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 www.nature.com/nrmicro Reviews 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 www.nature.com/nrmicro Reviews 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 volume 16 | OCTOBER 2018 | 619 Reviews 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 www.nature.com/nrmicro Reviews 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 volume 16 | OCTOBER 2018 | 621 Reviews 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. www.nature.com/nrmicro 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 Reviews 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 www.nature.com/nrmicro 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. 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L., Salvarezza, R. C. & de Mele, M. F. Nano/microscale order affects the early stages of biofilm formation on metal surfaces. Langmuir 23, 11206–11210 (2007). 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. volume 16 | OCTOBER 2018 | 627