Download Bacterial Growth and Metabolism on Surfaces in the Large Intestine

ORIGINAL ARTICLE
Bacterial Growth and Metabolism on Surfaces in the
Large Intestine
S. Macfarlane, M. J. Hopkins and G. T. Macfarlane
From the University of Dundee MRC Microbiology and Gut Biology Group, Ninewells Hospital and Medical
School, Dundee, UK
Correspondence to: G. Macfarlane University of Dundee, Department of Molecular and Cellular Pathology,
Ninewells Hospital and Medical School, Dundee DD1 9SY, Scotland. Tel: » 44-1382 496250, Fax: » 44-1-1382
633952, E-mail: [email protected]
Microbial Ecology in Health and Disease 2000; Suppl 2: 64– 72
The large intestinal microbiota is characteristically viewed as being a homogeneous entity, yet the proximal colon and distal bowel differ
markedly in relation to their nutritional availabilities and physicochemical attributes. Moreover, individual species and assemblages of
microorganisms exist in a multiplicity of different microhabitats and metabolic niches in the large gut, on the mucosa and in the mucus
layer, as well as in the gut lumen. Examination of intestinal material by scanning electron microscopy and uorescent light microscopy
shows that most of the bacteria are not freely dispersed, but occur in clumps, and in aggregates attached to plant cell structures and other
solids. With respect to the numerically predominant species, bacteria attached to surfaces in the gut lumen appear to be phylogenetically
similar but physiologically distinct from non-adherent populations. These adherent organisms are more directly involved in the
breakdown of complex insoluble polymers than unattached bacteria, which provides a competitive advantage in the ecosystem. In healthy
people, mucosal populations are more difŽcult to study than faecal bacteria due to difŽculty in gaining access to the bowel, and has
restricted studies on these communities. Consequently, little information is available concerning the composition, metabolism and
health-related signiŽcance of bacteria growing at or near the mucosal surface. Key words: bioŽlms, mucosa, bacterial metabolism, mucus,
pathogens.
SUBSTRATE AVAILABILITY AND ENERGY
GENERATION IN THE MICROBIOTA
Due to gastric acid and the washout effect resulting from
rapid passage of digestive substances through the stomach
and small bowel, the principal areas of permanent microbial colonisation of the human gut are the terminal ileum
and large intestine. This is primarily a result of the slowing
down of movement of digestive material in the colon,
which provides time for a complex and stable microbial
ecosystem to develop (1).
The main dietary sources of carbon and energy for
intestinal microorganisms are resistant starches, plant cell
wall polysaccharides and oligosaccharides, together with
peptides, proteins and other lower molecular weight substances that escape digestion and absorption in the small
gut (2). These, together with proteins and other complex
polymers formed by the host such as pancreatic secretions
and mucopolysaccharides, are degraded by a wide range of
hydrolytic enzymes to their component sugars and amino
acids, which are then fermented by the bacteria. Through
fermentation of carbohydrates (Fig. 1) and proteins
(Fig. 2), and the absorption and utilisation of short chain
fatty acids and other metabolic products, the large intestinal microora plays an important role in host digestive processes, enabling energy to be salvaged from unab© Taylor & Francis 2000. ISSN 1403-4174
sorbed dietary residues, as well as body tissues and secretions.
Intestinal bacterial fermentations are regulated by the
need to maintain redox balance, principally through the
reduction and oxidation of ferredoxins, avins and pyridine nucleotides. To a large degree, this affects the ow of
carbon through the bacteria, the energy yield obtained
from the substrate, and the fermentation products that can
be formed. Synthesis of reduced substances such as H2,
lactate, succinate, butyrate and ethanol is used to effect
redox balance during fermentation, whereas the production of more oxidized substances, such as acetate is associated with ATP generation. Conversely, more reduced
fermentation products result in comparatively low ATP
yields (3). Many of these electron sink products formed by
carbohydrate fermenting bacteria, especially H2, succinate
and lactate, serve as energy sources for non-saccharolytic
species in the microbiota, thereby making a major contribution to species diversity in the ecosystem.
Except in very broad terms, little is known of the
metabolic relationships that exist between individual bacterial communities in the colon or of the ecology and
multicellular organisation of the microbiota. A number of
molecular studies have shown that only a small fraction of
bacterial species in natural communities are culturable (4);
Microbial Ecology in Health and Disease
Bacterial growth on surfaces
(5) thus, while we can determine that the ecosystem contains large numbers of phylogenetically and physiologically
different bacteria, the relative population sizes and types of
non-culturable organisms present in the microbiota are
largely unknown.
Many host and environmental factors affect the composition of the gut microbiota (see Table I), including dietary
inuences, transit time of colonic contents and their pH
(6). However, ecological and metabolic interactions between the bacteria themselves are also signiŽcant, and are
largely dependent on the type of environment in which the
organisms exist. While bacterial species diversity largely
derives from the multiplicity of carbon and energy sources
available for fermentation in the colon, many different
types of ecological interaction occur between intestinal
microorganisms, including commensalism, where one species is stimulated by a second, which itself is unaffected by
the growth and activities of the Žrst; neutralism, where
bacterial communities co-exist but have no signiŽcant
metabolic effect on each other; antagonism, in which one
population is repressed by inhibitory substances produced
by another, and symbiosis, where two species have an
obligate dependence on each other. Of particular importance is the ability to compete for limiting nutrients and, in
some circumstances, adhesion sites on food particles,
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colonic mucus or the mucosa. Unsuccessful contenders in
these events are rapidly eliminated from the ecosystem.
BIOFILMS IN THE LARGE GUT
While the large intestinal microbiota is conveniently
viewed as being a homogeneous entity, the reality is quite
different, because the bacteria exist in a multiplicity of
different microhabitats and metabolic niches that are associated with the mucosa, the mucus layer and particle
surfaces in the colonic lumen. Furthermore, these microenvironments are constantly changing as resources are consumed or produced. Bacteria in the large bowel may occur
individually, but it is more likely they exist in microcolonies or in associations with other species on the surfaces of particulate materials. In fact, where there are
suitable surfaces, bacteria and other microorganisms seem
to have an innate tendency to form bioŽlms. In some
circumstances, these structures may be composed of a
single species, as in infections of catheters, heart valves and
other medical prosthetics, but bioŽlms usually comprise
complex multi-species consortia that develop in response
to the chemical composition of the substratum and other
environmental determinants (7).
Observational and modelling studies suggest that the
initial colonisation of surfaces occurs through the attach-
Fig. 1. Pathways of polysaccharide
breakdown, and the major routes
of fermentation of dietary carbohydrate in the gut microbiota,
showing the principal end products
of metabolism. NSP, non-starch
polysaccharides; PK, phosphoketolase; TA, transaldolase.
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S. Macfarlane et al.
Fig. 2. Mechanisms of protein breakdown by bacteria in the large
intestine and fates of the products of dissimilatory amino acid
metabolism.
ment of individual bacteria or small groups of organisms,
followed by non-linear proliferation of the cells that ultimately leads to formation of the mature bioŽlm. While
there is still some debate on this subject, it appears that
initial attachment occurs due to either electrostatic forces
on the bacterial surface or to the production of a sticky
glycocalyx by some cells.
Bacteria growing in bioŽlms often behave differently
from their non-adherent counterparts, and in particular,
the nature and efŽciency of their metabolism is changed,
while many species exhibit greater resistance to antibiotics,
and other inhibitory factors that have deleterious effects
on planktonic bacteria (8– 10). Bacterial bioŽlms occur in
a variety of natural environments including sediments,
soils, the oral cavity and the gastrointestinal tracts of
animals, but their study has been neglected in the human
gut. However, particle-associated and mucosal bacterial
populations in the large bowel are probably components
of highly evolved assemblages, analogous to those in oral
bioŽlm communities, where partner recognition appears to
be very speciŽc during the formative stages of co-aggregation (11).
BioŽlm communities often exhibit highly coordinated
multicellular behaviour, within and between species, and
many bioŽlm properties are dependent on local cell population densities. A good example of this is provided by
quorum sensing transcriptional activation in Gram-nega-
tive bacteria (12) (see also a review by Swift et al. in this
supplement). Close spatial relationships between bacterial
cells growing on surfaces are important in other ways,
particularly in relation to metabolic communication between microorganisms in the microbiota. They are ecologically signiŽcant in that they minimize potential growth
limiting effects on secondary cross-feeding populations,
that are associated with mass transfer resistance, as for
example between H2-producing bacteria and H2-consuming syntrophs (13). This is apparent in Fig. 3 where an
autouorescing microcolony of the archaeon Methanobrevibacter smithii can be seen growing in mucus. The obligate
H2-utilising organism cannot digest mucus glycoprotein by
itself and must therefore rely on H2 and CO2 formation
during mucinolysis by saccharolytic and amino acid-fermenting bacteria growing in close proximity, to provide
substrate.
Another facet of bacterial growth in bioŽlms in the
colon is that species colonising surfaces in the gut lumen
are likely to be more directly involved in the breakdown of
complex insoluble polymeric substances than non-adherent
organisms, giving them a signiŽcant advantage in competing for nutrients in the ecosystem (14).
MUCOSAL COMMUNITIES IN THE LARGE
INTESTINE
The compositions of mucosal bacterial communities in the
gastrointestinal tracts of animals have been relatively well
characterized, and in ruminants (15), termites (16) and
chickens (17), speciŽc microoras have been identiŽed on
epithelial surfaces. Secretory intestinal epithelia are covered in a mucus coating ranging between 100– 200 vm in
Fig. 4. Scanning electron micrograph of bacterial microcolonies
on the human rectal mucosa. The Žbrous-like matrix is the mucus
layer that has been dehydrated during sample preparation.
Bacterial growth on surfaces
Fig. 3. Light micrograph of an autouorescing microcolony of
Methanobrevibacter smithii in intestinal mucus.
Fig. 5. Bacteroides microcolonies on the rectal mucosa hybridized
with a speciŽc 16S rRNA probe labelled with Cy3.
Fig. 6. BiŽdobacterium microcolonies hybridized with a speciŽc
16S rRNA probe labelled with Cy5.
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Fig. 7. Live:dead Baclight (SYTO 9:propidium iodide) stain of a
rectal microcolony. Yellow cells are live, red cells are dead.
Fig. 8. Spirochaete-like organisms on the colonic wall. The bacteria are stained with a eubacterial 16S rRNA probe, labelled with
FITC.
thickness (18), and the mucus gel seems to play a role in
stabilising microbial communities growing in association
with the mucosa (19). Due to the presence of facultative
anaerobes, O2 does not appear to be a major factor
affecting the growth of anaerobic organisms at the epithelial surface.
Mucosal populations are difŽcult to study in healthy
people, mainly because of the inaccessibility of the
colon, and this has restricted work on these bacterial
communities. Despite this, some reports suggest that gut
epithelial populations in man are generally similar to
those present in the gut lumen (20). Bacteroides occur on
the mucosal surface, but other gut anaerobes including
eubacteria, biŽdobacteria, clostridia and a variety of
Gram-positive cocci are also present (21, 22). This can
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S. Macfarlane et al.
be seen in Fig. 4, which is an electron micrograph of the
rectal epithelium, and in Figs. 5 and 6 which are light
micrographs that respectively show Bacteroides and
biŽdobacterial microcolonies on a rectal biopsy. Fig. 7
illustrates a live:dead stained rectal microcolony, comprising a mixture of live (yellow) and dead (red) cells. Other
investigations involving the use of colonic and rectal biopsies have indicated that Bacteroides, particularly B. vulgatus and B. thetaiotaomicron, and biŽdobacteria, are the
major anaerobes associated with mucosal surfaces in the
gut (23– 25). Some mucosal bacteria that cannot be seen
or cultured from lumenal contents have unusual morphological properties (26). Scanning electron micrographs of
biopsy specimens show large helical cells, with lengths in
excess of 60 vm residing in the mucus layer (21). A
number of species growing in the mucus layer or in
association with the epithelial surface are Žlamentous or
spiral-shaped gliding bacteria (27). Fig. 8 is a uorescent
light micrograph showing swarms of spirochaete-like organisms, unusually arranged in parallel on the surface of
a colonic biopsy specimen. Mucin, other host secretions
and epithelial cells may be particularly important substrates for these mucosal species.
COLONISATION OF THE GUT MUCOSA
The structure and composition of bacterial communities
growing on the gut epithelium, as well as those existing in
the mucus layer, are probably determined by a variety of
host factors, including cellular and humoral immunity
(IgA), together with elements of the innate immune system, e.g., antimicrobial peptides such as defensins, which
are formed by polymorphonuclear cells and some enterocytes, and are active against Gram-positive and Gramnegative bacteria (28). The rate of synthesis and chemical
composition of mucus, turnover rates of intestinal epithelial cells, availability of adhesion sites, lysozyme production, pancreatic secretions, especially pancreatic endopeptidases, colonisation resistance mediated by components of the microbiota and gut motility are also likely to
be important.
Bacteria growing on the epithelial surface also affect
mucosal and systemic immunity in the host, involving
intestinal epithelial cells, blood leukocytes, B and T
lymphocytes, and other cells of the immune system (29).
Bacterial products with immunomodulatory properties include lipoteichoic acids (LTA), endotoxic lipopolysaccharide and peptidoglycans (30). BiŽdobacterial LTA possess
high binding afŽnity for human epithelial cell membranes,
and also serve as carriers for other antigens, binding them
to target tissues, where they provoke an immune reaction
(31). Maintenance of immune system homeostasis depends
to some degree on cell-cell contacts, intuitively therefore,
bacteria colonising the epithelial surface would seem to be
particularly involved in modulation of its activity.
In addition, phenotypic and antigenic variation by the
parasite are ongoing events during the colonisation process that often facilitates the evasion of host immune
system surveillance (32, 33). It is also of interest that
commensal and parasitic species living in close association
with host tissues often directly exploit the nutritional
potential of the substratum, examples include bacterial
utilisation of complex host macromolecules such as
mucins (34), as well as cell matrix constituents like vitronectin (35) and Žbronectin (36). Recent developments
have demonstrated that some adhesive bacteria are able
to recruit a variety of structurally diverse host proteins,
adhesive glycoproteins, growth factors and cytokines, by
initially binding heparin and functionally similar sulphated polysaccharides to their surfaces, whence they
serve as non-speciŽc, secondary recruiting sites for other
host molecules (37).
Several biŽdobacterial isolates of human origin have
been reported to exhibit protein-mediated adherence to
enterocyte-like Caco-2 cells in vitro (38). In these investigations, adherent B. longum, B. breve and B. infantis
variously inhibited cell-association and invasion of several
gut pathogens, including Escherichia coli, Salmonella typhimurium and Yersinia pseudotuberculosis. B. infantis and
some strains of B. longum and B. breve seemed to attach
strongly, while other B. breve and B. longum isolates were
poorly adherent.
With respect to other Gram-positive rods, some,
though not all lactobacilli are able to attach to human
intestinal epithelial cells (39). Species that colonize the gut
in this way characteristically exhibit high surface hydrophobicities (40), although protein-mediated adherence also
seems to play a role (41, 42). As with the biŽdobacteria,
protein-dependent adherence of L. acidophilus to Caco-2
cells inhibited binding of S. typhimurium, Y. pseudotuberculosis and E. coli (43). Lactobacillus antimicrobial activity, stimulation of enterocyte production of antimicrobial
substances with defensin-like characteristics, as well as
occupation of enterocyte receptors by the lactobacilli were
suggested as possible mechanisms associated with pathogen exclusion. Similar effects have been observed in vivo,
where in human volunteer feeding trials, probiotic lactobacilli were observed to temporarily colonize the gut surface, and displace other organisms including clostridia
and enterobacteria (44). Yogurt lactobacilli have also
been observed to bind to circulating peripheral blood
CD4 and CD8 T lymphocytes, though not to B cells (45),
while lactobacilli that adhere to human gut epithelial cells
(39) are capable of macrophage activation (46).
PATHOGENIC BACTERIA AND MUCUS
SURFACES IN THE GUT
In the initial stages of colonisation or infection of mucosal
surfaces in the large bowel, bacteria must be able to
Bacterial growth on surfaces
withstand the ow of the intestinal contents, and thereby
avoid being physically removed from the epithelial surface (47). However, epithelial cells in the gut are covered
by a layer of mucus, which prevents most microorganisms reaching the mucosal surface (48). This mucus
forms a viscoelastic gel (49), and it is these gel-like properties that are mainly protective against adhesion and
invasion by pathogenic microbes, bacterial toxins and
end-products of metabolism, pancreatic endopeptidases,
microbial antigens and other damaging agents present in
the lumen of the bowel. Pathogenic bacteria in the gut
deal with mucus barriers in different ways. For example,
mucus has a protective role against Yersinia enterocolit ica by reducing binding of the organisms to brush border membranes (50), while sulphomucins prevent
colonisation of the gastric mucosa by Helicobacter (51).
In contrast, some Gram-negative pathogens that colonize
the mouse intestine depend on their abilities to adhere to
mucus (52). Several motile species are chemotactic (14,
53, 54) or possibly viscotaxic to mucin (55). Thus, virulent strains of Serpulina hyodysenteriae are considerably
more chemotactic towards mucin than non-virulent isolates (56, 57). Moreover, some pathogens such as
Campylobacter do not degrade mucus, but bind to the
glycoprotein by means of speciŽc adhesins, as a prelude
to gaining access to cell membrane receptors (57). In
other gut pathogens, mucus has an important nutritional
function, and neuraminidase has been shown to be important for survival of Bacteroides fragilis in both in vivo
and in vitro model systems (58).
In some pathogens, colonisation of the mucosa is
achieved following invasion of the overlying mucus,
whilst others proceed through the mucus, adhering to
and colonising underlying epithelial cells. The bacterial
adhesins involved are diverse, but are usually cell surface
protein structures, and the eukaryotic cell receptors are
generally carbohydrate residues. It is likely that these
pathogenic bacteria possess a number of different adhesins, that may be used at different stages in the infection process. For example, strains of E. coli can express
several different Žmbriae which function as adhesins (see
also review by Adlerberth et al. in this supplement). It
has been suggested (59) that E. coli type 1 Žmbriae,
which bind to D -mannose residues on eukaryotic cells,
play an important role in colonisation of the urinary
tract and large bowel. A high percentage of E. coli
strains isolated from pyelonephritis produce a different
type of structure termed P Žmbriae, while a variety of
other bacteria also produce Žmbriae which are likely to
function as adhesins, including Salmonella spp., Neisseria
spp. and Pseudomonas spp (59). It has also been reported (60) that some strains of C. difŽcile are Žmbriated, but it remains to be determined whether these
structures are associated with adhesion and pathogenesis.
Using scanning electron microscopy Newell et al. (61),
69
observed in vitro adherence of C. jejuni to intestinal epithelial cells (62). Bacterial agella have been implicated
as carrying adhesins of C. jejuni for eukaryotic cell (61);
this was conŽrmed by studies of McSweegan and Walker
(62) who also suggested the involvement of a number of
bacterial adhesins, notably a bacterial surface protein.
Other microbial adhesins that have been reported include
the Žlamentous haemagglutinin of Bordetella pertussis
(59), the mannose-resistant haemagglutinin of S. typhimurium (63), and the bacterial cell surface proteins
that bind speciŽcally to Žbronectin on the surface of
eukaryotic cells.
Treponema pallidum, the aetiologic agent of syphilis,
uses three surface adhesins (P1, P2, P3) to speciŽcally
bind Žbronectin (59). Bacterial neuraminidases may also
facilitate adherence by exposing receptors on the surface
of eukaryotic cells. For example, the enzyme produced
by Bacteroides fragilis has been suggested to expose a
galactoside residue involved in adhesion (64). The capsule of B. fragilis is also believed to carry adhesins, as
does the capsule in Shigella spp. where the adhesins are
believed to be carbohydrate moieties (65). In addition to
these speciŽc interactions between bacterial adhesins and
host cell receptors, non-speciŽc mechanisms of cell attachment are also important. Hydrophobic interactions
are thought to facilitate adhesion by overcoming repulsive forces between host and microbial cells, and are an
important non-speciŽc adhesion mechanism in mucosal
association of bacteria. Indeed, many of the Žmbrial and
non-Žmbrial adhesins described have a high surface hydrophobicity. It has been proposed that the adhesiveness
of a pathogen is related to the hydrophobicity conferred
by its surface structures, and that whatever the receptor
attachment mechanism, surface hydrophobicity is involved (66).
BACTERIA ADHERING TO DIGESTIVE RESIDUES
IN THE GUT LUMEN AND MUCUS BREAKDOWN
Salivary, bronchial, gastric, small and large intestinal
mucins are all used as growth substrates by gut microorganisms. In small intestinal efuent, particulate substances such as partly digested plant cell materials are
entrapped in a viscoelastic mucus gel, which must be
broken down by bacteria in the colon to facilitate access
to the food residues. Microscopic examination of lumenal contents or faecal material shows that the majority
of bacteria are not freely dispersed, but occur in large
clumps and in aggregates attached to plant cell structures and other solids. While few studies have been
made on bacterial colonisation of particulate materials in
the gut lumen in humans, microbiological analysis of
partially digested food particles in faeces shows that bioŽlm populations growing on the surfaces of particulate
matter are members of complex multi-species consortia
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S. Macfarlane et al.
and, at genus level at least, the bioŽlm populations are
superŽcially similar to those in non-adherent microbiotas, with Bacteroides and biŽdobacteria predominating
(67).
Complete destruction of mucin and other complex
polymers in the gut is completed through the activities
of several different hydrolytic enzymes that can breakdown the protein backbone and carbohydrate side chains
of the macromolecule. Many of these enzymes are
catabolite regulated (67, 68) and their synthesis is therefore dependent on local concentrations of mucin and
other inducer and repressor substances. While some intestinal bacteria form a wide range of glycoside hydrolases, which in theory enables them to completely digest
heterogeneous polymers (69, 70), evidence suggests that
the breakdown of mucin and other complex organic
molecules in the bowel is a cooperative activity. In contrast to the depolymerisation of mucin and other host
mucopolysaccharides, there is undoubtedly severe competition between gut bacteria for the products of oligosaccharide hydrolysis. In fact, there are substantial
populations of saccharolytic bacteria on the rectal mucosa that are unable to digest the glycoprotein by themselves and they must exist by cross-feeding on
carbohydrate fragments produced by mucinolytic species
growing in nearby microcolonies (71).
Enzyme measurements have indicated that bioŽlms occurring on digestive substances in the gut lumen form
metabolically distinct assemblages in the ecosystem with
respect to the breakdown and metabolism of mucins and
other complex macromolecules (67). It was shown in
these studies that, with the exception of N-acetyl hgalactosaminidase, the vast majority of mucinolytic glycosidases were cell-associated in faecal material. Whilst
there was little difference in b -galactosidase and N-acetyl b-glucosaminidase in the bioŽlms, h-fucosidase and
N-acetyl h-galactosaminidase activities were manifestly
lower than in non-adherent populations. Proteases and
peptidases must also take part in mucin digestion, and
measurements of peptidolytic enzymes in lumenal bioŽlm
and non-adherent populations, using a range of protease
inhibitors, indicated that while the spectrum of proteolytic:peptidolytic activity was broadly similar in both
microbiotas (e.g. with respect to serine, thiol and aspartic protease proŽles), there were variations in trypsin,
chymotrypsin and to a lesser extent, metalloprotease activities. The higher trypsin and chymotrypsin in the bioŽlms were attributed to adsorbed pancreatic endopeptidases, whereas lower metalloprotease activities were
thought to reect differences in bacterial enzyme expression. Valyl alanyl:glycyl prolyl arylamidase, which can
be formed in very high levels by members of the Bacteroides fragilis group (34), was also markedly lower in
lumenal bioŽlms.
To conclude, because few studies have been made, little information is available concerning the structure and
function of bacterial bioŽlms in the large intestine or
their metabolic signiŽcance to the host. Moreover, because the investigation of bacterial growth on surfaces
and in aggregates is still in its infancy, many of the
analytical tools that have been used to study bioŽlm
composition and metabolism are innately destructive and
do not provide much information on organisation and
community structure in bioŽlms. However, the developing shift in emphasis away from culture-based studies,
and the introduction of methodologies involving measurements of ribosome abundance, using quantitative hybridisation techniques (72) should facilitate future work
on gut bioŽlms. Quantitation of both total rRNA and
that relating to speciŽc populations together with
uorescent labelling (73) and whole cell hybridisation:
confocal microscopy (74) will improve our understanding
of the temporal, metabolic and spatial organisation of
these microcosms.
ACKNOWLEDGEMENTS
This review has been carried out with Žnancial support from the
Commission of the European Communities, Agriculture, and
Fisheries (FAIR), speciŽc RTD programme PL98-4230 ‘Intestinal
Flora: Colonization Resistance and Other effects’. It does not
reects its views and in no way anticipates the Commission’s
future policy in this area. The careful assistance of Donatella
Lombardi in preparation of the manuscript is gratefully
acknowledged.
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