Helicobacter pylori and antimicrobial resistance: molecular mechanisms and clinical implications.

Abstract

Helicobacter pylori is an important human pathogen that colonises the stomach of about half of the world's population. The bacterium has now been accepted as the causative agent of several gastroduodenal disorders, ranging from chronic active gastritis and peptic ulcer disease to gastric cancer. The recognition of H pylori as a gastric pathogen has had a substantial effect on gastroenterological practice, since many untreatable gastroduodenal disorders with uncertain cause became curable infectious diseases. Treatment of H pylori infection results in ulcer healing and can reduce the risk of gastric cancer development. Although H pylori is susceptible to many antibiotics in vitro, only a few antibiotics can be used in vivo to cure the infection. The frequent indication for anti-H pylori therapy, together with the limited choice of antibiotics, has resulted in the development of antibiotic resistance in H pylori, which substantially impairs the treatment of H pylori-associated disorders. Antimicrobial resistance in H pylori is widespread, and although the prevalence of antimicrobial resistance shows regional variation per antibiotic, it can be as high as 95%. We focus on the treatment of H pylori infection and on the clinical relevance, mechanisms, and diagnosis of antimicrobial resistance.

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Helicobacter pylori and antimicrobial resistance: molecular
mechanisms and clinical implications
Monique M Gerrits, Arnoud H M van Vliet, Ernst J Kuipers, Johannes G Kusters
Helicobacter pylori is an important human pathogen that colonises the stomach of about half of the world’s population.
The bacterium has now been accepted as the causative agent of several gastroduodenal disorders, ranging from
chronic active gastritis and peptic ulcer disease to gastric cancer. The recognition of H pylori as a gastric pathogen has
had a substantial eff ect on gastroenterological practice, since many untreatable gastroduodenal disorders with
uncertain cause became curable infectious diseases. Treatment of H pylori infection results in ulcer healing and can
reduce the risk of gastric cancer development. Although H pylori is susceptible to many antibiotics in vitro, only a few
antibiotics can be used in vivo to cure the infection. The frequent indication for anti-H pylori therapy, together with
the limited choice of antibiotics, has resulted in the development of antibiotic resistance in H pylori, which substantially
impairs the treatment of H pylori-associated disorders. Antimicrobial resistance in H pylori is widespread, and
although the prevalence of antimicrobial resistance shows regional variation per antibiotic, it can be as high as 95%.
We focus on the treatment of H pylori infection and on the clinical relevance, mechanisms, and diagnosis of
antimicrobial resistance.
Introduction
In 1983, Warren and Marshall were the fi rst to report
the successful cultivation of the human pathogen
Helicobacter pylori from gastric biopsy samples.1,2 By
self-ingestion experiments, they showed that this
bacterium indeed caused gastroduodenal disorders,
thereby fulfi lling Koch’s postulates.3,4 This important
discovery, rewarded with the 2005 Nobel prize for
physiology or medicine, has changed peptic ulcer
disease from a chronic, relapsing disease of uncertain
cause into a curable infectious disease. Today, H pylori
is accepted as the causative agent of acute and chronic
gastritis, and a major predisposing factor for peptic
ulcer disease, gastric carcinoma, and gastric lymphoma
(fi gure 1).5,6
Infection with H pylori occurs worldwide, but there are
substantial diff erences in the prevalence of the infection
both within and between countries. In industrialised
countries, the overall carriage rate of H pylori infection in
middle-aged adults is 20–50%, compared with 80% or
more in many developing countries.7,8 Acquisition of
H pylori predominantly occurs during childhood, and
once acquired, the infection persists throughout life
unless specifi cally treated. In developing countries the
carriage rate of H pylori remains relatively stable, but in
the industrialised world these values have substantially
decreased over recent decades,9 probably as a result of
improved hygiene and sanitation, especially during
childhood,9 and active elimination of carriership via
antimicrobial treatment.
H pylori-associated disorders usually regress or heal
completely after successful treatment of H pylori
infection with antimicrobials. However, the available
antimicrobial therapies for H pylori infection have many
shortcomings—eg, side-eff ects, the need for combination
therapy, and limited effi cacy, in particular because of the
development of antimicrobial resistance. The continuous
increase in the prevalence of antimicrobial resistance in
H pylori, together with the lack of forthcoming novel
treatment options, already negatively aff ects eradication
of H pylori infection, and is predicted to lead to serious
problems for treatment of H pylori-associated disorders
in the near future.
Diagnosis of H pylori infection
H pylori infection can be diagnosed by a variety of invasive
and non-invasive tests.10 Invasive tests are based on
gastric samples, usually mucosal biopsies, which can be
screened by rapid urease test, histology, or culture.10 Non-
invasive tests require alternative clinical specimens—eg,
blood, breath, faeces, urine, or saliva. These samples can
Lancet Infect Dis 2006; 6:
699–709
Department of
Gastroenterology and
Hepatology, Erasmus MC -
University Medical Center
Rotterdam, Rotterdam,
Netherlands (M M Gerrits PhD,
A H M van Vliet PhD,
Prof E J Kuipers MD,
J G Kusters PhD)
Correspondence to:
Dr Johannes G Kusters,
Department of Gastroenterology
and Hepatology, room L-459,
Erasmus MC - University Medical
Center Rotterdam,
’s-Gravendijkwal 230, 3015 CE,
Rotterdam, Netherlands.
Tel +31 10 4632982;
fax +31 10 4632793;
j.g.kusters@erasmusmc.nl
High
Low
Acute
gastritis
Atrophic
gastritis
Pangastritis
Age (years)
Acid production
Chronic
gastritis
Asymptomatic
chronic gastritis
Duodenal ulcer
Gastric ulcer
Gastric cancer
>500
Figure 1: Schematic representation of the natural history of H pylori infection
Acquisition of H pylori usually occurs during childhood. Once acquired and left untreated, the infection persists for
life. After the acute phase, most H pylori-positive patients develop a chronic gastritis without symptoms. In some
patients, more severe manifestations will develop later in life. A normal or high acid secretion predisposes to
duodenal ulcers, whereas a low acid secretion predisposes to gastric ulcers and gastric cancer.

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be screened by serology, urea breath test, or stool antigen
test.10 The choice of a specifi c test depends on several
factors, including gastric complaints, age, local
availability, costs, and clinical information sought.
Patients with gastrointestinal bleeding, inexplicable
weight loss, or anaemia, as well as elderly patients
(>50 years) with newly developed dyspepsia, should
undergo endoscopic examination. If biopsy samples can
easily be obtained, the rapid urease test is the test of
choice.11 However, in patients with active or recent
bleeding, or in patients that are taking antimicrobials or
antisecretory compounds, histology is more reliable.12–14
Although culture of H pylori followed by antibiotic
susceptibility testing is the only method of diagnosis that
provides antibiotic resistance values for all agents used in
anti-H pylori therapy, this technique is not routinely done
at the initial diagnosis of H pylori infection.
When the alarm symptoms are absent, endoscopic
examination is usually not indicated,15 and diagnosis of
H pylori is limited to non-invasive methods, of which
the most widely used are the urea breath test and
serology. The urea breath test can be used for the initial
diagnosis of the H pylori as well as for treatment follow-
up.16 Serology, however, is more limited; it is not suited
to use for confi rming therapy success since a decrease
in the titre of anti-H pylori serum antibodies can only
reliably be seen several months after treatment.17 In
children younger than 6 years, breath samples cannot
easily be obtained, and serology is not always reliable;
thus the stool antigen tests for H pylori could provide
an alternative.10
Treatment of H pylori infections
Peptic ulcer disease and other H pylori-associated
disorders usually regress or even heal completely after
treatment of the H pylori infection.18,19 In vitro, H pylori is
susceptible to most antimicrobials20 but in vivo only a few
antimicrobials can be used to cure infected patients.21
The lack of activity in vivo is because of a combination of
factors—eg, inability of drugs to achieve appropriate
levels in the gastric mucus layer,22,23 inactivation of drugs
at low pH,24,25 and the slow growth rate of H pylori.
Metronidazole, clarithromycin, amoxicillin, tetracycline,
and bismuth (table 1) are the most widely used drugs for
the treatment of H pylori.26 Occasionally, ciprofl oxacin,
moxifl oxacin, levofl oxacin, furazolidone, and rifabutin
are used, in particular for third-line treatment.27 By
contrast with the treatment of many other gastrointestinal
bacterial infections, none of the above-mentioned
antibiotics is eff ective enough to eliminate H pylori when
given as monotherapy.28 Successful treatment of an
H pylori infection therefore requires a combination of
drugs, consisting of one or more antibiotics in
combination with an acid-suppressive drug (proton
pump inhibitors [PPIs] or H2-receptor antagonists) or a
bismuth component. Current guidelines for the
treatment of H pylori are given in table 2.
Inclusion of an acid-suppressive drug has been shown
to increase the effi cacy of the combination therapy.29
There seems to be a preference for the use of PPI-based
regimens rather than H2-receptor antagonist-based
regimens but the rationale for this is unclear. PPIs and
H2-receptor antagonists increase the gastric pH, which
Antimicrobial Commonly used compound Resistance rates* Mode of action Mechanism of resistance
NitroimidazolesMetronidazole, tinidazole 20–95% Reduction of prodrug by nitroreductases leads to
formation of nitro-anion radicals and imidazole
intermediates and subsequent DNA damage
Binds 23S rRNA ribosomal subunit, resulting in inhibition
of protein synthesis
Binding of beta-lactam antibiotic to penicillin-binding
proteins (PBP) inhibits cell division
Absence of imidazole reduction caused by reduced or
abolished activity of electron transport proteins (eg, RdxA,
FrxA, FdxB)
Point mutations in 23S rRNA genes MacrolidesClarithromycin,
erythromycin
Amoxicillin
0–50%
Penicillins 0–30%Decreased binding of amoxicillin to PBP D (tolerance) or
PBP1A (resistance caused by point mutation in the pbp1A
gene), and reduced membrane permeability (resistance)
Point mutations in 16S rRNA genes and reduced membrane
permeability
Point mutations in the DNA gyrase gene, gyrA
Tetracyclines Tetracycline 0–10%Binding to ribosome prevents association with aminoacyl-
tRNA and subsequent protein synthesis
Inhibition of DNA gyrase and topoisomerases, interfering
with DNA replication
Binding to RNA polymerase, resulting in transcription
inhibition
Reduction of prodrug by nitroreductases, leads to
formation of nitro anion radicals and subsequent DNA
damage
Inhibits the proton motive force of the bacterium, and
destabilises its site of colonisation in the stomach
Inhibits protein, ATP, and cell membrane synthesis
FluoroquinolonesCiprofl oxacin, moxifl oxacin,
levofl oxacin
Rifabutin
0–20%
Rifamycins0–2%
Point mutations in the RNA polymerase gene, rpoB
NitrofuransFurazolidone0–5%
Unknown
Proton pump
inhibitor
Bismuth
Omeprazole, lansoprazole,
pantoprazole
Bismuth subcitrate, bismuth
subsalicylate, ranitidine
bismuth citrate
Not reportedUnknown
Not reportedUnknown
*Prevalence of antimicrobial resistance in H pylori shows regional variation both within and between countries. In industrialised countries, the prevalence of resistance is lower than in developing countries.
Table 1: Mode of action, resistance mechanisms, and prevalence of resistance among antimicrobials used for treatment of H pylori infection

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extends the half-life of most antibiotics. Furthermore,
they potentially aff ect the microenvironment of the
bacterium by destabilisation of its site of colonisation.
Some PPIs have antimicrobial activity, since they directly
aff ect the proton motive force of the bacterium (table 1).30
Another benefi cial eff ect of acid-suppressive drugs is that
they decrease the severity of side-eff ects of a given
regimen,31 resulting in an increased compliance and
chance for successful treatment.32
Bismuth salts have been used in medicine since the
19th century, especially in the treatment of peptic
diseases. Colloidal bismuth
subsalicylate, and the newer ranitidine bismuth citrate
(acid inhibitor combined with a bismuth compound) are
commonly used agents in anti-H pylori therapy.33 The
mode of action of bismuth salts on H pylori is complex
and includes inhibition of the synthesis of proteins, ATP,
and cell walls (table 1).34 Although bismuth-based
monotherapy eff ectively suppresses the growth of
H pylori, the success rates of this therapy are low,35 but
when used in combination with one or two antibiotics,
bismuth displays synergistic activity, and thus increases
the effi cacy of the anti-H pylori therapy. Although
bismuth-based quadruple therapy, consisting of two
antibiotics, a PPI, and a bismuth compound is more
eff ective against H pylori than triple therapy based on
PPIs and H2-receptor antagonists, the quadruple regimen
is usually not prescribed as fi rst-line treatment because
of the more complex dosing schedule and side-eff ects.36
The most eff ective and best tolerated combinations
consist of amoxicillin (1000 mg, twice daily) with
clarithromycin (500 mg, twice daily), and a PPI (20–40 mg,
twice daily), or metronidazole (500 mg, twice daily) with
clarithromycin (500 mg, twice daily), and a PPI (20–40 mg,
twice daily) for 7–14 days (fi gure 2 and table 2).37–40 Diff erent
types of PPI can be used in these treatments, since they are
equally eff ective for this purpose.41,42 Although high success
rates have been obtained in clinical trials for these fi rst-line
therapies, in general practice 20–30% of the therapies fail,43
usually because of insuffi cient patient compliance or
development of antibiotic resistance. Patients who remain
subcitrate, bismuth
H pylori-positive after fi rst-line treatment are usually
re-treated with an adapted second-line regimen, by another
combination of antibiotics, prolonged treatment for
10–14 days, or addition of a bismuth component.44 A typical
second-line therapy consists of bismuth (120 mg, four
times a day), metronidazole (500 mg, three times a day),
tetracycline (500 mg, four times a day), and a PPI (20–40 mg,
Regimen*DoseDuration (days)
Triple therapy
Ranitidine bismuth citrate, clarithromycin, and amoxicillin
PPI, clarithromycin, and amoxicillin†
PPI, clarithromycin, and metronidazole
PPI, amoxicillin, and metronidazole
Quadruple therapy
PPI, bismuth, metronidazole, and tetracycline
400 mg, 500 mg, and 1 g, all twice daily
20–40 mg, 500 mg, and 1 g, all twice daily
20–40 mg, 500 mg, and 500 mg, all twice daily
20–40 mg, 1 g, and 500 mg, all twice daily
7–14
7–14
7–14
7–14
20–40 mg twice daily, 120 mg four times daily, 500 mg three
times daily, and 500 mg four times daily
7–10‡
PPI=proton pump inhibitor. *Metronidazole can be replaced by tinidazole. †Therapy approved by the Food and Drug Administration (FDA). ‡Antibiotics are given for
4–7 days; PPI usually started 3 days earlier.
Table 2: Current guidelines used for treatment of H pylori infections, based on the guidelines of the Maastricht 2-2000 Consensus, the National Institute
for Clinical Excellence (NICE), and the European Helicobacter pylori Study Group (EHPSG)
Dyspeptic patient
Amoxillin + clarithromycin + PPI
Metronidazole + clarithromycin + PPI
No further
treatment
No further
treatment
Metronidazole + tetracycline + PPI + bismuth
Invasive/non-invasive testing
Antibiotic susceptibility testing
Third-line treatment
First-line treatment
Second-line treatment
+
+
–
+
–
Figure 2: Management of H pylori treatment
If bismuth is not available, PPI-triple therapies should be used. Metronidazole
and tetracycline can be replaced by tinidazole and doxycycline, respectively.
+=H pylori positive. −=H pylori negative. PPI=proton pump inhibitor.

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twice daily) for 10 days (fi gure 2). There are no standard
third-line rescue therapies because of limited choice of
antibiotics that can be used, and interpatient and local
diff erences in primary and secondary antibiotic resistance.
To select an appropriate rescue treatment, endoscopy
followed by bacterial culture and antibiotic susceptibility
testing is advisable.
Prevalence of antimicrobial resistance
Resistance to nitroimidazoles is the most common form of
antimicrobial resistance in H pylori. Metronidazole and
tinidazole are the most frequently used nitroimidazoles for
treatment of H pylori infection, and there seems to be
substantial cross-resistance
imidazoles.45 In industrialised countries, about 35% of the
H pylori strains are resistant to nitroimidazoles (minimum
inhibitory concentration [MIC] ≥8 mg/L; susceptibility
breakpoint),46,47 whereas in developing countries, the
resistance rates for these drugs are much higher, and in
some areas, virtually all H pylori strains are nitroimidazole-
resistant (table 1).7 This diff erence in prevalence might be
caused by the common use of metronidazole and related
nitroimidazoles in developing countries for the treatment
of parasitic-related diseases, whereas in developed
countries these drugs are mainly used for treatment of
gynaecological and dental infections.48
The prevalence of macrolide resistance in H pylori is
much lower than that of nitroimidazole resistance. Again,
there is considerable cross-resistance between diff erent
macrolides (eg, clarithromycin and erythromycin).49 In
the late 1990s, about 10% of western H pylori isolates
were macrolide resistant (MIC ≥2 mg/L),46,47,50 whereas in
developing countries, the prevalence of macrolide
resistance was higher, and varied between 25% and 50%
(table 1).51–53
Until the end of the 20th century, resistance to penicillins
(eg, amoxicillin) and tetracyclines (eg, tetracycline and
doxycycline) was very rare in H pylori. By contrast, many
other bacteria exhibit widespread resistance to these
antibiotics. However, the incidence of amoxicillin and
tetracycline resistance (MIC ≥0·5 mg/L and MIC ≥4 mg/L,
respectively) in H pylori seems to increase in geographic
regions where these antibiotics can be obtained without
prescription (eg, Italy, Brazil, El Salvador, India, and
Lithuania).51,54–56 Resistance rates of 72% and 59% have
been reported for amoxicillin and tetracycline,
respectively; however, these estimates are based on a
single report and thus require further confi rmation.57 In
general, the prevalence of amoxicillin resistance varies
between 1% and 2%, whereas the prevalence of
tetracycline resistance is estimated to be less than 1%
(table 1).46,47 For the less frequently used antibiotics
furazolidone, rifabutin, ciprofl oxacin, and related
fl uoroquinolones, resistance has also been reported;51,58–63
however, accurate rates on the prevalence of resistance to
these drugs are missing, since the rates have been
determined in a limited number of studies only.
between these nitro-
Clinical eff ect of antibiotic resistance
Numerous studies have shown that antibiotic resistance
substantially impairs the effi cacy of anti-H pylori
therapy.64–69 Nevertheless, the clinical relevance of
antibiotic resistance in H pylori-associated diseases is still
challenged. The extent to which antibiotic resistance
reduces the success rates of H pylori treatment depends
on a variety of factors—eg, the components used in
therapy, the dose of the antimicrobial drugs, the duration
of the therapy, and the level of resistance present in the
H pylori strain.70,71
Several studies have shown that the success rates of
nitroimidazole-containing PPI-based triple therapies
drops from 90% (95% CI 85–96%) for nitroimidazole-
susceptible strains to 73% (66–79%) for resistant
strains.66,68–70,72 The success rates of nitroimidazole-
containing bismuth-based triple therapies are lower than
those of the nitroimidazole-containing PPI-based triple
therapies: 89% (83–95%) in susceptible strains versus
53% (42–66%) in resistant strains.64,66–68,72 Addition of a
bismuth component to a PPI-based triple therapy
(quadruple therapy) increased the effi cacy of the therapy
to 92% (87–97%) for nitroimidazole-susceptible strains
versus 83% (66–100%) for resistant strains.64,66–68,72
Although data on ranitidine bismuth citrate-based
therapies and nitroimidazole resistance are limited, the
ranitidine bismuth citrate-based therapies seem to have a
slightly higher effi cacy than do the PPI-based quadruple
therapies: 95% (92–99%) for nitroimidazole-susceptible
strains versus 89% (69–100%) for resistant strains.40,66,72
Most studies that have assessed the eff ect of macrolide
resistance on therapy effi cacy included a limited number
of cases only (one to 24 macrolide-resistant H pylori
isolates).64,66–68,72 Despite this limitation, macrolide
resistance seems to substantially reduce the effi cacy of
all macrolide-containing regimens.67 The success rates
of macrolide-containing dual therapies (with a PPI or
bismuth compound) decreased from 68% (61–75%)
for macrolide-susceptible strains to 33% (22–45%) for
macrolide-resistant strains.66–68,72 The success rates
for macrolide-containing triple therapies (with a PPI and
amoxicillin, metronidazole, or tinidazole) is high in
patients with macrolide-susceptible strains (86%,
80–92%), but remains low (25%, 12–38%) for macrolide-
resistant strains.66–69,72
Resistances to amoxicillin, tetracycline, furazolidone,
rifabutin, ciprofl oxacin, or other related fl uoroquinolones
have also been held responsible for therapy failure with
these drugs;54,61,67,73,74 however, there are not enough data
available yet to make an accurate estimate of the eff ect of
these resistances on treatment success.
Antibiotic activity and mechanisms of
resistance
In view of the large number of individuals colonised with
H pylori, the proportion of individuals who develop
disease, and the relative diffi culty to treat the infection, it

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is not surprising that a lot of eff ort has been spent on
understanding the mechanisms of antibiotic resistance.
The obtained knowledge (fi gure 3) could allow for rapid
testing of resistance and can provide clues to decrease
the spread of antibiotic resistance. In many bacteria,
antibiotic resistance mechanisms are located on plasmids,
transposons, or integrons.75 In H pylori, however, the
antibiotic resistance mechanisms are mainly based on
point mutations located on the bacterial chromosome
(table 1). In H pylori, antibiotic resistance easily develops
de novo, although horizontal gene transfer via natural
transformation among susceptible and resistant strains
cannot be excluded.76
Nitroimidazoles
Metronidazole and the closely related compound
tinidazole are bactericidal antibiotics that belong to the
nitroimidazole group of drugs. They are actively released
into the gastric juice,23 and their antimicrobial activity is
only marginally aff ected by low pH.25 Nitroimidazoles are
given as a prodrug that needs to be activated within the
target cell by one or two electron transfer processes. This
reduction leads to the formation of nitro-anion radicals
and imidazole intermediates that cause lethal damage to
subcellular structures and DNA.77 In theory, any protein
that possesses a low redox potential can accept electrons
from metronidazole and tinidazole and thus activate these
drugs. In anaerobic bacteria and protozoa, nitroimidazole
resistance is mediated by a reduced or abolished activity
of one electron acceptor, especially pyruvate:ferredoxin
oxidoreductase and ferredoxin itself.78 In H pylori, several
putative electron acceptors have been identifi ed, including
ferredoxin (FdxA), fl avodoxin (FldA), ferredoxin-like
protein (FdxB), NAD(P)H fl avin nitroreductase (FrxA),
2-oxoglutarate oxidoreductase (OorD), pyruvate:ferredoxin
oxidoreductase (PorD), and oxygen-insensitive NAD(P)H
nitroreductase (RdxA).79,80
An important step in the elucidation of the molecular
mechanism of nitroimidazole resistance (table 1) in
H pylori came with the discovery that null mutations in
the rdxA gene were suffi cient to confer metronidazole
resistance to a metronidazole-susceptible strain.81 Soon
afterwards, it became apparent that the mechanism of
resistance in H pylori is more complex, since there are
indications that other reducing factors are involved (eg,
FrxA, FdxB, RibF, MdaB).82–85 Regardless of whether
mutations in these factors alter the expression of the
corresponding protein, or result in a truncated protein or
changed aminoacid sequence, they reduce the activity of
the nitroimidazoles.81–83,85,86 The fi nding that mutations in
multiple proteins all result in an increased MIC of
nitroimidazole might explain the wide range in levels of
resistance.
Macrolides
Clarithromycin is a bacteriostatic antibiotic that belongs
to the group of macrolides that bind reversibly to the
peptidyl transferase loop of domain V of the 23S
ribosomal RNA (rRNA) molecule. This binding interferes
with protein elongation, and thus eff ectively blocks
bacterial protein synthesis. The antibacterial activity of
clarithromycin is much the same as that of other
macrolides, but clarithromycin is better absorbed in the
gastric mucus layer and is more acid-stable.87 In most
Gram-negative bacteria, resistance to clarithromycin or
related macrolides is mediated either by target
modifi cation (ie, 23S rRNA nucleotide mutations or post-
transcriptional methylation of the 23S rRNA) or through
overexpression of an effl ux mechanism.88 In H pylori,
resistance to clarithromycin and other related macrolides
is mostly because of point mutations in one of two
adjacent 23S rRNA nucleotides (table 1)—namely A2142→
Reduced or
abolished activity of
electron transporters
(metronidazole
resistance)
rRNA mutations
Alteration in PBPs
(amoxicillin
resistance)
Decreased membrane permeability
(amoxicillin and tetracycline resistance)
(clarithromycin and
tetracycline resistance)
C
AB
D
Figure 3: Mechanisms of antibiotic resistance in H pylori
(A) metronidazole, (B) amoxicillin, (C) clarithromycin and tetracycline, and (D) amoxicillin and tetracycline.
(A) Reduced or abolished activity of electron transport proteins—eg RdxA, FrxA, or FdxB. (B) Alteration in the
penicillin-binding proteins PBP-D and PBP1A. (C) Point mutations in the rRNA genes for 16S and 23S rRNA.
(D) Decreased membrane permeability.

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G/C and A2143→G.89, 90 These substitutions cause decreased
affi nity of the ribosomes for macrolides, resulting in
increased resistance.91 The A2142→G/C substitutions are
linked to high-level macrolide resistance (MIC >64 mg/L),
whereas the A2143→G substitution is linked to high-level
erythromycin resistance
intermediate levels of resistance to clarithromycin,
clindamycin, and streptogramin (MIC 2–64 mg/L).49,89,91–93
Occasionally, other 23S rRNA mutations have been
reported in macrolide-resistant H pylori isolates, and
A2515G and T2717C have been shown to confer macrolide
resistance,94,95 whereas A2116G, G2141A, A2144T, T2182C, G2224A,
C2245T, and T2289C have been associated with macrolide
resistance.96–99
(MIC >64 mg/L) and
Penicillins
Amoxicillin is a bactericidal antibiotic that belongs to the
penicillin group of drugs. The drug binds to penicillin-
binding proteins (PBPs) and interferes with bacterial cell
wall synthesis, resulting in lysis of replicating bacteria.
The antibacterial activity of amoxicillin is much the same
as that of other penicillins, but amoxicillin is better
released in the gastric juice, and displays increased
stability in acidic conditions compared with other
penicillins.100 In Gram-negative bacteria, resistance to
penicillins is mostly because of the activity of beta-
lactamase,101 an enzyme that disrupts the beta-lactam ring
of penicillins, whereas in Gram-positive bacteria,
resistance is mainly mediated by mutational changes in
one or more PBPs. In H pylori, however, there are no
indications that amoxicillin resistance is because of beta-
lactamase activity,55,102–106 and resistance seems to be
mainly mediated by alterations to PBPs (table 1).103,106
Many H pylori isolates described as being amoxicillin
resistant are often only tolerant to penicillins—ie, their
resistance is only transient and not stable.55 Directly after
isolation from H pylori-positive patients, the isolates have
very high MIC values for amoxicillin, but the values drop
below breakpoint (MIC <0·5 mg/L) upon freezing at
–80°C or repeated subculturing in the absence of
amoxicillin. PBP-binding studies with ³H-benzyl-
penicillin have shown that amoxicillin resistance in these
amoxicillin-tolerant isolates is mediated by the absence
of PBP D (also called PBP4).103 Whether the decreased
labelling of PBP D is caused by reduced expression or
decreased affi nity of the protein for amoxicillin was not
further characterised.
Stable amoxicillin resistance in H pylori is rare. This
form of resistance is mediated by mutational changes in,
or adjacent to, the second and third PBP-motif of
PBP1A.104–107 By use of biotinylated amoxicillin, the
mutations were shown to result in reduced affi nity of
PBP1A for amoxicillin.105,108 In addition to changes in
PBPs, reduced membrane permeability or active effl ux of
amoxicillin in H pylori have been suggested as possible
mechanisms that might contribute to higher levels of
amoxicillin resistance. Both aspects have been tested
with the proton translocator CCCP.104,108 Amoxicillin-
resistant H pylori strains accumulated less than 60%
[14C]-penicillin G compared with amoxicillin-susceptible
H pylori strains (100%), both in the presence and absence
of CCCP, thus excluding a role for an active effl ux
mechanism. These fi ndings suggest that amoxicillin
resistance in H pylori is partly because of an increased
diff usional barrier (table 1),104,108 an eff ect that could be
explained by alterations in outer membrane protein
composition.104
Tetracyclines
Tetracycline is a bacteriostatic antibiotic that binds to the
16S rRNA, thus interfering with the attachment of
aminoacyl-tRNA to the ribosome, resulting in inhibition
of protein synthesis and bacterial growth.109 In many
bacteria, tetracycline resistance is mediated either by
overexpression of effl ux proteins or changes in ribosomal
protection proteins.110 In H pylori, however, there are no
indications that tetracycline resistance is mediated by
either of these mechanisms.111,112 The main mechanism of
tetracycline resistance in H pylori seems to be based on
single, double, and triple basepair substitutions in the 16S
rRNA primary binding site of tetracycline (table 1).56,112–116
High-level tetracycline resistance seems to be related to
the triple basepair substitution AGA926–928→TTC in the 16S
rRNA gene,112–114,117 whereas low-level tetracycline resistance
(MIC <4 mg/L) is associated with single and double
basepair substitutions in the same area.56,114–116 Since some
of the tetracycline-resistant H pylori isolates retain the
tetracycline-susceptible AGA926–928 sequence, but showed
decreased accumulation of tetracycline, it has been
postulated that reduced membrane permeability could
contribute to tetracycline resistance.116
Fluoroquinolones, nitrofurans, and rifamycins
Since resistance against the commonly used antibiotics
is increasing, fl uoroquinolones (eg, ciprofl oxacin,
moxifl oxacin, and levofl oxacin),
furazolidone), and rifamycins (eg, rifabutin) are
occasionally being used in second and third-line
therapies.27 Initial results obtained with these antibiotics
were promising, but antibiotic resistance against these
drugs soon emerged.51,58–60,62,63,118
Fluoroquinolones are bactericidal antibiotics that exert
their antimicrobial activity by inhibition of DNA gyrase.
This enzyme is a tetramer that consists of two A subunits
and two B subunits, encoded by the gyrA and gyrB genes,
respectively.60 The main function of this enzyme is to
catalyse the negative supercoiling of DNA.119 Most H pylori
isolates are susceptible to fl uoroquinolones, but the
incidence of fl uoroquinolone resistance seems to be
increasing58 and can reach up to 10–20%.120 In H pylori,
resistance to fl uoroquinolones is caused by point
mutations in the DNA gyrase-encoding gene gyrA at the
positions encoding aminoacids 87, 88, 91, and 97
(table 1).60,62
nitrofurans (eg,

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Furazolidone and nitrofurantoin are bactericidal
antibiotics that share similarities with metronidazole
both in their structures and modes of action. H pylori is
usually susceptible to furazolidone and nitrofurantoin,
but isolates with an increased MIC have been reported
occasionally.51,63 The mechanism of resistance is not yet
known, but clearly diff ers from that of metronidazole,
since inactivation of the rdxA, frxA, and fdxB genes did
not result in furazolidone or nitrofurantoin resistance
(table 1), and furazolidone and nitrofurantoin are active
against metronidazole-resistant H pylori isolates.63
Rifabutin and several other rifampicin derivates are
bactericidal antibiotics that bind to the β-subunit of DNA-
dependent RNA polymerase, resulting in inhibition of
transcription.121 The β-subunit of this complex is encoded
by the rpoB gene.121 Until a few years ago, resistance
against rifamycins and rifabutin in vivo was very rare;
however, the incidence of rifamycin and rifabutin
resistance in H pylori clinical isolates is now increasing.58
In H pylori, resistance to these antibiotics is linked to
point mutations in the rpoB gene that correspond to the
aminoacids 149, 524–545, and 586 (table 1).122
Detection of antibiotic resistance
Numerous techniques have been developed to detect
antibiotic resistance in H pylori. These methods can be
divided in culture and nucleic acid-based assays (fi gure 4).
Antibiotic susceptibility in H pylori is usually assessed by
culture-based methods (eg, agar dilution, disc diff usion,
E-test, breakpoint susceptibility
microdilution method), but since knowledge of antibiotic
resistance mechanisms in H pylori is growing, several
(non-)invasive nucleic acid-based tests have been
developed—eg, restriction
polymorphism (RFLP), mismatch PCR, immunoassays,
real-time PCR, and fl uorescent in-situ hybridisation
(FISH).
Agar dilution is a reliable method to assess antibiotic
susceptibility in H pylori. The US National Committee
for Clinical Laboratory Standards has approved this
technique as the method of choice to detect resistance to
all commonly used antibiotics for anti-H pylori therapy.123
In the agar dilution assay, antibiotic susceptibility is
assessed by growing H pylori on agar plates containing
two-fold serial dilutions of the antibiotic. This method
can also be done in broth, by the so-called broth
microdilution method. Since both methods are time
consuming and not used on a daily basis, the protocols
have been simplifi ed; bacteria are grown either in agar or
broth containing a critical concentration of the antibiotic
necessary to defi ne antibiotic resistance (breakpoint
susceptibility testing).
Another cheap and simple method to assess antibiotic
susceptibility is the disc diff usion method. In this assay,
antibiotic discs are placed on an agar plate with bacteria,
and, after incubation, antibiotic susceptibility is
determined by measuring the inhibition zone. E-test is a
testing, broth
fragment length
commercially available quantitative variant of the disc
diff usion method.124,125
Studies that compare agar dilution, disc diff usion, and
E-test have shown that results are not always
consistent.124,125 All these methods are slow (data are
usually obtained after 6–10 days), cumbersome, and fail
in about 10% of cases because of contamination of biopsy
samples or growth failure of H pylori. Nucleic acid-based
methods could off er an alternative. Such methods are
faster, independent of living or growing bacteria, give
reproducible results, and are easily standardised.
Although several (non-)invasive nucleic acid-based tests
are currently available for the detection of clarithromycin,
tetracycline, and ciprofl oxacin resistance,117,126–129 these
methods are not routinely used because they are still
expensive, labour intensive, and require specifi c expertise
and equipment.
PCR-RFLP is a PCR-based assay that is commonly used
to detect mutational changes in H pylori obtained from
biopsy samples or faeces.126,128 The method is relatively
simple and is based on the presence or absence of a
restriction site within the amplifi ed DNA fragment. If
there is no suitable restriction enzyme available,
mutations can be detected using mismatch PCR.130 Other
methods—eg, PCR-DNA enzyme immunoassay, PCR
oligonucleotide ligation assay, preferential homoduplex
formation, and PCR-line probe assay—include an
additional hybridisation step with labelled oligonucleotide
probes, specifi c antibodies, or streptavidin-alkaline
phosphatase after the PCR step.90,92,131,132 Real-time PCR
hybridisation assays, however, amplify the DNA fragment
of interest in the presence of one or two fl uorescently
labelled probes. After completion of PCR, the temperature
is increased to determine the melting point of the
probe(s). When there are mismatches present in the
Susceptibility testing
Nucleic acid-based techniques
(biopsies and faeces)
PCR-RFLP
Mismatch PCR
Immunoassays
Real-time PCR
FISH
Culture-based techniques
(biopsies)
Agar dilution
Broth microdilution
Breakpoint testing
Disc diffusion
E-test
Figure 4: Detection of antibiotic resistance in H pylori
Antibiotic resistance in H pylori can be assessed by culture and nucleic acid-based
techniques. PCR-RFLP=PCR-restriction fragment length polymorphism.
FISH=fl uorescent in-situ hybridisation.

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target sequence, lower melting temperatures are obtained
compared with the matched hybrid. This technique can
be done on biopsy samples as well as faeces.115,127,129,133,134
Fluorescent in-situ hybridisation can identify H pylori
and antibiotic resistance can be detected in biopsy
samples without doing PCR. In this assay, intact H pylori
is hybridised, with fl uorescently labelled H pylori-specifi c
probes containing the mutation of interest, and visualised
by fl uorescence microscopy.135
Alternative treatment options
In the past few years, much time and eff ort has been spent
in developing alternative anti-H pylori treatments,
especially via vaccine development. Prophylactic as well as
therapeutic vaccination could potentially save millions of
lives and reduce the costs related to the treatment of
H pylori-associated diseases. Several virulence factors (eg,
urease), the vacuolating toxin A (VacA), the cytotoxin-
associated antigen (CagA), and the blood-group-antigen
binding adhesin (BabA), in combination with cholera
toxin (AB5 toxin), the heat-labile toxin of Escherichia coli,
or Freunds adjuvants, have been used to induce a
protective immune response in animal models.136 Although
phase I and II clinical trials have been done in human
beings,137–139 a commercial vaccine is still not available.
Other potential developments in anti-H pylori
treatment have been made. Antimicrobial peptides—eg,
magainins, LL-37/hCAP18, and defensins140–142—par-
ticipate in the innate and adaptive immunity by attracting
monocytes or neutrophils, and induce the migration of
human naive T cells and immature dendritic cells.143
Porphyrins—a class of naturally occurring compounds
that exhibit antimicrobial activity through the catalysis
of peroxidase and oxidase reactions144—are another
option, as are new diets, based on essential oils such as
those isolated from Cinnamomum
Cymbopagon citratus, and Lippia citriodora,145,146 or
probiotics such as Lactobacillus spp and Bifi dobacterium
spp.147,148 These components could be used either as
monotherapy or synergistically in combination with
other antimicrobials, thus resulting in more eff ective
anti-H pylori therapy. Initial studies with antimicrobial
peptides, porphyrins, essential oils, and probiotics are
promising, but there is still a long way to go before they
can be used in clinical practice.
zeylanicum,
Confl icts of interest
We declare that we have no confl icts of interest.
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