138.
Non-​coding small RNAs (sRNAs) regulate adhesion via the post-​transcriptional control of flagellar genes as well as
adhesion genes, such as those required for the production of curli and exopolysaccharide (reviewed in Ref.139). CsrB,
which was the first sRNA shown to control bacterial adhesion, targets the RNA-​binding protein carbon storage regulator
A (CsrA)140. CsrA is a master regulator that binds to the promoters of genes involved in adhesion and motility and is
regulated via binding to the CsrB and CsrC sRNAs141. Homologous systems of Csr have been identified in a number of
bacteria, including E. coli, Salmonella, Pseudomonas or Vibrio species142. The transcriptional regulator CsgD, which is
involved in the regulation of curli fimbriae production and export as well as flagellar synthesis, is another sRNA target143.
The RNA-​binding chaperone Hfq and its associated sRNAs regulate the expression of CsgD, as well as several other genes
involved in the motile-​to-sessile transition and in fimbriae production, which affect cell attachment144. sRNAs also control
the levels of c-​di-GMP, directly or indirectly, and Hfq, CsgB and CrsA regulate the expression of diguanylate cyclases that
synthesize c-​di-GMP145.
Stringent response
A stress response in bacteria,
whereby starvation induces the
production of the small
molecule guanosine
tetraphosphate and
pentaphosphate ((p)ppGpp),
which leads to the rapid
transcriptional changes and
growth arrest.
can be explained by two phenomena: cells that are
bound by their cell pole are able to spin along their axis,
facilitating their detachment from the surface58, and
longitudinal positioning maximizes the contact area
between the bacterial cell and the surface4 (Fig. 3Aa).
For example, in P. aeruginosa, the exopolysaccharide
Pel helps the cells to transition from polar adhesion to
longitudinal attachment and strengthens the adhesion59.
In P. fluorescens and P. putida, the protein LapA
enhances the interaction between the bacterial cell body
and the surface60 (Fig. 3Ba). Mutants unable to produce
LapA on the cell surface still undergo initial attachment
at the pole but are no longer capable of lateral adhesion60. LapA is a multidomain protein that is involved in
adherence to a wide range of surfaces60,61, but different
domains of this protein have a specific role in attachment to hydrophilic surfaces62. Accumulation of LapA
on the cell surface63 is regulated post-​translationally by
c-​di-GMP. When adhesion to the surface is favourable,
such as when inorganic phosphate levels are high in the
environment, the level of intracellular c-​di-GMP is high,
resulting in LapD-​mediated inhibition of the LapG protease, preventing the cleavage of LapA, thereby securing its localization on the cell surface and promoting
longitudinal adhesion64. The diguanylate cyclases GcbB
and GcbC are involved in modulating c-​di-GMP levels
in this process64, and the c-​di-GMP effector FleQ is a
positive regulator of lapA expression when c-​di-GMP
concentration is high65. However, transitioning from
622 | OCTOBER 2018 | volume 16
an initial polar adhesion to a horizontal interaction
with the surface is not the only strategy developed by
bacteria to reach irreversible adhesion: a number of
Alphaproteobacteria stay attached via their pole and
achieve permanent adhesion via a strong localized polar
adhesin7, as discussed below66–69 (Fig. 3Ab).
The rearrangement of cell surface structures has an
important role in mediating irreversible attachment in
some species. For example, the FimH adhesin protein
present at the tip of type I pili in E. coli70 experiences
conformational changes that enable the cells to undergo
tight adherence to surfaces under high flow. The type I
pilus is composed of several proteins that form a filament that is 1–2 µm long and 7 nm wide71. The main
pilus is composed of more than 1,000 subunits of FimA
tightly arranged in a right-​handed helical rod, while
FimF, FimG and FimH form the 3 nm fibrillum at the
tip of the main pilus7. FimH is the distal protein of
the pilus tip and is composed of two subunits: a pilin
domain that anchors it to the pilus and a lectin domain
that specifically recognizes terminal mannose moieties of target epithelial host cells or mannose-​coated
surfaces72 (Fig. 3B). The binding of FimH to mannose
residues is dependent on shear stress73: at low shear,
FimH forms weak, short-​lived bonds with the mannose residues on the surface, causing the bacterium
to move downstream as bonds break. At higher shear,
mannose-​bound FimH changes conformation, causing the bacterium to attach firmly to the surface 73.
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Reviews
A
Aa
Flagellum
Reversible
adhesion
Irreversible
adhesion
Extracellular matrix
Multicellular
community
Surface
Ab
Reversible
adhesion
Multicellular
community
Irreversible
adhesion
Adhesin
B
FimH protein capping the
type I pili from E. coli
LapA from P. fluorescens
Ba
Discrete polysaccharide
holdfast from
C. crescentus
Bc
Bb
500 nm
1 μm
150 nm
100 nm
Fig. 3 | The transition from reversible to irreversible attachment. A | Models for the
switch from reversible to irreversible adhesion. Aa | For some bacteria, such as Pseudomonas
fluorescens or Escherichia coli, initial surface contact is mediated by flagella and pili, which
leads to polar adhesion. These bacteria transition from reversible to irreversible adhesion by
repositioning the cell body to a longitudinal position. The adhesion is enhanced by the
synthesis of protein or polysaccharide adhesins (LapA for P. fluorescens (red) or Pel for E. coli).
Irreversible adhesion leads to the formation of a multicellular community embedded in an
extracellular matrix composed of polysaccharides, proteins and DNA. Ab | Bacteria, such as
Caulobacter crescentus or Agrobacterium tumefaciens, initiate surface attachment through
the polar flagellum. Cells stay attached via their pole and achieve permanent adhesion
through the secretion of a polar adhesin (red dots: holdfast for C. crescentus and UPP for
A. tumefaciens). Consequently, the incipient multicellular community is composed of cells
mainly oriented polarly and is devoid of an extracellular matrix. For simplicity, the presence
of a sinusoidal flagellum illustrates motility of free-​swimming bacteria, and the flagellum is
the only external appendage shown. B | Examples of adhesins for permanent attachment.
To generate permanent adhesion to the surface, bacteria synthesize adhesin molecules.
The most common types of such adhesins are protein and polysaccharide molecules.
Shown are three different examples of adhesins. Ba | A 3D-​structure illumination
microscopy (SIM) image of the discrete non-​fimbrial adhesin protein LapA from
P. fluorescens (red). The localization of LapA-​haemagglutinin (HA) is indicated (using an
HA-specific antibody, with AlexaFluor-488 conjugated secondary antibody (green)).
Bb | The FimH protein capping the type I pili from E. coli. Shown is an immuno-​electron
microscopy image, using 8 nm gold-​conjugated FimH antibodies. Bc | A transmission
electron microscope (TEM) image of the discrete polysaccharide holdfast (see inset) from
C. crescentus at the tip of the thin cylindrical stalk. Part Ba adapted from Ivanov, I. E. et al.
Atomic force and super-​resolution microscopy support a role for LapA as a cell-​surface
biofilm adhesin of Pseudomonas fluorescens. Res. Microbiol. (2012) 163, 685–691.
Copyright © 2018 Elsevier Masson SAS. All rights reserved. Part Bb adapted with permission
from Ref.71, Elsevier. Image in part Bc courtesy of C. Berne, Indiana University, USA.
This force-​dependent mechanism is known as a catch
bond and enables E. coli to colonize gastrointestinal cells in a high pulsatile flow environment while
adapting to variable flow conditions.
NATuRE REvIEwS | MICRoBIology
Another example of surface structures that undergo
rearrangements upon extended surface interaction
include curli found on E. coli cell surfaces57. Specifically,
the major curlin subunit CsgA exhibits local flexible
regions that contain aromatic residues that promote
hydrophobic interactions, which strengthen adhesion to
surfaces over time. Flexibility within the protein and the
resulting loss of secondary structure enable more accessibility of these residues to the surface and thus tighter
surface interaction.
Surface sensing and stimulation of adhesin production. Surface contact-​stimulated adhesin production
occurs in diverse bacteria74 and can be triggered by surface sensing through the cell envelope or extracellular
appendages75 (Fig. 4); however, the underlying molecular
mechanisms are not yet fully understood.
Once a bacterium is in contact with a surface,
adhesion forces trigger mechanical stress and slight
deformations of the cell envelope. In some cases, the
deformation of the membrane upon contact with a
surface can stimulate production of adhesin molecules
(Fig. 4a). For example, in E. coli, contact with hydrophobic surfaces induces stress in the cell membrane, which
triggers the Cpx two-​component system and positively
regulates curli transcription and promotes adhesion76.
Surface-​stimulated activation of the Cpx system requires
the outer membrane lipoprotein NlpE in a manner that
is independent of protein accumulation or pH-​induced
stress. However, NlpE-​dependent surface activation of
Cpx may be promoted by changes in osmolarity, which
could mimic surface contact 77,78. S. aureus can also
recognize contact with the surface by cell wall deformation upon contact with the surface79, and the bacterium
expresses adhesin molecules at the cell–surface interface
to strengthen the adhesion80.
The flagellum has been implicated as a surface sensor in many species, though its exact role in this process
has remained unclear. In B. subtilis, either tethering of
rotating flagella using antibodies or the deletion of flagella genes stimulates adhesin production and biofilm
formation74. In Vibrio parahaemolyticus, the addition
of viscous compounds that increase load force on polar
flagellar rotation triggers the expression of lateral flagella
required for surface-​associated motility, and deletion
of polar flagellar genes induces the same response74.
Collectively, these results suggest a mechanism by which
increased load on flagellar rotation stimulates bacterial
surface sensing (Fig. 4b).
Interestingly, at least two mechanisms of surface
sensing may exist in C. crescentus. Newly divided cells
initiate surface attachment through the polar flagellum
and rapidly produce an adhesive holdfast that mediates permanent attachment within seconds of surface
contact in a pilus-​dependent manner81,82. Recent work
demonstrates a role for the flagellar motor in permanent
adhesion by C. crescentus83 that is independent of flagellar rotation (Fig. 4c). Although the flagellum motor seems
to be important for attachment, the external flagellum
filament is not, which suggests a role for the proton
motive force that drives flagellar rotation in the surface
contact response. Another study showed that the
volume 16 | OCTOBER 2018 | 623
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a
Adhesion forces
Deformation of
the cell envelope
Adhesin production
Adhesin
Surface
b
Flagellum
Repression of
flagellar rotation
Adhesin production
c
Flagellar motor
d
Adhesin production
Rotor-dependent
surface sensing
Pilus
Pilus retraction
Adhesin production
Fig. 4 | Production of adhesins in response to surface contact. a | Cell envelope
deformation. Upon contact with the surface, the bacterial cell is subjected to adhesion
forces from the surface (blue arrow), which triggers a deformation of the cell envelope.
b | Repression of flagellar rotation. Initial contact with the surface increases the torque
applied to the flagellum and slows down rotation, which generates a signal to upregulate
adhesion-​related genes. c | Flagellar motor as a mechanosensor. Physical contact
between the cell and the surface triggers a conformational change of the motor, which
generates a burst of cyclic di-​GMP (c-​di-GMP), to stimulate adhesin production. d | Pilus
retraction. Interaction between the pili and the surface increases the load on
surface-bound, retracting pili, which leads to adhesin production.
resistance on retracting Tad pili can be sensed by
the cell to stimulate the synthesis of the holdfast in
C. crescentus50 (Fig. 4d) . How the flagellum and pili
together coordinate surface sensing in C. crescentus
remains to be elucidated. Other species, such as
P. aeruginosa, also harbour flagella and type IV pili at
the same pole. These pili also have a role in surface
sensing and surface-​induced virulence, which indicates
that this mechanism of coordinated action between pili
and flagella may be widespread among diverse clades
of bacteria84,85.
The use of a polar polysaccharide adhesin for irreversible adhesion is a common theme in the Alpha­
proteobacteria7, examples of which include the C.
crescentus holdfast and the unipolar polysaccharide (UPP)
produced by A. tumefaciens, Prosthecomicrobium hirschii
and Rhodopseudomonas palustris, among others7,66,68,69
(Fig. 3B). The C. crescentus polar holdfast is required for
irreversible adhesion to surfaces86 and is produced in
624 | OCTOBER 2018 | volume 16
both a developmental and a surface-​stimulated manner.
This adhesin is a gel-​like material partly composed of
β-1,4-N-​acetyl-glucosamine residues87,88, protein and
DNA89. The holdfast exhibits an impressive adhesive
force in the micro Newton range, making it one of the
strongest biological adhesives yet described90. The adhesion strength of the holdfast increases logarithmically
with time upon surface contact56, likely to be due to a
curing mechanism56,91. The synthesis of the holdfast is
regulated temporally during the developmental life cycle
of cells that do not encounter a surface. When in the bulk
liquid environment, a newborn C. crescentus cell spends
approximately one-​third of its life as a motile swarmer
cell bearing pili at its flagellar pole before differentiating
into a stalked cell. During this genetically programmed
transition, the cell sheds its polar flagellum, retracts its
pili and synthesizes a holdfast followed by a stalk at the
same pole66. The production of the holdfast is regulated
by c-​di-GMP in this developmental pathway92. Another
developmental regulator of holdfast production is the
small hydrophobic protein HfiA, which inactivates
the holdfast polysaccharide glycosyltransferase HfsJ by
direct protein–protein interaction and prevents holdfast
formation93. HfiA is stabilized by the chaperone DnaK
and is regulated by the major cell cycle regulators CtrA,
GcrA and StaR, ensuring the proper timing of holdfast production during the developmental program94.
In addition, HfiA is regulated by environmental inputs,
which prevents the formation of the holdfast and
therefore irreversible adhesion of C. crescentus when
nutrients are limited93. By contrast, blue-​light stimulation enhances holdfast production via the lovK–lovR
system95. Moreover, swarmer cells that reach a surface
bypass the developmental pathway and synthesize a
holdfast within seconds of surface contact irrespective
of the time since cell division81,82.
Although it is well established that adhesin molecules
help maintain biofilm structure and function as major
components of the biofilm matrix at the population
level96, how those molecules mediate the transition from
reversible to irreversible adhesion at the single-​cell level
is less documented. The advent of new single-​cell techniques (Box 1) in the past decade enables us to better
appreciate the role of various exopolysaccharide components in single-​cell adhesion. For example, AFM
experiments performed on single cells show that
lipopolysaccharides,type I pili and capsular colanic
acid in E. coli97 and the polysaccharide adhesin PIA in
methicillin-​resistant S. aureus (MRSA)98 have a crucial
role in the irreversible adhesion of a single cell.
Conclusions and perspectives
In recent years, many collaborations between biologists,
chemists and physicists have led to the development
of new ways to successfully study early-​stage bacterial attachment at the single-​cell level41, for example,
using microfluidic devices99 and AFM33,100. As a result,
we are beginning to better appreciate the mechanisms
underlying the first moments of the complex process
of adhesion. However, as we discussed in this Review,
the diverse mechanisms that single bacterial cells use to
attach to surfaces complicate our ability to draw general
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Reviews
conclusions on how adhesion is achieved at the single-​
cell level. The bacterial cell, the liquid environment
and the surface itself are dynamic, and modification of
one can have major repercussions on how these factors
interact with each other. Different bacterial species
harbour different cellular features that aid or hinder
attachment to surfaces under varying conditions, and
even a clonal population exhibits heterogeneity at
the population level. Differences between sister cells
from the same population may provide advantages
for coping with fluctuations in the environment and
surface properties, although this possibility remains to
be investigated.
The fate of a single cell, once it is firmly attached to
the surface, is to multiply and form multicellular communities, which is achieved by numerous interactions
of individual bacteria with the surface and with each
other. These aggregates continue to grow and mature
to form complex biofilms. The transition of attached
single cells into a biofilm community often triggers
important physiological changes for individual cells
that contribute to the formation of an extracellular
matrix to protect the colony and strengthen adhesion
to the surface96. The biofilm community is a dynamic
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Acknowledgements
The authors thank the members of the Brun laboratory for
critical reading of the manuscript. Work in the authors’ laboratory is supported by grants R01GM102841 and
R35GM122556 from the National Institutes of Health (to
Y.V.B.) and by National Science Foundation fellowship
1342962 (to C.K.E.).
Author contributions
C.B., C.K.E. and A.D. researched data for the article. Y.V.B.,
C.B. and C.K.E. made substantial contributions to discussions
of the content. C.B. and C.K.E. wrote the article. Y.V.B., C.B.
and C.K.E. reviewed and/or edited the manuscript before
submission.
Competing interests
The authors declare no competing interests.
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional
claims in published maps and institutional affiliations.
Reviewer information
Nature Reviews Microbiology thanks Y. Dufrene, M. Parsek
and the other anonymous reviewer(s) for their contribution to
the peer review of this work.
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