Mucin Degrading Bacteria in Biofilms 439
439
36
Growth of Mucin Degrading Bacteria in Biofilms
George T. Macfarlane and Sandra Macfarlane
1. Introduction
Mucins are important sources of carbohydrate for bacteria growing in the human
large intestine. As well as being produced by goblet cells in the colonic mucosa, sali-
vary, gastric, biliary, bronchial, and small intestinal mucins also enter the colon in
effluent from the small bowel. Particulate matter, such as partly digested plant cell
materials, are entrapped in this viscoelastic gel, which must be broken down to facili-
tate access of intestinal microorganisms to the food residues. It is estimated that
between 2 to 3 g of mucin enter the large bowel each day from the upper digestive tract
(1), however, the rate of colonic mucus formation is unknown. Complex polymers,
such as mucin must be degraded by a wide range of hydrolytic enzymes to smaller
oligomers and their component sugars and amino acids before they can be assimilated
by intestinal bacteria.
Pure and mixed culture studies have established that in many intestinal bacteria,
synthesis of these enzymes, particularly β-galactosidase, N-acetyl β-glucosaminidase,
and neuraminidase (2–4), is catabolite regulated, and is therefore dependent on local
concentrations of mucin and other carbohydrates. Although some colonic microorgan-
isms can produce several different glycosidases, which allows them to completely
digest heterogeneous polymers (5–8), the majority of experimental data points to the
fact that the breakdown of mucin and other complex organic molecules is a coopera-
tive activity.
In the large bowel, bacteria occur in a multiplicity of different microhabitats and
metabolic niches, on the mucosa, in the mucous layer, and in the colonic lumen, where
they exist in microcolonies, as free-living organisms, or on the surfaces of particulate
materials (9,10).
Wherever there are surfaces, bacteria form biofilms. They are usually complex
microbial assemblages that develop in response to the chemical composition of the
substratum and other environmental constraints. The available evidence shows that in
the colon, these microbiotas are heterogeneous entities that form rapidly on the sur-
From:
Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The Mucins
Edited by: A. Corfield © Humana Press Inc., Totowa, NJ
440 Macfarlane and Macfarlane
faces of partly digested foodstuffs in the intestinal lumen, and in the mucous layer
covering the mucosa (9). Sessile microorganisms in biofilms often behave very differ-
ently from their nonadherent counterparts, and, in particular, the nature and efficiency
of their metabolism may be changed (10–12). Close spatial relationships between bac-
terial cells in biofilms are ecologically important in that they minimize potential growth
limiting effects on crossfeeding populations (13).
Although the large gut is often likened to a continuous culture system, this is an
oversimplification. Consideration of colonic motility and the way in which intestinal
material is processed suggests that only the cecum and ascending colon exhibit char-
acteristics of a continuous culture. The inaccessibility of the large bowel for experi-
ments on the digestion of mucin by colonic microorganisms inevitably means that the
majority of studies are made in vitro. A variety of models are available that enable
pure and mixed populations of intestinal bacteria to be grown under anaerobic condi-
tions, ranging from small screw-capped or serum bottles to more complex batch and
continuous fermentation systems (chemostats).
The effectiveness of in vitro model systems varies depending on the problem to be
investigated, and each method has advantages and disadvantages. For example, fer-
mentation experiments made using serum bottles are inexpensive, allow screening of a
number of substrates and/or fecal samples from different individuals, and require small
amounts of substrate and test sample (14). Depending on bacterial cell numbers, the
experiments can be designed to be of short duration, thereby minimising potential
distortions in the data resulting from the selection of nonrepresentative populations of
microorganisms. Longer-term experiments effectively become enrichment cultures,
selecting bacteria that are most efficient at utilizing the test substrates.
However, these fermentations are uncontrolled, and yield little information on bac-
terial metabolism, the organisms involved, or how the processes are regulated. The
limited data provided by such experiments relate simply to the input of substrates and
the output of products. Other problems may also be encountered; for example, if high-
substrate concentrations are used, strong nonphysiological buffers are needed to con-
trol pH, which may have unpredictable effects on bacterial metabolism. If culture pH
is not regulated, the environment in the vessel will change rapidly such that the fer-
mentation conditions become physiologically irrelevant.
Another factor to be considered with batch fermentations is that they are closed
systems, in which bacterial metabolic activities and environmental conditions in the
cultures are constantly changing. Thus, at the beginning of bacterial growth, substrate
concentrations are high, and become depleted as the cells grow, whereas bacterial
fermentation products and other autoinhibitory metabolites progressively accumulate
in the culture.
Many of these problems are avoided in continuous cultures, since they are open
systems that work efficiently at high bacterial population densities. Cell growth is
strictly controlled by the concentrations of limiting nutrients in the feed medium. For
this reason, the organisms grow suboptimally at specific growth rates (µ) set by the
experimenter, through alterations in dilution rate (D), which is regulated by varying
the rate at which culture medium is fed to the fermentation vessel.
Mucin Degrading Bacteria in Biofilms 441
The principal advantage of the chemostat in physiologic and ecologic studies on
microorganisms is that it enables long-term detailed investigations to be made under a
multitude of externally imposed steady-state conditions that are not possible with
closed batch-type cultures. A two-stage continuous culture model can be used for
studying the formation of mucin-degrading biofilms under nutrient-rich and nutrient-
depleted conditions. The system comprises paired glass fermentation vessels fitted
with modified lids (Fig. 1), and several extra sampling ports fitted to the vessel sides
to which removable mucin baits or mucin gel cassette holders (Fig. 2) are attached.
The fermenters are connected in series, with fresh culture medium being fed to ves-
sel 1 (V1), and spent culture from this vessel being pumped into vessel 2 (V2). This
facilitates the study of mucin colonization under relatively carbohydrate-rich V1 and
extremely carbon-limited (V2) environmental conditions, comparable to the proximal
and distal colons.
Three main protocols are outlined in this chapter for studying (1) mucous-degrad-
ing bacterial consortia occurring in biofilms on the rectal mucosa, (2) mucinolytic
species growing in artificial mucin biofilms in continuous culture models of the colon
in the laboratory, and (3) mucinolytic microorganisms colonizing the surfaces of food
particles in fecal material.
Methods outlined for the isolation of bacteria from the rectal mucosa are essentially
destructive, and primarily provide details of the types and numbers of different species
that take part in this process. They do not contribute information concerning the mul-
ticellular organization of biofilm communities. However, the chemostat modeling pro-
tocols afford useful comparative data on the enzymology and physiology of the
breakdown of mucin by adherent (mucin baits) and planktonic bacterial communities,
under varying environmental conditions, while the use of mucin-coated glass slides
attached to cassettes facilitates microscopic examination of biofilm development.
2. Materials
1. Samples for scanning electron microscopy (SEM) are placed in 3% (v/v) glutaraldehyde
in 1 M PIPES buffer, pH 7.0. Then fix the samples with 4% (w/v) aqueous OsO
4
, dehy-
drated stepwise in ethanol, which involves three changes (10 min) in each of 50, 75, 95,
and, finally, 100% ethanol. Then dry the samples on a Poleron E 5000 critical-point drier,
place on stubs, and gold-coat to a depth of 30 nm.
2. Glass tubes, mucin gel cassettes, and fermentation vessels for baiting studies are manu-
factured by Soham Glass, Ely, Cambs, UK.
3. All formulated bacteriologic culture media and growth supplements are supplied by
Oxoid. Use as per manufacturer’s instructions. Unless stated otherwise, all chemicals are
obtained from Sigma Aldrich Ltd. (Poole, UK). Agars for isolating specific bacterial
groups in mucin-degrading consortia are as follows:
a. Nutrient agar (total facultative anaerobes).
b. MaConkey agar no. 2 (lactose-fermenting and nonlactose-fermenting enterobacteria,
enterococci).
c. Azide blood agar base (facultative anaerobic cocci, some Gram-positive anaerobic
cocci).
d. Wilkins-Chalgren agar (total anaerobe counts).
442 Macfarlane and Macfarlane
e. Wilkins-Chalgren agar plus nonsporing supplements (nonsporing anaerobes). The
supplements contain hemin, menadione, sodium pyruvate, and nalidixic acid.
f. Wilkins-Chalgren plus Gram-negative supplements (Gram-negative anaerobes). The
selective agents in this culture medium are hemin, menadione, sodium succinate,
nalidixic acid, and vancomycin.
g. MRS agar (lactobacilli).
h. Perfringens agar and supplements (Clostridium perfringens and certain other
clostridia). The selective supplements (A and B) contain sulfadiazine, oleandomycin
phosphate, and polymyxin B. All antibiotic additions are added at 50°C after auto-
claving for 121°C at 15 min.
Fig. 1. Two-stage continuous culture model used to study mucin-degrading biofilms under
carbon-excess (vessel 1, left) and carbon-limited (vessel 2, right) conditions.
Mucin Degrading Bacteria in Biofilms 443
i. Fusobacterium agar (fusobacteria). This comprises: 37.0 g/L Brucella agar base, 5.0
g/L Na
2
HPO
4
, 1.0 g/L NaH
2
PO
4
, 1.0 g/L MgSO
4
·7H
2
O, 0.005 g/L hemin, at pH 7.6.
This is autoclaved, cooled to 50°C and the following antibiotics are then added after
filter sterilisation in 5 mL distilled water: 20 mg/L neomycin, 10 mg/L vancomycin,
6.0 mg/L josamycin (ICN Biomedicals, Aurora, Ohio).
j. Beerens Agar for selective isolation of bifidobacteria is made as follows: 42.5 g/L
Columbia agar, 5.0 g/L glucose, 0.5 g/L cysteine HCl, 1.5 g/L purified bacteriologic
agar. Five milliliters of propionic acid is added to these constituents, after they have
been boiled and cooled to 70°C. The pH of the medium is then adjusted to 5.0 before
pouring the agar into Petri plates.
Fig. 2. Mucin gel cassette used for investigating bacterial colonization of mucous surfaces
in continuous culture experiments. The glass frame contains several slots into which are fitted
removable mucin-coated glass plates or microscope cover slips.
444 Macfarlane and Macfarlane
k. Bacteroides mineral salts medium for selective isolation of members of the B. fragilis
group consists of: 1.5 g/L KH
2
PO
4
, 1.0 g/L K
2
HPO
4
, 9.0 g/L NaCl, 1.2 g/L cysteine
HCl, 1.2 g/L NaHCO
3
, 0.1 g/L CaCl
2
·2H
2
O, 0.15 g/L MgCl
2
·6H
2
O, 0.05 g/L
MnCl
2
·4H
2
O, 0.05 g/L CoCl
2
·6H
2
O, 0.001 g/L FeSO
4·
7H
2
O, 0.005 g/L hemin, 0.005
g/L vitamin B
12
, 1.0 g/L NH
4
SO
4
, 5.0 g/L glucose, 20 g/L purified bacteriologic agar.
After autoclaving and cooling to 50°C, 5 mL of a filter sterilized antibiotic solution is
added, containing: 3.0 mg/L vancomycin, 10.0 mg/L nalidixic acid.
4. Freezer vials containing Wilkins-Chalgren broth supplemented with 10% glycerol and
2% porcine gastric mucin (Sigma Type III, partially purified), with pH adjusted to 6.5.
5. Neuraminidase substrate (1 mg/mL N-acetylneuraminlactose). The neuraminidase stan-
dard is 1 mg/mL N-acetylneuraminic acid (NANA). Make both in 0.1 M acetate buffer
(pH 5.5).
a. Solution A: 0.2 M sodium periodate (meta) in 9 M phosphoric acid.
b. Solution B: 10% sodium arsenite in 0.5 M sodium sulfate/0.2 M H
2
SO
4
.
c. Solution C: 0.6% thiobarbituric acid in 0.5 M sodium sulfate.
Store solutions A and B at room temperature, and make solution C fresh daily.
6. Make glycosidase assays using the following p-nitrophenyl substrates: N-acetyl α-
D
-
galactosaminide, α-
L
-fucopyranoside, N-acetyl β-
D
-glucosaminide, and β-
D
-galacto-
pyranoside, all prepared as 15 mM solutions in 0.01 M Tris buffer, pH 6.5. The stop
solution is a mixture of 0.5 M Na
2
CO
3
and 0.5 M NaHCO
3
.A standard curve using vary-
ing dilutions of p-nitrophenol is used to calculate enzyme activities.
7. PYG broth: 20 g/L glucose, 10.0 g/L Yeast extract, 5.0 g/L Tryptone Soya broth, 5.0 g/L
Peptone water, 0.5 g/L cysteine HCl, 0.005 g/L hemin (see item 8). Add 40 mL of PYG
salt solution, 0.2 mL of vitamin K
1
solution (see item 8), and 10 mL of Tween-80 to
950 mL of distilled water.
a. To make PYG salt solution, add 0.2 g of CaCl
2
·2H
2
0 and 0.2 g of MgSO
4
to 300 mL
of distilled H
2
O and dissolve by mixing. Then add a further 500 mL of H
2
O, together
with 1.0 g K
2
HPO
4
, 1.0 g of KH
2
PO
4
, 10.0 g of NaHCO
3
, and 2.0 g of NaCl. Finally,
make up the volume to 1 L with distilled water.
8. MIDI PYG broth is made as follows: 5.0 g/L peptone water, 5.0 g/L Pepticase (Quest
International, Norwich, New York), 10.0 g/L Yeast extract, 0.5 g/L cysteine HCl, 10.0 g/L
glucose. In addition, the following solutions are added: 40.0 mL salts solution, 10 mL
hemin solution, 0.2 mL vitamin K
1
. Add the hemin solution, vitamin K
1
, and cysteine
after the medium is boiled, but before it is dispensed into metal capped glass Universal
bottles at 100°C and autoclaved. The salt solution is made in the same way as for normal
PYG medium, but the NaCl concentration is increased to 50 g. The haemin solution is
made as follows: Dissolve 50 mg of hemin in 1 mL of 1 M NaOH; make to 100 mL with
distilled water, then autoclave at 121°C for 15 min. Store at 4°C. Vitamin K
1
: Dissolve
0.15 mg in 30 mL 95% of ethanol. Store at 4°C in a brown bottle. Discard after 1 mo. For
identification of Gram-positive organisms, add 2.5 mL of 1:10 Tween-80 in distilled water
at the same time as cysteine to the medium.
9. To make Balch trace elements solution (15), add the following constituents to 600 mL of
distilled water: 3.0 g MgSO
4
·7H
2
O, 0.45 g MnCl
2
·4H
2
O, 1.0 g NaCl, 0.10 g FeSO
4
·7H
2
O,
0.18 g CoSO
4
·7H
2
O, 0.10 g CaCl
2
·2H
2
O, 0.18 g ZnSO
4
·7H
2
O, 0.01 g CuSO
4
·5H
2
O, 0.018
g Al(SO
4
)
2
·12H
2
O, 0.01 g H
3
BO
4
, 0.01 g NaMoO
4
·2H
2
O, 0.19 g Na
2
SeO
4
, 0.092 g
NiCl
2
·6H
2
O. Adjust the solution to pH 7.0 with 1M KOH, then make up to 1 L. Store at
4°C until use.
Mucin Degrading Bacteria in Biofilms 445
3. Methods
3.1. Enumeration and Identification
of Mucinolytic Bacteria in Rectal Biopsies
1. Rectal biopsy material is obtained from hospital out-patients. Tissue samples should be
immediately placed in preweighed sterile Bijoux bottles containing 4 mL of a suitable
anaerobic transport medium, such as Wilkins-Chalgren broth (see Note 1).
2. Weights and sizes of the samples are measured before placing them in an anaerobic cabi-
net (atmosphere 10% H
2
, 10% CO
2
, 80% N
2
) at 37°C. Speed is important during this step
(see Note 2).
3. Mascerate the biopsy material using a sterile glass tissue homogeniser. One mL of this
sample is serially diluted (10-fold dilutions to 10
–5
) in test-tubes containing 9 mL half-
strength sterile anaerobic Peptone water (see Note 1).
4. Plate out 50 µL of the original sample and 100 µL of all dilutions to 10
–5
in triplicate, onto
a range of selective and nonselective culture media, using sterile tips and glass spreaders
(see Subheading 2., item 3). Plates for aerobic incubation are removed from the anaero-
bic cabinet and incubated at 37°C.
5. Aerobic plates are incubated for 2 d, and anaerobic plates for up to 5 d, with periodic
examination, before counting of colonies.
6. The bacteria are then characterized on the basis of their Gram staining characteristics,
cellular morphology, fermentation products (16), and cellular fatty acid methyl ester
(FAME) profiles (see Note 3).
7. Fermentation products (short chain fatty acids, lactate, succinate) are analysed by growing
the organisms as pure cultures in PYG broth (see Subheading 2., item 7) for 24 h, then
centrifuging (13,000g, 10 min) to obtain a clear supernatant for GC or HPLC analysis.
8. Bacterial cellular fatty acids are extracted from overnight cultures of the organisms in MIDI
PYG broth (see Subheading 2., item 8). After centrifugation to obtain a cell pellet, FAMEs
are produced by saponification, methylation, and finally, solvent extraction. FAMEs are then
separated using a 5898 A Microbial Identification System. (Microbial ID, Newark, DE).
9. FAMEs are automatically integrated and numerical analysis done using standard MIS
Library Generation Software which identifies the organisms.
10. Colonies for further study are grown on agar plates and removed with sterile swabs into
2-mL freezer vials which are then stored at –80°C (see Subheading 2., item 4).
3.2. Mucin-Degrading Enzymes in Mucosal Bacteria
1. Grow individual isolates at 37°C in Wilkins-Chalgren Broth, supplemented with 5 g/L
partially purified porcine gastric mucin, in anaerobic Universal bottles (prepare by boil-
ing and dispensing the media into the bottles at 100°C, and then autoclaving).
2. After the cultures have grown, keep a portion of the whole culture, and harvest some of
the bacteria by centrifugation (13,000g, 30 min). Retain the cell-free supernatants and the
whole-cell cultures for comparative determinations of cell-bound and extracellular mu-
cin-degrading enzymes.
3. Calculate culture dry weights (see Note 4) by spinning down 1 mL of the culture in a
microcentrifuge at 13,000g for 5 min. Discard the supernatant and add a further 1 mL,
repeating the process until a total of 5 mL of culture have been collected. Finally, wash
the bacterial pellets with distilled water. Place the microcentrifuge tubes containing the
bacteria in a drying oven at 90°C for 3 d, or until dry. Determine the culture weights by
weighing the sample and calculating the dry weight per milliliter of original culture.
446 Macfarlane and Macfarlane
4. Neuraminidase assay: Test solution (0.05 mL) and boiled controls are incubated with
0.1 mL N-acetylneuraminlactose for 1–2 h at 37·C. Stop the reaction by boiling at 100·C
for 2 min. Add solution A (0.1 mL), mix, and allow to stand for 20 min at room tempera-
ture. Next add solution B (0.4 mL) and mix until the yellow colour disappears. Then add
solution C (1.0 mL), and heat the mixture in a boiling water bath for 15 min, before
cooling in cold water for 5 min. After centrifuging at 13,000g for 5 min to remove the
precipitate, read the absorbance of the supernatant at 549 nm. Prepare a standard curve by
using known amounts of NANA and developing these with the test after the boiling stage
(see Subheading 2., item 5).
5. In glycosidase assays, incubate 0.5 mL of test solution at 37°C with 0.25 mL of substrate,
until a yellow color begins to appear, or for 1 h. Terminate the reaction by adding 0.75
mL of stop solution and then centrifuge at 13,000g for 5 min before reading the absor-
bance at 420 nm (see Subheading 2., item 6).
3.3. In Vitro Modeling System Using Mucin Baits
and Mucin Gel Cassettes
1. Use glass fermentation vessels (560 mL working volume) with modified lids, containing
several extra sampling ports in these experiments (see Fig. 1).
2. Use fresh feces to prepare 20% (w/v) inocula in 100 mM anaerobic sodium phosphate buffer
(pH 6.0), by macerating the stool in a stomacher for 5 min and then sequentially filtering
through 500- and a 250-µm metal sieves to remove particulate material. Add 200 mL of this
inocula to 200 mL of double-strength culture medium (see step 4) in the fermenter.
3. Constantly stir the fermentation vessels and set the dilution rates at 0.1/h (see Note 5)
operating pH at 6.0, and temperatures at 37°C. Maintain anaerobic conditions by sparging
cultures with O
2
-free N
2
at a low gas flow rate (2.4 L/h).
4. A suitable culture medium (see Note 6) comprises: 2.0 g/L starch (soluble), 0.5 g/L pec-
tin (citrus), 0.5 g/L inulin, 0.5 g/L xylan (oatspelt) 0.5 g/L arabinogalactan (larchwood),
0.5 g/L guar gum, 2.0 g/L mucin, 3.0 g/L Tryptone, 3.0 g/L Peptone water, 4.5 g/L Yeast
extract, 0.015 g/L hemin, 4.5 g/L NaCl, 2.5 g/L KCl, 0.45 g/L MgCl
2
·6H
2
O, 0.2 g/L
CaCl
2
·6H
2
O, 0.4 g/L KH
2
PO
4
, 0.8 g/L cysteine, 0.4 g/L Bile salts No.3, 20 mL Balch
trace elements (see Subheading 2., item 9); 0.5 mL Tween-80.
5. When the chemostats reach steady state, at least 10 turnovers in culture volume, as indi-
cated by analysis of short chain fatty acid (SCFA) profiles (see Note 7), extracellular and
cell-associated samples of the lumenal populations, for comparative purposes, are taken
for both chemical and enzymic measurements, and for viable counts of bacteria, using a
range of selective and nonselective agars (see Subheading 2., item 3). The range of dilu-
tions of samples for plating should be increased to 10
–3
–10
–8
, to take into account the
greater numbers of bacteria in these samples.
6. Place sterile mucin gels in glass tubes (17 × 12 mm, 2 mL/vol), or mucin gel cassettes in
the fermenters, in each of the five side sample ports. Prepare the baits by placing the glass
tubes in a covered glass beaker, autoclave them, and when cool, pour over 2% (w/v)
porcine gastric mucin with the addition of 0.2% (w/v) purified bacteriologic agar, after
autoclaving and cooling. Then place the gels in an anaerobic cabinet to set. Make the gel
cassettes by autoclaving a solution containing 0.8% (w/v) purified bacteriologic agar and
2% (w/v) mucin in distilled water, then aseptically coating sterile glass microscope cov-
erslips, or custom made glass plates, with this solution at 60°C, before fitting them with
sterile forceps to the cassette holder.
7. Remove gels periodically over 48 h for analysis. Wash the surfaces gently with 100 mM
anaerobic sodium phosphate buffer, pH 6.0 to remove loosely adherent planktonic micro-
Mucin Degrading Bacteria in Biofilms 447
organisms. Resuspend the gel material in 10 mL anaerobic glycosidase buffer at pH 6.5
for enzymic analysis, and bacterial enumeration (methods as for biopsy samples). Use the
mucin-coated glass coverslips directly for microscopic analysis. Take the samples and
freeze at –20°C for carbohydrate analysis (see below).
8. Keep samples of the planktonic populations and fermenter media for measurements of
mucin carbohydrate uptake. Rates are calculated as follows: q
s
= D (S
o
–S)/x, where D =
dilution rate, S
o
= substrate entering fermenter, S = residual substrate in fermenter, and x
= community dry weight (q
s
= substrate utilized/[min·mg dry weight bacteria]).
9. Mucin oligosaccharides are determined by hydrolysing samples in 2 M H
2
SO
4
for 2 h at
100°C. A standard sugar mix containing 1 mg/mL of fucose, galactosamine, glucosamine,
galactose, glucose and mannose is also hydrolyzed in 2 M of H
2
SO
4
. To 100 mL of
hydrolysate, or standard sugar mix (in 2 M of H
2
SO
4
), add 5 mL of internal standard
solution (0.02 mg/mL deoxygalactose in high purity water), mix, and then run on a Dionex
DX 500/ED 40 analytical system.
10. Neutral and amino sugars are separated by high-pressure anion exchange chromatogra-
phy with pulsed amperometric detection (HPAEC-PAD) on a Dionex CarboPac PA 10
(4 × 250 mm) column equipped with a Dionex PA 10 guard column (4 × 50 mm) and a
Dionex ED 40 detector using the Dionex DX500 system (see Note 8). High-purity
deionized water (18 MΩ cm) should be employed in these tests, after being filtered
through 2-mm filters. Sodium hydroxide (50%, low in carbonate) is purchased from BDH,
Poole, Dorest, UK. Solution 1 is 0.2 M NaOH, and solution 2 is
distilled water. During
preparation of these solutions the water is sparged with helium for 15 min before and
during the addition of NaOH. Carry out monosaccharide detection using a gold cell and
preset carbohydrate waveforms. Achieve isocratic separation of neutral and amino sugars
at 1.0 mL/min with 30 mM NaOH. After 20 min, the column is purged with 100 mM
NaOH for 10 min, then re-equilibrated with the starting conditions for 10 min before the
next sample is injected. Use a PC 10 Pneumatic controller to introduce 0.3 M NaOH at a
flow rate of 0.5 mL/min to the column effluent, before the PAD cell, which minimizes
baseline drift and increases the analytical signal. Use a Dionex Eluant De-gas Module to
saturate the eluants with helium gas to minimize CO
2
absorption. Transfer the samples to
polyvials with 20
-
mm filters and inject with a Dionex AS40 Automatic sampler via a
Dionex high pressure valve. Use a Dionex Peaknet Software data handling system to plot
and integrate results.
11. Determine NANA by hydrolyzing samples in 0.05 M of H
2
SO
4
for 1 h at 80°C. Then
visualize released NANA colorimetrically as in the neuraminidase assay (see Subhead-
ing 3.2., item 4), after the boiling stage.
3.4. Short-Term Fermentation Studies
on Biofilm and Lumenal Populations in Chemostats
1. Take culture from both culture vessels, together with material from mucin baits or gel
cassetes. At this time, also remove the biofilms that form on the vessel walls. After wash-
ing and resuspension in 0.1 M sodium phosphate buffer, pH 6.0, the samples are centri-
fuged at 13,000g for 20 min. Resuspend each of the resulting pellets in 20 mL anaerobic
0.4 M phosphate buffer, pH 6.0. Add 10 mL of each suspension to 40 mL of chemostat
medium in 70 mL Wheaton serum, bottles under N
2
, at 37°C. Take samples hourly for 6 h
(see Note 9), centrifuge at 13,000g for 10 min then freeze the supernantants at –20°C for
subsequent measurement of SCFA and other organic acids. Also freeze samples for analy-
sis of residual mucin. Make dry weight determinations on the samples as in Subheading
3.2., item 3 for calculations of specific rates of substrate uptake and utilization.
448 Macfarlane and Macfarlane
3.5. Desorption of Mucinolytic Bacteria from Food Materials in Feces
1. Fresh fecal samples are homogenized in anaerobic 0.1 mol/L sodium phosphate buffer
(pH 6.5) to give 10% (w/v) slurries. Pass fecal slurries sequentially through 500- and
250-mm diameter sieves. Retain filtrates containing nonadherent bacteria under anaero-
bic conditions for enzymic analysis, fermentation studies, and bacterial counts.
2. Material retained on the filters is washed twice with 500 mL of the anaerobic buffer to
remove loosely adherent organisms. Washed food particles are subsequently incubated at
37°C under anaerobic conditions (O
2
-free N
2
atmosphere) in phosphate buffer in the pres-
ence of a surfactant such as 0.001% (w/v) cetyltrimethylammonium bromide (CTAB)
(BDH) for 30 min, with mixing. Samples are then refiltered to remove food materials.
Retain filtrates containing adherent bacterial populations and residual food materials
under anaerobic conditions (see Note 10).
3. Place samples of particulate material, washed particulate material and CTAB treated par-
ticles in 3% (v/v) glutaraldehyde in PIPES buffer (0.1 M, pH 7.4) at 4°C for SEM (see
Subheading 2., item 1).
4. Perform enzymic analysis on bacteria extracted directly from feces and organisms
removed from particulate materials with CTAB resuspended in 0.1 M sodium phosphate
buffer (pH 6.5), as described in step 2.
5. Serially dilute unattached fecal bacteria and organisms desorbed from particulate mate-
rial with 0.001% CTAB on a variety of selective and nonselective agars for enumeration
(see Subheading 2., item 3).
3.6. Mucin Fermentation Experiments
with Biofilm and Nonadherent Fecal Bacteria
1. Incubate biofilm and nonadherent faecal bacteria from fecal material at 37°C under O
2
-
free N
2
in 0.1 M sodium phosphate buffer (pH 6.5), in sealed 70-mL serum bottles
(Wheaton) with mucin. Take samples (2 mL) periodically over a period of 6 h (see Sub-
heading 3.4.) and freeze for analysis of fermentation products and residual mucin carbo-
hydrate. Determine culture dry weights (see Subheading 3.2., item 3) to calculate specific
rates of substrate utilization and fermentation product formation.
4. Notes
1. The benefits of using rectal biopsies to study mucosal bacterial populations are that, for
most of the time, the rectum is empty and the mucosa is clean, and uncontaminated with
lumenal material, and samples are relatively easy to obtain since the patients/volunteers
do not need to be cleaned, or otherwise specially prepared, as would be required when
removing tissue from the proximal or distal bowel during colonoscopy. Wilkins-Chalgren
broth is sterilized by autoclaving (121
o
C, 15 min). The bottles are prereduced by being
placed in an anaerobic chamber or gas jar (Don Whitley Scientific, Shipley, Yorks) with
loose lids, and allowed to cool. This is also used to prepare anaerobic peptone water for
the dilution series.
2. Weights and physical dimensions of the biopsy samples are needed to estimate bacterial
cell densities, either as per unit area or as per unit tissue weight. Rapid handling of samples
is essential to prevent growth of facultative anaerobes, and inactivation of strict anaer-
obes during transport.
3. Bacterial CFAs are highly stable and reproducible taxonomic markers. This allows phe-
notypic analysis of pure and mixed populations of intestinal microorganisms to be under-
taken by extracting their CFAs and comparing patterns of the methyl esters by GC, using
Mucin Degrading Bacteria in Biofilms 449
the MIDI system. This highly automated procedure is controlled by a microprocessor,
and contains several computer-generated libraries comprising data on over several hun-
dred different aerobic and anaerobic species.
4. Culture dry weights or, alternatively, the protein content of the sample (e.g., Lowry
method) are used for calculating specific enzyme activities.
5. The basic experimental setup of a semicontinuous or continuous culture type fermenta-
tion system, fitted with automatic pH control is shown in Fig. 3. The fermentation vessel
can be operated as either a batch or a continuous culture. The reactor is stirred magneti-
Fig. 3. Schematic diagram showing a simple single-stage continuous culture system.
450 Macfarlane and Macfarlane
cally, and in the continuous culture configuration, where sterile growth medium is con-
tinuously supplied to the fermenter, spent culture medium leaves the fermentation vessel
Fig. 4. Scanning electron micrographs of (A) bacterial biofilm colonising the surface of
digestive materials in human cecal contents, and (B) particulate material in which the bacteria
have been removed by surfactant treatment.
Mucin Degrading Bacteria in Biofilms 451
by gravity, via an overflow syphon. More sophisticated systems stir the cultures using a
direct-drive impeller, with spent culture medium and bacterial cells being removed from
the reactor using a dedicated pump. Dilution rate (D) is calculated as the working volume
of the fermentation vessel divided by the flow rate of growth medium into the system.
Specific growth rates (µ) of bacteria in a chemostat are equal to D, under steady-state
conditions. The mean residence time of organisms in the fermenter can be calculated as
the reciprocal of D, and mean doubling times of the cells (T
d
) are derived from In2/D.
6. The growth medium contains a complex mixture of proteins, carbohydrates and mucin, to
maximize species diversity in the ecosystems. When the chemostats reach steady-state,
sterile mucin gels in baits, or glass coverslips, are fitted to the fermenters and removed
periodically for analysis. Partially purified porcine gastric mucin is used in these experi-
ments because of its compositional and structural similarity to human mucins.
7. Bacteriologic analysis is time-consuming and labor-intensive, especially when working
with strict anaerobes. Daily measurements of SCFA (acetate, propionate, butyrate) in the
cultures provides a rapid and simple test of metabolic steady state.
8. Sulfate ions in the hydrolysate decrease retention times and result in coelution of peaks.
Incorporation of a AG5 guard column, with column switching after 2.4 min prevents
sulfate ions from reaching the analytical column.
9. Incubations should be kept as short as possible, to prevent major changes occurring in the
species composition of the samples.
10. Figure 4 shows the effectiveness of CTAB in removing strongly adherent bacteria from
digestive materials. Do not use higher concentrations of the surfactant, since it is inhibi-
tory to many intestinal bacteria.
References
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colon. Direct measurements of humans. Gastroenterology 85, 589–595.
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lence determinants in Clostridium septicum in relation to growth on mucin and the swarm
cell cycle. Biosci. Micro. 16, 28.
3. Macfarlane, G. T. and Gibson, G. R. (1991) Formation of glycoprotein degrading enzymes
by Bacteroides fragilis. FEMS Microbiol. Lett. 77, 289–294.
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culture system. J. Appl. Bacteriol. 66, 407–417.
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carbohydrates on growth, polysaccharidase and glycosidase production by Bacteroides
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fermentation of cellobiose by Ruminococcus flavifaciens. J. Gen. Microbiol. 110, 29–38.
7. Berg, J. O. (1981) Cellular location of glycoside hydrolases in Bacteroides fragilis. Curr.
Microbiol. 5, 13–17.
8. Degnan, B. A. (1993) Transport and Metabolism of Carbohydrate by Anaerobic Gut Bac-
teria. Ph.D Thesis, Cambridge University, Cambridge.
9. Englyst, H. N., Hay, S., and Macfarlane, G. T. (1987) Polysaccharide breakdown by mixed
populations of human faecal bacteria. FEMS Microbiol. Ecol. 95, 163–171.
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