Download Factors affecting growth and antibiotic susceptibility of Helicobacter

J. Med Microbiol. - Vol. 44 (1 996), 425433
0 1996 The Pathological Society of Great Britain and Ireland
BACTERIAL PATHOG EN IClTY
Factors affecting growth and antibiotic
susceptibility of Helicobacter pylori: effect of pH
and urea on the survival of a wild-type strain and a
urease-deficient mutant
J. E. SJOSTROM and H. LARSSON"
Departments of Cell Biology and *Gastrointestinal Pharmacology Preclinical Research and Development, Astra
Hassle AB, Molndal, Sweden
This study investigated how pH and the presence of urea affect the survival and growth
of Helicobacter pylori and whether these factors affect susceptibility to antibiotics in
vitro. The viability of a wild-type strain and a urease-deficient mutant of H. pylori was
studied after incubation for 1h in buffers at different pH values at 37°C under microaerophilic conditions. Viable counts were not affected at pH 5 and pH 7. In buffer at pH
3, there were no viable organisms, but urea (6.25 mM) protected the wild-type strain,
which survived well. At pH 9, urea further reduced the viability of H. pylori and
flurofamide almost abolished the effect of urea on the wild-type strain. Neither urea nor
flurofamide affected the viability of the urease-deficient mutant under the same
conditions. Growth was also pH dependent and was enhanced in shake-cultures. At pH
5, urea supported growth of the wild-type strain, but at pH 7 a toxic effect on the
bacteria was observed. Growth of H. pylori at pH 5.9 was poor, and susceptibility to
amoxycillin, erythromycin and clarithromycin was markedly less than at pH 7.2 and 7.9.
The bactericidal activities of metronidazole and tetracycline were similar at the different
pH values studied. At neutral pH the killing rates of amoxycillin and clarithromycin
were growth rate dependent. Susceptibility to metronidazole was enhanced in stationary
cultures. The interaction obtained between the proton pump inhibitor, omeprazole, and
amoxycillin at pH 7 was of additive type. These results suggest that pH and growth
conditions may be important in the antibacterial efficacy of different antibiotics in vivo
and also provide a possible explanation for the potentiating effect of omeprazole with
antibiotics in the treatment of H. pylori infections.
Introduction
Colonisation of the gastric antral mucosa by Helicobacter pylori is strongly associated with active and
chronic gastritis as well as with peptic ulcer diseases
[l, 21. Despite its ability to colonise the gastric mucosa,
the organism does not survive in-vitro conditions of
high acidity [3], and growth is restricted to a pH range
of 6.5-7.5 [4,5]. The pH for optimum growth of H.
pylori is reported to be c. 7.0 [6]. Recently, and as an
exception to what others have found, Kangatharalingam
and Amy [7] reported growth of H. pylori at a pH
value as low as 4.5 in a modified Brucella broth
medium without serum. In vitro, urea increased
survival at low pH [3] but Greig et al. [8] observed a
self-destruction of bacteria in the presence of urea at
Received 5 May. 1995; revised version accepted 6 Nov.
1995.
Corresponding author: Dr J. E. Sjostrom.
pH 6. The effect was prevented by addition of
hydroxyurea and was not seen at pH 7, showing that
the bactericidal effect was dependent on pH and urease
activity.
All clinical isolates of H. pylori show urease activity,
which is believed to facilitate colonisation [9, 101 by
generating ammonia to buffer gastric acidity [ll].
Administration of the urease inhibitor, flurofamide, to
rats infected with H. felis did not affect the growth of
H. felis, showing that there is no requirement for
urease activity after the bacteria have reached their
unique niche beneath the mucus layer of the gastric
mucosa, where pH may be close to neutrality [12, 131.
Antimicrobial agents generally display good activity
against H. pylori in vitro [14]. However, the
bactericidal activity of single antibiotics against H.
pylori in vivo is very poor [15-171 and they do not
succeed in eradicating the organism. Apart from
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J. E. SJOSTROM AND H. LARSSON
antibiotics combined with acid inhibitors, the most
effective treatment regimen for the eradication of H.
pylori [18, 191, given good compliance by patients
[20], is triple therapy with bismuth subcitrate and two
antimicrobial agents (tetracycline or amoxycillin and
metronidazole).
A possible explanation for the success of combined
therapy may reside in the synergy between metronidazole and amoxycillin or tetracycline that has been
described in vitro [21]. Bismuth subcitrate also
interacts synergically in vitro with several antimicrobial agents, including metronidazole and b-lactams
[22]. The antimicrobial activity of several antibiotics
in vitro is also affected by pH, with increased MICs
under acid conditions [23-251. The synergic effect
obtained in vivo between amoxycillin and the proton
pump inhibitor, omeprazole [26], is probably due to
the increase in pH in the vicinity of H. pylori,
improving the conditions for antibiotic activity. More
recently, optimal short-term treatment of H. pylori
infections with omeprazole in combination with two
antibiotics has been recommended [27,28]. In vitro,
the culture conditions, affecting growth rates [29], as
well as whether the bacteria are planktonic or adherent
to cells [30], are other important determinants of
antibiotic susceptibility.
The aim of the work reported here was to investigate
how pH affects the growth and survival of H. pylori
and its susceptibility to different antibiotics in vitro, in
order to provide a possible explanation for variations
in antibiotic efficacy in vivo.
Materials and methods
Bacterial strains
The strains of H. pylori used in this study are listed in
Table 1. Stock cultures were stored at -70°C in
Brucella Broth (Difco) with heat-inactivated (56"C,
30min) fetal calf serum (FCS) lo%, pH 7.0,
supplemented with glycerol 20%. The type strains of
H. pylori selected for this study were NTCC 11637 and
CCUG 15818. Strains AH 5 and AH 28 from our
collection were clinical isolates from duodenal ulcer
patients. Strains N6, wild-type, and N6Kmy its
isogenic urease-deficient mutant [311, were kindly
provided by A. Labigne, Institute Pasteur, Paris.
Table 1. H. pylori strains used
~~
Strain no.
~~
~~
Genotypelphenotype
NTCC 11637
CCUG 15818
N6
N6 KmV
AH5
AH28
*MtzR, metronidazole resistant.
MtzR*
MtzR
MtzR, KmR, ureBt
t Kanamycin-resistant transformant. The ureB gene is disrupted by
insertion of a mini-Tn3-Km transposon.
Media and buflers
Solid medium for H. pylori was Columbia blood agar
comprising Columbia Agar Base I1 (Oxoid) 42.5 g/L,
Bactoagar (Oxoid) 15 g/L, horse blood 7% and
IsoVitale X (BBL Microbiology System) 1%, pH
7.3 5 0.2. Growth medium was, if not stated otherwise,
Brucella Broth (Difco) supplemented with FCS lo%,
pH 7.0. Brucella broth pH 5.0 was adjusted to that pH
with HC1.
Brucella broth, pH 5.9 and 7.9, was prepared in
66 m~ sodium-phosphate buffer. Citrate-phosphate
H
b3.0, pH 5.0, pH 7.0) and glycine-NaOH
(pH 9.0) were used at 100 mM for studying the
survival of bacteria. Phosphate-buffered saline (PBS)
(pH 7.2) was used for washing and dilution of
bacteria.
Chemicals
Urea and antibiotics were purchased from Sigma.
Flurofamide was synthesised by Synthelec AB (Lund,
Sweden) and omeprazole was from Astra Hassle AB
(Molndal, Sweden). Other salts and solvents were from
commercial sources and were of the highest purity
available.
Growth conditions
Bacteria stored at -70°C were thawed and subcultured
twice on Columbia blood agar for 2-3 days at 37°C in
an automatic C02/02 incubator (Forma Scientific),
under micro-aerophilic conditions ( N 2 85%, C02 lo%,
0 2 5%) before being used in an experiment or as an
inoculum for broth cultures. Viable counts, expressed
as cfidml, were determined on blood agar after washing
and dilutions in PBS, and colonies were counted after
incubation for 3 days under the conditions described
above.
Determination of growth and p H
Growth of bacteria in liquid media was determined by
reading the optical density in a spectrophotometer
(Shimadzu UV-120-01) at 560 nm or in a microtitration
plate reader (Molecular Devices) at the same wavelength. pH was monitored with a glass pH electrode
(Radiometer, Copenhagen).
Determination of surviving bacteria in buSfer
H. pylori strains grown on Columbia blood agar plates
were harvested and washed in PBS by centrifugation at
4000 rpm for 10 min at room temperature and resuspended in tubes with 1.0 ml of sterile buffer at different
pH values. Cell density was adjusted to lo7-lo8 cfid
ml. Viable counts were determined by a modified
Miles and Misra technique [32] by dilution in PBS and
spreading on agar plates at time zero, and after
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EFFECT OF pH AND UREA ON SURVIVAL OF H. PYLORZ
different incubation times under microaerophilic conditions at 37°C. The results were expressed as mean
and SEM. The survival of bacteria under different
conditions was compared by the paired t test.
Determination of MBC and fractional inhibitory
concentration (FIC) index
Minimal bactericidal concentrations (MBCs) for amoxycillin, tetracycline, erythromycin, clarithromycin and
metronidazole dissolved in dimethylsulphoxide
(DMSO) were determined by serial dilutions in
Brucella broth plus FCS 10% at pH 5.9, 7.2 and 7.9,
at a cell density of lo6 cWml in 96-well microtitration
plates. After incubation for 72 h at 37°C under microaerophilic conditions, lop1 from each well were
applied with a replicator to large (120 mm X
120 mm X 17 mm) Columbia blood agar plates. The
plates were read after incubation for 72 h. MBC was
defined as the lowest concentration of the compound
427
giving >99.9% killing. The combined activity of
omeprazole and amoxycillin was quantified by the
simplified broth dilution checkerboard technique, in
Brucella broth plus FCS 10% at pH 7 against the type
strains NTCC 11637 and CCUG 15818, as described
by Krogstad and Moellering [33]. With this method
synergy is defined as a fractional inhibitory concentration (FIC) index S0.5; additivity as an FIC index of
0.5-1.0 and antagonism as an FIC index of 1.0-2.0.
Results
Survival of non-growing bacteria at diflerent p H
values and the effect of urea
The decrease in viability of H.pylori strains N6 and
N6KmV in buffers with and without urea is shown in
Fig. 1. When urea was added to buffer at a final
concentration of 6.25 m~ at pH 3 and pH 9, the cell
viability for the wild-type strain N6 was markedly
a
*
E
\
3
o
Y-
.-C
8
a
sa
U
b
8
7
6
5
4
3
2
1
0
3
I
5
I
7
I
9
I
pH value
Fig. 1. Loglo decrease in survival of bacteria after 1 h in buffers at different pH values at 37°C under micro-aerophilic
conditions. a, H. pylori N6 (wild-type); b, H. pylori N6 KmV (urease-deficient). 0, no additions; W, 4 0 p ~
M
+ 6.25 mM urea. Each bar represents the mean and SEM (n = 4).
flurofamide; , 6.25 mM urea; , 40 ~ U flurofamide
*p < 0.05, **p < 0.01, ***p < 0.001.
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428
J. E. SJOSTROM AND H. LARSSON
affected during the incubation period of 1 h. At pH 3,
survival was significantly increased (p < O.OOl), probably depending on the ammonium ions formed by the
urease activity, which results in an increase in pH
adjacent to the bacteria. This was reflected by an
increase in buffer pH to 3.65 (data not shown).
Addition of flurofamide, an inhibitor of urease, reduced
the protective effect exerted by the urease reaction
(p < 0.01). In contrast, at pH 9, urea further significantly reduced viability (p < 0.05) and all bacteria
were killed. However, if flurofamide was added
simultaneously to a final concentration of 40,uM, the
effects of urea at both low and high pH values were
reduced. At pH 5 and 7, the numbers of viable bacteria
a
0.70
0.60
0.50
pH5
-
PH 7
-
0.40
0.30
0.20
0.10
0.00
0
0
72
8
2
Om70
1
b
PH 7
0
0
72
72
Time (h)
Fig. 2. Growth of H. pylori in Brucella broth plus FCS 10% at 37°C under micro-aerophilic conditions at pH 5 and 7
a, Strain N6 (wild-type); b, N6 KmV (urease-deficient). 0, Brucella broth; U, Brucella broth + 6.25 mM urea.
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EFFECT OF pH AND UREA ON SURVIVAL OF H. PYLORZ
were unaffected by addition of urea or flurofamide. The
urease-deficient mutant was not significantly affected
by urea or flurofamide under any of the test conditions,
and no alterations in pH values were observed.
Effect of urea on growth of H. pylori at different
pH values
H. pylori strains N6 and N6KmV were grown in
microtitration plates in Brucella broth with FCS 10% at
pH 5 and pH 7 in the presence or absence of 6.25 m~
urea. The absorbance at 560 nm and the pH were read
at the start and after incubation for 72 h at 37°C under
micro-aerophilic conditions. The results in Fig. 2
show that neither the wild-type strain nor the ureasedeficient mutant grew at pH 5 , and that growth was
obtained for the wild-type strain at pH 5 only in the
presence of urea. During incubation, the medium pH
increased to 7.1 (data not shown). However, at a
starting pH of 7, there was a reduction in growth of the
wild-type strain when urea was added, and pH increase
to 7.9 compared to 7.5 for the urease-deficient mutant
(data not shown). Growth of the urease-deficient
mutant at pH 5 and at pH 7 was not affected by
addition of urea.
Growth and antibiotic susceptibility at different
pH values
Four strains of H. pylori were grown in microtitration
plates in Brucella broth plus FCS 10% at different pH
values at 37°C under micro-aerophilic conditions. The
increase in absorbance was determined after incubation
for 72 h. At pH 5.9, growth was slower than at pH 7.2
(Table 2). At pH 7.9 the bacteria were again affected,
and growth was similar to that obtained at pH 5.9.
At pH 5.9, the bactericidal activities of amoxycillin,
clarithromycin and erythromycin were poor, but at pH
7.2 and 7.9, the bacterial susceptibility was increased,
resulting in much lower MBCs (Table 2). The
susceptibility to amoxycillin increased 10-2O-fold,
and even more for the macrolides. The lowest MBCs
were achieved at pH 7.9. The MBCs for tetracycline
and metronidazole were not appreciably affected by
pH. Only strain A H 5 was sensitive to metronidazole.
Growth of H. pylori in flasks with and without
shaking
H. pylori strains NTCC 11637 and AH 28 were
incubated in 125-ml flasks with 20 ml of Brucella
broth plus FCS 10% at pH 7.0, and the effect of
shaking on growth was studied by incubating the flasks
with and without shaking at ISOrpm, at 37°C under
micro-aerophilic conditions. Growth was followed by
determination of viable counts (cfidml). Fig. 3 shows
the results from three different experiments. There was
an increase of >1 loglo cfidml during the first 12 h of
incubation with shaking. Mean Alog for strains NTCC
11637 and AH 28 was 1.528 SEM 0.1043 and 1.246
SEM 0.1429, respectively. The numbers of growing
bacteria were then rapidly reduced, especially for strain
AH 28. The growth pattern in stationary cultures was
different and probably reflects a steady state. The
increase in viable counts was poor, if any, and no
reduction in viability was observed during the incubation period of 72 h studied. The difference in growth
measured after incubation for 12 h between shaken and
non-shaken cultures was significant (NCTT 11637,
p < 0.01 and AH 28, p < 0.01). The fact that growth
was much better in shaking flasks suggests that a good
transfer of the gas mixture into the broth medium
improves growth of H. pylori.
Killing activity of amoxycillin, clarithromycin and
metronidazole in diflerent bacterial growth
conditions
The difference in growth of H. pylori with or without
shaking (Fig. 3) was used to study whether the killing
Table2. MBCs for different antibiotics against H.pylori at pH 5.9, 7.2 and 7.9
~~
Strain no.
Cla
~
pH value
AA560
Amox
Ery
N6
N6 KmV
AH5
NTCC1 1 637
5.9
5.9
5.9
5.9
0.23
0.09
0.04
0.10
0.2
0.1
N6
N6 KmV
AH5
NTCC 1 1637
7.2
7.2
7.2
7.2
0.55
0.60
0.14
0.32
0.025
0.012
0.05
0.1
0.025
0.012
0.025
0.1
0.12
0.25
0.12
N6
N6 KmV
AH5
NTCC 1 1637
7.9
7.9
7.9
7.9
0.03
0.16
0.03
0.13
0.012
0.0062
0.0031
0.0031
0.0016
0.0031
0.031
0.031
0.016
0.062
~
Tet
Met
0.5
0.5
ND
ND
1
ND
~
>0.4
>0.4
0.05
0.05
429
B0.4
0.4
>0.4
B0.4
>4
>4
>4
>4
1
2
ND
ND
1
ND
0.5
~
Amox, amoxycilljn; Cla, clarithromycin; Ery, erythromycin; Tet, tetracycline; Met, metronidazole.
ND, not done (strains are MtzR).
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0.25
0.25
1
1
ND
ND
1
ND
430
J. E. SJOSTROM AND H. LARSSON
9 1
a
8
7
6
5
4
3
2
1
-
0
I
E
I
12
\
3
0
I
I
I
I
I
24
36
48
60
72
.c
I
9
CJ)
1
0
b
9
8
7
6
5
4
3
2
1
1 -
0 '
I
I
I
I
1
1
12
24
36
48
60
72
1
Time (h)
Fig. 3. Growth patterns from three experiments with: a H.pylori NTCC 11637; b, AH 28, in Brucella broth plus FCS
lo%, pH 7.0. 0 , Shake-cultures; 0, stationary cultures. The difference in growth rate (12 h) between shake-cultures
and stationary cultures is statistically significant (p < 0.01 for both strains).
rate for amoxycillin, clarithromycin and metronidazole
was growth dependent at pH 7. The results obtained for
H. pylori strain AH 28 (Fig. 4) show that the
bactericidal activity exerted by amoxycillin was slow
in comparison with that of clarithromycin and metronidazole. The effect of amoxycillin and clarithromycin
on slow or non-growing bacteria in the stationary
culture was reduced compared with the effect on fastgrowing bacteria in the shake-culture, indicating that
the effect of amoxycillin and clarithromycin was
growth rate dependent. For metronidazole, the highest
killing rate was obtained in flasks without shaking,
where the reduction in viable bacteria was almost
4 loglo cWml during incubation for 12 h. The results
from this and other experiments with two different
strains (NTCC 11374 and AH 28) show that the
differences in killing rate between fast- and slowgrowing bacteria are significant: amoxycillin (n = 4)
p < 0.05, clarithromycin (n = 3) p < 0.05, and metronidazole (n = 3) p < 0.001.
Interaction between omeprazole and amoxycillin
The results obtained in checkerboard titration for the
type strain of H. pylori NTCC 11637 and strain CCUG
15818 (FIC index = 0.625) indicated that the combined
activity of the proton pump inhibitor, omeprazole
(MIC = 64 pg/ml), and amoxycillin (MIC = 0.1 pg/ml)
was additive rather than synergic.
Discussion
Although H. pylori is a fastidious organism requiring
special culture conditions in vitro, i.e., micro-aerophilic
or COz 5-10% environments with an optimum temperature of 35"-37"C at pH 6.5-7.5 [34,35], it adapts
to a unique niche beneath the mucus layer of the
gastric mucosa. Studies of the pH at this location in
experimental animals indicate a pH of c. 7.0 [12, 131,
and, therefore, protection against acid may not be
required after adhesion of bacteria to epithelial cells.
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EFFECT OF pH AND UREA ON SURVIVAL OF H. PYLORZ
431
9
8
7
6
-
2€0
z
0
3
5
4
3
2
1
0
I
I
I
I
I
I
I
1
3
5
7
9
11
13
Time (h)
Fig. 4. Growth and killing rates for different antibiotics in shaken (0)and stationary (0)cultures of H. pylori strain
AH 28 grown in Brucella broth plus FCS 10% at pH 7.0 (-) and with antibiotics (- -) - a, amoxycillin 0.25 pglml; c,
clarithromycin 0.25 pglml; my metronidazole 4 pglml. The results of a single experiment are shown.
-
However, the passage through the acid lumen requires
protective bacterial urease activity [9, lo]. These results
clearly show that, in vitro, H. pylori is sensitive to acid
both in buffer and in broth, and that supplementation
with urea increased survival at pH 3. We suggest that
survival at this low pH is accomplished by a sufficient
increase in the pH immediately surrounding the
bacteria by the action of urease on urea, resulting in
ammonium ion production. This was also reflected by
an increase in medium pH to 3.65. These results are in
agreement with those previously described by Marshall
et al. [3]. Comparison of the effects of the potent
urease inhibitor, flurofamide, and wild-type strain on a
urease-deficient mutant suggests that the protection is
mediated by urease.
to urea supplementation. To increase the buffer
capacity of Brucella broth and keep pH at a relatively
stable level, the broth was prepared in 66 mM sodiumphosphate buffer, and in this medium the lowest pH at
which H. pylori grew was 5.9. The optimum pH for
growth of H. pylori in vitro has been reported to be c.
7.0 [5,6], which fits with the data in the present
study. However, Kangatharalingam and Amy [7]
recently reported growth of H. pylori at pH as low
as 4.5, and urea neither stimulated growth further nor
led to an increase in pH. Whether these contradictory
results depend on the modified Brucella medium used
by the group or other factors was not discussed by the
authors.
In contrast to the finding at pH 3, supplementation
with urea at both pH 7 and pH 9 adversely affected
growth and cell viability. A suicidal effect reported by
Greig et al. [8] was also suggested to be due to the
urease activity. The killing mechanism at high pH in
the presence of urea may, according to Neithercut et
al. [36], depend on an imbalance in ammonium
metabolism, resulting in a lack of a-ketoglutarate,
which is a necessary intermediate in the biosynthesis
of the glutamate family of amino acids.
H. pylori does not grow aerobically [37] and is
categorised as an obligate micro-aerophile [7]. Shahmat et al. [38] compared a stationary growth system
with an agitated system, and growth in shaken culture
was much less than that in the stationary system.
However, the present results are in agreement with
those reported by Morgan et al. [6], showing only
slight growth in flasks incubated without shaking.
These data indicate that a good dispersion of the gas
mixture throughout the liquid medium is essential for
optimal growth of H. pylori.
Wild-type H. pylori did not grow in Brucella broth at
pH 5 except in the presence of 6.25 m~ urea,
resulting in an increase in pH to 7 after incubation
for 72 h. The urease-deficient mutant did not respond
MBCs for amoxycillin, erythromycin and clarithromycin were much higher at low pH. The reason for these
differences in antibacterial activity depend either on
pH effects directly on the antimicrobial agents, on
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J. E. SJOSTROM AND H. LARSSON
lower expression of bacterial targets or on lower
binding. The effects of pH on the antibacterial activity
of a range of antibiotics against H. pylori and other
bacteria have been studied previously [14,39] and, in
accordance with what was reported, the present results
show that susceptibility to tetracycline and metronidazole was not pH-dependent and not affected by the
poor growth obtained at pH 5.9 and 7.9. On the
contrary, slow-growing bacteria were more susceptible
to metronidazole than fast-growing ones. However,
this could be due to more anaerobic conditions
prevailing in the flask without shaking, thus improving
the anaerobic killing mechanism of metronidazole.
The results of the present study further show that, at a
higher growth rate at neutral pH, the bactericidal
activity of amoxycillin and clarithromycin was enhanced, possibly due to an increase in target expression. However, the susceptibilities for amoxycillin,
erythromycin and clarithromycin seem to be more pHdependent than growth-dependent, as the poor growth
at pH 7.9 compared to 7.2 did not increase MBCs.
Growth patterns of strain N6 (the wild-type strain),
and the isogenic urease-deficient mutant (N6KmV)
seem to be different. At pH 5.9, growth of the wildtype strain was better than that of the mutant.
However, at pH 7.9 the opposite was true, which
further strengthens the difference in response to pH
for the wild-?ype and the mutant strains.
However, these results clearly show that both the pH
of growth medium and the growth rate of H. pylori
affect antibiotic susceptibility in vitro, and we suggest
that these factors may also play an important role in
antibacterial activity in vivo.
The data provide further support for the hypothesis
that, in vivo, omeprazole exerts a synergic effect with
amoxycillin and other antibiotics [26,40], probably by
improving growth condition for H. pylori and thus
improving the conditions for antibacterial action in
vivo. Furthermore, the interaction obtained between
omeprazole and amoxycillin in vitro was additive
rather than synergic, a fact that would refbte the
suggestion that synergy in vivo is exerted via a direct
action on the bacterium. The poor antibiotic effect
exerted in vivo by a single drug may be attributed to
the fact that important factors forgrowth of H. pylori
or for antibacterial efficacy vary substantially between
different parts of the gastric and duodenal mucosa.
These results suggest that pH, growth rate and
possibly the partial pressure of oxygen, affecting the
killing mechanism of antibiotics, are such important
factors. This interpretation provides support for the
findings that omeprazole, in combination with antibiotics, enhances the efficacy of eradication of H.
pylori.
We thank Dr J. Fryklund for valuable discussions, M-L. Berglund for
excellent technical assistance, U. Edvardsson for statistical analysis
and L. Ruehl for typing the manuscript.
References
1. Graham DY. Helicobacter,pylori: its epidemiology and its role
in duodenal ulcer disease. J Gastroenterol Hepatol 1991; 6:
105-1 13.
2. Graham DY, Malaty HM, Evans DG, Evans DJ, Klein PD,
Adam E. Epidemiology of Helicobacter pylori in an asymptomatic population in the United States. Effect of age, race, and
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