Emergent heterogeneous microenvironments in biofilms: substratum surface heterogeneity and bacterial adhesion force-sensing

Emergent heterogeneous microenvironments in biofilms: substratum surface heterogeneity and... Abstract Phenotypically heterogeneous microenvironments emerge as biofilms mature across different environments. Phenotypic heterogeneity in biofilm sub-populations not obeying quorum sensing-dictated, collective group behavior may be considered as a strategy allowing non-conformists to survive hostile conditions. Heterogeneous phenotype development has been amply studied with respect to gene expression and genotypic changes, but ‘biofilm genes’ responsible for preprogrammed development of heterogeneous microenvironments in biofilms have never been discovered. Moreover, the question of what triggers the development of phenotypically heterogeneous microenvironments has never been addressed. The definition of biofilms as ‘surface-adhering and surface-adapted’ microbial communities contains the word ‘surface’ twice. This leads us to hypothesize that phenotypically heterogeneous microenvironments in biofilms develop as an adaptive response of initial colonizers to their adhering state, governed by the forces through which they adhere to a substratum surface. No surface is entirely homogeneous, while adhering bacteria can substantially contribute to stochastically occurring surface heterogeneity. Accordingly, bacterial adhesion forces sensed by initial colonizers differ across a substratum surface, leading to differential mechanical deformation of the cell wall and membrane, where many environmental sensors are located. Bacteria directly adhering to heterogeneous substratum domains therewith formulate their own local responses to their adhering state and command non-conformist behavior, leading to phenotypically heterogeneous microenvironments in biofilms. quorum sensing, environmental sensing, swarming, antibiotic resistance, cooperativity, biosurfactants ABBREVIATIONS ABBREVIATIONS AFM atomic force microscopy DDS dichlorodimethylsilane eDNA extracellular DNA EPS extracellular polymeric substances HA hydroxyapatite PE polyethylene PEG polyethylene glycol PEO poly(ethylene) oxide PET polyethylene terephthalate PIA polysaccharide intercellular adhesin PDMS polydimethylsiloxane PMMA polymethyl methacrylate PS polystyrene QA quaternary ammonium SS stainless steel SR silicone rubber WCA water contact angle INTRODUCTION Bacterial adhesion and biofilm formation Bacteria adhere to surfaces in most industrial and natural environments, regardless of whether the surfaces are of synthetic or biological origin, and the latter includes the surfaces of prokaryotic and eukaryotic cells. Bacterial adhesion clearly marks the start of ‘biofilm’ formation, but it still remains a challenge to define the end of biofilm formation. Biofilms are defined as surface-adhering and surface-adapted communities of microorganisms (Tolker-Nielsen 2015), which grow embedded in their self-produced matrix of extracellular polymeric substances (EPS: see Text Box 1) (Flemming and Wingender 2010). Note that this definition includes cell-to-cell adhesion and therefore also encompasses planktonic aggregates (Vert et al.2012). Text Box 1. Extracellular polymeric substances Polymers, such as polysaccharides, proteins, extracellular DNA (eDNA) or nucleic acids, secreted by bacteria and forming a ‘glue’ that holds a biofilm together, possibly serving other functions such as nutrient trapping and protection against antimicrobial challenges (Flemming and Wingender 2010). Emergent biofilm properties The biofilm phenotype of bacteria is distinguished from the planktonic state by emergent properties (‘localized gradients, sorption and retention, cooperation and competition, tolerance and resistance’: see Text Box 2) (Flemming et al.2016). Text Box 2. Emergent biofilm properties New properties which emerge in a biofilm that is not predictable from the properties of free-living bacterial cells (Flemming et al.2016). Biofilm phenotypes do not emerge homogeneously across a biofilm. Heterogeneous microenvironments with different microbial composition, pH, live-dead ratios of bacteria, EPS production, including eDNA-rich or -poor domains, differential penetrability, density, water content and channelization have been observed in biofilms using fluorescent probes (Stewart and Franklin 2008) or optical coherence tomography (Wagner et al.2010). Phenotypically heterogeneous microenvironments are present in biofilms of both Gram-negative and Gram-positive species in different environments (Fig. 1), where non-conformists represent a bacterial sub-population that does not obey quorum-sensing commands (see Text Box 3), generally thought to coordinate a homogeneous response in an entire biofilm (Grote, Krysciak and Streit 2015). Possession of heterogeneous microenvironments can be considered as a deliberate strategy of biofilm inhabitants, with the potential of offering multiple mechanisms to combat hostile conditions and therewith facilitate survival of non-conformists. Figure 1. View largeDownload slide Examples of heterogeneously developing microenvironments in biofilms. (A) Red-fluorescent patches of EPS in a Strep. mutans (green-fluorescent) biofilm on saliva-coated HA. (Gao et al.2016, reprinted with permission from Elsevier Ltd). (B) Scattered red-fluorescent patches corresponding to EPS in 43 h S. mutans (green-fluorescent) biofilm grown on saliva-coated HA discs with orthogonal distribution of catalytic nanoparticles (white) (Gao et al.2016, reprinted with permission from Elsevier Ltd). (C) Live (green-fluorescent) and dead (red-fluorescent) Mycobacterium smegmatis scattered through a biofilm on a hydrophobic PS surface after 72 h exposure to ciprofloxacin (Muñoz-Egea et al.2015, reprinted with permission from BioMed Central), indicating differential susceptibility to ciprofloxacin and presumably reflecting a variation in physiological state. (D) Distribution of bacteria and EPS after live-dead staining in a multispecies oral biofilm with Strep. mutans, Strep. sanguinis and Strep. gordonii, formed on a dental adhesive surface (Ge et al.2017, reprinted with permission from MDPI). (E) Evolution of spatially segregated communities in Burkholderia cenocepacia biofilms on PS, with different colony morphotypes showing differently colored fluorescence (Poltak and Cooper 2011, reprinted with permission from the Nature Publishing group). Three distinct colony morphotypes reproducibly emerged within biofilms inoculated with a single ancestor. (F) Uneven pattern of penetration and accumulation of Nile-red loaded micelles into a staphylococcal biofilm grown on glass (Liu et al.2016, reprinted with permission from American Chemical Society). The micelle carriers have a poly(ethylene)glycol shell and are biologically invisible allowing them to enter a biofilm, where they acquire a cationic charge at low pH to interact electrostatically with the bacterial cell surface. Thus, the observed distribution of Nile red likely demonstrates heterogeneity with respect to channelization and possibly low pH microenvironments within the biofilm. (G) In vitro grown Strep. mutans biofilm on HA, with green-fluorescent bacteria and blue-fluorescent EPS patches occurring unevenly across the biofilm (Stoodley et al.2008, reprinted with permission from Elsevier Ltd). Figure 1. View largeDownload slide Examples of heterogeneously developing microenvironments in biofilms. (A) Red-fluorescent patches of EPS in a Strep. mutans (green-fluorescent) biofilm on saliva-coated HA. (Gao et al.2016, reprinted with permission from Elsevier Ltd). (B) Scattered red-fluorescent patches corresponding to EPS in 43 h S. mutans (green-fluorescent) biofilm grown on saliva-coated HA discs with orthogonal distribution of catalytic nanoparticles (white) (Gao et al.2016, reprinted with permission from Elsevier Ltd). (C) Live (green-fluorescent) and dead (red-fluorescent) Mycobacterium smegmatis scattered through a biofilm on a hydrophobic PS surface after 72 h exposure to ciprofloxacin (Muñoz-Egea et al.2015, reprinted with permission from BioMed Central), indicating differential susceptibility to ciprofloxacin and presumably reflecting a variation in physiological state. (D) Distribution of bacteria and EPS after live-dead staining in a multispecies oral biofilm with Strep. mutans, Strep. sanguinis and Strep. gordonii, formed on a dental adhesive surface (Ge et al.2017, reprinted with permission from MDPI). (E) Evolution of spatially segregated communities in Burkholderia cenocepacia biofilms on PS, with different colony morphotypes showing differently colored fluorescence (Poltak and Cooper 2011, reprinted with permission from the Nature Publishing group). Three distinct colony morphotypes reproducibly emerged within biofilms inoculated with a single ancestor. (F) Uneven pattern of penetration and accumulation of Nile-red loaded micelles into a staphylococcal biofilm grown on glass (Liu et al.2016, reprinted with permission from American Chemical Society). The micelle carriers have a poly(ethylene)glycol shell and are biologically invisible allowing them to enter a biofilm, where they acquire a cationic charge at low pH to interact electrostatically with the bacterial cell surface. Thus, the observed distribution of Nile red likely demonstrates heterogeneity with respect to channelization and possibly low pH microenvironments within the biofilm. (G) In vitro grown Strep. mutans biofilm on HA, with green-fluorescent bacteria and blue-fluorescent EPS patches occurring unevenly across the biofilm (Stoodley et al.2008, reprinted with permission from Elsevier Ltd). Text Box 3. Quorum sensing Intra- and interspecific bacterial communication by producing, releasing and detecting small, diffusible molecular auto-inducers. When auto-inducers reach a threshold concentration, it is commonly accepted that a whole population collectively obeys with homogeneous gene expression. Non-conformists represent a bacterial sub-population that does not obey quorum-sensing commands (Grote, Krysciak and Streit 2015). Phenotypically heterogeneous, emergent microenvironments Heterogeneous gene expression or genotypic changes form the basis for the development of phenotypically heterogeneous microenvironments in biofilms. Gene expression is traditionally studied as an average behavioral property in a bacterial population. However, phenotypic heterogeneity occurs also already at the single-bacterium level (Dubnau and Losick 2006), and it could be argued that phenotypic heterogeneities at the single-bacterium level form the basis of heterogeneously emerging properties in biofilms. The development of heterogeneous phenotypes at the level of biofilm communities, as well as at the level of single bacteria, has been amply studied and reviewed with respect to gene expression and genotypic changes in planktonic bacterial aggregates and biofilms grown in well plates or on agar (Wolska et al.2016). However, the question of what actually triggers the emergence of heterogeneous microenvironments in biofilms remains unanswered. Hypothesis on the development of phenotypically heterogeneous, emergent microenvironments Despite their frequent observation, heterogeneous microenvironments are usually taken for granted, without wondering why one only sees patches of EPS (Nuryastuti et al.2011), polysaccharide intercellular adhesin (PIA) (Arciola et al.2015) or other compounds (Dueholm and Nielsen 2016) appear in a microscopic image, why isolated regions of dead bacteria occur (Muñoz-Egea et al.2015), why pH varies across a biofilm (Hidalgo et al.2009), why penetrability varies at different locations in a biofilm (Liu et al.2016) or why some adhering bacteria develop motility while others remain non-motile (Prüss 2017)? Are these heterogeneous responses that emerge stochastically distributed by coincidence, are they a transient state in a kinetic process, are they a response to an environmental trigger or do they develop as a genetically preprogrammed, deterministic property in the transition from an adhering bacterium to a mature biofilm? Since ‘biofilm genes’ responsible for preprogrammed development of heterogeneous microenvironments in mature biofilms have consistently not been discovered (O’Toole, Kaplan and Kolter 2000), emergent phenotypic heterogeneity in biofilms is likely governed by environmental triggers (Vlamakis et al.2008) and physical cues (O’Toole and Wong 2016; Chew and Yang 2017). However, the precise nature of the actual trigger or physical cue has not been addressed. The word ‘surface’ occurs twice in the definition of biofilms by Tolker-Nielsen: ‘surface-adhering’ and ‘surface-adapted’ communities of microorganisms (Tolker-Nielsen 2015). This leads us to hypothesize that phenotypically heterogeneous, emergent microenvironments in biofilms develop as a response of bacteria to their adhering state and are governed by the local properties of the substratum surface. Aim of this review In this review, we summarize the events that stimulate different emergent phenotypes during biofilm formation on different non-biological materials with the aim of identifying substratum surface-associated triggers for the development of phenotypically heterogeneous, emergent microenvironments in a biofilm. MICROENVIRONMENTS IN BIOFILMS ON DIFFERENT SUBSTRATUM SURFACES In Table 1, we summarize events stimulating emergent phenotypes across a wide variety of different bacterial strains and species and on different substrata. Data in the table are literature-derived without the intention of representing a complete overview of the literature. Instead, the table serves to identify substratum surface-associated triggers for emergent phenotypes, as discussed below. Opposite to the discussion below which is phenomenologically organized, the table is organized alphabetically for different strains. Table 1. Summary of observations involving the emergence of different phenotypes across a wide variety of different bacterial strains and species and on different substrata. Strain Substrata Observations Relevant details References Single species studies Caulobacter crescentus Glass Bacteria made multiple surface contact before transitioning from reversible to irreversible adhesion. WCA < 30º; microfluidic flow conditions Hoffman et al.2015 Escherichia coli Micron-scale patterned PDMS Surface appendages enable bacteria to overcome unfavorable surface patterns Static conditions Friedlander et al.2013 E. coli PS well plates pH heterogeneity within biofilms Type of PS and WCA not reported; shaking conditions (30 rpm) Hidalgo et al.2009 E. coli Hydrophobic glass beads Cpx pathway regulates adhesion-induced gene expression Otto and Silhavy 2002 Lactobacillus plantarum Lectin monolayer and hydrophobic coatings Time-dependent binding to lectin layers; fast, time-independent binding to hydrophobic coatings Beaussart et al.2013 Mycobacteria Hydrophobic slides Biofilm viability and structure affected by antibiotic presence 30 min initial adhesion; orbital shaking (80 rpm) Muñoz-Egea et al.2015 Pseudomonas aeruginosa Glass, SS, PET, hydrophobic SS, hydrophilic PET Flagella increase adhesion on hydrophobic surfaces; straight and long flagella on PET and SS; curved and short flagella on glass WCA and surface roughness provided for all surfaces Bruzaud et al.2015 Staphylococcus aureus PE, SS Adhesion force and nisin efflux pump efficacy was highest on hydrophobic PE surfaces WCA for PE 85º and for SS 35º; static conditions Carniello et al.2018 S. aureus PE, SS, Ti–6Al–4V alloy, HA Adhesion forces, bacterial retention and viability are substratum related WCA for SS 49º, for PE 82º, for Ti–6Al–4V 69º and for HA 95º Alam and Balani 2017 S. aureus PE, SS, PMMA Matrix production and icaA gene expression is inversely related with adhesion forces WCA for SS 33º, for PMMA 69ºº and for PE 84º; submicron roughness Harapanahalli et al.2015 S. aureus Glass Cell wall deformation and long-range adhesion forces are related Chen et al.2014 S. aureus Glass Heterogeneous pattern of penetration and accumulation of Nile-red loaded micelles into biofilms Liu et al.2016 Staphylococcus epidermidis QA coatings Strong adhesion forces cause bacterial death Surfaces carry a positive charge Asri et al.2014 S. epidermidis SS, PMMA, PE Substratum-dependent EPS production and gentamicin susceptibility Nuryastuti et al.2011 Streptococcus sobrinus DDS coatings Substratum hydrophobicity determines bacterial retention, with less impact on adhesion WCA for DDS coatings 90º and glass 20º Bos et al.2000 Multiple species studies S. aureus E. coli Nanoporous or nanopillared, hydrophobized aluminum oxide Adhesion to hydrophobic, nanopillared surfaces smaller than to hydrophilic or nanoporous surfaces WCA varies from 0–162º; static and flow conditions Hizal et al.2017 S. aureus P. aeruginosa Plasma etched black silicon Smaller, more densely packed pillars exhibited the greatest bactericidal activity WCA varies from 8 to 160º; pillar heights of 212, 475–610 nm Linklater et al.2017 S. aureus S. epidermidis Nanopillared-Si wafers Nanopatterning stimulates EPS production and yields bacterial killing Regular patterning with sharply pointed pillars; flow conditions Hizal et al.2016 P. aeruginosa S. aureus Graphene nanosheets Graphene nanosheets creates pores in bacterial cell walls, causing bacterial death Roughness of the graphene sheets varies between 19 and 44 nm. Pham et al.2015 Branhamella catarrhalisBacillus subtilisE. coli P. aeruginosa Pseudomonas fluorescens Pseudomonas. maritimus S. aureus Cicada wing, nanopatterned surfaces Nanopatterning kills only Gram-negative bacteria Hasan et al.2013 Asticcacaulis biprosthecum Agrobacterium tumefaciens C. crescentus glass Reversible attachment of bacterial cells is mediated by motile cells bearing pili triggering adhesin production. Li et al.2012 S. aureus S. epidermidis P. aeruginosa SR; SR with Pluronic brush Adhesion forces dictated the transition from a planktonic to a biofilm mode of growth Flow conditions; WCA for SR 110º Muszanska et al.2012 Actinomyces naeslundii Lactobacillus acidophilus Streptococcus mitis Streptococcus mutans Streptococcus oralis Streptococcus sanguinis S. sobrinus SS, bovine enamel Salivary conditioning films reduce adhesion forces Salivary films reduced WCA of SS to 23º and of enamel to 26º; sub-micron roughness Mei et al.2009 S. aureus S. epidermidis Various substrata Staphylococcal biofilms show four distinct states, growing aerobically, growing fermentatively, dead and dormant, contributing to their tolerance to antimicrobials Different reactor systems Rani et al.2007 P. aeruginosa S. epidermidis PEO coatings PEO-brush coating reduced adhesion of all strains and species Flow conditions Roosjen et al.2004 Marinobacter hydrocarbonoclasticus Psychrobactersp. Halomonas pacifica Glass Dissolved organic carbon alters surface properties with an impact on adhesion Flow conditions; surfaces conditioned with natural seawater Bakker et al.2003 Strain Substrata Observations Relevant details References Single species studies Caulobacter crescentus Glass Bacteria made multiple surface contact before transitioning from reversible to irreversible adhesion. WCA < 30º; microfluidic flow conditions Hoffman et al.2015 Escherichia coli Micron-scale patterned PDMS Surface appendages enable bacteria to overcome unfavorable surface patterns Static conditions Friedlander et al.2013 E. coli PS well plates pH heterogeneity within biofilms Type of PS and WCA not reported; shaking conditions (30 rpm) Hidalgo et al.2009 E. coli Hydrophobic glass beads Cpx pathway regulates adhesion-induced gene expression Otto and Silhavy 2002 Lactobacillus plantarum Lectin monolayer and hydrophobic coatings Time-dependent binding to lectin layers; fast, time-independent binding to hydrophobic coatings Beaussart et al.2013 Mycobacteria Hydrophobic slides Biofilm viability and structure affected by antibiotic presence 30 min initial adhesion; orbital shaking (80 rpm) Muñoz-Egea et al.2015 Pseudomonas aeruginosa Glass, SS, PET, hydrophobic SS, hydrophilic PET Flagella increase adhesion on hydrophobic surfaces; straight and long flagella on PET and SS; curved and short flagella on glass WCA and surface roughness provided for all surfaces Bruzaud et al.2015 Staphylococcus aureus PE, SS Adhesion force and nisin efflux pump efficacy was highest on hydrophobic PE surfaces WCA for PE 85º and for SS 35º; static conditions Carniello et al.2018 S. aureus PE, SS, Ti–6Al–4V alloy, HA Adhesion forces, bacterial retention and viability are substratum related WCA for SS 49º, for PE 82º, for Ti–6Al–4V 69º and for HA 95º Alam and Balani 2017 S. aureus PE, SS, PMMA Matrix production and icaA gene expression is inversely related with adhesion forces WCA for SS 33º, for PMMA 69ºº and for PE 84º; submicron roughness Harapanahalli et al.2015 S. aureus Glass Cell wall deformation and long-range adhesion forces are related Chen et al.2014 S. aureus Glass Heterogeneous pattern of penetration and accumulation of Nile-red loaded micelles into biofilms Liu et al.2016 Staphylococcus epidermidis QA coatings Strong adhesion forces cause bacterial death Surfaces carry a positive charge Asri et al.2014 S. epidermidis SS, PMMA, PE Substratum-dependent EPS production and gentamicin susceptibility Nuryastuti et al.2011 Streptococcus sobrinus DDS coatings Substratum hydrophobicity determines bacterial retention, with less impact on adhesion WCA for DDS coatings 90º and glass 20º Bos et al.2000 Multiple species studies S. aureus E. coli Nanoporous or nanopillared, hydrophobized aluminum oxide Adhesion to hydrophobic, nanopillared surfaces smaller than to hydrophilic or nanoporous surfaces WCA varies from 0–162º; static and flow conditions Hizal et al.2017 S. aureus P. aeruginosa Plasma etched black silicon Smaller, more densely packed pillars exhibited the greatest bactericidal activity WCA varies from 8 to 160º; pillar heights of 212, 475–610 nm Linklater et al.2017 S. aureus S. epidermidis Nanopillared-Si wafers Nanopatterning stimulates EPS production and yields bacterial killing Regular patterning with sharply pointed pillars; flow conditions Hizal et al.2016 P. aeruginosa S. aureus Graphene nanosheets Graphene nanosheets creates pores in bacterial cell walls, causing bacterial death Roughness of the graphene sheets varies between 19 and 44 nm. Pham et al.2015 Branhamella catarrhalisBacillus subtilisE. coli P. aeruginosa Pseudomonas fluorescens Pseudomonas. maritimus S. aureus Cicada wing, nanopatterned surfaces Nanopatterning kills only Gram-negative bacteria Hasan et al.2013 Asticcacaulis biprosthecum Agrobacterium tumefaciens C. crescentus glass Reversible attachment of bacterial cells is mediated by motile cells bearing pili triggering adhesin production. Li et al.2012 S. aureus S. epidermidis P. aeruginosa SR; SR with Pluronic brush Adhesion forces dictated the transition from a planktonic to a biofilm mode of growth Flow conditions; WCA for SR 110º Muszanska et al.2012 Actinomyces naeslundii Lactobacillus acidophilus Streptococcus mitis Streptococcus mutans Streptococcus oralis Streptococcus sanguinis S. sobrinus SS, bovine enamel Salivary conditioning films reduce adhesion forces Salivary films reduced WCA of SS to 23º and of enamel to 26º; sub-micron roughness Mei et al.2009 S. aureus S. epidermidis Various substrata Staphylococcal biofilms show four distinct states, growing aerobically, growing fermentatively, dead and dormant, contributing to their tolerance to antimicrobials Different reactor systems Rani et al.2007 P. aeruginosa S. epidermidis PEO coatings PEO-brush coating reduced adhesion of all strains and species Flow conditions Roosjen et al.2004 Marinobacter hydrocarbonoclasticus Psychrobactersp. Halomonas pacifica Glass Dissolved organic carbon alters surface properties with an impact on adhesion Flow conditions; surfaces conditioned with natural seawater Bakker et al.2003 Relevant experimental details are included, when available in the references used. View Large Table 1. Summary of observations involving the emergence of different phenotypes across a wide variety of different bacterial strains and species and on different substrata. Strain Substrata Observations Relevant details References Single species studies Caulobacter crescentus Glass Bacteria made multiple surface contact before transitioning from reversible to irreversible adhesion. WCA < 30º; microfluidic flow conditions Hoffman et al.2015 Escherichia coli Micron-scale patterned PDMS Surface appendages enable bacteria to overcome unfavorable surface patterns Static conditions Friedlander et al.2013 E. coli PS well plates pH heterogeneity within biofilms Type of PS and WCA not reported; shaking conditions (30 rpm) Hidalgo et al.2009 E. coli Hydrophobic glass beads Cpx pathway regulates adhesion-induced gene expression Otto and Silhavy 2002 Lactobacillus plantarum Lectin monolayer and hydrophobic coatings Time-dependent binding to lectin layers; fast, time-independent binding to hydrophobic coatings Beaussart et al.2013 Mycobacteria Hydrophobic slides Biofilm viability and structure affected by antibiotic presence 30 min initial adhesion; orbital shaking (80 rpm) Muñoz-Egea et al.2015 Pseudomonas aeruginosa Glass, SS, PET, hydrophobic SS, hydrophilic PET Flagella increase adhesion on hydrophobic surfaces; straight and long flagella on PET and SS; curved and short flagella on glass WCA and surface roughness provided for all surfaces Bruzaud et al.2015 Staphylococcus aureus PE, SS Adhesion force and nisin efflux pump efficacy was highest on hydrophobic PE surfaces WCA for PE 85º and for SS 35º; static conditions Carniello et al.2018 S. aureus PE, SS, Ti–6Al–4V alloy, HA Adhesion forces, bacterial retention and viability are substratum related WCA for SS 49º, for PE 82º, for Ti–6Al–4V 69º and for HA 95º Alam and Balani 2017 S. aureus PE, SS, PMMA Matrix production and icaA gene expression is inversely related with adhesion forces WCA for SS 33º, for PMMA 69ºº and for PE 84º; submicron roughness Harapanahalli et al.2015 S. aureus Glass Cell wall deformation and long-range adhesion forces are related Chen et al.2014 S. aureus Glass Heterogeneous pattern of penetration and accumulation of Nile-red loaded micelles into biofilms Liu et al.2016 Staphylococcus epidermidis QA coatings Strong adhesion forces cause bacterial death Surfaces carry a positive charge Asri et al.2014 S. epidermidis SS, PMMA, PE Substratum-dependent EPS production and gentamicin susceptibility Nuryastuti et al.2011 Streptococcus sobrinus DDS coatings Substratum hydrophobicity determines bacterial retention, with less impact on adhesion WCA for DDS coatings 90º and glass 20º Bos et al.2000 Multiple species studies S. aureus E. coli Nanoporous or nanopillared, hydrophobized aluminum oxide Adhesion to hydrophobic, nanopillared surfaces smaller than to hydrophilic or nanoporous surfaces WCA varies from 0–162º; static and flow conditions Hizal et al.2017 S. aureus P. aeruginosa Plasma etched black silicon Smaller, more densely packed pillars exhibited the greatest bactericidal activity WCA varies from 8 to 160º; pillar heights of 212, 475–610 nm Linklater et al.2017 S. aureus S. epidermidis Nanopillared-Si wafers Nanopatterning stimulates EPS production and yields bacterial killing Regular patterning with sharply pointed pillars; flow conditions Hizal et al.2016 P. aeruginosa S. aureus Graphene nanosheets Graphene nanosheets creates pores in bacterial cell walls, causing bacterial death Roughness of the graphene sheets varies between 19 and 44 nm. Pham et al.2015 Branhamella catarrhalisBacillus subtilisE. coli P. aeruginosa Pseudomonas fluorescens Pseudomonas. maritimus S. aureus Cicada wing, nanopatterned surfaces Nanopatterning kills only Gram-negative bacteria Hasan et al.2013 Asticcacaulis biprosthecum Agrobacterium tumefaciens C. crescentus glass Reversible attachment of bacterial cells is mediated by motile cells bearing pili triggering adhesin production. Li et al.2012 S. aureus S. epidermidis P. aeruginosa SR; SR with Pluronic brush Adhesion forces dictated the transition from a planktonic to a biofilm mode of growth Flow conditions; WCA for SR 110º Muszanska et al.2012 Actinomyces naeslundii Lactobacillus acidophilus Streptococcus mitis Streptococcus mutans Streptococcus oralis Streptococcus sanguinis S. sobrinus SS, bovine enamel Salivary conditioning films reduce adhesion forces Salivary films reduced WCA of SS to 23º and of enamel to 26º; sub-micron roughness Mei et al.2009 S. aureus S. epidermidis Various substrata Staphylococcal biofilms show four distinct states, growing aerobically, growing fermentatively, dead and dormant, contributing to their tolerance to antimicrobials Different reactor systems Rani et al.2007 P. aeruginosa S. epidermidis PEO coatings PEO-brush coating reduced adhesion of all strains and species Flow conditions Roosjen et al.2004 Marinobacter hydrocarbonoclasticus Psychrobactersp. Halomonas pacifica Glass Dissolved organic carbon alters surface properties with an impact on adhesion Flow conditions; surfaces conditioned with natural seawater Bakker et al.2003 Strain Substrata Observations Relevant details References Single species studies Caulobacter crescentus Glass Bacteria made multiple surface contact before transitioning from reversible to irreversible adhesion. WCA < 30º; microfluidic flow conditions Hoffman et al.2015 Escherichia coli Micron-scale patterned PDMS Surface appendages enable bacteria to overcome unfavorable surface patterns Static conditions Friedlander et al.2013 E. coli PS well plates pH heterogeneity within biofilms Type of PS and WCA not reported; shaking conditions (30 rpm) Hidalgo et al.2009 E. coli Hydrophobic glass beads Cpx pathway regulates adhesion-induced gene expression Otto and Silhavy 2002 Lactobacillus plantarum Lectin monolayer and hydrophobic coatings Time-dependent binding to lectin layers; fast, time-independent binding to hydrophobic coatings Beaussart et al.2013 Mycobacteria Hydrophobic slides Biofilm viability and structure affected by antibiotic presence 30 min initial adhesion; orbital shaking (80 rpm) Muñoz-Egea et al.2015 Pseudomonas aeruginosa Glass, SS, PET, hydrophobic SS, hydrophilic PET Flagella increase adhesion on hydrophobic surfaces; straight and long flagella on PET and SS; curved and short flagella on glass WCA and surface roughness provided for all surfaces Bruzaud et al.2015 Staphylococcus aureus PE, SS Adhesion force and nisin efflux pump efficacy was highest on hydrophobic PE surfaces WCA for PE 85º and for SS 35º; static conditions Carniello et al.2018 S. aureus PE, SS, Ti–6Al–4V alloy, HA Adhesion forces, bacterial retention and viability are substratum related WCA for SS 49º, for PE 82º, for Ti–6Al–4V 69º and for HA 95º Alam and Balani 2017 S. aureus PE, SS, PMMA Matrix production and icaA gene expression is inversely related with adhesion forces WCA for SS 33º, for PMMA 69ºº and for PE 84º; submicron roughness Harapanahalli et al.2015 S. aureus Glass Cell wall deformation and long-range adhesion forces are related Chen et al.2014 S. aureus Glass Heterogeneous pattern of penetration and accumulation of Nile-red loaded micelles into biofilms Liu et al.2016 Staphylococcus epidermidis QA coatings Strong adhesion forces cause bacterial death Surfaces carry a positive charge Asri et al.2014 S. epidermidis SS, PMMA, PE Substratum-dependent EPS production and gentamicin susceptibility Nuryastuti et al.2011 Streptococcus sobrinus DDS coatings Substratum hydrophobicity determines bacterial retention, with less impact on adhesion WCA for DDS coatings 90º and glass 20º Bos et al.2000 Multiple species studies S. aureus E. coli Nanoporous or nanopillared, hydrophobized aluminum oxide Adhesion to hydrophobic, nanopillared surfaces smaller than to hydrophilic or nanoporous surfaces WCA varies from 0–162º; static and flow conditions Hizal et al.2017 S. aureus P. aeruginosa Plasma etched black silicon Smaller, more densely packed pillars exhibited the greatest bactericidal activity WCA varies from 8 to 160º; pillar heights of 212, 475–610 nm Linklater et al.2017 S. aureus S. epidermidis Nanopillared-Si wafers Nanopatterning stimulates EPS production and yields bacterial killing Regular patterning with sharply pointed pillars; flow conditions Hizal et al.2016 P. aeruginosa S. aureus Graphene nanosheets Graphene nanosheets creates pores in bacterial cell walls, causing bacterial death Roughness of the graphene sheets varies between 19 and 44 nm. Pham et al.2015 Branhamella catarrhalisBacillus subtilisE. coli P. aeruginosa Pseudomonas fluorescens Pseudomonas. maritimus S. aureus Cicada wing, nanopatterned surfaces Nanopatterning kills only Gram-negative bacteria Hasan et al.2013 Asticcacaulis biprosthecum Agrobacterium tumefaciens C. crescentus glass Reversible attachment of bacterial cells is mediated by motile cells bearing pili triggering adhesin production. Li et al.2012 S. aureus S. epidermidis P. aeruginosa SR; SR with Pluronic brush Adhesion forces dictated the transition from a planktonic to a biofilm mode of growth Flow conditions; WCA for SR 110º Muszanska et al.2012 Actinomyces naeslundii Lactobacillus acidophilus Streptococcus mitis Streptococcus mutans Streptococcus oralis Streptococcus sanguinis S. sobrinus SS, bovine enamel Salivary conditioning films reduce adhesion forces Salivary films reduced WCA of SS to 23º and of enamel to 26º; sub-micron roughness Mei et al.2009 S. aureus S. epidermidis Various substrata Staphylococcal biofilms show four distinct states, growing aerobically, growing fermentatively, dead and dormant, contributing to their tolerance to antimicrobials Different reactor systems Rani et al.2007 P. aeruginosa S. epidermidis PEO coatings PEO-brush coating reduced adhesion of all strains and species Flow conditions Roosjen et al.2004 Marinobacter hydrocarbonoclasticus Psychrobactersp. Halomonas pacifica Glass Dissolved organic carbon alters surface properties with an impact on adhesion Flow conditions; surfaces conditioned with natural seawater Bakker et al.2003 Relevant experimental details are included, when available in the references used. View Large Phenotypic drug tolerance and resistance Phenotypic heterogeneity with respect to drug tolerance and resistance has been observed frequently in bacterial bulk cultures. Correct mechanistic distinction between tolerance and resistance is difficult (see Text Box 4). Phenotypic resistance is thought to be mainly due to environmentally triggered changes in bacterial cell wall permeability impeding drug access, activation of efflux pumps and release of drug-deactivating enzymes (Kester and Fortune 2014). Examples of environmentally triggered events are the reversible change in porin expression levels in enteric bacteria in response to high osmolarity or temperature (Dupont et al.2007) or the reduced antibiotic sensitivity of Enterobacter aerogenes which results from reduced porin expression under antibiotic pressure (Bornet et al.2000). Phenotypic tolerance, on the other hand, involves an environmental trigger of bacterial dormancy, persistence, differentiation and biofilm formation, including EPS production (Kester and Fortune 2014; Kaldalu, Hauryliuk and Tenson 2016). Although the mechanisms of phenotypic heterogeneity with respect to tolerance and resistance likely unite in a biofilm, the role of the substratum surface and its specific properties as an environmental trigger for the development of biofilm heterogeneity has not been considered (Olsen 2015; Brauner et al.2016). Text Box 4. Resistance and tolerance Antibiotic resistance generally means an increase in the minimum inhibitory concentration of an antibacterial agent due to a permanent change in the bacterium, e.g. by mutation or through horizontal gene transfer. Antibiotic tolerance is the ability of bacteria to survive the effect of an antibiotic due to a reversible phenotypic state. Two main forms of tolerance have been identified: ‘tolerance by slow growth’ (occurs at steady state) and ‘tolerance by lag’ (a transient state that is induced by starvation or stress) (Olsen 2015; Brauner et al.2016). Staphylococcus epidermidis and S. aureus biofilms grown on polycarbonate filters on agar possessed at least four distinct phenotypes: bacteria growing either aerobically or fermentatively, dead or dormant (Rani et al.2007). Multiple strains of S. epidermidis containing the ica locus, which encodes for PIA, were found to produce biofilms on hydrophobic polyethylene (PE) surfaces (water contact angle [WCA] of 84º) which contained large patches of EPS. Alternatively, on more hydrophilic acrylic and stainless steel surfaces (WCA of 69º and 33º, respectively), heterogeneously occurring EPS production was less and concurrently, ica-gene expression was low in these biofilms as compared with biofilms on PE (Nuryastuti et al.2011). Similarly, EPS production in biofilms of S. aureus and S. epidermidis on hydrophobic silicone rubber (SR) surfaces (WCA of 110 degrees) was massive and yielded resistance to gentamicin, whereas on hydrophilic polyethylene glycol (PEG), polymer-brush-coated SR (WCA of around 40º), EPS production was absent and bacteria remained susceptible to gentamicin. To a lesser extent, such differences were also observed in biofilms of the Gram-negative bacterium, P. aeruginosa (Roosjen et al.2004; Muszanska et al.2012). Expression of the membrane located sensor, NsaS and the NsaA two-component efflux pump in S. aureus SH1000, responsible for nisin resistance in the planktonic state, was enhanced when the organism was adhering to a substratum surface. Moreover, adhesion to a hydrophobic PE surface triggered a greater expression of nsaS and nsaA than adhesion to a more hydrophilic stainless steel surface (Carniello et al.2018). Despite the influence that the specific properties of the substratum surface have on emergent biofilm properties, most experiments are reported in the literature without reference to the substratum material. In many cases, biofilm assays are performed in multiwell polystyrene (PS) plates and the type of PS is not specified even though this will affect surface properties: for example, bacterial-grade PS is more hydrophobic in the absence of surface treatment (WCA 78º) than tissue culture-grade PS after physical treatment (WCA 43º), and these differences may severely impact on bacterial adaptive behavior. Moreover, often conclusions on surface adaptation are extrapolated from results obtained in biofilms grown on aqueous agar, which may not accurately reflect the conditions encountered on solid substratum surfaces. Collectively, these examples demonstrate that the substratum surface, most notably its hydrophobicity or hydrophilicity (see Text Box 5), provides an environmental trigger for the development of antibiotic resistance and tolerance in biofilms. Importantly, in most of these examples, a uniform response of the entire biofilm has been inferred without evidence that the biofilm is homogeneous over its entire volume. However, where available, closer inspection of micrographs in the published literature (see Fig. 1 for specific examples) clearly shows stochastically occurring non-conformists, providing clear evidence of heterogeneity. Text Box 5. Surface hydrophobicity ‘Surface hydrophobicity’ and its opposite ‘surface hydrophilicity’ literally indicate the ‘fear’ or ‘love’ of a surface for water. Surface hydrophobicity can be quantitated by placing a small water droplet on a surface and measuring its degree of spreading, full spreading being characterized by a 0º WCA (hydrophilic surface). On super-hydrophobic materials, such as nanostructured hydrophobic surfaces, air can become entrapped and water has an almost 180º WCA (Hizal et al.2017), making it behave like a mercury droplet. Swarming behavior Swarming is another drug-resistance mechanism allowing bacteria to explore and subsequently escape an antibiotic-laden or otherwise hostile environment (Lai, Tremblay and Déziel 2009), and also enables bacteria to actively search for nutrients (Daniels, Vanderleyden and Michiels 2004). Swarming phenotypes are often characterized by being hyperflagellated, elongated, multinucleate (Toguchi et al.2000) and antibiotic-resistant. In Paenibacillus vortex biofilms, antibiotic-refractory, swarming phenotypes function to explore the environment for antibiotic-laden regions that should be avoided by the ‘builders’ of the biofilm community (Roth et al.2013). Swarming bacteria either reside in (i) bulk suspension, where they are unlikely to experience any effects from a substratum surface, (ii) surface-constrained, near the surface but still in suspension and experiencing hydrodynamic shear or (iii) in direct interaction with the substratum surface (Tuson and Weibel 2013). Swarming in the surface-constrained regime requires reversible adhesion on the one hand, but in order to prevent detachment back into the bulk suspension, bacteria must have a means to rapidly transit between reversible and irreversible adhesion. Indeed, studies on single cells of C. crescentus demonstrated that transitioning from reversible to irreversible adhesion is not a single event and most cells reversibly contact a surface multiple times before a final transition to irreversible adhesion takes place, with pili playing an important role in this transition (Hoffman et al.2015). Bacteria can sense the presence of a surface by obstruction of surface appendages such as flagella, pili or fimbriae (Friedlander et al.2013; Ellison and Brun 2015) and subsequent activation of membrane-located sensors (Belas 2014). In C. crescentus, arrest of flagellum rotation and concurrent stimulation of ‘just-in-time’ polysaccharide adhesive occurs to maximize adhesion and prevent untimely detachment back into suspension (Li et al.2012). The presence of P. aeruginosa flagella and type IV pili increased bacterial adhesion to highly hydrophobic substratum surfaces (Bruzaud et al.2015), suggesting a role for substratum surface properties on development of bacterial swarming phenotypes. HOW BACTERIA DIFFERENTIATE BETWEEN DIFFERENT SUBSTRATUM SURFACES Adhesion forces between bacteria and substratum surfaces The observation that bacteria adapt differently to adhesion on different substratum surfaces immediately raises the question of how bacteria sense that they are on a surface, and more importantly, how they tailor their adaptive response to the characteristic properties of the surface they adhere to. Adhesion, whether arising from specific, molecular ligand-receptor or non-specific interactions (Bos, Van der Mei and Busscher 1999), is an interplay between ever present attractive Lifshitz-Van der Waals forces, attractive or repulsive acid–base interactions as a generalized form of hydrogen bonding, electrostatic forces with a magnitude depending on pH and ionic strength of the fluid environment and Brownian motion forces. The attractive Lifshitz-Van der Waals forces are the most long-ranged ones, acting over distances of up to 1 μm and becoming increasingly stronger when the interacting surfaces become closer. The sum total of these different forces determine the force by which a bacterium adheres to a substratum surface and this varies on different surfaces (Alam and Balani 2017), while at close approach Lifshitz-Van der Waals forces are usually able to overcome electrostatic barriers (Puddu and Perry 2012; Paula et al.2014). Distinguishing three adhesion force regimes (Busscher and Van der Mei 2012), it was proposed that extremely weakly adhering bacteria (adhesion forces less than 1 nN) do not realize they are in an adhering state and therefore do not show any adaptive response to a substratum surface. Alternatively, when adhering very strongly (proposed adhesion forces above 10 nN) as on quaternary ammonium (QA)-coated surfaces (Muszanska et al.2012), cell wall damage is inferred resulting in bacterial cell death (Tiller et al.2001; Asri et al.2014). The intermediate regime comprising adhesion forces between 1 and 10–15 nN, as occurs on most common substratum surfaces across a wide variety of bacterial strains and species (Van der Mei et al.2008; Beaussart et al.2013; Sullan et al.2014; Thewes et al.2015), invokes bacterial adaptation with production of EPS according to the magnitude of the adhesion forces experienced (Harapanahalli et al.2015). The ability to measure bacterial adhesion forces using the AFM (see Text Box 6) creates an awareness of the enormous magnitude of bacterial adhesion forces as compared with the gravitational forces they experience (see Text Box 7). Thus, it is not surprising that a lethal regime exists in which bacteria die due to cell wall damage as result of experiencing adhesion forces that are 106–108-fold higher than the gravitational force they experience. It has been argued that bacterial cell walls are rigid to resist large internal pressures, but remarkably plastic in order to adapt to a wide range of external forces (Amir et al.2014), including adhesion forces. In fact, it has been demonstrated using AFM (Chen et al.2014) and surface enhanced fluorescence (see Text Box 8) that the bacterial cell wall deforms under the influence of the relatively large adhesion forces arising from a substratum surface (Fig. 2), despite the rigidity provided to bacteria by their peptidoglycan layer. Also, AFM imaging of S. epidermidis trapped in a filter has shown structural and mechanical deformation of the cell wall (Méndez-Vilas, Gallardo-Moreno and González-Martín 2007). Figure 2. View largeDownload slide Bacterial cell wall deformation under the influence of adhesion forces arising from a substratum surface (Chen et al.2014, reprinted with permission from American Society for Microbiology). An undeformed bacterium with a radius R approaching a substratum surface comes under the influence of the adhesion forces arising from the substratum. It gradually deforms, which brings more molecules (solid red region) under the influence of the adhesion forces, stimulating further adhesion until opposing forces arising from the rigid bacterial cell wall and increased intracellular pressure fully counteract the adhesion force. Figure 2. View largeDownload slide Bacterial cell wall deformation under the influence of adhesion forces arising from a substratum surface (Chen et al.2014, reprinted with permission from American Society for Microbiology). An undeformed bacterium with a radius R approaching a substratum surface comes under the influence of the adhesion forces arising from the substratum. It gradually deforms, which brings more molecules (solid red region) under the influence of the adhesion forces, stimulating further adhesion until opposing forces arising from the rigid bacterial cell wall and increased intracellular pressure fully counteract the adhesion force. Text Box 6. Bacterial adhesion force measurement Bacterial adhesion can be measured using atomic force microscopy (AFM). In bacterial probe AFM, a bacterium is attached to a highly flexible cantilever and brought into contact with a substratum surface, allowing contact between the bacterium and the surface for a defined time period and applied loading force. Upon retraction of the cantilever from the surface, the force required to break the bond between the bacterium and the substratum surface is recorded from the bending of the flexible cantilever. In this way, bacterial adhesion forces to biological and non-biological surfaces in the picoNewton (pN) to nanoNewton (nN) range have been measured (Dufrêne 2015). Text Box 7. On the magnitude of bacterial adhesion forces to surfaces Most forces by which bacteria adhere to surfaces are reportedly in the nN-range (Van der Mei et al.2008; Beaussart et al.2013; Sullan et al.2014; Thewes et al.2015), which is large compared to the gravity force experienced by bacteria. In air, the gravity force experienced by a bacterium is around 10−6 nN, while due to buoyancy, this force reduces in an aqueous suspension to around 10−8 nN. Assuming an adhesion force of around 1 nN, this implies that the forces by which bacteria adhere to a substratum surface are 106–108-fold higher than the gravity forces they experience. Text Box 8. Surface-enhanced bacterial fluorescence Surface-enhanced fluorescence is the phenomenon in which fluorophores within 20–30 nm from a metal surface show a stronger fluorescence intensity than expected for the same fluorophore in solution (Lee et al.2011). Surface-enhanced bacterial fluorescence of fluorescent bacteria adhering to metallic surfaces can be exploited to demonstrate bacterial cell wall deformation, because more of the fluorescent, intracellular content of a bacterium is brought into the close vicinity of the surface upon adhesion and subsequent cell wall deformation, and therewith subject to surface-enhanced fluorescence (Li et al.2014). Cell wall deformation and surface adaptation The role of cell wall deformation in triggering bacterial responses is difficult to demonstrate experimentally, as bacterial cell wall deformation is small due to the rigidity provided by the bacterial peptidoglycan layer surrounding the membrane. In mammalian cells, however, lacking a rigid cell wall, the influence of substratum hydrophobicity is more obvious and many different types of tissue cells remained ‘cauliflower’ shaped on hydrophobic substratum surfaces while deforming to a ‘pancake’ shape on hydrophilic ones (Schakenraad et al.1986). Also in mammalian cells, sensors located in the cell membrane have been described, which control the subsequent differentiation of stem cells in a substratum-dependent fashion (Engler et al.2006). Deliberate compression of bacteria between AFM cantilevers and substratum surfaces has demonstrated that the bacterial cell wall deforms in a viscoelastic way (Vadillo-Rodriguez, Beveridge and Dutcher 2008; Vadillo-Rodriguez and Dutcher 2009), although it should be noted that deformation under such conditions is not exactly the same as ‘spontaneous’ deformation under the influence of adhesion forces arising from a substratum surface. Escherichia coli and Bacillus subtilis behaved like elastic rods when subjected to external forces, but deformed permanently in the plastic regime of viscoelastic deformation when cell wall synthesis occurred while the force was applied (Amir et al.2014). Moreover, the offspring of plastically deformed bacteria always recovered their shape, but this required conditions allowing cell wall synthesis (Sliusarenko et al.2010; Amir et al.2014) over several generations (Si et al.2015). Bacterial cell wall deformation changes the pressure profile across the lipid membrane (Perozo et al.2002), which is laden with environmental sensors that can become activated by such changes (Kocer 2015) through gating of mechanosensitive channels (Haswell, Phillips and Rees 2011) or directly by conformational changes in membrane-located receptors (Otto and Silhavy 2002). Thus, adhesion-force sensing and subsequent cell wall deformation provide an important mechanism for adhering bacteria to realize they are on a surface and begin the process of surface adaptation. The role of rigid bacterial peptidoglycan layers in adhesion force-sensing and subsequent cell wall deformation is probably large, since an S. aureus Δpbp4 mutant, which lacks peptidoglycan cross-linking, seemed unable to adapt its response in line with the adhesion forces arising from a substratum surface (Harapanahalli et al.2015). HETEROGENEOUS SURFACES AND BACTERIAL INTERACTIONS Surface heterogeneity due to protein adsorption All naturally occurring and synthetic surfaces are heterogeneous, either on a micro- or nanoscopic scale and will exert different local adhesion forces on adhering bacteria to trigger different adaptive responses. Dental enamel is an excellent example of a naturally occurring heterogeneous surface with distinct crystalline hydroxyapatite (HA) structures comprised in an organic matrix, which in the oral cavity become covered within seconds with a conditioning film of adsorbed salivary proteins forming a network structure over the enamel surface (Busscher et al.1989; Simmons et al.2011). Although the network structure of adsorbed proteins is a heterogeneous surface structure in itself, saliva contains many different proteins (Marsh et al.2016) that adsorb and displace each other in succession, which further contributes to surface heterogeneity. In the oral cavity, formation and composition of salivary conditioning films varies on different surfaces (Aroonsang et al.2014) and precedes adhesion of bacteria and subsequently influences bacterial adhesion forces and biofilm detachment (Song, Koo and Ren 2015). A similar succession of protein adsorption and desorption occurs on cellular and synthetic graft surfaces exposed to blood (Vroman 2008). Note that, in the marine and other aqueous environments, conditioning films are often described as adsorbed films composed of dissolved organic carbon (Bakker et al.2003). Since bacteria diffuse more slowly than proteins, bacteria mostly adhere to such heterogeneous, adsorbed conditioning films, regardless of whether in the oral cavity or in any other environment. Surface charge heterogeneity Strong electrostatic attraction between positively charged QA-coated surfaces and negatively charged bacterial cell surfaces is reported to cause cell wall damage and subsequent cell death (Asri et al.2014). Charge heterogeneity on glass surfaces, often thought to be homogeneous, became evident by repetitively allowing negatively charged, 1-μm diameter PS particles to adhere to the same glass surface. Under low ionic strength conditions, particles always adhered first to the same, previously occupied microscopic location through strong, local electrostatic attraction (Wit and Busscher 1998), demonstrating the existence of positively charged heterogeneities on an overall negatively charged glass surface. Heterogeneity in surface hydrophobicity and roughness Heterogeneity in surface hydrophobicity and roughness at the sub-micrometer scale are easily demonstrable by the measurement of WCA hysteresis on material surfaces (see Text Box 9). Large differences between advancing and receding contact angles on ‘smooth’ surfaces with a roughness less than 0.1 μm indicate regions with a large difference in surface hydrophobicity. Roughened, hydrophobic surfaces may appear as ‘superhydrophobic’, while roughened, hydrophilic surfaces possess smaller WCAs than expected based on the hydrophobicity, respectively the hydrophilicity of their smooth counterparts. Text Box 9. Contact angle hysteresis When a water droplet advances over a perfectly smooth surface, it can be stopped by a small, more hydrophobic heterogeneity or rugosity, which causes the contact angle to be higher than when the droplet is in an equilibrium state. Equally so, when receding over an already wetted surface, water tends to remain behind on a hydrophilic heterogeneity and the contact angle appears smaller than in an equilibrium state. The difference in advancing and receding contact angles is called ‘contact angle hysteresis’ (Timmons and Zisman 1966). Only perfectly smooth and chemically homogeneous surfaces have a 0º contact angle hysteresis, which makes the measurement of contact angle hysteresis suitable for the measurement of surface heterogeneity in general at a sub-micrometer scale. Bacteria themselves are in fact also ideal to demonstrate heterogeneity in substratum surface hydrophobicity due to differential interaction with hydrophobic and hydrophilic regions on a substratum surface. Micro-patterned substratum surfaces consisting of hydrophobic lines separated by wide hydrophilic spacings, for instance, attracted equal numbers of streptococci over its entire surface, but when challenged with a detachment force, streptococci were retained only on the hydrophobic lines (Bos et al.2000), suggesting that the strength of bacterial adhesion is higher to hydrophobic regions. Adhesion force measurement using AFM on a patterned substratum consisting of square arrays of non-adhesive PEG hydrogels comparable in size to a bacterial cell on a hydrophobic, silanized glass surface showed that S. aureus adhesion was decreased at the hydrogel spacings as these presumably impeded contact between the bacterial cell and the hydrophobic surface (Wang et al.2011). Nanoscopically heterogeneous substratum surfaces Nanotechnological advances have enabled the production of nanoscopically heterogeneous surfaces, which are often bioinspired (Tripathy et al.2017) most notably by the so called ‘lotus effect’ (Huang et al.2016). Such plant leaves, and also certain insect wings, remain free of bacteria through self-cleaning and antibacterial properties, thought to be mediated by nanopillared arrays (Hasan et al.2013) that inherently represent a nanoscopically heterogeneous substratum surface. Electron micrographs have clearly demonstrated that the bacterial cell wall can locally severely deform under the influence of the adhesion forces arising from extruding random (Svensson et al.2014) and periodic (Hizal et al.2016) nanostructures to yield pressure-induced EPS production and even bacterial cell death in Gram-positive staphylococci. This is supported by observations that killing of P. aeruginosa and S. aureus on graphene nanosheets related with density of the edges of the graphene (Pham et al.2015). Approximately 98% of P. aeruginosa cells and 97% of S. aureus cells were killed on superhydrophilic and superhydrophobic black silicon surfaces with well-defined surface geometries and wettability, smaller, more densely packed pillars exhibiting the greatest bactericidal activity (Linklater et al.2017). It is speculated that the bactericidal activity is due to irreversible membrane bulging. In antibiotic-challenged E. coli, pores in the peptidoglycan network with a critical radius of around 20 nm, the typical distance between neighboring peptides and glycan strands, are required to cause bulging of the cytoplasmic membrane out through the pore. This bulging is irreversible and leading to loss of cell viability (Daly et al.2011). SUBSTRATUM SURFACE HETEROGENEITIES INDUCED BY ADHERING BACTERIA During adhesion, bacteria can create heterogeneities as a means of communication (Fig. 3) to allow localized positive or negative cooperation in colonizing a substratum surface, that is, stimulate or discourage adhesion of other bacteria in their immediate surroundings (Sjollema et al.1990). In a broader sense, bacteria have been suggested to leave ‘footprints’ when adhering to and detaching from a substratum surface (Neu 1992) that will contribute to substratum surface heterogeneity. Figure 3. View largeDownload slide Bacterially induced substratum surface heterogeneities as a means of communication and interaction between initially adhering bacteria. (A) Certain strains of bacteria excrete biosurfactants that spread over the substratum surface, modifying the immediate surrounding surface so that it is less favorable (red colored) for adherence by other bacteria. (B) Positive cooperativity is the mechanism by which an adhering bacterium changes the conformation of adsorbed proteins in its immediate surroundings or produces adhesive EPS, generating a more favorable surface (green colored) for adherence by other bacteria. Figure 3. View largeDownload slide Bacterially induced substratum surface heterogeneities as a means of communication and interaction between initially adhering bacteria. (A) Certain strains of bacteria excrete biosurfactants that spread over the substratum surface, modifying the immediate surrounding surface so that it is less favorable (red colored) for adherence by other bacteria. (B) Positive cooperativity is the mechanism by which an adhering bacterium changes the conformation of adsorbed proteins in its immediate surroundings or produces adhesive EPS, generating a more favorable surface (green colored) for adherence by other bacteria. Localized cooperative phenomena and biosurfactant release Biosurfactants (see Text Box 10), by their amphiphilic nature, are ideal molecules to be transported over large distances to reach remote areas of a substratum surface as a means to interact with other initial colonizers (Fig. 3A). Streptococcus mitis strains excrete biosurfactants that modify their immediate surroundings to make it less attractive for their competitors to adhere (Van Hoogmoed et al.2000; Loozen et al.2014) and the spreading of oral biosurfactants excreted by initial colonizers such as S. mitis over dental enamel surfaces reduced the adhesion forces of other colonizers (Van Hoogmoed et al.2006). Lactobacilli also claim substratum surface area by excretion of biosurfactants that discourage adhesion of enterococci and other uropathogens (Velraeds et al.1996). Text Box 10. Biosurfactants Biosurfactants are amphiphilic compounds produced by living organisms, mostly microorganisms, and excreted extracellularly, which contain hydrophobic and hydrophilic moieties, accumulating at an interface and reducing interfacial tensions versus air, a liquid surrounding or another material (Cochis et al.2012; Sambanthamoorthy et al.2014). Quorum-sensing controlled expression of phenol-soluble modulin surfactants in S. aureus (Periasami et al.2012) and rhamnolipids in P. aeruginosa (Davey, Caiazza and O’Toole 2003) biofilms has been shown to mediate biofilm structuring and detachment. For P. aeruginosa, siderophores, eDNA and biosurfactants play multiple roles in the interaction between different sub-populations in a biofilm and influence its structural development, as related to biosurfactants concentration and composition (Pamp and Tolker-Nielsen 2007). Bacterially induced changes in adsorbed protein conformation and positive cooperativity Bacteria also have other means to modify their immediate surroundings on a substratum surface to exert positive cooperativity (Nesbitt et al.1982; Van der Mei et al.1993): several initial colonizers of protein-conditioned surfaces have the ability to induce conformational changes in the adsorbed protein film that surrounds them (Fig. 3B), making the film more attractive for their peers to adhere. Initial colonizers of oral surfaces in vivo have slightly stronger adhesion forces with salivary conditioning films than later colonizers (Mei et al.2009), which may be underlying their ability to induce conformational changes in the adsorbed proteins to which they adhere. Since clinically, the relative prevalence of initially colonizing strains on a surface depends on the forces by which specific bacterial strains are attracted to their substratum surface (Wessel et al.2014), local induced changes in the conformation of adsorbed proteins may yield biofilm regions with a different bacterial composition. Cooperativity through EPS production EPS production can be considered as another cooperative phenomenon offering advantages in adhesion to neighboring bacteria by creating local surface heterogeneity around an adhering organism (see also Fig. 3B) (Nadell and Bassler 2011) but, like for positive cooperativity in general, at the obvious expense of impairing dispersal of adhering bacteria to new locations. Psl, for instance, is a cell wall anchored polysaccharide in P. aeruginosa (Ma et al.2009), promoting aggregate formation between neighboring bacteria in microenvironments of a biofilm, which does not occur and subsequently yields less biofilm in strains lacking Psl (Wang et al.2013). Mixed species oral biofilms on saliva-coated surfaces possess acidic niches in their EPS matrix that selectively stimulate the localized growth of pathogenic Strep. mutans (Xiao et al.2012; Koo and Yamada 2016). THE COMMANDING ROLE OF INITIAL COLONIZERS IN BIOFILM FORMATION Bacterial responses to prevailing environmental conditions is virtually always a survival strategy to maintain their adhering state in competition with others or under mechanical attack, while the production of EPS as an adaptive response embeds adhering bacteria in a matrix that also offers protection against chemical attacks (de la Fuente-Núñez et al.2013; Carniello et al.2016). Initially, adhering bacteria have various ways to influence the development of microenvironments in the biofilm that grows on top of them, in which adhesion force-sensing plays a crucial role. Adhesion force sensing and biofilm composition In the sequence of events that lead to a full-grown biofilm with heterogeneously occurring microenvironments, the initially adhering bacteria firstly have various ways to induce local heterogeneities on a substratum surface to which they adhere. Newcomers can recognize these heterogeneities by the strength of the local adhesion forces they experience and interpret them as signs to ‘stay away’ or ‘welcome, adhere here’. This, in turn, will create microenvironments in a biofilm with different microbial composition. Therewith, the basis of cooperation, and possible conflicts, in a mature biofilm (Xavier and Foster 2007) is commanded by the initially adhering bacteria. Adhesion force sensing and EPS production Emergent EPS production follows initial adhesion in the sequence of events leading to a mature biofilm, and is arguably one of the most important adaptive responses within a biofilm. Adhesion force sensing constitutes an environmental trigger for EPS production. The production of the matrix molecule, poly-N-acetylglucosamine, and the secretion of eDNA decrease with increasing adhesion force, suggesting that adhering staphylococci adjust their adaptive response to environmental need (Harapanahalli et al.2015) to prevent unnecessary costs to their fitness (Brooks and Jefferson 2014). Similarly, EPS production by bacteria adhering under fluid shear conditions is more extensive than under stagnant conditions, suggesting that its expression is induced only when required (Nivens et al.1993; Hou et al.2017). Since the effective range of adhesion forces is limited to maximally 1 μm, it is impossible for bacteria other than the initial colonizers to directly sense a substratum, while their immediate neighbors reside at distances between 1–3 μm and are embedded in an EPS matrix (Drescher et al.2016). Accordingly, only initially adhering bacteria are able to sense and adapt to the adhesion forces exerted by a substratum surface and, in fact, the majority of bacteria in a biofilm have never contacted the substratum surface (Zhao et al.2013). Since the same will be true for the bacteria in emergent heterogeneous microenvironments, this leads to the conclusion that initially adhering bacteria command the development of emerging heterogeneous microenvironments by sensing and adapting to the substratum and communicating with neighboring bacteria information about that surface (see Fig. 4). Stochastically occurring environmental triggers have been suggested before as being causative to phenotypic heterogeneity (Vega and Gore 2014), but have never been associated with triggers derived from stochastically occurring substratum surface heterogeneity. Figure 4. View largeDownload slide The commanding role in adaptive responses of initial colonizers in a biofilm. Initially, adhering bacteria sense different local adhesion forces which trigger different adaptive responses that spread through the biofilm by diffusion of quorum-sensing molecules until their concentration is below a detectable threshold and the commands given are lost, limiting heterogeneous microenvironment development in space and time. Microenvironments, including the adhesion forces that trigger differential responses, the commanding organisms and obeying inhabitants of the microenvironment, are indicated by different colors. Figure 4. View largeDownload slide The commanding role in adaptive responses of initial colonizers in a biofilm. Initially, adhering bacteria sense different local adhesion forces which trigger different adaptive responses that spread through the biofilm by diffusion of quorum-sensing molecules until their concentration is below a detectable threshold and the commands given are lost, limiting heterogeneous microenvironment development in space and time. Microenvironments, including the adhesion forces that trigger differential responses, the commanding organisms and obeying inhabitants of the microenvironment, are indicated by different colors. The surface adaptation (Text Box 11) of initial colonizers in response to direct contact with a substratum surface likely do not disappear with the first generation of later colonizers, not in direct contact with the surface, but will most probably disappear only after a number of generations (Si et al.2015), and the progeny returns to a more planktonic phenotype. Return to a planktonic phenotype does not necessarily imply bacterial return back into suspension, but may also occur in a biofilm, where bacteria are ‘suspended’ or ‘free floating’ in an EPS matrix at average distances of 1–3 μm from neighboring organisms (Drescher et al.2016), i.e. more specifically formulated, outside the influence of adhesion forces exerted by their neighbors. Text Box 11. Surface adaptation Bacterial surface adaptation comprises the particular response of a bacterium to the surface properties of the substratum to which it adheres. Adhesion force sensing and quorum sensing Identifying initial colonizers that are in direct contact with a substratum surface as ‘commanding’ bacteria implies that there must be a communication means available within a biofilm to pass information derived from adhesion force sensing to bacteria that are not in direct contact with the substratum enabling them to indirectly sense the surface. The initially adhering bacteria likely pass substratum information by producing and releasing auto-inducing molecules to which later biofilms colonizers respond. Since the distance over which auto-transducers can be transported and remain detectable is limited by diffusion (Vega and Gore 2014), quorum sensing is eventually quenched which restricts the adaptive response to microenvironments in a biofilm, although ‘calling distances’ between Gram-negative bacteria extending up to 78 μm have been reported (Elias and Banin 2012). However, most effective calling distances for producing and releasing, sensing and responding to auto-transducer gradients are suggested to be between 4 and 5 μm (Gantner et al.2006; Elias and Banin 2012), and bacteria can optimize the use of auto-inducers by being in each other's close vicinity. Myxococcus xanthus, E. coli, B. subtilis and lactobacilli, for instance, use contact-dependent signaling for communication (Blango and Mulvey 2009). Direct physical contact between bacteria in a biofilm is generally absent, unless co-adhering bacterial pairs are involved, which occur mostly in the oral cavity (Rickard et al.2003). SUMMARY In summary, all surfaces are heterogeneous with respect to hydrophobicity, charge and/or the possession of micro- or nanoscopic structures. Such stochastically occurring heterogeneities exert different adhesion forces upon adhering bacteria. Bacteria sense these adhesion forces through cell wall deformation, which subsequently activates membrane-located sensors to stimulate phenotypic responses in initially adhering bacteria in direct contact with the surface. The local adaptive response of initial colonizers is conveyed to other biofilm inhabitants through diffusion of auto-inducers produced by the initial colonizers and their first generation progeny. Later, generation progeny will lose the surface-adapted phenotype of the initial colonizers, while diffusion of auto-inducers occurs only over limited distances. This puts initial colonizers in command of the development of localized, stochastically occurring heterogeneous domains in a biofilm. The role of adhesion force sensing in cell wall deformation as local triggers for the development of heterogeneous microenvironments in biofilms puts a strong emphasis on the substratum surface on which biofilms are grown. Hitherto, in research on adaptive responses of bacteria to environmental triggers, conclusions are frequently extrapolated from agar-grown ‘biofilms’ and biofilms on undefined well-plate materials to biofilms in general. Realization of the role of substratum properties in localized, adaptive responses of adhering bacteria and subsequent properties of a biofilm may accelerate development of much needed insight in the mechanisms of heterogeneous microenvironment development in biofilms. FUNDING This study was supported by the University Medical Center Groningen-University of Groningen, Groningen both in The Netherlands. HJB is also director-owner of SASA BV. Opinions and assertions contained herein are those of the authors and are not meant to be construed as the representing views of the organizations to which the authors are affiliated. Conflict of interest. None declared. REFERENCES Alam F , Balani K . Adhesion force of Staphylococcus aureus on various biomaterial surfaces . 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Emergent heterogeneous microenvironments in biofilms: substratum surface heterogeneity and bacterial adhesion force-sensing

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Blackwell
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© FEMS 2018.
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0168-6445
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1574-6976
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10.1093/femsre/fuy001
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Abstract

Abstract Phenotypically heterogeneous microenvironments emerge as biofilms mature across different environments. Phenotypic heterogeneity in biofilm sub-populations not obeying quorum sensing-dictated, collective group behavior may be considered as a strategy allowing non-conformists to survive hostile conditions. Heterogeneous phenotype development has been amply studied with respect to gene expression and genotypic changes, but ‘biofilm genes’ responsible for preprogrammed development of heterogeneous microenvironments in biofilms have never been discovered. Moreover, the question of what triggers the development of phenotypically heterogeneous microenvironments has never been addressed. The definition of biofilms as ‘surface-adhering and surface-adapted’ microbial communities contains the word ‘surface’ twice. This leads us to hypothesize that phenotypically heterogeneous microenvironments in biofilms develop as an adaptive response of initial colonizers to their adhering state, governed by the forces through which they adhere to a substratum surface. No surface is entirely homogeneous, while adhering bacteria can substantially contribute to stochastically occurring surface heterogeneity. Accordingly, bacterial adhesion forces sensed by initial colonizers differ across a substratum surface, leading to differential mechanical deformation of the cell wall and membrane, where many environmental sensors are located. Bacteria directly adhering to heterogeneous substratum domains therewith formulate their own local responses to their adhering state and command non-conformist behavior, leading to phenotypically heterogeneous microenvironments in biofilms. quorum sensing, environmental sensing, swarming, antibiotic resistance, cooperativity, biosurfactants ABBREVIATIONS ABBREVIATIONS AFM atomic force microscopy DDS dichlorodimethylsilane eDNA extracellular DNA EPS extracellular polymeric substances HA hydroxyapatite PE polyethylene PEG polyethylene glycol PEO poly(ethylene) oxide PET polyethylene terephthalate PIA polysaccharide intercellular adhesin PDMS polydimethylsiloxane PMMA polymethyl methacrylate PS polystyrene QA quaternary ammonium SS stainless steel SR silicone rubber WCA water contact angle INTRODUCTION Bacterial adhesion and biofilm formation Bacteria adhere to surfaces in most industrial and natural environments, regardless of whether the surfaces are of synthetic or biological origin, and the latter includes the surfaces of prokaryotic and eukaryotic cells. Bacterial adhesion clearly marks the start of ‘biofilm’ formation, but it still remains a challenge to define the end of biofilm formation. Biofilms are defined as surface-adhering and surface-adapted communities of microorganisms (Tolker-Nielsen 2015), which grow embedded in their self-produced matrix of extracellular polymeric substances (EPS: see Text Box 1) (Flemming and Wingender 2010). Note that this definition includes cell-to-cell adhesion and therefore also encompasses planktonic aggregates (Vert et al.2012). Text Box 1. Extracellular polymeric substances Polymers, such as polysaccharides, proteins, extracellular DNA (eDNA) or nucleic acids, secreted by bacteria and forming a ‘glue’ that holds a biofilm together, possibly serving other functions such as nutrient trapping and protection against antimicrobial challenges (Flemming and Wingender 2010). Emergent biofilm properties The biofilm phenotype of bacteria is distinguished from the planktonic state by emergent properties (‘localized gradients, sorption and retention, cooperation and competition, tolerance and resistance’: see Text Box 2) (Flemming et al.2016). Text Box 2. Emergent biofilm properties New properties which emerge in a biofilm that is not predictable from the properties of free-living bacterial cells (Flemming et al.2016). Biofilm phenotypes do not emerge homogeneously across a biofilm. Heterogeneous microenvironments with different microbial composition, pH, live-dead ratios of bacteria, EPS production, including eDNA-rich or -poor domains, differential penetrability, density, water content and channelization have been observed in biofilms using fluorescent probes (Stewart and Franklin 2008) or optical coherence tomography (Wagner et al.2010). Phenotypically heterogeneous microenvironments are present in biofilms of both Gram-negative and Gram-positive species in different environments (Fig. 1), where non-conformists represent a bacterial sub-population that does not obey quorum-sensing commands (see Text Box 3), generally thought to coordinate a homogeneous response in an entire biofilm (Grote, Krysciak and Streit 2015). Possession of heterogeneous microenvironments can be considered as a deliberate strategy of biofilm inhabitants, with the potential of offering multiple mechanisms to combat hostile conditions and therewith facilitate survival of non-conformists. Figure 1. View largeDownload slide Examples of heterogeneously developing microenvironments in biofilms. (A) Red-fluorescent patches of EPS in a Strep. mutans (green-fluorescent) biofilm on saliva-coated HA. (Gao et al.2016, reprinted with permission from Elsevier Ltd). (B) Scattered red-fluorescent patches corresponding to EPS in 43 h S. mutans (green-fluorescent) biofilm grown on saliva-coated HA discs with orthogonal distribution of catalytic nanoparticles (white) (Gao et al.2016, reprinted with permission from Elsevier Ltd). (C) Live (green-fluorescent) and dead (red-fluorescent) Mycobacterium smegmatis scattered through a biofilm on a hydrophobic PS surface after 72 h exposure to ciprofloxacin (Muñoz-Egea et al.2015, reprinted with permission from BioMed Central), indicating differential susceptibility to ciprofloxacin and presumably reflecting a variation in physiological state. (D) Distribution of bacteria and EPS after live-dead staining in a multispecies oral biofilm with Strep. mutans, Strep. sanguinis and Strep. gordonii, formed on a dental adhesive surface (Ge et al.2017, reprinted with permission from MDPI). (E) Evolution of spatially segregated communities in Burkholderia cenocepacia biofilms on PS, with different colony morphotypes showing differently colored fluorescence (Poltak and Cooper 2011, reprinted with permission from the Nature Publishing group). Three distinct colony morphotypes reproducibly emerged within biofilms inoculated with a single ancestor. (F) Uneven pattern of penetration and accumulation of Nile-red loaded micelles into a staphylococcal biofilm grown on glass (Liu et al.2016, reprinted with permission from American Chemical Society). The micelle carriers have a poly(ethylene)glycol shell and are biologically invisible allowing them to enter a biofilm, where they acquire a cationic charge at low pH to interact electrostatically with the bacterial cell surface. Thus, the observed distribution of Nile red likely demonstrates heterogeneity with respect to channelization and possibly low pH microenvironments within the biofilm. (G) In vitro grown Strep. mutans biofilm on HA, with green-fluorescent bacteria and blue-fluorescent EPS patches occurring unevenly across the biofilm (Stoodley et al.2008, reprinted with permission from Elsevier Ltd). Figure 1. View largeDownload slide Examples of heterogeneously developing microenvironments in biofilms. (A) Red-fluorescent patches of EPS in a Strep. mutans (green-fluorescent) biofilm on saliva-coated HA. (Gao et al.2016, reprinted with permission from Elsevier Ltd). (B) Scattered red-fluorescent patches corresponding to EPS in 43 h S. mutans (green-fluorescent) biofilm grown on saliva-coated HA discs with orthogonal distribution of catalytic nanoparticles (white) (Gao et al.2016, reprinted with permission from Elsevier Ltd). (C) Live (green-fluorescent) and dead (red-fluorescent) Mycobacterium smegmatis scattered through a biofilm on a hydrophobic PS surface after 72 h exposure to ciprofloxacin (Muñoz-Egea et al.2015, reprinted with permission from BioMed Central), indicating differential susceptibility to ciprofloxacin and presumably reflecting a variation in physiological state. (D) Distribution of bacteria and EPS after live-dead staining in a multispecies oral biofilm with Strep. mutans, Strep. sanguinis and Strep. gordonii, formed on a dental adhesive surface (Ge et al.2017, reprinted with permission from MDPI). (E) Evolution of spatially segregated communities in Burkholderia cenocepacia biofilms on PS, with different colony morphotypes showing differently colored fluorescence (Poltak and Cooper 2011, reprinted with permission from the Nature Publishing group). Three distinct colony morphotypes reproducibly emerged within biofilms inoculated with a single ancestor. (F) Uneven pattern of penetration and accumulation of Nile-red loaded micelles into a staphylococcal biofilm grown on glass (Liu et al.2016, reprinted with permission from American Chemical Society). The micelle carriers have a poly(ethylene)glycol shell and are biologically invisible allowing them to enter a biofilm, where they acquire a cationic charge at low pH to interact electrostatically with the bacterial cell surface. Thus, the observed distribution of Nile red likely demonstrates heterogeneity with respect to channelization and possibly low pH microenvironments within the biofilm. (G) In vitro grown Strep. mutans biofilm on HA, with green-fluorescent bacteria and blue-fluorescent EPS patches occurring unevenly across the biofilm (Stoodley et al.2008, reprinted with permission from Elsevier Ltd). Text Box 3. Quorum sensing Intra- and interspecific bacterial communication by producing, releasing and detecting small, diffusible molecular auto-inducers. When auto-inducers reach a threshold concentration, it is commonly accepted that a whole population collectively obeys with homogeneous gene expression. Non-conformists represent a bacterial sub-population that does not obey quorum-sensing commands (Grote, Krysciak and Streit 2015). Phenotypically heterogeneous, emergent microenvironments Heterogeneous gene expression or genotypic changes form the basis for the development of phenotypically heterogeneous microenvironments in biofilms. Gene expression is traditionally studied as an average behavioral property in a bacterial population. However, phenotypic heterogeneity occurs also already at the single-bacterium level (Dubnau and Losick 2006), and it could be argued that phenotypic heterogeneities at the single-bacterium level form the basis of heterogeneously emerging properties in biofilms. The development of heterogeneous phenotypes at the level of biofilm communities, as well as at the level of single bacteria, has been amply studied and reviewed with respect to gene expression and genotypic changes in planktonic bacterial aggregates and biofilms grown in well plates or on agar (Wolska et al.2016). However, the question of what actually triggers the emergence of heterogeneous microenvironments in biofilms remains unanswered. Hypothesis on the development of phenotypically heterogeneous, emergent microenvironments Despite their frequent observation, heterogeneous microenvironments are usually taken for granted, without wondering why one only sees patches of EPS (Nuryastuti et al.2011), polysaccharide intercellular adhesin (PIA) (Arciola et al.2015) or other compounds (Dueholm and Nielsen 2016) appear in a microscopic image, why isolated regions of dead bacteria occur (Muñoz-Egea et al.2015), why pH varies across a biofilm (Hidalgo et al.2009), why penetrability varies at different locations in a biofilm (Liu et al.2016) or why some adhering bacteria develop motility while others remain non-motile (Prüss 2017)? Are these heterogeneous responses that emerge stochastically distributed by coincidence, are they a transient state in a kinetic process, are they a response to an environmental trigger or do they develop as a genetically preprogrammed, deterministic property in the transition from an adhering bacterium to a mature biofilm? Since ‘biofilm genes’ responsible for preprogrammed development of heterogeneous microenvironments in mature biofilms have consistently not been discovered (O’Toole, Kaplan and Kolter 2000), emergent phenotypic heterogeneity in biofilms is likely governed by environmental triggers (Vlamakis et al.2008) and physical cues (O’Toole and Wong 2016; Chew and Yang 2017). However, the precise nature of the actual trigger or physical cue has not been addressed. The word ‘surface’ occurs twice in the definition of biofilms by Tolker-Nielsen: ‘surface-adhering’ and ‘surface-adapted’ communities of microorganisms (Tolker-Nielsen 2015). This leads us to hypothesize that phenotypically heterogeneous, emergent microenvironments in biofilms develop as a response of bacteria to their adhering state and are governed by the local properties of the substratum surface. Aim of this review In this review, we summarize the events that stimulate different emergent phenotypes during biofilm formation on different non-biological materials with the aim of identifying substratum surface-associated triggers for the development of phenotypically heterogeneous, emergent microenvironments in a biofilm. MICROENVIRONMENTS IN BIOFILMS ON DIFFERENT SUBSTRATUM SURFACES In Table 1, we summarize events stimulating emergent phenotypes across a wide variety of different bacterial strains and species and on different substrata. Data in the table are literature-derived without the intention of representing a complete overview of the literature. Instead, the table serves to identify substratum surface-associated triggers for emergent phenotypes, as discussed below. Opposite to the discussion below which is phenomenologically organized, the table is organized alphabetically for different strains. Table 1. Summary of observations involving the emergence of different phenotypes across a wide variety of different bacterial strains and species and on different substrata. Strain Substrata Observations Relevant details References Single species studies Caulobacter crescentus Glass Bacteria made multiple surface contact before transitioning from reversible to irreversible adhesion. WCA < 30º; microfluidic flow conditions Hoffman et al.2015 Escherichia coli Micron-scale patterned PDMS Surface appendages enable bacteria to overcome unfavorable surface patterns Static conditions Friedlander et al.2013 E. coli PS well plates pH heterogeneity within biofilms Type of PS and WCA not reported; shaking conditions (30 rpm) Hidalgo et al.2009 E. coli Hydrophobic glass beads Cpx pathway regulates adhesion-induced gene expression Otto and Silhavy 2002 Lactobacillus plantarum Lectin monolayer and hydrophobic coatings Time-dependent binding to lectin layers; fast, time-independent binding to hydrophobic coatings Beaussart et al.2013 Mycobacteria Hydrophobic slides Biofilm viability and structure affected by antibiotic presence 30 min initial adhesion; orbital shaking (80 rpm) Muñoz-Egea et al.2015 Pseudomonas aeruginosa Glass, SS, PET, hydrophobic SS, hydrophilic PET Flagella increase adhesion on hydrophobic surfaces; straight and long flagella on PET and SS; curved and short flagella on glass WCA and surface roughness provided for all surfaces Bruzaud et al.2015 Staphylococcus aureus PE, SS Adhesion force and nisin efflux pump efficacy was highest on hydrophobic PE surfaces WCA for PE 85º and for SS 35º; static conditions Carniello et al.2018 S. aureus PE, SS, Ti–6Al–4V alloy, HA Adhesion forces, bacterial retention and viability are substratum related WCA for SS 49º, for PE 82º, for Ti–6Al–4V 69º and for HA 95º Alam and Balani 2017 S. aureus PE, SS, PMMA Matrix production and icaA gene expression is inversely related with adhesion forces WCA for SS 33º, for PMMA 69ºº and for PE 84º; submicron roughness Harapanahalli et al.2015 S. aureus Glass Cell wall deformation and long-range adhesion forces are related Chen et al.2014 S. aureus Glass Heterogeneous pattern of penetration and accumulation of Nile-red loaded micelles into biofilms Liu et al.2016 Staphylococcus epidermidis QA coatings Strong adhesion forces cause bacterial death Surfaces carry a positive charge Asri et al.2014 S. epidermidis SS, PMMA, PE Substratum-dependent EPS production and gentamicin susceptibility Nuryastuti et al.2011 Streptococcus sobrinus DDS coatings Substratum hydrophobicity determines bacterial retention, with less impact on adhesion WCA for DDS coatings 90º and glass 20º Bos et al.2000 Multiple species studies S. aureus E. coli Nanoporous or nanopillared, hydrophobized aluminum oxide Adhesion to hydrophobic, nanopillared surfaces smaller than to hydrophilic or nanoporous surfaces WCA varies from 0–162º; static and flow conditions Hizal et al.2017 S. aureus P. aeruginosa Plasma etched black silicon Smaller, more densely packed pillars exhibited the greatest bactericidal activity WCA varies from 8 to 160º; pillar heights of 212, 475–610 nm Linklater et al.2017 S. aureus S. epidermidis Nanopillared-Si wafers Nanopatterning stimulates EPS production and yields bacterial killing Regular patterning with sharply pointed pillars; flow conditions Hizal et al.2016 P. aeruginosa S. aureus Graphene nanosheets Graphene nanosheets creates pores in bacterial cell walls, causing bacterial death Roughness of the graphene sheets varies between 19 and 44 nm. Pham et al.2015 Branhamella catarrhalisBacillus subtilisE. coli P. aeruginosa Pseudomonas fluorescens Pseudomonas. maritimus S. aureus Cicada wing, nanopatterned surfaces Nanopatterning kills only Gram-negative bacteria Hasan et al.2013 Asticcacaulis biprosthecum Agrobacterium tumefaciens C. crescentus glass Reversible attachment of bacterial cells is mediated by motile cells bearing pili triggering adhesin production. Li et al.2012 S. aureus S. epidermidis P. aeruginosa SR; SR with Pluronic brush Adhesion forces dictated the transition from a planktonic to a biofilm mode of growth Flow conditions; WCA for SR 110º Muszanska et al.2012 Actinomyces naeslundii Lactobacillus acidophilus Streptococcus mitis Streptococcus mutans Streptococcus oralis Streptococcus sanguinis S. sobrinus SS, bovine enamel Salivary conditioning films reduce adhesion forces Salivary films reduced WCA of SS to 23º and of enamel to 26º; sub-micron roughness Mei et al.2009 S. aureus S. epidermidis Various substrata Staphylococcal biofilms show four distinct states, growing aerobically, growing fermentatively, dead and dormant, contributing to their tolerance to antimicrobials Different reactor systems Rani et al.2007 P. aeruginosa S. epidermidis PEO coatings PEO-brush coating reduced adhesion of all strains and species Flow conditions Roosjen et al.2004 Marinobacter hydrocarbonoclasticus Psychrobactersp. Halomonas pacifica Glass Dissolved organic carbon alters surface properties with an impact on adhesion Flow conditions; surfaces conditioned with natural seawater Bakker et al.2003 Strain Substrata Observations Relevant details References Single species studies Caulobacter crescentus Glass Bacteria made multiple surface contact before transitioning from reversible to irreversible adhesion. WCA < 30º; microfluidic flow conditions Hoffman et al.2015 Escherichia coli Micron-scale patterned PDMS Surface appendages enable bacteria to overcome unfavorable surface patterns Static conditions Friedlander et al.2013 E. coli PS well plates pH heterogeneity within biofilms Type of PS and WCA not reported; shaking conditions (30 rpm) Hidalgo et al.2009 E. coli Hydrophobic glass beads Cpx pathway regulates adhesion-induced gene expression Otto and Silhavy 2002 Lactobacillus plantarum Lectin monolayer and hydrophobic coatings Time-dependent binding to lectin layers; fast, time-independent binding to hydrophobic coatings Beaussart et al.2013 Mycobacteria Hydrophobic slides Biofilm viability and structure affected by antibiotic presence 30 min initial adhesion; orbital shaking (80 rpm) Muñoz-Egea et al.2015 Pseudomonas aeruginosa Glass, SS, PET, hydrophobic SS, hydrophilic PET Flagella increase adhesion on hydrophobic surfaces; straight and long flagella on PET and SS; curved and short flagella on glass WCA and surface roughness provided for all surfaces Bruzaud et al.2015 Staphylococcus aureus PE, SS Adhesion force and nisin efflux pump efficacy was highest on hydrophobic PE surfaces WCA for PE 85º and for SS 35º; static conditions Carniello et al.2018 S. aureus PE, SS, Ti–6Al–4V alloy, HA Adhesion forces, bacterial retention and viability are substratum related WCA for SS 49º, for PE 82º, for Ti–6Al–4V 69º and for HA 95º Alam and Balani 2017 S. aureus PE, SS, PMMA Matrix production and icaA gene expression is inversely related with adhesion forces WCA for SS 33º, for PMMA 69ºº and for PE 84º; submicron roughness Harapanahalli et al.2015 S. aureus Glass Cell wall deformation and long-range adhesion forces are related Chen et al.2014 S. aureus Glass Heterogeneous pattern of penetration and accumulation of Nile-red loaded micelles into biofilms Liu et al.2016 Staphylococcus epidermidis QA coatings Strong adhesion forces cause bacterial death Surfaces carry a positive charge Asri et al.2014 S. epidermidis SS, PMMA, PE Substratum-dependent EPS production and gentamicin susceptibility Nuryastuti et al.2011 Streptococcus sobrinus DDS coatings Substratum hydrophobicity determines bacterial retention, with less impact on adhesion WCA for DDS coatings 90º and glass 20º Bos et al.2000 Multiple species studies S. aureus E. coli Nanoporous or nanopillared, hydrophobized aluminum oxide Adhesion to hydrophobic, nanopillared surfaces smaller than to hydrophilic or nanoporous surfaces WCA varies from 0–162º; static and flow conditions Hizal et al.2017 S. aureus P. aeruginosa Plasma etched black silicon Smaller, more densely packed pillars exhibited the greatest bactericidal activity WCA varies from 8 to 160º; pillar heights of 212, 475–610 nm Linklater et al.2017 S. aureus S. epidermidis Nanopillared-Si wafers Nanopatterning stimulates EPS production and yields bacterial killing Regular patterning with sharply pointed pillars; flow conditions Hizal et al.2016 P. aeruginosa S. aureus Graphene nanosheets Graphene nanosheets creates pores in bacterial cell walls, causing bacterial death Roughness of the graphene sheets varies between 19 and 44 nm. Pham et al.2015 Branhamella catarrhalisBacillus subtilisE. coli P. aeruginosa Pseudomonas fluorescens Pseudomonas. maritimus S. aureus Cicada wing, nanopatterned surfaces Nanopatterning kills only Gram-negative bacteria Hasan et al.2013 Asticcacaulis biprosthecum Agrobacterium tumefaciens C. crescentus glass Reversible attachment of bacterial cells is mediated by motile cells bearing pili triggering adhesin production. Li et al.2012 S. aureus S. epidermidis P. aeruginosa SR; SR with Pluronic brush Adhesion forces dictated the transition from a planktonic to a biofilm mode of growth Flow conditions; WCA for SR 110º Muszanska et al.2012 Actinomyces naeslundii Lactobacillus acidophilus Streptococcus mitis Streptococcus mutans Streptococcus oralis Streptococcus sanguinis S. sobrinus SS, bovine enamel Salivary conditioning films reduce adhesion forces Salivary films reduced WCA of SS to 23º and of enamel to 26º; sub-micron roughness Mei et al.2009 S. aureus S. epidermidis Various substrata Staphylococcal biofilms show four distinct states, growing aerobically, growing fermentatively, dead and dormant, contributing to their tolerance to antimicrobials Different reactor systems Rani et al.2007 P. aeruginosa S. epidermidis PEO coatings PEO-brush coating reduced adhesion of all strains and species Flow conditions Roosjen et al.2004 Marinobacter hydrocarbonoclasticus Psychrobactersp. Halomonas pacifica Glass Dissolved organic carbon alters surface properties with an impact on adhesion Flow conditions; surfaces conditioned with natural seawater Bakker et al.2003 Relevant experimental details are included, when available in the references used. View Large Table 1. Summary of observations involving the emergence of different phenotypes across a wide variety of different bacterial strains and species and on different substrata. Strain Substrata Observations Relevant details References Single species studies Caulobacter crescentus Glass Bacteria made multiple surface contact before transitioning from reversible to irreversible adhesion. WCA < 30º; microfluidic flow conditions Hoffman et al.2015 Escherichia coli Micron-scale patterned PDMS Surface appendages enable bacteria to overcome unfavorable surface patterns Static conditions Friedlander et al.2013 E. coli PS well plates pH heterogeneity within biofilms Type of PS and WCA not reported; shaking conditions (30 rpm) Hidalgo et al.2009 E. coli Hydrophobic glass beads Cpx pathway regulates adhesion-induced gene expression Otto and Silhavy 2002 Lactobacillus plantarum Lectin monolayer and hydrophobic coatings Time-dependent binding to lectin layers; fast, time-independent binding to hydrophobic coatings Beaussart et al.2013 Mycobacteria Hydrophobic slides Biofilm viability and structure affected by antibiotic presence 30 min initial adhesion; orbital shaking (80 rpm) Muñoz-Egea et al.2015 Pseudomonas aeruginosa Glass, SS, PET, hydrophobic SS, hydrophilic PET Flagella increase adhesion on hydrophobic surfaces; straight and long flagella on PET and SS; curved and short flagella on glass WCA and surface roughness provided for all surfaces Bruzaud et al.2015 Staphylococcus aureus PE, SS Adhesion force and nisin efflux pump efficacy was highest on hydrophobic PE surfaces WCA for PE 85º and for SS 35º; static conditions Carniello et al.2018 S. aureus PE, SS, Ti–6Al–4V alloy, HA Adhesion forces, bacterial retention and viability are substratum related WCA for SS 49º, for PE 82º, for Ti–6Al–4V 69º and for HA 95º Alam and Balani 2017 S. aureus PE, SS, PMMA Matrix production and icaA gene expression is inversely related with adhesion forces WCA for SS 33º, for PMMA 69ºº and for PE 84º; submicron roughness Harapanahalli et al.2015 S. aureus Glass Cell wall deformation and long-range adhesion forces are related Chen et al.2014 S. aureus Glass Heterogeneous pattern of penetration and accumulation of Nile-red loaded micelles into biofilms Liu et al.2016 Staphylococcus epidermidis QA coatings Strong adhesion forces cause bacterial death Surfaces carry a positive charge Asri et al.2014 S. epidermidis SS, PMMA, PE Substratum-dependent EPS production and gentamicin susceptibility Nuryastuti et al.2011 Streptococcus sobrinus DDS coatings Substratum hydrophobicity determines bacterial retention, with less impact on adhesion WCA for DDS coatings 90º and glass 20º Bos et al.2000 Multiple species studies S. aureus E. coli Nanoporous or nanopillared, hydrophobized aluminum oxide Adhesion to hydrophobic, nanopillared surfaces smaller than to hydrophilic or nanoporous surfaces WCA varies from 0–162º; static and flow conditions Hizal et al.2017 S. aureus P. aeruginosa Plasma etched black silicon Smaller, more densely packed pillars exhibited the greatest bactericidal activity WCA varies from 8 to 160º; pillar heights of 212, 475–610 nm Linklater et al.2017 S. aureus S. epidermidis Nanopillared-Si wafers Nanopatterning stimulates EPS production and yields bacterial killing Regular patterning with sharply pointed pillars; flow conditions Hizal et al.2016 P. aeruginosa S. aureus Graphene nanosheets Graphene nanosheets creates pores in bacterial cell walls, causing bacterial death Roughness of the graphene sheets varies between 19 and 44 nm. Pham et al.2015 Branhamella catarrhalisBacillus subtilisE. coli P. aeruginosa Pseudomonas fluorescens Pseudomonas. maritimus S. aureus Cicada wing, nanopatterned surfaces Nanopatterning kills only Gram-negative bacteria Hasan et al.2013 Asticcacaulis biprosthecum Agrobacterium tumefaciens C. crescentus glass Reversible attachment of bacterial cells is mediated by motile cells bearing pili triggering adhesin production. Li et al.2012 S. aureus S. epidermidis P. aeruginosa SR; SR with Pluronic brush Adhesion forces dictated the transition from a planktonic to a biofilm mode of growth Flow conditions; WCA for SR 110º Muszanska et al.2012 Actinomyces naeslundii Lactobacillus acidophilus Streptococcus mitis Streptococcus mutans Streptococcus oralis Streptococcus sanguinis S. sobrinus SS, bovine enamel Salivary conditioning films reduce adhesion forces Salivary films reduced WCA of SS to 23º and of enamel to 26º; sub-micron roughness Mei et al.2009 S. aureus S. epidermidis Various substrata Staphylococcal biofilms show four distinct states, growing aerobically, growing fermentatively, dead and dormant, contributing to their tolerance to antimicrobials Different reactor systems Rani et al.2007 P. aeruginosa S. epidermidis PEO coatings PEO-brush coating reduced adhesion of all strains and species Flow conditions Roosjen et al.2004 Marinobacter hydrocarbonoclasticus Psychrobactersp. Halomonas pacifica Glass Dissolved organic carbon alters surface properties with an impact on adhesion Flow conditions; surfaces conditioned with natural seawater Bakker et al.2003 Strain Substrata Observations Relevant details References Single species studies Caulobacter crescentus Glass Bacteria made multiple surface contact before transitioning from reversible to irreversible adhesion. WCA < 30º; microfluidic flow conditions Hoffman et al.2015 Escherichia coli Micron-scale patterned PDMS Surface appendages enable bacteria to overcome unfavorable surface patterns Static conditions Friedlander et al.2013 E. coli PS well plates pH heterogeneity within biofilms Type of PS and WCA not reported; shaking conditions (30 rpm) Hidalgo et al.2009 E. coli Hydrophobic glass beads Cpx pathway regulates adhesion-induced gene expression Otto and Silhavy 2002 Lactobacillus plantarum Lectin monolayer and hydrophobic coatings Time-dependent binding to lectin layers; fast, time-independent binding to hydrophobic coatings Beaussart et al.2013 Mycobacteria Hydrophobic slides Biofilm viability and structure affected by antibiotic presence 30 min initial adhesion; orbital shaking (80 rpm) Muñoz-Egea et al.2015 Pseudomonas aeruginosa Glass, SS, PET, hydrophobic SS, hydrophilic PET Flagella increase adhesion on hydrophobic surfaces; straight and long flagella on PET and SS; curved and short flagella on glass WCA and surface roughness provided for all surfaces Bruzaud et al.2015 Staphylococcus aureus PE, SS Adhesion force and nisin efflux pump efficacy was highest on hydrophobic PE surfaces WCA for PE 85º and for SS 35º; static conditions Carniello et al.2018 S. aureus PE, SS, Ti–6Al–4V alloy, HA Adhesion forces, bacterial retention and viability are substratum related WCA for SS 49º, for PE 82º, for Ti–6Al–4V 69º and for HA 95º Alam and Balani 2017 S. aureus PE, SS, PMMA Matrix production and icaA gene expression is inversely related with adhesion forces WCA for SS 33º, for PMMA 69ºº and for PE 84º; submicron roughness Harapanahalli et al.2015 S. aureus Glass Cell wall deformation and long-range adhesion forces are related Chen et al.2014 S. aureus Glass Heterogeneous pattern of penetration and accumulation of Nile-red loaded micelles into biofilms Liu et al.2016 Staphylococcus epidermidis QA coatings Strong adhesion forces cause bacterial death Surfaces carry a positive charge Asri et al.2014 S. epidermidis SS, PMMA, PE Substratum-dependent EPS production and gentamicin susceptibility Nuryastuti et al.2011 Streptococcus sobrinus DDS coatings Substratum hydrophobicity determines bacterial retention, with less impact on adhesion WCA for DDS coatings 90º and glass 20º Bos et al.2000 Multiple species studies S. aureus E. coli Nanoporous or nanopillared, hydrophobized aluminum oxide Adhesion to hydrophobic, nanopillared surfaces smaller than to hydrophilic or nanoporous surfaces WCA varies from 0–162º; static and flow conditions Hizal et al.2017 S. aureus P. aeruginosa Plasma etched black silicon Smaller, more densely packed pillars exhibited the greatest bactericidal activity WCA varies from 8 to 160º; pillar heights of 212, 475–610 nm Linklater et al.2017 S. aureus S. epidermidis Nanopillared-Si wafers Nanopatterning stimulates EPS production and yields bacterial killing Regular patterning with sharply pointed pillars; flow conditions Hizal et al.2016 P. aeruginosa S. aureus Graphene nanosheets Graphene nanosheets creates pores in bacterial cell walls, causing bacterial death Roughness of the graphene sheets varies between 19 and 44 nm. Pham et al.2015 Branhamella catarrhalisBacillus subtilisE. coli P. aeruginosa Pseudomonas fluorescens Pseudomonas. maritimus S. aureus Cicada wing, nanopatterned surfaces Nanopatterning kills only Gram-negative bacteria Hasan et al.2013 Asticcacaulis biprosthecum Agrobacterium tumefaciens C. crescentus glass Reversible attachment of bacterial cells is mediated by motile cells bearing pili triggering adhesin production. Li et al.2012 S. aureus S. epidermidis P. aeruginosa SR; SR with Pluronic brush Adhesion forces dictated the transition from a planktonic to a biofilm mode of growth Flow conditions; WCA for SR 110º Muszanska et al.2012 Actinomyces naeslundii Lactobacillus acidophilus Streptococcus mitis Streptococcus mutans Streptococcus oralis Streptococcus sanguinis S. sobrinus SS, bovine enamel Salivary conditioning films reduce adhesion forces Salivary films reduced WCA of SS to 23º and of enamel to 26º; sub-micron roughness Mei et al.2009 S. aureus S. epidermidis Various substrata Staphylococcal biofilms show four distinct states, growing aerobically, growing fermentatively, dead and dormant, contributing to their tolerance to antimicrobials Different reactor systems Rani et al.2007 P. aeruginosa S. epidermidis PEO coatings PEO-brush coating reduced adhesion of all strains and species Flow conditions Roosjen et al.2004 Marinobacter hydrocarbonoclasticus Psychrobactersp. Halomonas pacifica Glass Dissolved organic carbon alters surface properties with an impact on adhesion Flow conditions; surfaces conditioned with natural seawater Bakker et al.2003 Relevant experimental details are included, when available in the references used. View Large Phenotypic drug tolerance and resistance Phenotypic heterogeneity with respect to drug tolerance and resistance has been observed frequently in bacterial bulk cultures. Correct mechanistic distinction between tolerance and resistance is difficult (see Text Box 4). Phenotypic resistance is thought to be mainly due to environmentally triggered changes in bacterial cell wall permeability impeding drug access, activation of efflux pumps and release of drug-deactivating enzymes (Kester and Fortune 2014). Examples of environmentally triggered events are the reversible change in porin expression levels in enteric bacteria in response to high osmolarity or temperature (Dupont et al.2007) or the reduced antibiotic sensitivity of Enterobacter aerogenes which results from reduced porin expression under antibiotic pressure (Bornet et al.2000). Phenotypic tolerance, on the other hand, involves an environmental trigger of bacterial dormancy, persistence, differentiation and biofilm formation, including EPS production (Kester and Fortune 2014; Kaldalu, Hauryliuk and Tenson 2016). Although the mechanisms of phenotypic heterogeneity with respect to tolerance and resistance likely unite in a biofilm, the role of the substratum surface and its specific properties as an environmental trigger for the development of biofilm heterogeneity has not been considered (Olsen 2015; Brauner et al.2016). Text Box 4. Resistance and tolerance Antibiotic resistance generally means an increase in the minimum inhibitory concentration of an antibacterial agent due to a permanent change in the bacterium, e.g. by mutation or through horizontal gene transfer. Antibiotic tolerance is the ability of bacteria to survive the effect of an antibiotic due to a reversible phenotypic state. Two main forms of tolerance have been identified: ‘tolerance by slow growth’ (occurs at steady state) and ‘tolerance by lag’ (a transient state that is induced by starvation or stress) (Olsen 2015; Brauner et al.2016). Staphylococcus epidermidis and S. aureus biofilms grown on polycarbonate filters on agar possessed at least four distinct phenotypes: bacteria growing either aerobically or fermentatively, dead or dormant (Rani et al.2007). Multiple strains of S. epidermidis containing the ica locus, which encodes for PIA, were found to produce biofilms on hydrophobic polyethylene (PE) surfaces (water contact angle [WCA] of 84º) which contained large patches of EPS. Alternatively, on more hydrophilic acrylic and stainless steel surfaces (WCA of 69º and 33º, respectively), heterogeneously occurring EPS production was less and concurrently, ica-gene expression was low in these biofilms as compared with biofilms on PE (Nuryastuti et al.2011). Similarly, EPS production in biofilms of S. aureus and S. epidermidis on hydrophobic silicone rubber (SR) surfaces (WCA of 110 degrees) was massive and yielded resistance to gentamicin, whereas on hydrophilic polyethylene glycol (PEG), polymer-brush-coated SR (WCA of around 40º), EPS production was absent and bacteria remained susceptible to gentamicin. To a lesser extent, such differences were also observed in biofilms of the Gram-negative bacterium, P. aeruginosa (Roosjen et al.2004; Muszanska et al.2012). Expression of the membrane located sensor, NsaS and the NsaA two-component efflux pump in S. aureus SH1000, responsible for nisin resistance in the planktonic state, was enhanced when the organism was adhering to a substratum surface. Moreover, adhesion to a hydrophobic PE surface triggered a greater expression of nsaS and nsaA than adhesion to a more hydrophilic stainless steel surface (Carniello et al.2018). Despite the influence that the specific properties of the substratum surface have on emergent biofilm properties, most experiments are reported in the literature without reference to the substratum material. In many cases, biofilm assays are performed in multiwell polystyrene (PS) plates and the type of PS is not specified even though this will affect surface properties: for example, bacterial-grade PS is more hydrophobic in the absence of surface treatment (WCA 78º) than tissue culture-grade PS after physical treatment (WCA 43º), and these differences may severely impact on bacterial adaptive behavior. Moreover, often conclusions on surface adaptation are extrapolated from results obtained in biofilms grown on aqueous agar, which may not accurately reflect the conditions encountered on solid substratum surfaces. Collectively, these examples demonstrate that the substratum surface, most notably its hydrophobicity or hydrophilicity (see Text Box 5), provides an environmental trigger for the development of antibiotic resistance and tolerance in biofilms. Importantly, in most of these examples, a uniform response of the entire biofilm has been inferred without evidence that the biofilm is homogeneous over its entire volume. However, where available, closer inspection of micrographs in the published literature (see Fig. 1 for specific examples) clearly shows stochastically occurring non-conformists, providing clear evidence of heterogeneity. Text Box 5. Surface hydrophobicity ‘Surface hydrophobicity’ and its opposite ‘surface hydrophilicity’ literally indicate the ‘fear’ or ‘love’ of a surface for water. Surface hydrophobicity can be quantitated by placing a small water droplet on a surface and measuring its degree of spreading, full spreading being characterized by a 0º WCA (hydrophilic surface). On super-hydrophobic materials, such as nanostructured hydrophobic surfaces, air can become entrapped and water has an almost 180º WCA (Hizal et al.2017), making it behave like a mercury droplet. Swarming behavior Swarming is another drug-resistance mechanism allowing bacteria to explore and subsequently escape an antibiotic-laden or otherwise hostile environment (Lai, Tremblay and Déziel 2009), and also enables bacteria to actively search for nutrients (Daniels, Vanderleyden and Michiels 2004). Swarming phenotypes are often characterized by being hyperflagellated, elongated, multinucleate (Toguchi et al.2000) and antibiotic-resistant. In Paenibacillus vortex biofilms, antibiotic-refractory, swarming phenotypes function to explore the environment for antibiotic-laden regions that should be avoided by the ‘builders’ of the biofilm community (Roth et al.2013). Swarming bacteria either reside in (i) bulk suspension, where they are unlikely to experience any effects from a substratum surface, (ii) surface-constrained, near the surface but still in suspension and experiencing hydrodynamic shear or (iii) in direct interaction with the substratum surface (Tuson and Weibel 2013). Swarming in the surface-constrained regime requires reversible adhesion on the one hand, but in order to prevent detachment back into the bulk suspension, bacteria must have a means to rapidly transit between reversible and irreversible adhesion. Indeed, studies on single cells of C. crescentus demonstrated that transitioning from reversible to irreversible adhesion is not a single event and most cells reversibly contact a surface multiple times before a final transition to irreversible adhesion takes place, with pili playing an important role in this transition (Hoffman et al.2015). Bacteria can sense the presence of a surface by obstruction of surface appendages such as flagella, pili or fimbriae (Friedlander et al.2013; Ellison and Brun 2015) and subsequent activation of membrane-located sensors (Belas 2014). In C. crescentus, arrest of flagellum rotation and concurrent stimulation of ‘just-in-time’ polysaccharide adhesive occurs to maximize adhesion and prevent untimely detachment back into suspension (Li et al.2012). The presence of P. aeruginosa flagella and type IV pili increased bacterial adhesion to highly hydrophobic substratum surfaces (Bruzaud et al.2015), suggesting a role for substratum surface properties on development of bacterial swarming phenotypes. HOW BACTERIA DIFFERENTIATE BETWEEN DIFFERENT SUBSTRATUM SURFACES Adhesion forces between bacteria and substratum surfaces The observation that bacteria adapt differently to adhesion on different substratum surfaces immediately raises the question of how bacteria sense that they are on a surface, and more importantly, how they tailor their adaptive response to the characteristic properties of the surface they adhere to. Adhesion, whether arising from specific, molecular ligand-receptor or non-specific interactions (Bos, Van der Mei and Busscher 1999), is an interplay between ever present attractive Lifshitz-Van der Waals forces, attractive or repulsive acid–base interactions as a generalized form of hydrogen bonding, electrostatic forces with a magnitude depending on pH and ionic strength of the fluid environment and Brownian motion forces. The attractive Lifshitz-Van der Waals forces are the most long-ranged ones, acting over distances of up to 1 μm and becoming increasingly stronger when the interacting surfaces become closer. The sum total of these different forces determine the force by which a bacterium adheres to a substratum surface and this varies on different surfaces (Alam and Balani 2017), while at close approach Lifshitz-Van der Waals forces are usually able to overcome electrostatic barriers (Puddu and Perry 2012; Paula et al.2014). Distinguishing three adhesion force regimes (Busscher and Van der Mei 2012), it was proposed that extremely weakly adhering bacteria (adhesion forces less than 1 nN) do not realize they are in an adhering state and therefore do not show any adaptive response to a substratum surface. Alternatively, when adhering very strongly (proposed adhesion forces above 10 nN) as on quaternary ammonium (QA)-coated surfaces (Muszanska et al.2012), cell wall damage is inferred resulting in bacterial cell death (Tiller et al.2001; Asri et al.2014). The intermediate regime comprising adhesion forces between 1 and 10–15 nN, as occurs on most common substratum surfaces across a wide variety of bacterial strains and species (Van der Mei et al.2008; Beaussart et al.2013; Sullan et al.2014; Thewes et al.2015), invokes bacterial adaptation with production of EPS according to the magnitude of the adhesion forces experienced (Harapanahalli et al.2015). The ability to measure bacterial adhesion forces using the AFM (see Text Box 6) creates an awareness of the enormous magnitude of bacterial adhesion forces as compared with the gravitational forces they experience (see Text Box 7). Thus, it is not surprising that a lethal regime exists in which bacteria die due to cell wall damage as result of experiencing adhesion forces that are 106–108-fold higher than the gravitational force they experience. It has been argued that bacterial cell walls are rigid to resist large internal pressures, but remarkably plastic in order to adapt to a wide range of external forces (Amir et al.2014), including adhesion forces. In fact, it has been demonstrated using AFM (Chen et al.2014) and surface enhanced fluorescence (see Text Box 8) that the bacterial cell wall deforms under the influence of the relatively large adhesion forces arising from a substratum surface (Fig. 2), despite the rigidity provided to bacteria by their peptidoglycan layer. Also, AFM imaging of S. epidermidis trapped in a filter has shown structural and mechanical deformation of the cell wall (Méndez-Vilas, Gallardo-Moreno and González-Martín 2007). Figure 2. View largeDownload slide Bacterial cell wall deformation under the influence of adhesion forces arising from a substratum surface (Chen et al.2014, reprinted with permission from American Society for Microbiology). An undeformed bacterium with a radius R approaching a substratum surface comes under the influence of the adhesion forces arising from the substratum. It gradually deforms, which brings more molecules (solid red region) under the influence of the adhesion forces, stimulating further adhesion until opposing forces arising from the rigid bacterial cell wall and increased intracellular pressure fully counteract the adhesion force. Figure 2. View largeDownload slide Bacterial cell wall deformation under the influence of adhesion forces arising from a substratum surface (Chen et al.2014, reprinted with permission from American Society for Microbiology). An undeformed bacterium with a radius R approaching a substratum surface comes under the influence of the adhesion forces arising from the substratum. It gradually deforms, which brings more molecules (solid red region) under the influence of the adhesion forces, stimulating further adhesion until opposing forces arising from the rigid bacterial cell wall and increased intracellular pressure fully counteract the adhesion force. Text Box 6. Bacterial adhesion force measurement Bacterial adhesion can be measured using atomic force microscopy (AFM). In bacterial probe AFM, a bacterium is attached to a highly flexible cantilever and brought into contact with a substratum surface, allowing contact between the bacterium and the surface for a defined time period and applied loading force. Upon retraction of the cantilever from the surface, the force required to break the bond between the bacterium and the substratum surface is recorded from the bending of the flexible cantilever. In this way, bacterial adhesion forces to biological and non-biological surfaces in the picoNewton (pN) to nanoNewton (nN) range have been measured (Dufrêne 2015). Text Box 7. On the magnitude of bacterial adhesion forces to surfaces Most forces by which bacteria adhere to surfaces are reportedly in the nN-range (Van der Mei et al.2008; Beaussart et al.2013; Sullan et al.2014; Thewes et al.2015), which is large compared to the gravity force experienced by bacteria. In air, the gravity force experienced by a bacterium is around 10−6 nN, while due to buoyancy, this force reduces in an aqueous suspension to around 10−8 nN. Assuming an adhesion force of around 1 nN, this implies that the forces by which bacteria adhere to a substratum surface are 106–108-fold higher than the gravity forces they experience. Text Box 8. Surface-enhanced bacterial fluorescence Surface-enhanced fluorescence is the phenomenon in which fluorophores within 20–30 nm from a metal surface show a stronger fluorescence intensity than expected for the same fluorophore in solution (Lee et al.2011). Surface-enhanced bacterial fluorescence of fluorescent bacteria adhering to metallic surfaces can be exploited to demonstrate bacterial cell wall deformation, because more of the fluorescent, intracellular content of a bacterium is brought into the close vicinity of the surface upon adhesion and subsequent cell wall deformation, and therewith subject to surface-enhanced fluorescence (Li et al.2014). Cell wall deformation and surface adaptation The role of cell wall deformation in triggering bacterial responses is difficult to demonstrate experimentally, as bacterial cell wall deformation is small due to the rigidity provided by the bacterial peptidoglycan layer surrounding the membrane. In mammalian cells, however, lacking a rigid cell wall, the influence of substratum hydrophobicity is more obvious and many different types of tissue cells remained ‘cauliflower’ shaped on hydrophobic substratum surfaces while deforming to a ‘pancake’ shape on hydrophilic ones (Schakenraad et al.1986). Also in mammalian cells, sensors located in the cell membrane have been described, which control the subsequent differentiation of stem cells in a substratum-dependent fashion (Engler et al.2006). Deliberate compression of bacteria between AFM cantilevers and substratum surfaces has demonstrated that the bacterial cell wall deforms in a viscoelastic way (Vadillo-Rodriguez, Beveridge and Dutcher 2008; Vadillo-Rodriguez and Dutcher 2009), although it should be noted that deformation under such conditions is not exactly the same as ‘spontaneous’ deformation under the influence of adhesion forces arising from a substratum surface. Escherichia coli and Bacillus subtilis behaved like elastic rods when subjected to external forces, but deformed permanently in the plastic regime of viscoelastic deformation when cell wall synthesis occurred while the force was applied (Amir et al.2014). Moreover, the offspring of plastically deformed bacteria always recovered their shape, but this required conditions allowing cell wall synthesis (Sliusarenko et al.2010; Amir et al.2014) over several generations (Si et al.2015). Bacterial cell wall deformation changes the pressure profile across the lipid membrane (Perozo et al.2002), which is laden with environmental sensors that can become activated by such changes (Kocer 2015) through gating of mechanosensitive channels (Haswell, Phillips and Rees 2011) or directly by conformational changes in membrane-located receptors (Otto and Silhavy 2002). Thus, adhesion-force sensing and subsequent cell wall deformation provide an important mechanism for adhering bacteria to realize they are on a surface and begin the process of surface adaptation. The role of rigid bacterial peptidoglycan layers in adhesion force-sensing and subsequent cell wall deformation is probably large, since an S. aureus Δpbp4 mutant, which lacks peptidoglycan cross-linking, seemed unable to adapt its response in line with the adhesion forces arising from a substratum surface (Harapanahalli et al.2015). HETEROGENEOUS SURFACES AND BACTERIAL INTERACTIONS Surface heterogeneity due to protein adsorption All naturally occurring and synthetic surfaces are heterogeneous, either on a micro- or nanoscopic scale and will exert different local adhesion forces on adhering bacteria to trigger different adaptive responses. Dental enamel is an excellent example of a naturally occurring heterogeneous surface with distinct crystalline hydroxyapatite (HA) structures comprised in an organic matrix, which in the oral cavity become covered within seconds with a conditioning film of adsorbed salivary proteins forming a network structure over the enamel surface (Busscher et al.1989; Simmons et al.2011). Although the network structure of adsorbed proteins is a heterogeneous surface structure in itself, saliva contains many different proteins (Marsh et al.2016) that adsorb and displace each other in succession, which further contributes to surface heterogeneity. In the oral cavity, formation and composition of salivary conditioning films varies on different surfaces (Aroonsang et al.2014) and precedes adhesion of bacteria and subsequently influences bacterial adhesion forces and biofilm detachment (Song, Koo and Ren 2015). A similar succession of protein adsorption and desorption occurs on cellular and synthetic graft surfaces exposed to blood (Vroman 2008). Note that, in the marine and other aqueous environments, conditioning films are often described as adsorbed films composed of dissolved organic carbon (Bakker et al.2003). Since bacteria diffuse more slowly than proteins, bacteria mostly adhere to such heterogeneous, adsorbed conditioning films, regardless of whether in the oral cavity or in any other environment. Surface charge heterogeneity Strong electrostatic attraction between positively charged QA-coated surfaces and negatively charged bacterial cell surfaces is reported to cause cell wall damage and subsequent cell death (Asri et al.2014). Charge heterogeneity on glass surfaces, often thought to be homogeneous, became evident by repetitively allowing negatively charged, 1-μm diameter PS particles to adhere to the same glass surface. Under low ionic strength conditions, particles always adhered first to the same, previously occupied microscopic location through strong, local electrostatic attraction (Wit and Busscher 1998), demonstrating the existence of positively charged heterogeneities on an overall negatively charged glass surface. Heterogeneity in surface hydrophobicity and roughness Heterogeneity in surface hydrophobicity and roughness at the sub-micrometer scale are easily demonstrable by the measurement of WCA hysteresis on material surfaces (see Text Box 9). Large differences between advancing and receding contact angles on ‘smooth’ surfaces with a roughness less than 0.1 μm indicate regions with a large difference in surface hydrophobicity. Roughened, hydrophobic surfaces may appear as ‘superhydrophobic’, while roughened, hydrophilic surfaces possess smaller WCAs than expected based on the hydrophobicity, respectively the hydrophilicity of their smooth counterparts. Text Box 9. Contact angle hysteresis When a water droplet advances over a perfectly smooth surface, it can be stopped by a small, more hydrophobic heterogeneity or rugosity, which causes the contact angle to be higher than when the droplet is in an equilibrium state. Equally so, when receding over an already wetted surface, water tends to remain behind on a hydrophilic heterogeneity and the contact angle appears smaller than in an equilibrium state. The difference in advancing and receding contact angles is called ‘contact angle hysteresis’ (Timmons and Zisman 1966). Only perfectly smooth and chemically homogeneous surfaces have a 0º contact angle hysteresis, which makes the measurement of contact angle hysteresis suitable for the measurement of surface heterogeneity in general at a sub-micrometer scale. Bacteria themselves are in fact also ideal to demonstrate heterogeneity in substratum surface hydrophobicity due to differential interaction with hydrophobic and hydrophilic regions on a substratum surface. Micro-patterned substratum surfaces consisting of hydrophobic lines separated by wide hydrophilic spacings, for instance, attracted equal numbers of streptococci over its entire surface, but when challenged with a detachment force, streptococci were retained only on the hydrophobic lines (Bos et al.2000), suggesting that the strength of bacterial adhesion is higher to hydrophobic regions. Adhesion force measurement using AFM on a patterned substratum consisting of square arrays of non-adhesive PEG hydrogels comparable in size to a bacterial cell on a hydrophobic, silanized glass surface showed that S. aureus adhesion was decreased at the hydrogel spacings as these presumably impeded contact between the bacterial cell and the hydrophobic surface (Wang et al.2011). Nanoscopically heterogeneous substratum surfaces Nanotechnological advances have enabled the production of nanoscopically heterogeneous surfaces, which are often bioinspired (Tripathy et al.2017) most notably by the so called ‘lotus effect’ (Huang et al.2016). Such plant leaves, and also certain insect wings, remain free of bacteria through self-cleaning and antibacterial properties, thought to be mediated by nanopillared arrays (Hasan et al.2013) that inherently represent a nanoscopically heterogeneous substratum surface. Electron micrographs have clearly demonstrated that the bacterial cell wall can locally severely deform under the influence of the adhesion forces arising from extruding random (Svensson et al.2014) and periodic (Hizal et al.2016) nanostructures to yield pressure-induced EPS production and even bacterial cell death in Gram-positive staphylococci. This is supported by observations that killing of P. aeruginosa and S. aureus on graphene nanosheets related with density of the edges of the graphene (Pham et al.2015). Approximately 98% of P. aeruginosa cells and 97% of S. aureus cells were killed on superhydrophilic and superhydrophobic black silicon surfaces with well-defined surface geometries and wettability, smaller, more densely packed pillars exhibiting the greatest bactericidal activity (Linklater et al.2017). It is speculated that the bactericidal activity is due to irreversible membrane bulging. In antibiotic-challenged E. coli, pores in the peptidoglycan network with a critical radius of around 20 nm, the typical distance between neighboring peptides and glycan strands, are required to cause bulging of the cytoplasmic membrane out through the pore. This bulging is irreversible and leading to loss of cell viability (Daly et al.2011). SUBSTRATUM SURFACE HETEROGENEITIES INDUCED BY ADHERING BACTERIA During adhesion, bacteria can create heterogeneities as a means of communication (Fig. 3) to allow localized positive or negative cooperation in colonizing a substratum surface, that is, stimulate or discourage adhesion of other bacteria in their immediate surroundings (Sjollema et al.1990). In a broader sense, bacteria have been suggested to leave ‘footprints’ when adhering to and detaching from a substratum surface (Neu 1992) that will contribute to substratum surface heterogeneity. Figure 3. View largeDownload slide Bacterially induced substratum surface heterogeneities as a means of communication and interaction between initially adhering bacteria. (A) Certain strains of bacteria excrete biosurfactants that spread over the substratum surface, modifying the immediate surrounding surface so that it is less favorable (red colored) for adherence by other bacteria. (B) Positive cooperativity is the mechanism by which an adhering bacterium changes the conformation of adsorbed proteins in its immediate surroundings or produces adhesive EPS, generating a more favorable surface (green colored) for adherence by other bacteria. Figure 3. View largeDownload slide Bacterially induced substratum surface heterogeneities as a means of communication and interaction between initially adhering bacteria. (A) Certain strains of bacteria excrete biosurfactants that spread over the substratum surface, modifying the immediate surrounding surface so that it is less favorable (red colored) for adherence by other bacteria. (B) Positive cooperativity is the mechanism by which an adhering bacterium changes the conformation of adsorbed proteins in its immediate surroundings or produces adhesive EPS, generating a more favorable surface (green colored) for adherence by other bacteria. Localized cooperative phenomena and biosurfactant release Biosurfactants (see Text Box 10), by their amphiphilic nature, are ideal molecules to be transported over large distances to reach remote areas of a substratum surface as a means to interact with other initial colonizers (Fig. 3A). Streptococcus mitis strains excrete biosurfactants that modify their immediate surroundings to make it less attractive for their competitors to adhere (Van Hoogmoed et al.2000; Loozen et al.2014) and the spreading of oral biosurfactants excreted by initial colonizers such as S. mitis over dental enamel surfaces reduced the adhesion forces of other colonizers (Van Hoogmoed et al.2006). Lactobacilli also claim substratum surface area by excretion of biosurfactants that discourage adhesion of enterococci and other uropathogens (Velraeds et al.1996). Text Box 10. Biosurfactants Biosurfactants are amphiphilic compounds produced by living organisms, mostly microorganisms, and excreted extracellularly, which contain hydrophobic and hydrophilic moieties, accumulating at an interface and reducing interfacial tensions versus air, a liquid surrounding or another material (Cochis et al.2012; Sambanthamoorthy et al.2014). Quorum-sensing controlled expression of phenol-soluble modulin surfactants in S. aureus (Periasami et al.2012) and rhamnolipids in P. aeruginosa (Davey, Caiazza and O’Toole 2003) biofilms has been shown to mediate biofilm structuring and detachment. For P. aeruginosa, siderophores, eDNA and biosurfactants play multiple roles in the interaction between different sub-populations in a biofilm and influence its structural development, as related to biosurfactants concentration and composition (Pamp and Tolker-Nielsen 2007). Bacterially induced changes in adsorbed protein conformation and positive cooperativity Bacteria also have other means to modify their immediate surroundings on a substratum surface to exert positive cooperativity (Nesbitt et al.1982; Van der Mei et al.1993): several initial colonizers of protein-conditioned surfaces have the ability to induce conformational changes in the adsorbed protein film that surrounds them (Fig. 3B), making the film more attractive for their peers to adhere. Initial colonizers of oral surfaces in vivo have slightly stronger adhesion forces with salivary conditioning films than later colonizers (Mei et al.2009), which may be underlying their ability to induce conformational changes in the adsorbed proteins to which they adhere. Since clinically, the relative prevalence of initially colonizing strains on a surface depends on the forces by which specific bacterial strains are attracted to their substratum surface (Wessel et al.2014), local induced changes in the conformation of adsorbed proteins may yield biofilm regions with a different bacterial composition. Cooperativity through EPS production EPS production can be considered as another cooperative phenomenon offering advantages in adhesion to neighboring bacteria by creating local surface heterogeneity around an adhering organism (see also Fig. 3B) (Nadell and Bassler 2011) but, like for positive cooperativity in general, at the obvious expense of impairing dispersal of adhering bacteria to new locations. Psl, for instance, is a cell wall anchored polysaccharide in P. aeruginosa (Ma et al.2009), promoting aggregate formation between neighboring bacteria in microenvironments of a biofilm, which does not occur and subsequently yields less biofilm in strains lacking Psl (Wang et al.2013). Mixed species oral biofilms on saliva-coated surfaces possess acidic niches in their EPS matrix that selectively stimulate the localized growth of pathogenic Strep. mutans (Xiao et al.2012; Koo and Yamada 2016). THE COMMANDING ROLE OF INITIAL COLONIZERS IN BIOFILM FORMATION Bacterial responses to prevailing environmental conditions is virtually always a survival strategy to maintain their adhering state in competition with others or under mechanical attack, while the production of EPS as an adaptive response embeds adhering bacteria in a matrix that also offers protection against chemical attacks (de la Fuente-Núñez et al.2013; Carniello et al.2016). Initially, adhering bacteria have various ways to influence the development of microenvironments in the biofilm that grows on top of them, in which adhesion force-sensing plays a crucial role. Adhesion force sensing and biofilm composition In the sequence of events that lead to a full-grown biofilm with heterogeneously occurring microenvironments, the initially adhering bacteria firstly have various ways to induce local heterogeneities on a substratum surface to which they adhere. Newcomers can recognize these heterogeneities by the strength of the local adhesion forces they experience and interpret them as signs to ‘stay away’ or ‘welcome, adhere here’. This, in turn, will create microenvironments in a biofilm with different microbial composition. Therewith, the basis of cooperation, and possible conflicts, in a mature biofilm (Xavier and Foster 2007) is commanded by the initially adhering bacteria. Adhesion force sensing and EPS production Emergent EPS production follows initial adhesion in the sequence of events leading to a mature biofilm, and is arguably one of the most important adaptive responses within a biofilm. Adhesion force sensing constitutes an environmental trigger for EPS production. The production of the matrix molecule, poly-N-acetylglucosamine, and the secretion of eDNA decrease with increasing adhesion force, suggesting that adhering staphylococci adjust their adaptive response to environmental need (Harapanahalli et al.2015) to prevent unnecessary costs to their fitness (Brooks and Jefferson 2014). Similarly, EPS production by bacteria adhering under fluid shear conditions is more extensive than under stagnant conditions, suggesting that its expression is induced only when required (Nivens et al.1993; Hou et al.2017). Since the effective range of adhesion forces is limited to maximally 1 μm, it is impossible for bacteria other than the initial colonizers to directly sense a substratum, while their immediate neighbors reside at distances between 1–3 μm and are embedded in an EPS matrix (Drescher et al.2016). Accordingly, only initially adhering bacteria are able to sense and adapt to the adhesion forces exerted by a substratum surface and, in fact, the majority of bacteria in a biofilm have never contacted the substratum surface (Zhao et al.2013). Since the same will be true for the bacteria in emergent heterogeneous microenvironments, this leads to the conclusion that initially adhering bacteria command the development of emerging heterogeneous microenvironments by sensing and adapting to the substratum and communicating with neighboring bacteria information about that surface (see Fig. 4). Stochastically occurring environmental triggers have been suggested before as being causative to phenotypic heterogeneity (Vega and Gore 2014), but have never been associated with triggers derived from stochastically occurring substratum surface heterogeneity. Figure 4. View largeDownload slide The commanding role in adaptive responses of initial colonizers in a biofilm. Initially, adhering bacteria sense different local adhesion forces which trigger different adaptive responses that spread through the biofilm by diffusion of quorum-sensing molecules until their concentration is below a detectable threshold and the commands given are lost, limiting heterogeneous microenvironment development in space and time. Microenvironments, including the adhesion forces that trigger differential responses, the commanding organisms and obeying inhabitants of the microenvironment, are indicated by different colors. Figure 4. View largeDownload slide The commanding role in adaptive responses of initial colonizers in a biofilm. Initially, adhering bacteria sense different local adhesion forces which trigger different adaptive responses that spread through the biofilm by diffusion of quorum-sensing molecules until their concentration is below a detectable threshold and the commands given are lost, limiting heterogeneous microenvironment development in space and time. Microenvironments, including the adhesion forces that trigger differential responses, the commanding organisms and obeying inhabitants of the microenvironment, are indicated by different colors. The surface adaptation (Text Box 11) of initial colonizers in response to direct contact with a substratum surface likely do not disappear with the first generation of later colonizers, not in direct contact with the surface, but will most probably disappear only after a number of generations (Si et al.2015), and the progeny returns to a more planktonic phenotype. Return to a planktonic phenotype does not necessarily imply bacterial return back into suspension, but may also occur in a biofilm, where bacteria are ‘suspended’ or ‘free floating’ in an EPS matrix at average distances of 1–3 μm from neighboring organisms (Drescher et al.2016), i.e. more specifically formulated, outside the influence of adhesion forces exerted by their neighbors. Text Box 11. Surface adaptation Bacterial surface adaptation comprises the particular response of a bacterium to the surface properties of the substratum to which it adheres. Adhesion force sensing and quorum sensing Identifying initial colonizers that are in direct contact with a substratum surface as ‘commanding’ bacteria implies that there must be a communication means available within a biofilm to pass information derived from adhesion force sensing to bacteria that are not in direct contact with the substratum enabling them to indirectly sense the surface. The initially adhering bacteria likely pass substratum information by producing and releasing auto-inducing molecules to which later biofilms colonizers respond. Since the distance over which auto-transducers can be transported and remain detectable is limited by diffusion (Vega and Gore 2014), quorum sensing is eventually quenched which restricts the adaptive response to microenvironments in a biofilm, although ‘calling distances’ between Gram-negative bacteria extending up to 78 μm have been reported (Elias and Banin 2012). However, most effective calling distances for producing and releasing, sensing and responding to auto-transducer gradients are suggested to be between 4 and 5 μm (Gantner et al.2006; Elias and Banin 2012), and bacteria can optimize the use of auto-inducers by being in each other's close vicinity. Myxococcus xanthus, E. coli, B. subtilis and lactobacilli, for instance, use contact-dependent signaling for communication (Blango and Mulvey 2009). Direct physical contact between bacteria in a biofilm is generally absent, unless co-adhering bacterial pairs are involved, which occur mostly in the oral cavity (Rickard et al.2003). SUMMARY In summary, all surfaces are heterogeneous with respect to hydrophobicity, charge and/or the possession of micro- or nanoscopic structures. Such stochastically occurring heterogeneities exert different adhesion forces upon adhering bacteria. Bacteria sense these adhesion forces through cell wall deformation, which subsequently activates membrane-located sensors to stimulate phenotypic responses in initially adhering bacteria in direct contact with the surface. The local adaptive response of initial colonizers is conveyed to other biofilm inhabitants through diffusion of auto-inducers produced by the initial colonizers and their first generation progeny. Later, generation progeny will lose the surface-adapted phenotype of the initial colonizers, while diffusion of auto-inducers occurs only over limited distances. This puts initial colonizers in command of the development of localized, stochastically occurring heterogeneous domains in a biofilm. The role of adhesion force sensing in cell wall deformation as local triggers for the development of heterogeneous microenvironments in biofilms puts a strong emphasis on the substratum surface on which biofilms are grown. Hitherto, in research on adaptive responses of bacteria to environmental triggers, conclusions are frequently extrapolated from agar-grown ‘biofilms’ and biofilms on undefined well-plate materials to biofilms in general. Realization of the role of substratum properties in localized, adaptive responses of adhering bacteria and subsequent properties of a biofilm may accelerate development of much needed insight in the mechanisms of heterogeneous microenvironment development in biofilms. FUNDING This study was supported by the University Medical Center Groningen-University of Groningen, Groningen both in The Netherlands. HJB is also director-owner of SASA BV. Opinions and assertions contained herein are those of the authors and are not meant to be construed as the representing views of the organizations to which the authors are affiliated. Conflict of interest. None declared. REFERENCES Alam F , Balani K . Adhesion force of Staphylococcus aureus on various biomaterial surfaces . 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FEMS Microbiology ReviewsOxford University Press

Published: Jan 9, 2018

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