Antimicrobial susceptibility testing of Helicobacter pylori in a large multicenter trial: the MACH 2 study.
ABSTRACT Culture and susceptibility testing of Helicobacter pylori strains was performed in a large multinational, multicenter randomized clinical trial. Culture was carried out on gastric biopsy samples obtained from 516 patients at entry and had a sensitivity of 99% when the [(13)C]urea breath test was used as a reference. Susceptibility testing was performed for clarithromycin and metronidazole on 485 strains by an agar dilution method and the epsilometer test (Etest) and for amoxicillin by an agar dilution method only. Resistance to clarithromycin (>1 microgram/ml) was found in 3% of the H. pylori strains, with a perfect correlation between Etest and agar dilution methods. Resistance to metronidazole (>8 microliter/ml) was found in 27% of the strains by agar dilution, but there were important discrepancies between it and the Etest method. No resistance to amoxicillin was found. The logarithms of the MICs of the three antibiotics against susceptible strains had a distribution close to normal. The impact of resistance was tested in the four arms of the trial. There were not enough clarithromycin-resistant strains to evaluate the impact of resistance on the cure rate of clarithromycin-based regimens. For metronidazole-resistant strains, the impact noted in the clarithromycin-metronidazole arm was partially overcome when omeprazole was added (76% eradication for resistant strains versus 95% for susceptible strains). Secondary resistance to clarithromycin occurred in strains from 12 of 105 patients (11.4%) after the failure of a clarithromycin-based regimen to effect eradication. The detection of point mutations in clarithromycin-resistant strains was performed by a combination of PCR and restriction fragment length polymorphism. Mutations (A2142G and 2143G) were found in all strains tested except one. This study stresses the importance of performing susceptibility tests in clinical trials in order to explain the results of different treatments.
- SourceAvailable from: Shaukat Ali Shahid[Show abstract] [Hide abstract]
ABSTRACT: This study evaluates in vivo and in vitro anti-Helicobacter pylori (H. pylori) efficacy of silver nanoparticles (Ag-NPs) prepared via a cost-effective green chemistry route wherein Peganum harmala L. seeds extract was used as a reducing and capping agent.The structural features, as elucidated by surface plasmon resonance spectrophotometry, transmission electron microscopy, and powder X-ray diffraction spectroscopy, revealed the Ag-NPs synthesized to be polydispersed in nature and spherical in shape with 5–40nm size. A typical Ag-NPs suspension (S5), with size being 15 nm, when tested in vitro against forty-two local isolates and two reference strains, showed a considerable anti-H. pylori activity. In case of in vivo trial against H. pylori induced gastritis, after oraladministration of 16mg/kg body weight of S5 for seven days, a complete clearance was recorded in male albino rates. In comparative time-killing kinetics, S5 exhibited dose- and time-dependent anti-H. pylori activity that was almost similar to tetracycline and clarithromycin, less than amoxicillin, but higher than metronidazole. Furthermore, S5 was found to be an equally effective anti-H. pylori agent at low (≤4) and high pH with no drug resistance observed even up to 10 repeated exposures while a significant drug resistance was recorded for most of the standard drugs employed. The present results revealed the potential of the synthesized Ag-NPs as safer bactericidal agents for the treatment of H. pylori induced gastritis.Bioinorganic Chemistry and Applications 08/2014; 2014:1-11. · 1.66 Impact Factor
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ABSTRACT: Resistance to metronidazole, a key component of therapies against Helicobacter pylori, is common in clinical isolates. Resistance generally requires inactivation of rdxA (HP0954), and sometimes also frxA (HP0642), two related nitroreductase genes. Here we studied the effect of resistance to metronidazole on fitness of the gastric pathogen H. pylori. The effect of metronidazole resistance for H. pylori in culture was assessed first by looking at colonies formed by freshly constructed mutant derivatives of H. pylori strain 26695. Mutations resulting in metronidazole resistance caused premature death of H.pylori in stationary phase, but had no significant effect on early exponential growth. The effect of nitroreductase deficiencies on fitness in vivo was tested by infecting C57BL/6 mice with 1:1 mixtures of SS1 wild type and its isogenic metronidazole resistant derivatives. Inactivation of rdxA caused an inability to colonize mice in SS1 H. pylori strain. Derivatives of a metronidazole resistant strain that survived better in stationary phase, although remaining metronidazole resistant, could again colonize mice. In conclusion, metronidazole resistance diminishes H. pylori's fitness, but their costs can be suppressed by additional mutation.Journal of Life Science. 01/2005; 15(6).
- Science against microbial pathogens: communicating current research and technological advances (Microbiology Book Series, number # 3), Edited by A. Méndez-Vilas, 01/2011: chapter Helicobacter pylori resistance to antibiotics; Formatex.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY,
Copyright © 1999, American Society for Microbiology. All Rights Reserved.
Nov. 1999, p. 2747–2752Vol. 43, No. 11
Antimicrobial Susceptibility Testing of Helicobacter pylori
in a Large Multicenter Trial: the MACH 2 Study
FRANCIS ME´GRAUD,1* NORBERT LEHN,2TORE LIND,3EKKEHARD BAYERDO ¨RFFER,4
COLM O’MORAIN,5ROBIN SPILLER,6PETER UNGE,7SANDER VELDHUYZEN VAN ZANTEN,8
MICHAEL WRANGSTADH,9AND CARL FREDRIK BURMAN9
Laboratoire de Bacte ´riologie, Ho ˆpital Pellegrin, Bordeaux, France1; Institut fu ¨r Medizinische Mikrobiologie und Hygiene,
University of Regensburg, Regensburg,2and Medizinische Klinik I, Gastroenterologie, Universita ¨tsklinikum
Carl Gustav Carus, Dresden,4Germany; Department of Surgery, Ka ¨rnsjukhuset, Sko ¨vde,3
Department of Internal Medicine, Sandviken Hospital, Sandviken,7and Astra Ha ¨ssle,
Mo ¨lndal,9Sweden; Adelaide & Meath Hospital, Trinity College Dublin, Tallaght,
Dublin, Ireland5; Division of Gastroenterology, Queen’s Medical Centre,
Nottingham, United Kingdom6; and Department of Medicine,
Queen Elizabeth II Hospital, Halifax, Nova Scotia, Canada8
Received 4 January 1999/Returned for modification 10 May 1999/Accepted 28 August 1999
Culture and susceptibility testing of Helicobacter pylori strains was performed in a large multinational,
multicenter randomized clinical trial. Culture was carried out on gastric biopsy samples obtained from 516
patients at entry and had a sensitivity of 99% when the [13C]urea breath test was used as a reference.
Susceptibility testing was performed for clarithromycin and metronidazole on 485 strains by an agar dilution
method and the epsilometer test (Etest) and for amoxicillin by an agar dilution method only. Resistance to
clarithromycin (>1 ?g/ml) was found in 3% of the H. pylori strains, with a perfect correlation between Etest
and agar dilution methods. Resistance to metronidazole (>8 ?l/ml) was found in 27% of the strains by agar
dilution, but there were important discrepancies between it and the Etest method. No resistance to amoxicillin
was found. The logarithms of the MICs of the three antibiotics against susceptible strains had a distribution
close to normal. The impact of resistance was tested in the four arms of the trial. There were not enough
clarithromycin-resistant strains to evaluate the impact of resistance on the cure rate of clarithromycin-based
regimens. For metronidazole-resistant strains, the impact noted in the clarithromycin-metronidazole arm was
partially overcome when omeprazole was added (76% eradication for resistant strains versus 95% for suscep-
tible strains). Secondary resistance to clarithromycin occurred in strains from 12 of 105 patients (11.4%) after
the failure of a clarithromycin-based regimen to effect eradication. The detection of point mutations in
clarithromycin-resistant strains was performed by a combination of PCR and restriction fragment length
polymorphism. Mutations (A2142G and 2143G) were found in all strains tested except one. This study stresses
the importance of performing susceptibility tests in clinical trials in order to explain the results of different
The eradication of Helicobacter pylori has been the subject of
numerous clinical trials these last 10 years in order to find the
optimum therapy. However, few of these trials have been large
multicenter randomized clinical trials, and moreover the cul-
ture of H. pylori which allows the testing of antimicrobial sus-
ceptibility has rarely been performed (3, 15, 27, 39).
There are several limitations to previous studies of the an-
timicrobial characteristics of H. pylori strains, which include
small sample size, no details about the history of the patients,
a geographical distribution limited to specific areas, and inad-
equate methodology, with few exceptions (13).
Despite the fact that culture is the standard detection
method for most pathogenic bacteria, the specific require-
ments of H. pylori in terms of transport and growth render it
difficult to culture. Other methods such as histological detec-
tion have even been proposed as the “gold standard” (1).
Moreover, the impact of primary resistance on the eradication
rate and the occurrence of secondary resistance have been
challenged (17, 28) and deserve to be studied further in the
context of large trials.
The aim of our study was to perform culture on gastric
biopsies from a large, double-blind, randomized, multina-
tional, multicenter clinical trial performed with a homoge-
neous group of duodenal ulcer patients in remission in Europe
(i) to study the distribution of MICs of antibiotics against H.
pylori strains, (ii) to compare the results obtained by agar
susceptibility testing and epsilometer test (Etest), (iii) to detect
the point mutations associated with resistance to macrolides,
and (iv) to measure the impact of primary resistance on treat-
ment outcome as well as the occurrence of secondary resis-
tance in H. pylori.
MATERIALS AND METHODS
Detection of H. pylori. Patients with duodenal ulcer disease at 47 centers in six
countries (France, Germany, Ireland, Norway, Sweden, and the United King-
dom) were included in the study. Only patients with no or at the most one
previous H. pylori eradication attempt were enrolled in the study. The details of
the clinical trial design are provided elsewhere (18).
The antibiotics tested were amoxicillin, clarithromycin, and metronidazole
since the drugs used in the trial were either amoxicillin (1 g) plus clarithromycin
(500 mg) with (OAC) and without (AC) omeprazole (20 mg) or metronidazole
(400 mg) plus clarithromycin (250 mg) with (OMC) and without (MC) omepra-
zole (20 mg). All of the drugs were given twice daily for 7 days.
* Corresponding author. Mailing address: Laboratoire de Bacte ´ri-
ologie, Ho ˆpital Pellegrin, 33076 Bordeaux cedex, France. Phone: 33-5 56
79 59 10. Fax: 33-5 56 79 60 18. E-mail: francis.me ´graud@chu-aquitaine
At the first visit, patients underwent both a [13C]urea breath test (UBT), as
previously described (19), and endoscopy. The UBT kit used was made by
Utandningsester Sverize AB (Go ˆteborg, Sweden), and the cutoff value was 5 ?
0/00. During endoscopy, a screening test (CLO test) was performed and biopsies
were taken for culture from the antrum (two biopsies 1 to 2 cm from the pylorus
on the anterior and posterior walls) and the corpus (two biopsies approximately
10 cm from the cardia along the greater curvature). They were immediately
introduced into a transport medium (Portagerm pylori; bioMe ´rieux, Marcy
l’Etoile, France) and sent by courier to a local bacteriology laboratory within 24 h
in a cool transport container (Sarstedt, Nu ¨mbrecht, Germany). There was one
bacteriology laboratory per country except in Ireland, where the biopsies were
sent to the United Kingdom.
The following procedure was used in all laboratories. The biopsies were first
ground in 0.5 ml of brucella broth in an electric homogenizer. The suspension
was then plated on three different media. Two were made in-house: Wilkins-
Chalgren agar (Oxoid, Basingstoke, United Kingdom) enriched with 10% human
blood and supplemented with vancomycin (10 ?g/ml), cefsulodin (5 ?g/ml),
trimethoprim (5 ?g/ml), and actidione (100 ?g/ml) and Columbia agar enriched
with 10% human blood without antibiotics; the third was a commercial selective
medium: pylori agar (bioMe ´rieux). The plates were incubated for 12 days in a
microaerobic atmosphere (jars with GasPaks). Colonies suspected to be H. pylori
were identified by the presence of urease, catalase, and oxidase activities. The
strains were kept frozen at ?70°C in brucella broth with 25% glycerol and sent
in dry ice by courier at regular intervals to a central laboratory in Bordeaux,
France, for further study.
The UBT was repeated 4 and 8 weeks after the end of treatment. Only the
patients positive by UBT after treatment underwent a second endoscopy and had
a culture for H. pylori performed.
Eradication was defined as the conversion of a positive test at entry (culture or
UBT) to two negative UBTs 4 and 8 weeks after cessation of therapy.
Determination of MICs of antibiotics against H. pylori by the agar dilution
method. All of the plates were prepared extemporaneously. McFarland 3 sus-
pensions were prepared in brucella broth from 48-h agar plate cultures and
inoculated with a Steers apparatus onto Wilkins-Chalgren agar enriched with
10% sheep blood and containing the antibiotic at the following concentrations:
0.0035 to 4 ?g/ml (amoxicillin), 0.0035 to 128 ?g/ml (clarithromycin), and 0.03 to
128 ?g/ml (metronidazole). The plates were immediately incubated at 37°C in a
microaerobic atmosphere (jars with GasPaks) and read after 48 h. The MIC was
defined as the lowest concentration with complete growth inhibition.
The quality control strains used were CCUG 38770, CCUG 38771, and CCUG
38772, proposed in Europe for this purpose. In the absence of standard break-
points, the breakpoints used to define a resistant strain in this study were based
on a previous publication (18) and on our experience in clinical trials. They were
the following: metronidazole, ?8 ?g/ml (11); clarithromycin, ?1 ?g/ml (0.25 to
1 ?g/ml, intermediate). No breakpoint was predefined for amoxicillin.
Determination of MICs of antibiotics against H. pylori by the Etest. Clarithro-
mycin and metronidazole were tested. McFarland 3 suspensions were prepared
in brucella broth from 48-h agar plate cultures; 0.5 ml was used to flood Wilkins-
Chalgren agar plates enriched with 10% human blood but without antibiotics,
and the excess was removed. After the plates were dried, Etest strips were placed
on them and they were incubated for 48 h in a microaerobic atmosphere (jars
with GasPaks) without anaerobic preincubation and read according to the sup-
Detection of point mutations associated with clarithromycin resistance by
PCR-RFLP. One pair of primers was used to amplify one fragment of the
peptidyltransferase region of the 23S rRNA. The sequences of the primers were
based on the published sequence of the 23S rRNA gene of H. pylori (GenBank
accession no., U27270). A primer extending from position 1820 to 1839 (5?-CCA
CAG CG CTC AG-3?) and a reverse primer from position 2244 to 2225 (5?-CTC
CAT AAG AGC CAA AGC CC-3?) were used to amplify a fragment of 425 bp.
PCR amplification of DNA was performed in a final volume of 50 ?l containing
1 ?g of H. pylori genomic DNA, 67 mM Tris-HCl (pH 8.8), 16 mM (NH4)2SO4,
0.01% Tween 20, 1.5 mM MgCl2, a 0.2 mM concentration of deoxynucleoside
triphosphate mixture, 0.2 ?M concentrations of primers, and 1 U of Taq DNA
polymerase. Each reaction mixture was overlaid with 50 ?l of mineral oil. The
cycling program was 1 cycle at 94°C for 5 min; 40 cycles of 94°C for 1 min, 55°C
for 1 min, and 72°C for 1 min; and a final elongation step at 72°C for 7 min. The
two reactions resulted in a single fragment of the expected size.
Advantage was taken of the occurrence of restriction sites for specific enzymes
to perform PCR-restriction fragment length polymorphism (RFLP) as a rapid
method of detection of the mutation. The method used to detect the mutations
without sequencing involved restriction of the PCR products. Ten microliters of
amplicon (425 bp) was treated with enzyme BsaI or BbsI (New England Biolabs,
Beverly, Mass.). The fragments were incubated for 24 h at 50°C for BsaI and at
37°C for BbsI in order to detect the restriction site occurring when the mutation
was A3G at position 2143 and at position 2142, respectively.
Statistical analysis. Differences in eradication rates were tested with the
Fisher exact test. The influence of the metronidazole MICs on the eradication
probability in the OMC group was tested by a stepwise procedure based on a
permutation test for trend. The underlying assumption for the procedure was
that the probability of treatment failure, as a function of the MIC, is nonde-
creasing, i.e., that higher MICs are not associated with higher success probability.
In the first step, the permutation test, with the logarithms of the MICs as scores,
was applied to all OMC data to test for a trend towards lower eradication rates
for higher MICs. In case of statistical significance, it was concluded that (at least)
the highest MIC (128 ?g/ml) was associated with a decreased eradication rate
and the stepwise procedure was then continued. In the second step, the trend
analysis was repeated excluding the strains for which the MIC was 128 ?g/ml. A
MIC of 64 ?g/ml was also interpreted as a significant result giving a decreased
probability of eradication. The stepwise procedure was continued in this way,
gradually excluding the highest MICs, until the first nonsignificant result was
encountered. The procedure was then stopped. This stepwise procedure is a kind
of closed test (20) that strongly protects the overall type I error rate.
There were 539 patients randomized in the study of whom
514 were included in the intention to treat analysis and 449
were included in the per protocol analysis.
A comparison between UBT and culture was made for only
529 patients at entry, since one test was missing in 10 cases.
With UBT as the reference, the sensitivity of culture was 99%.
There were 485 strains available for determination of the
MICs. The strains were isolated from Sweden (203), Norway
(93), Germany (85), the United Kingdom and Ireland (60), and
France (44). The distribution of the MICs of clarithromycin,
metronidazole, and amoxicillin against H. pylori determined by
the agar dilution method are presented in Fig. 1 to 3, respec-
tively. Only 14 strains (3%) were categorized as intermediate
or resistant to clarithromycin, while 131 strains (27%) were
resistant to metronidazole. The distribution of metronidazole
resistance rates varied between countries from 16 to 42%, the
highest rate occurring in Norway (Table 1). The results of the
Etest were compared to those of the agar dilution method for
clarithromycin and metronidazole (Table 2). There was a per-
fect correlation for clarithromycin. In contrast, there was a
large proportion of discrepancies (22%) for metronidazole: the
Etest led to higher MICs, and more strains were categorized as
resistant. When the dilution steps were compared (Fig. 4), it
was found that the percentage of agreement within plus or
minus 1 dilution was 57% and within plus or minus 2 dilutions
The impact of primary resistance of H. pylori on the clinical
outcome in the different treatment groups was determined for
470 patients. For clarithromycin, it was difficult to evaluate this
impact because of the low number of resistant strains. How-
ever, none of the resistant strains was eradicated in the two
arms whose patients received only the antibiotics without ome-
FIG. 1. Distribution of the MICs of amoxicillin against 485 strains of H.
pylori. MIC at which 50% of the strains were inhibited (MIC50), 0.03 ?g/ml;
MIC90, 0.125 ?g/ml; range, 0.035 to 0.5 ?g/ml.
2748ME´GRAUD ET AL.ANTIMICROB. AGENTS CHEMOTHER.
prazole. For metronidazole, there was a clear impact of resis-
tance on the cure of the infection, which was partly overcome
by the addition of omeprazole. The eradication rate increased
from 76 (95% confidence interval [CI], 58 to 89%) to 91%
(95% CI, 82 to 96%) when it was added (P ? 0.007). In the
OAC group, considered a control group, no difference between
strains susceptible and resistant to metronidazole was ob-
Metronidazole MICs of 128, 64, and 32 ?g/ml were respon-
sible for a significantly higher risk of failure (P ? 0.001, P ?
0.001, and P ? 0.05, respectively) but not 16 ?g/ml (P ? 0.06).
The failure of OAC was not associated with a higher MIC of
amoxicillin against H. pylori. With an MIC of 0.03 ?g/ml, there
was one failure among 39 cases (2.5%); with an MIC of 0.06
?g/ml, there were three failures among 27 cases (11%); and
with an MIC of 0.125 ?g/ml, there was one failure among 12
cases (8%). The distribution of failures was similar in the
groups which did not receive amoxicillin.
Fifteen percent of the patients had received previous erad-
ication therapy: 57 patients had received amoxicillin, 22 had
received macrolides, and 15 had received metronidazole. An
impact of this previous therapy was found only for macrolides
(P ? 0.001).
Secondary resistance of H. pylori to clarithromycin, defined
as a strain which converted from below (at inclusion) to above
(at the posttreatment control) the defined breakpoint, oc-
curred in 12 of 105 patients (11.4%) receiving clarithromycin;
secondary resistance to metronidazole occurred in 12 patients
receiving metronidazole. In addition, for the patients receiving
amoxicillin and clarithromycin alone, there were discrepancies
between the results obtained with metronidazole: nine suscep-
tible strains became resistant to metronidazole, and six resis-
tant strains became susceptible to metronidazole.
PCR-RFLP to detect point mutations associated with clar-
ithromycin resistance was performed for 10 strains which were
resistant pretreatment and for the 12 strains which became
resistant after treatment. The distribution of the mutation ac-
cording to the pre- or posttreatment isolation is presented in
Table 3. Only one pretreatment strain did not have one of the
two mutations sought for; this strain possibly had an A2142C
mutation. For those strains not eradicated, the same mutation
was found before and after treatment. A mixed population of
resistant wild-type bacteria was only found in one case, both
before and after treatment. The median MIC was 16 ?g/ml for
the strains with the A2143G mutation and 64 ?g/ml for those
with the A2142G mutation.
In contrast to what has been previously published and rec-
ommended (1), we found that culture, which is a prerequisite
FIG. 2. Distribution of the MICs of clarithromycin against 485 strains of H.
pylori. MIC at which 50% of the strains were inhibited (MIC50), 0.03 ?g/ml;
MIC90, 0.125 ?g/ml; range, 0.07 to 128 ?g/ml.
FIG. 3. Distribution of the MICs of metronidazole against 485 strains of H.
pylori. MIC at which 50% of the strains were inhibited (MIC50), 2 ?g/ml; MIC90,
64 ?g/ml; range, 0.25 to 128 ?g/ml.
TABLE 1. Distribution of primary resistance of H. pylori to
antibiotics in different countries determined by the
agar dilution method
Frequency (%) of resistance to:
aResistance as defined in this study, MIC ?8 ?g/ml (18).
bResistance as defined in this study, MIC ?1 ?g/ml. Intermediate strains with
clarithromycin MICs of 0.25 to 1 ?g/ml (18).
cNo breakpoint was defined.
dUK, United Kingdom.
TABLE 2. Comparison of the MIC results for clarithromycin and
metronidazole against H. pylori by agar dilution and Etest
Drug and Etest
No. of strains with indicated agar dilution result
VOL. 43, 1999H. PYLORI SUSCEPTIBILITY TESTING 2749
for antimicrobial susceptibility testing (23), is also an accurate
way to diagnose H. pylori infection. Despite the fact that there
were 47 centers involved in this study, the recovery rate was
higher than 90% and even slightly better than the sensitivity of
the UBT. The most probable explanation for this high yield
was the use of a rigorous and standardized protocol, identical
in the five laboratories involved in performing the bacterial
culture. The critical points were the grinding of biopsies, the
use of several kinds of agar media including two which were
freshly prepared, and the follow-up readings of the plates over
a 12-day period. Moreover, a transport system which main-
tained the biopsies at a low temperature, avoided dessication
and contact with air (26a), and assured delivery within 24 h was
We have consistently used Wilkins-Chalgren agar enriched
with human blood for culture for the past 10 years following an
initial comparative study of media (25). It has provided the
best results in clinical practice compared to other media, but
these findings have never been formally published (22). Hu-
man serum is considered the best growth supplement (38).
Cefsulodin is a good selective agent for gram-negative bacilli
including Pseudomonas aeruginosa (16) and has been proposed
in a commercially available selective supplement (6). The im-
proved growth capacity of fresh media has been stressed by
others (12), as well as the use of selective and nonselective
A reference method for studying the susceptibility of H.
pylori to clarithromycin was recommended by the National
Committee for Clinical Laboratory Standards (NCCLS) (29,
34) after this study was performed. The NCCLS method also
uses agar dilution but differs on the following points: Wilkins-
Chalgren agar was used instead of Mueller-Hinton agar; 10%
instead of 5% blood was used; there was a larger bacterial
inoculum (109instead of 107to 108CFU/ml), which was pre-
pared in brucella broth from a 48-h plate instead of in saline
from a 72-h plate; and a shorter incubation time (48 instead of
72 h) was used. The higher inoculum was used in order to
detect possible resistant mutant colonies and to be able to read
the plates within 48 h. The agar dilution method, which is
considered to be the reference method for performing suscep-
tibility tests for most other bacteria, was also used to test
metronidazole and amoxicillin.
This is the first time that a frequency distribution of the
logarithms of the MICs for the three antibiotics has been
obtained on such a large random sample of strains cultured
from duodenal ulcer disease patients who are possibly repre-
sentative of those in northern Europe.
The MIC distribution for amoxicillin against H. pylori was
normal, with the highest MIC at 0.5 ?g/ml. It is necessary to
seek a resistance mechanism, such as a modification in peni-
cillin binding proteins, before categorizing these strains as re-
sistant. We found no resistant strains, in contrast to the find-
ings of van Zwet et al. (35), or tolerant strains, as recently
identified by Dore et al. (7).
The MIC distribution for clarithromycin against H. pylori
was normal for 97% of the strains. A point mutation on the 23S
rRNA gene (30, 37) was detected in all resistant strains except
one by PCR-RFLP, leading one to think that an A2142C mu-
tation could be present. The proportion of resistant strains was
very low in this study. This may reflect the limited use of
macrolides in countries from the northern part of Europe,
especially Scandinavia. However, the resistance rate was also
surprisingly low in France, which is contrary to results obtained
in several other studies involving hundreds of strains, where it
was in the range of 10% (24). Another explanation may be that
strains isolated from patients with duodenal ulcer disease are
more likely to be susceptible to macrolides. Further studies
should be performed to address this question.
The MIC distribution of metronidazole against H. pylori
showed a normal distribution for part of the strains. A ten-
dency for a second mode was observed for strains with MICs
higher than 8 ?g/ml. The resistance mechanism of H. pylori to
metronidazole is not well known. It may concern the enzymes
involved in the reduction of the nitro group, but alternate
pathways may exist, and therefore the observed MICs would be
the result of a complex phenomenon (32). The breakpoint of 8
?g/ml has been proposed for metronidazole resistance based
on studies using bismuth-based triple therapies (11), and this
figure was applied in this study. This breakpoint was valid for
the MC group but not for the OMC group. The stepwise test
indicated that the threshold of 32 ?g/ml would be more ap-
propriate if the latter regimen is used.
In contrast to clarithromycin resistance, resistance to met-
ronidazole (?8 ?g/ml) was found in 27% of the strains, with a
marked variation between the different countries. Surprisingly,
the frequency of resistance was only 16% in France; in previ-
ous studies the frequency was double this value. In Norway it
was 42%. The reason for this high resistance rate in Norway is
The Etest has proven to be an accurate method to test the
susceptibilities of fastidious organisms, including H. pylori, to
antibiotics. However, there is some concern regarding the
value of Etest results for testing the effect of metronidazole on
this bacterium. While we found an excellent agreement be-
tween the agar dilution method and the Etest for clarithromy-
cin, there was an unacceptable discrepancy rate of 22% for
metronidazole. The reason is unclear, but in this study, the
plates were not preincubated in an anaerobic atmosphere (4).
On the other hand, for the agar dilution test, the plates were
FIG. 4. Differences of dilution steps for MICs determined by Etest and the
agar dilution method (n ? 469).
TABLE 3. Distribution of point mutations associated with
clarithromycin resistance in H. pylori strains
isolated pre- and posttreatment
No. of strains showing:
No restriction with
BsaI and BbsI
2750 ME´GRAUD ET AL.ANTIMICROB. AGENTS CHEMOTHER.
prepared extemporaneously and therefore they were more
likely to have a constant redox potential than the plates used
for the Etest. In previously performed studies, the correlation
was indeed far from perfect: the percentage of agreement
within plus or minus 1 dilution was 89 (31) and 83% (8), but
only 56% in the study of Cederbrant (5). Contrary to these
results, Hirschl et al. found the Etest to be accurate and precise
The clinical relevance of H. pylori resistance to antibiotics is
an important aspect to consider (26). Unfortunately, it was not
possible to draw any conclusions for clarithromycin because of
the low number of resistant strains encountered. With regard
to metronidazole, there was an important impact of the resis-
tance to this drug when it was used only with clarithromycin
but the addition of omeprazole had a beneficial effect. The
reason is not clear since metronidazole activity is not supposed
to be pH dependent and, even more importantly, the diffusion
of the drug in the gastric lumen decreases when the pH in-
creases (9, 36). Therefore, it is most likely that the beneficial
effect involves clarithromycin, whose activity is pH dependent,
and/or a synergism between the two compounds. In another
study, where amoxicillin was given instead of clarithromycin
with metronidazole and lansoprazole, there was a lower cure
A consequence of the high eradication rate is a low occur-
rence of secondary resistance. Indeed, in the group receiving
OAC, no secondary resistance was noted. In the group treated
with OMC, four strains resistant to clarithromycin and four
strains resistant to metronidazole were observed, and even
more were observed in the group treated with MC, with five
strains resistant to clarithromycin and eight resistant to met-
The occurrence of resistance to metronidazole in the group
receiving AC was unexpected. In fact, the determination of
these MICs was repeated and most of them were found to be
in the susceptible range. The finding of resistance was probably
due to technical problems related to the method of metroni-
dazole testing. Even with agar dilution, the reproducibility is
not 100% satisfactory. The recent discovery that mutations of
the nitroreductase gene can result in resistance to metronida-
zole gives us some hope that in the near future molecular tests
will be developed (10).
In conclusion, culture of H. pylori proved to be an achievable
goal even in a large randomized clinical trial and, as expected,
susceptibility testing is important in explaining the results ob-
served. Since the prevalence of primary resistance varies be-
tween different areas, it is of paramount importance to include
culture and susceptibility testing in future trials. Even though
the Etest has proved to be a very satisfactory test for clarithro-
mycin, its use for metronidazole requires further standardization.
We acknowledge Astra Ha ¨ssle, Mo ¨lndal, Sweden, which financially
supported this study.
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