Journal of Medical Microbiology (2004), 53, 807–811
DOI 10.1099/jmm.0.45636-0
Chemotactic response of Helicobacter pylori to
human plasma and bile
Mulugeta L. Worku,1 Q. Najma Karim,1 John Spencer2
and Ramon L. Sidebotham3
Correspondence
1,3
Ramon L. Sidebotham
[email protected]
Received 17 February 2004
Accepted 4 March 2004
2
Departments of Medical Microbiology1 and Gastroenterology3 , Imperial College School of Medicine
at St Mary’s, Norfolk Place, London W2 1NY, UK
Department of Surgery, Imperial College School of Medicine at Hammersmith, DuCane Road,
London W12 0NN, UK
To clarify further the role of chemotaxis in Helicobacter pylori colonization, the in vitro bacterium
response to human plasma and bile (secretions containing chemoeffector compounds that are
present in the gastric mucus layer) was examined. Human plasma, after dilution to 1 % (v/v) with
buffer, was found to be a chemoattractant for the motile bacillus. Human gall-bladder bile, after
dilution to 2 % (v/v) with buffer, was found to be a chemorepellent, but did not cause the motility of
the bacillus to be diminished after prolonged exposure. The basis of the chemoattractant effect of
plasma was explored by examining how urea and 12 amino acids found in plasma affected the taxis of
H. pylori. Urea and the amino acids histidine, glutamine, glycine and arginine were the strongest
chemoattractants. Other amino acids were chemoattractants, with the exceptions of aspartic and
glutamic acids, which were chemorepellents. The basis of the chemorepellent effect of bile was
explored by examining how the six most abundant conjugated bile acids in human bile affected the
taxis of H. pylori. All the bile acids were chemorepellents, with the greatest effects being
demonstrated by taurocholic and taurodeoxycholic acids. The implications of these findings for
H. pylori colonization of gastric epithelium are discussed.
INTRODUCTION
Within the gastric microenvironment, Helicobacter pylori is
found to be most frequently congregating at inter-epithelial
junctions, and associating with the epithelial surface by cell
attachment and mucus entrapment (Hazell et al., 1986;
Thomsen et al., 1990). The incentive to identify the factors
that control this distinctive pattern of colonization is considerable, since it has been shown that infection density at the
epithelial surface correlates directly with the severity of
gastritis and risk of peptic ulceration (Khulusi et al., 1995;
Talamini et al., 1997).
Freter (1980) has indicated that effective colonization of the
epithelial surface only occurs when bacteria with normal
motility and chemotaxis behaviour are directed towards the
mucosa by an appropriate chemotactic stimulus. In the H.
pylori-infected stomach, the important chemotactic gradients across the mucus layer, which draw the bacterium into
the epithelial surface, have yet to be identified. However, they
are likely to be formed from biliary surfactants refluxing into
the stomach, and constituents of plasma transuding from
gastric mucosa.
Abbreviations: CLV, curvilinear velocity; CR, chemotactic response.
The reflux of bile into the stomach post-prandially is a
normal occurrence (King et al., 1984; Schindlbeck et al.,
1987), and is reported to be increased in patients with
gastroduodenal disease (DuPlessis, 1965; Sobala et al.,
1993). In the stomach, refluxed bile does not remove (Bell
et al., 1985), but diffuses through the protective mucus layer
to alter (Dixon et al., 1986; DuPlessis, 1965) and eventually to
be absorbed (Davenport, 1967; Ivey et al., 1970) by the
underlying mucosa. Thus, H. pylori colonizing the gastric
microenvironment (especially in the pyloric antrum) will be
exposed to biliary surfactants and, more particularly, should
exist within a bile concentration gradient that decreases from
the luminal to the epithelial surface of the mucus layer. This
gradient might be expected to draw the bacterium into the
epithelial surface, since biliary surfactants are reported to act
as chemorepellents for other bacteria (Hugdahl et al., 1988).
The leakage or transudation of plasma from microcapillaries
into interstitial spaces, and then into the lumen of the
stomach, is a normal occurrence (Brassinne, 1979;
Hollander & Horowitz, 1962), and is increased considerably
when gastric mucosa is damaged experimentally (Brassinne,
1979; Sober et al., 1950) or through disease (Brassinne, 1974;
Jarnum & Jensen, 1972). Thus, H. pylori colonizing the
gastric microenvironment will be exposed to plasma, and
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45636 & 2004 SGM Printed in Great Britain
807
M. L. Worku and others
more particularly should exist within a plasma concentration
gradient that decreases from the epithelial to the luminal
surface of the mucus layer. This gradient might be expected
to draw the bacterium into the epithelial surface, since amino
acids found in plasma are reported to act as chemoattractants
for other bacteria (Hugdahl et al., 1988; Mesibov & Adler,
1972; Moulton & Montie, 1979).
In this preliminary communication we have tested these
ideas, by examining how the movements of H. pylori in an in
vitro chemotaxis system are affected by human plasma and
bile and some of their constituents.
METHODS
Gall-bladder bile was collected by routine puncture from a prospective
(adult, male) surgical patient. The sample was sterilized by filtration
through a 0.22 ìm syringe filter (Acrodiscs) and stored at 4 8C until
required. Blood plasma was obtained from a healthy volunteer and used
immediately. Conjugated bile acids, amino acids and urea were from
Sigma.
Bacteria. H. pylori was isolated from endoscopic biopsies of patients
with duodenal ulcer or non-ulcer dyspepsia. The bacterium was initially
plated out on 7 % (v/v) defibrinated horse blood agar, which included
H. pylori-selective supplement (Oxoid), and incubated in a microaerobic environment (10 % CO2 , 18 % O2 and 72 % N2 ) within a
Campypak gas jar (Oxoid) at 37 8C for 48 h. Colonies from these plates
were suspended in sterile saline to a turbidity equivalent to that of
McFarland’s No. 4 standard (108 bacteria ml 1 ). From this suspension,
100 ìl was transferred to 2.9 ml brain heart infusion broth (BBL)
supplemented with 10 % (v/v) new born calf serum (Sigma) and
H. pylori-selective supplement. The broth was then incubated with
agitation in a 50 ml capacity loose-capped container (Bibby Sterilin) in a
microaerobic atmosphere (see above) at 37 8C for about 48 h (bacilli) or
over 72 h (cocci). Aliquots were withdrawn periodically for assessment
of bacterial motility and viable count by the method of Miles et al.
(1938). Gram stain, oxidase and catalase tests were used to confirm
absence of contamination.
Motility measurements. The culture (containing bacilli or cocci) was
initially diluted with fresh medium to a concentration not exceeding
107 bacteria ml 1 . Aliquots (10 ìl) of the diluted culture were then
added to 90 ìl of 100 mM phosphate/citrate buffer pH 6.5, or gallbladder bile [diluted to 2.2 % (v/v) with phosphate/citrate buffer]. The
resulting bacterial suspension was drawn by capillary action into an
optical microslide (CamLab) of 0.1 mm path length. The microslide was
sealed at one end with vinyl plastic putty (Oxford Labware), transferred
to the warm stage (37 8C) of a phase-contrast microscope (340
magnification) and allowed to equilibrate for 5 min before observations
commenced with a Hobson BacTracker (Hobson Tracking Systems).
In this study, a single representative parameter was used to assess
H. pylori motility: curvilinear velocity (CLV); the distance in ìm
travelled along the path of the bacterium in each second between two
stops. To ascertain the contribution of Brownian movement to bacterial
motility, H. pylori was killed (no growth on horse blood agar) by
exposure to 10 % (v/v) formalin for 10 min at room temperature, before
examination with the BacTracker. Data collected with the BacTracker
were analysed by Mann–Whitney U-test with an SPSS (Social Sciences
Version 6) statistics package. A detailed description of the Hobson
BacTracker and its operation may be found in the manufacturer’s
literature.
Chemotaxis measurements. The technique used to determine how
808
H. pylori accumulates in gradients formed by bile, plasma or their
constituents was a modification of that described by Adler (1973).
Microcapillary tubes of 10 ìl capacity (Sigma) were filled with blood
plasma or gall-bladder bile, diluted to 1 and 2 % (v/v), respectively, in
100 mM phosphate/citrate buffer pH 6.5, or with blood plasma components (amino acids and urea) or selected bile acids, at concentrations
of 1 and 2 mM, respectively, in phosphate/citrate buffer, and then sealed
at the upper end with vinyl plastic putty (Oxford Labware). Control
microcapillaries, used to assess background accumulations of bacteria,
were filled with phosphate/citrate buffer alone. The open end of each
microcapillary tube was inserted into an Eppendorf tube of 0.5 ml
capacity, containing a suspension of the bacterium (bacilli or cocci) in
buffer (300 ìl) at a concentration of 106 cells ml 1 , and incubated
horizontally under microaerobic conditions for 45 min at 37 8C. After
this time, the contents of the upper two-thirds of the tube were expelled
onto a glass slide, and numbers of bacteria that had accumulated into the
microcapillary counted with a Hobson BacTracker. Each assay was
performed on seven different isolates (unless stated otherwise) and
repeated at least six times. To normalize data from different experiments, results were expressed as the chemotactic response (CR),
calculated as the number of bacteria in the test microcapillary, divided
by the number of bacteria in the control microcapillary.
Statistical analyses. The significance of results was tested using the
Mann–Whitney U-test. Differences at the P , 0.05 level were considered to be statistically significant.
RESULTS
Bacterial motility
The motility of the H. pylori bacillus was not significantly
diminished (when compared to that in phosphate/citrate
buffer), when the bacterium was exposed to diluted gallbladder bile for up to 6 h (Fig. 1).
Accumulation of bacilli and cocci in microcapillary
tubes
Bacillary and coccoidal forms of H. pylori were isolated from
the same culture, and their accumulations in microcapillary
tubes, containing phosphate/citrate buffer, 1 % blood plasma
or 2 % gall-bladder bile, were compared under identical test
conditions. Results are summarized in Fig. 2. The accumulation of the motile bacillary form of H. pylori in plasma was
found to be significantly greater (P , 0.001) and in gallbladder bile was found to be significantly less (P , 0.01) than
in buffer. In contrast, the accumulation of the non-motile,
non-flagellate (Worku et al., 1999) coccoidal form of H.
pylori was not significantly different in buffer, plasma or bile.
Chemotactic response to human plasma, urea and
some amino acids
Human plasma, diluted to 1 % (v/v) in phosphate/citrate
buffer, was found to be a significant chemoattractant for the
H. pylori bacillus (Table 1). The responsiveness of the bacillus
was also tested towards urea, which is a major constituent of
plasma (Snook, 1986), and 12 amino acids that occur in
human plasma (Stein & Moore, 1954). Results are sum-
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Journal of Medical Microbiology 53
H. pylori chemotaxis to plasma and bile
40
Table 1. Chemotactic response of H. pylori to human plasma,
human gall-bladder bile and selected constituents
Chemotaxis was measured under microaerobic conditions with a
modified Adler apparatus in which the control microcapillary contained
100 mM phosphate/citrate buffer, pH 6.5. Plasma and bile were diluted
in buffer. Amino acids and urea were at a concentration of 1 mM, and
bile acids were at a concentration of 2 mM in buffer. Data are results
from seven different isolates. Abbreviations: GCA, glycocholic acid;
TCA, taurocholic acid; GDCA, glycodeoxycholic acid; TDCA, taurodeoxycholic acid; GCDCA, glycochenodeoxycholic acid; TCDCA,
taurochenodeoxycholic acid.
CLV (µm s⫺1)
30
20
Substrate
10
0
Buffer
Bile 1
Bile 2
Bile 3
Fig. 1. Mean CLV of H. pylori in 100 mM phosphate/citrate buffer,
pH 6.5 (buffer), and after exposure to human gall-bladder bile diluted
to 2 % (v/v) in buffer for 1 h (bile 1), 3 h (bile 2) and 6 h (bile 3) at
37 8C. The histogram shows mean results from six different isolates.
Differences in mean CLV were without significance.
8⫻
104
Bacteria (ml⫺1)
6 ⫻ 104
Plasma 1 % (v/v)
Bile 2 % (v/v)
Plasma constituents
Urea
L-Alanine
L-Arginine
L-Aspartic acid
L-Asparagine
L-Glutamic acid
L-Glutamine
Glycine
L-Histidine
L-Leucine
L-Proline
L-Tyrosine
L-Valine
Bile acids
GCA
TCA
GDCA
TDCA
GCDCA
TCDCA
CR (mean 6 SD)
P value*
4.6 0.4
0.64 0.2
, 0.001
, 0.01
3.6 0.4
2.3 0.3
4.8 0.5
0.7 0.25
2.3 0.4
0.9 0.2
6.4 0.5
5.2 0.4
6.1 0.7
1.6 0.35
2.2 0.35
1.3 0.2
1.7 0.4
, 0.01
, 0.01
, 0.001
, 0.05
, 0.01
0.65 0.14
0.53 0.13
0.69 0.15
0.34 0.10
0.66 0.11
0.72 0.19
, 0.01
, 0.01
, 0.01
, 0.01
, 0.01
, 0.01
NS
, 0.001
, 0.001
, 0.001
, 0.05
, 0.01
NS
, 0.05
*Mann–Whitney U-test of means compared with control.
significant.
NS,
not
4 ⫻ 104
marized in Table 1. Urea and all the amino acids (apart from
aspartic acid and glutamic acid) acted as chemoattractants at
a concentration of 1 mM in phosphate/citrate buffer.
2 ⫻ 104
Chemotactic response to human bile and some bile
acids
0
Buffer
Plasma
Bile
Fig. 2. Accumulation of bacillary (filled bars) and coccoidal forms
(open bars) of H. pylori in microcapillary tubes containing 100 mM
phosphate/citrate buffer, pH 6.5, 1 % (v/v) plasma in buffer or 2 %
(v/v) gall-bladder bile in buffer. Mean SD CLV 32.1 4.7 ìm s 1
(bacilli) and 5.0 1.0 ìm s 1 (cocci). Mean CLV of bacilli killed by
exposure to formalin solution 5.5 1.2 ìm s 1 . The histogram shows
mean results from two different isolates.
http://jmm.sgmjournals.org
Human gall-bladder bile, diluted to 2 % (v/v) in phosphate/
citrate buffer, was found to be a significant chemorepellent
for the H. pylori bacillus (Table 1). The responsiveness of the
bacillus was also tested towards the six most abundant
conjugated bile acids in human bile (Lentner, 1981). Results
are summarized in Table 1. All the bile acids were found to be
significant chemorepellents at a concentration of 2 mM in
phosphate/citrate buffer, with the strongest chemorepellent
being taurodeoxycholic acid.
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809
M. L. Worku and others
DISCUSSION
H. pylori is a chemotactic bacterium in which sensor proteins
on the cell membrane are coupled to the flagellar motors
through two CheY response regulator proteins, three CheV
proteins (of uncertain function) and a histidine kinase sensor
CheA (Foynes et al., 2000; Pittman et al., 2001). The
importance of this chemoreceptor complex for effective
gastric colonization has been demonstrated in the failure of
motile, but non-chemotactic deletion mutants to survive in
an animal model (Foynes et al., 2000). In this study, we have
sought to clarify further the role of chemotaxis in H. pylori
colonization by examining how the bacterium responds
in vitro to human plasma and bile, secretions containing
chemoeffector compounds that are present in the gastric
mucus layer.
Comparative accumulation of bacillary and coccoidal forms
of H. pylori in microcapillary tubes, containing buffer,
plasma and gall-bladder bile, indicated that the motile
bacillus responds chemotactically (in our test system) to
both human plasma and bile. Highly diluted human plasma
was found to be a significant chemoattractant for the bacillus,
and highly diluted gall-bladder bile a significant chemorepellent.
Bile is reported to be bactericidal for H. pylori, although at
higher concentrations than those used in our experiments
(Tompkins & West, 1987). Our finding, that the motility of
the H. pylori bacillus is not significantly diminished on
prolonged exposure to diluted bile, is therefore of importance, because it established that the chemorepellent effect of
bile in our experiments was not simply an artefact due to loss
of bacterial viability.
The basis of the chemoattractant effect of diluted plasma was
explored by examining the chemotactic behaviour of urea
and several plasma amino acids. Measurements were conducted at a concentration of 1 mM, selected because it lies
well below the mean concentration range for free amino acids
[2.5–4 mM (Stein & Moore, 1954)] and for urea [5–
10.5 mM (Snook, 1986)] in human plasma, and was the
amino acid concentration that most often gave an optimum
chemotactic response with other bacteria (Mesibov & Adler,
1972; Moulton & Montie, 1979).
Urea and the amino acids histidine, glutamine, glycine and
arginine were the most potent chemoattractants amongst the
plasma components tested. All the other amino acids were
more moderate chemoattractants, except for aspartic and
glutamic acids, which were chemorepellents.
For most of the amino acids we could discern no relationship
between the magnitude of the mean chemotactic response
and the nature of the substituent group R in the generalized
amino acid formula: R.CH.NH2 .COOH. Exceptions were
the chemorepellents aspartic and glutamic acids, where R
includes a carboxyl group. Our experiments suggest that the
carboxylamide group, -CO.NH2 , might be an important
binding structure for H. pylori receptors, because (i) conversion of aspartic and glutamic acids to their respective
810
amides turned chemorepellents into chemoattractants and
(ii) this functional group is present in urea, one of the
strongest chemoattractants tested.
Our finding that arginine and urea are significant attractants
for H. pylori agrees with earlier reports (Mizote et al., 1997;
Cerda et al., 2003). The response of the bacterium to these
two plasma constituents (and to bicarbonate ion) has
recently been linked with expression of the HP0099 gene
(Cerda et al., 2003).
The basis of the chemorepulsive effect of diluted gall-bladder
bile was explored by examining the chemotactic behaviour of
the six most abundant conjugated bile acids in the secretion
(Lentner, 1981). Measurements were conducted at a concentration of 2 mM, reflecting the mean concentration range
for combined bile acids in human gall-bladder bile (Arianoff,
1966; Nakayama, 1967) after dilution to 2 % (v/v) in buffer.
All the bile acids were significant chemorepellents for the
H. pylori bacillus at this concentration, with the greatest
effects being demonstrated by taurocholic and taurodeoxycholic acids. We are uncertain of the structural basis of this
repellent action, although our findings with aspartic and
glutamic acids (see above) suggest that there might be an
involvement of free or esterified carboxyl groups in these
surfactant molecules.
Implications for gastric colonization
The manner in which H. pylori colonizes the gastric microenvironment (Hazell et al., 1986; Thomsen et al., 1990)
implies that chemotactic gradients, which draw the bacterium into the epithelial surface, are present across the mucus
layer.
Our in vitro experiments strengthen the view that an
important chemotactic gradient across the mucus layer is
formed from components of plasma that transude from
microcapillaries in the gastric mucosa. They showed, for
instance, that plasma is a significant chemoattractant for the
H. pylori bacillus at a dilution greater than that which
normally occurs in the gastric lumen [between two and four
times, when based on a urea marker (Piper et al., 1967;
Snook, 1986)], and therefore at a dilution greater than that
which must occur at the epithelial surface of the H. pyloriinfected mucus layer.
Our in vitro experiments also strengthen the view that an
important chemotactic gradient across the mucus layer is
formed from components of bile that reflux into the
stomach. They showed, for instance, that bile acids are
significant chemorepellents for the H. pylori bacillus at a
concentration of 2 mM. This concentration is likely to be
attained post-prandially by combined bile acids in the pyloric
antrum (Han et al., 1996), although less so in the body of the
normal acid-secreting stomach (Dixon et al., 1986;
Schindlbeck et al., 1987)
Bile and plasma chemotactic gradients across the gastric
mucus layer may, therefore, be influential in directing
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Journal of Medical Microbiology 53
H. pylori chemotaxis to plasma and bile
H. pylori to the pyloric antrum, and in enabling this
important pathogen to attain high population densities at
the epithelial surface of the gastric mucus layer.
Jarnum, S. & Jensen, K. B. (1972). Plasma protein turnover (albumin,
transferrin, IgG, IgM) in Menetrier’s disease (giant hypertrophic
gastritis): evidence of non-selective protein loss. Gut 13, 128–137.
Khulusi, S., Mendall, M. A., Patel, P., Levy, J., Badve, S. & Northfield,
T. C. (1995). Helicobacter pylori infection density and gastric inflamma-
ACKNOWLEDGEMENTS
tion in duodenal ulcer and non-ulcer subjects. Gut 37, 319–324.
We wish to thank Abbott Laboratories and the University of London for
their gifts of equipment used in this work.
King, P. M., Adam, R. D., Pryde, A., McDicken, W. N. & Heading, R. C.
(1984). Relationships of human antroduodenal motility and transpy-
loric fluid movement: non-invasive observations with real-time ultrasound. Gut 25, 1384–1391.
REFERENCES
Adler, J. (1973). A method for measuring chemotaxis and use of the
method to determine optimum conditions for chemotaxis by Escherichia coli. J Gen Microbiol 74, 77–91.
Arianoff, A. A. (1966). Etude de la composition de la bile humaine
normale et pathologique. T-sels billiares cholestero electrolytes. Acta
Gastro-ent Belg 29, 733–738 (in French).
Lentner, C. (1981). Bile. In Geigy Scientific Tables: Units of Measurement,
Body Fluids, Composition of the Body, Nutrition, vol. 1, pp. 134–146.
Edited by C. Lentner. Basle: Ciba-Geigy.
Mesibov, R. & Adler, J. (1972). Chemotaxis toward amino acids in
Escherichia coli. J Bacteriol 112, 315–326.
Miles, A. A., Misra, S. S. & Irwin, J. O. (1938). The estimation of the
bactericidal power of blood. J Hyg 38, 732–748.
Bell, A. E., Sellers, L. A., Allen, A., Cunliffe, W. J., Morris, E. R. & RossMurphy, S. B. (1985). Properties of gastric and duodenal mucus: effects
Mizote, T., Yoshiyama, H. & Nakazawa, T. (1997). Urease-independent
of proteolysis, disulfide reduction, bile, acid, ethanol and hypertonicity
on mucus gel structure. Gastroenterology 88, 269–280.
chemotactic responses of Helicobacter pylori to urea, urease inhibitors,
and sodium bicarbonate. Infect Immun 65, 1519–1521.
Brassinne, A. (1974). Gastric clearance of serum albumin in normal
Moulton, R. C. & Montie, T. C. (1979). Chemotaxis by Pseudomonas
man and in certain gastroduodenal disorders. Gut 15, 194–199.
Brassinne, A. (1979). Effect of ethanol on plasma protein shedding in
the stomach. Dig Dis Sci 24, 44–47.
aeruginosa. J Bacteriol 137, 274–280.
Nakayama, F. (1967). Quantitative microanalysis of bile. J Lab Clin Med
69, 594–609.
Cerda, O., Rivas, A. & Toledo, H. (2003). Helicobacter pylori strain
ATCC700392 encodes a methyl-accepting chemotaxis receptor protein
(MPC) for arginine and sodium bicarbonate. FEMS Microbiol Lett 224,
175–181.
Davenport, H. W. (1967). Absorption of taurocholate-24-14 C through
the canine gastric mucosa. Proc Soc Exp Biol Med 125, 670–673.
Dixon, M. F., O’Connor, H. J., Axon, A. T. R., King, R. F. J. G. & Johnson, D.
(1986). Reflux gastritis: distinct histopathological entity? J Clin Pathol
39, 524–530.
DuPlessis, D. J. (1965). Pathogenesis of gastric ulceration. Lancet i,
974–978.
Piper, D. W., Fenton, B. H. & Goodman, L. R. (1967). Lactic, pyruvic,
citric, and uric acid and urea content of human gastric juice. Gastroenterology 53, 42–48.
Pittman, M. S., Goodwin, M. & Kelly, D. J. (2001). Chemotaxis in the
human gastric pathogen Helicobacter pylori: different roles for CheW
and the three CheV paralogues, and evidence for CheV2 phosphorylation. Microbiology 147, 2493–2504.
Schindlbeck, N. E., Heinrich, C., Stellaard, F., Paumgartner, G. &
Muller-Lissner, S. A. (1987). Healthy controls have as much bile reflux
as gastric ulcer patients. Gut 28, 1577–1583.
Foynes, S., Dorrell, N., Ward, S. J., Stabler, R. A., McColm, A. A., Rycroft,
A. N. & Wren, B. W. (2000). Helicobacter pylori possesses two CheY
Snook, J. (1986). Urea/creatinine ratio and gastrointestinal haemor-
response regulators and a histidine kinase sensor, CheA, which are
essential for chemotaxis and colonization of the gastric mucosa. Infect
Immun 68, 2016–2023.
Sobala, G. M., O’Connor, H. J., Dewar, E. P., King, R. F. G., Axon, A. T. R. &
Dixon, M. F. (1993). Bile reflux and intestinal metaplasia in gastric
Freter, R. (1980). Prospects for preventing the association of harmful
Sober, H. A., Hollander, F. & Sonnenblick, B. T. (1950). Response of
bacteria with host mucosal surfaces. In Bacterial Adherence: Receptors
and Recognition, series B, vol. 6, pp. 441–458. Edited by E. H. Beachey.
London: Chapman & Hall.
Han, S. W., Evans, D. G., el-Zaatari, F. A. K., Go, M. F. & Graham, D. Y.
(1996). The interaction of pH, bile and Helicobacter pylori may explain
rhage. Lancet ii, 400.
mucosa. J Clin Pathol 46, 235–240.
gastric mucous barrier in pouch dogs to repeated topical application of
eugenol. Am J Physiol 162, 120–130.
Stein, W. H. & Moore, S. (1954). The free amino acids of human blood
plasma. J Biol Chem 211, 915–926.
duodenal ulcer. Am J Gastroenterol 91, 1135–1137.
Talamini, G., Zamboni, G. & Cavallini, G. (1997). Antral mucosal
Hazell, S. L., Lee, A., Brady, L. & Hennessy, W. (1986). Campylobacter
Helicobacter pylori infection density as a risk factor of duodenal ulcer.
Digestion 58, 211–217.
pyloridis and gastritis: association with intercellular spaces and adaptation to an environment of mucus as important factors in colonization of
the gastric epithelium. J Infect Dis 153, 658–663.
Hollander, F. & Horowitz, M. I. (1962). Serum proteins in gastric mucus
Thomsen, L. L., Gavin, J. B. & Tasman-Jones, C. (1990). Relation of
Helicobacter pylori to the human gastric mucosa in chronic gastritis of
the antrum. Gut 31, 1230–1236.
and other secretions. Implications in relation to the protein-losing
enteropathies. Gastroenterology 43, 75–83.
Tompkins, D. S. & West, A. P. (1987). Campylobacter pylori, acid and
Hugdahl, M. B., Beery, J. T. & Doyle, M. P. (1988). Chemotactic behavior
Worku, M. L., Sidebotham, R. L., Walker, M. M., Keshavarz, T. & Karim,
Q. N. (1999). The relationship between Helicobacter pylori motility,
of Campylobacter jejuni. Infect Immun 56, 1560–1566.
Ivey, K. J., DenBesten, L. & Bell, S. (1970). Absorption of bile salts from
human gastric mucosa. J Appl Physiol 29, 806–808.
http://jmm.sgmjournals.org
bile. J Clin Pathol 40, 1387.
morphology and phase of growth: implications for gastric colonization
and pathology. Microbiology 145, 2803–2811.
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