Rendueles. 2012. Environmental Microbiol
Rendueles. 2012. Environmental Microbiol
doi:10.1111/j.1462-2920.2012.02810.x
Minireview
Antibiofilm polysaccharides
emi_2810
1..13
Introduction
Biofilm is the predominant mode of growth for bacteria in
most natural, industrial and clinical environments. Biofilms
typically consist of densely packed, multispecies populations of cells, encased in a self-synthesized polymeric
matrix, and attached to a tissue or surface (Costerton et al.,
1987; Stoodley et al., 2002). The biofilm lifestyle is associated with a high tolerance to exogenous stress, and
treatment of biofilms with antibiotics or other biocides is
usually ineffective at eradicating them (Hall-Stoodley and
Stoodley, 2009). Biofilm formation is therefore a major
problem in many fields, ranging from industrial corrosion
and biofouling (Lopez et al., 2010) to chronic and nosocomial infections (Francolini and Donelli, 2010; Hoiby et al.,
2011).
Received 29 March, 2012; revised 12 May, 2012; accepted 15 May,
2012. *For correspondence. E-mail [email protected]; Tel.
(+33) 01 40 61 34 18; Fax (+33) 01 45 68 88 36.
One of the hallmarks of biofilm formation is the production of an extracellular matrix composed of 90% water and
10% extracellular polymeric substances (EPS) (Flemming
and Wingender, 2010). Extracellular polymeric substance
components mediate most of the cell-to-cell and cell-tosurface interactions that are necessary for the formation
and stabilization of biofilm colonies. Structural components of the EPS matrix include cellsurface proteins,
proteinaceous pili, DNA, RNA, lipids and polysaccharides
(Flemming and Wingender, 2010). Among these, polysaccharides, often identical to cell-bound capsular polysaccharides produced by free-floating or planktonic bacteria,
constitute a major component of the biofilm matrix in
many bacteria (Sutherland, 2001; Bazaka et al., 2011).
More than 30 different biofilm matrix polysaccharides
have been characterized so far (Flemming and Wingender, 2010). Well-known examples include alginate,
Pel and Psl produced by Pseudomonas aeruginosa
(Davies and Geesey, 1995; Ramsey and Wozniak, 2005;
Qin et al., 2009); poly-N-acetylglucosamine (PNAG)
produced by Escherichia coli (Wang et al., 2004), Staphylococcus aureus (OGara, 2007), Staphylococcus epidermidis (Gerke et al., 1998), Acinetobacter baumannii (Choi
et al., 2009), Burkholderia spp. (Yakandawala et al., 2011)
and Bordetella spp. (Parise et al., 2007); cellulose
secreted by E. coli (Zogaj et al., 2001), Pseudomonas
fluorescens (Spiers et al., 2003) and Salmonella spp.
(Solano et al., 2002); and glucans produced by Streptococcus mutans (Bowen and Koo, 2011). Mutant strains
unable to synthesize or export these exopolysaccharides
usually exhibit decreased adherence, decreased biofilm
formation, and increased sensitivity to killing by biocides
and host defences. These results highlight the importance
of exopolysaccharides in maintaining the integrity of the
biofilm and in mediating the pathogenic potential of the
biofilm lifestyle.
Bacterial exopolysaccharides exhibit highly variable
structures and it is likely that they also perform additional
functions besides their implied function in matrix stabilization and energy storage (Flemming and Wingender,
2010; Bazaka et al., 2011). In fact, several studies
showed that certain bacterial mutants deficient in capsular
polysaccharide production exhibit increased biofilm formation. Examples include E. coli (Valle et al., 2006),
Polysaccharide
Molecular
weight (kDa)
A101
> 500
Ec111p
> 500
Ec300p
> 500
K2
500
PAM
Pel
PI80 EPS
Psl
> 300
Unknown
280
46
r-EPS
SP1 EPS
Unknown
1800
Components
Reference
Galacturonic acid
Glucuronic acid
Rhamnose
Glucosamine
Mannose
Glucose
Galactose
Glucuronic acid
Mannose
Glucose
Galactose
Glucuronic acid
Galactose
Glycerol
Phosphate
Acetate
Galactofuranose
Glucose-rich
Arabinose
Mannose
Glucose
Rhamnose
Unknown
Galactose
Glycerol
Phosphate
E. coli Ec111
E. coli Ec300
E. coli CFT073
K. kingae PYKK081
P. aeruginosa PAO1
S. phocae PI80
P. aeruginosa PAO1
L. acidophilus A4
B. licheniformis SP1
Streptococcus pneumoniae (Moscoso et al., 2006; Domenech et al., 2009), Staphylococcus haemolyticus (Flahaut
et al., 2008), Vibrio vulnificus (Joseph and Wright, 2004),
Shewanella oneidensis (Kouzuma et al., 2010), Tannerella forsythia (Honma et al., 2007) and Porphyromonas
gingivalis (Davey and Duncan, 2006). These observations
suggest that some bacterial exopolysaccharides can
perform functions that inhibit or destabilize the biofilm.
This review focuses on a series of recent studies (Table 1)
that investigated the physical and biological characteristics of non-biocidal bacterial antibiofilm polysaccharides,
and their role in interspecies interactions in mixed, multispecies biofilms.
Bacterial antibiofilm polysaccharides
The first antibiofilm polysaccharide was discovered while
studying interactions between uropathogenic and commensal strains of E. coli in mixed in vitro biofilms. Valle
and colleagues (2006) found that the biomass of biofilms
produced by the commensal MG1655 strain was reduced
in the presence of uropathogenic CFT073 strain (Fig. 1A).
The biofilm reducing activity was present in CFT073
culture supernatant, and was shown to be due to group II
capsular polysaccharide commonly produced by extraintestinal E. coli of phylogenetic group B2 or D. A screen of
culture supernatants derived from 110 E. coli strains of
diverse origin revealed that only supernatants from 39
2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
adhesins of fimbriae and pili. For example, lectindependent adhesion of pathogenic P. aeruginosa to
human cells is efficiently inhibited by galactomannans
(Zinger-Yosovich and Gilboa-Garber, 2009).
Alteration of abiotic surface properties
Biosurfactants and bioemulsifiers have been shown to
alter the physico-chemical properties of surfaces by modifying the wettability and charge of the surface and hence
affecting the interaction of bacteria with the surface (Neu,
1996; Banat et al., 2010). This mechanism of biofilm inhibition is similar to the mode of action of rhamnolipid surfactants produced by P. aeruginosa (Davey et al., 2003)
as well as several biosurfactants and bioemulsifiers produced by marine bacteria that display antibiofilm activity
against pathogenic bacteria (Kiran et al., 2010). Physical
measurements have directly demonstrated that bacterial
antibiofilm polysaccharides can alter the properties of
abiotic surfaces. For example, cationic colloids brought
into contact with E. coli K2 culture supernatants led to a
rapid charge inversion, indicative of their highly anionic
nature (Valle et al., 2006). In addition, both K2 and
Ec300p polysaccharides lowered the interfacial energy of
glass surfaces, increasing the hydrophilicity of the surface
(Valle et al., 2006; Rendueles et al., 2011) (Fig. 2A). Similarly, purified S. phocae PI80 EPS, which is highly viscous
in solution, exhibited emulsifying activity against
n-hexadecane and flocculating activity against an activated carbon suspension (Kanmani et al., 2011), both of
which are indicative of biosurfactant activity.
Studies utilizing culture supernatants or purified antibiofilm polysaccharides as surface coatings have provided
further evidence that antibiofilm polysaccharides modify
the physical properties of abiotic surfaces. Precoating
microtiter plate wells with B. licheniformis SP1 culture
supernatants, for example, inhibited biofilm formation by
E. coli (Sayem et al., 2011). Similarly, pretreatment of
glass surfaces with E. coli K2 supernatants reduced
biofilm formation by E. coli, S. aureus, S. epidermidis,
E. faecalis, K. pneumoniae and P. aeruginosa in microfermentors (Valle et al., 2006), and pretreatment of glass
slides with purified E. coli Ec300p inhibited S. aureus
biofilm formation in a flow reactor (Rendueles et al.,
2011). Evaporation coating of K. kingae colony biofilm
extract onto polystyrene surfaces also produced an antiadhesive film that resisted biofilm formation by S. epidermidis and A. actinomycetemcomitans (Fig. 2B).
Alteration of biotic surface properties
Evidence suggests that antibiofilm polysaccharides not
only modify abiotic surfaces but also alter the physical
properties of Gram-negative and Gram-positive bacterial
2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
Signalling
In plants, rhizobial polysaccharides and oligosaccharides
are well-known communication molecules able to induce
2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
Human
Human
Inhibits binding of H. pylori and Campylobacter jejuni to human stomach tissue in vitro. Inhibits binding of
C. jejuni to chicken stomach tissue in vitro.
Inhibits binding of H. pylori to human gastric cancer cells in vitro.
Inhibits adhesion of S. mutans to saliva-coated hydroxyapatite beads.
Milk oligosaccharides inhibit binding of N. meningitidis pili to bovine thyroglobulin.
Inhibition of H. pylori, Propionibacterium acnes and S. aureus to host cell lines.
Inhibits binding of H. pylori to porcine gastric mucin in vitro.
Inhibits binding of S. mutans, S. sobrinus, P. gingivalis, F. nucleatum and Actinomyces to saliva-coated
hydroxyapatite in vitro. Prevents colonization of rats by S. cricetus and S. sobrinus, and reduces caries
score. Inhibits plaque development in human subjects when administered as a chewing gum.
Inhibits binding of H. pylori to human stomach tissue in vitro. Inhibits binding of P. gingivalis to rat
oesophageal tissue in vitro.
Polysulfated polysaccharides inhibit binding of several H. pylori strains to murine macrophage cell lines.
Heparin blocks adhesion of H. pylori and enterohaemorrhagic E. coli to macrophages and colonic
epithelium respectively.
Inhibits agglutination of human erythrocytes by H. pylori, P. gingivalis, A. actinomycetemcomitans, P. acnes
and S. aureus. Inhibits binding of H. pylori to human gastric cancer cells in vitro.
Inhibits binding of H. pylori to human stomach tissue in vitro.
Inhibits adhesion of enterotoxigenic E. coli to intestinal cells.
Inhibits binding of H. pylori to porcine gastric mucin in vitro. Inhibits colonization of mice by H. pylori.
Inhibits adhesion of S. mutans to saliva-coated hydroxyapatite beads.
Abelmoschus esculentus (okra fruit)
Activities
Potential applications
Source
References
2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
10
Future questions
Recent screens of culture supernatants and biofilm
extracts for antibiofilm activities have yielded hit rates as
high as 20% (Valle et al., 2006; Bendaoud et al., 2011;
Sayem et al., 2011), suggesting we have only begun to
explore the diversity of the contribution of bacterial antibiofilm polysaccharides in interspecies interactions
(Fig. 3). Whereas use of non-biocidal antibiofilm compounds in medicine and industry represents an appealing
strategy, much research is still needed to validate the use
of such molecules as alternative anti-infectious treatments in environmental or clinical settings. Focus should
for instance be put on clarifying the relationship between
polysaccharide structures and antibiofilm activities. While
determination of antibiofilm polysaccharide composition
and structure will enable their controlled industrial-scale
production, it could also clarify their mechanism of action
and provide leads for developing analogues with
enhanced antibiofilm activity. Further studies should also
address the biological roles of antibiofilm polysaccharides
and their impact on population dynamics in vivo. While
determinant for future applications, these studies could
also provide new and valuable insights into processes
driving co-evolution in multispecies biofilm consortia.
Acknowledgements
We thank C. Beloin, F. Stressmann and D. Lebeaux for critical
reading of the manuscript. O. R. was supported by a fellowship from the Network of Excellence EuroPathoGenomics
(European Community grant LSHB-CT-2005-512061).
J. B. K. was supported in part by NIH grant AI82392.
References
Anderl, J.N., Franklin, M.J., and Stewart, P.S. (2000) Role of
antibiotic penetration limitation in Klebsiella pneumoniae
biofilm resistance to ampicillin and ciprofloxacin. Antimicrob Agents Chemother 44: 18181824.
2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
11
2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
12
2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
13
2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology