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Antimicrobial Agents and Chemotherapy, July 1999, p. 1779-1782, Vol. 43, No. 7
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Rapid Detection, by PCR and Reverse Hybridization,
of Mutations in the Helicobacter pylori 23S rRNA Gene,
Associated with Macrolide Resistance
Leen-Jan
van
Doorn,1,*
Yvette J.
Debets-Ossenkopp,2
Armelle
Marais,3
Ricardo
Sanna,1
Francis
Mégraud,3
Johannes G.
Kusters,2 and
Wim G. V.
Quint1
Delft Diagnostic Laboratory,
Delft,1 and Department of Medical
Microbiology, Free University of Amsterdam,
Amsterdam,2 The Netherlands, and
Laboratoire de Bactériologie, Hôpital Pellegrin
and University Victor Ségalen Bordeaux 2, Bordeaux,
France3
Received 23 November 1998/Returned for modification 15 February
1999/Accepted 4 May 1999
 |
ABSTRACT |
A PCR-based reverse hybridization system (research prototype kit
INNO-LiPA for H. pylori resistance) was developed and
evaluated for simultaneous detection of 23S ribosomal DNA point
mutations, associated with macrolide resistance in Helicobacter
pylori. Fifty-seven H. pylori strains (51 natural, 6 laboratory-derived artificial, 52 resistant, and 5 susceptible strains)
were tested by PCR-LiPA (detecting mutations A2115
G, G2141A,
A2142G, A2142C, A2143G, A2143C, and A2143T), DNA
sequencing, restriction fragment length polymorphism, and/or
hybridization to oligonucleotide probes. Results were highly
concordant, but PCR-LiPA appears to be more sensitive for the
simultaneous detection of multiple mutants.
| |
TEXT |
Helicobacter pylori is a
gram-negative bacterium that colonizes the human stomach. Infection
with H. pylori is associated with gastritis and peptic ulcer
disease and may eventually result in the development of atrophic
gastritis and gastric cancer (1, 7).
Infection with H. pylori can be effectively treated by a
combination of proton pump inhibitors and/or H2 receptor
antagonists and antibiotics. Metronidazole, amoxicillin,
clarithromycin, and tetracycline are frequently included in the triple
or quadruple treatment regimens used to eradicate H. pylori
(5, 18). Resistance to antimicrobial agents is an important
factor for the clinical outcome of anti-Helicobacter
treatment. Resistance to metronidazole is observed in 10 to 50% of the
cases in developed countries but can be as high as 90% in developing
countries (12). Resistance to macrolides also is of
importance and was found in less than 2% of the strains in The
Netherlands (19) but in more than 10% of those from France
as well as some other countries (2, 6, 12, 14). The
prevalence of resistant strains appears to be increasing (6, 8,
11, 17).
The major cause of macrolide resistance in H. pylori is the
lack of binding of the macrolides to the 23S rRNA components of the
bacterial ribosome due to a modification of the target site by
methylation or point mutations in the peptidyltransferase region of
domain V of the 23S rRNA (21). H. pylori contains
two copies of the 23S ribosomal DNA (rDNA) gene, and at least five
distinct point mutations have been reported that are associated with
macrolide resistance. Versalovic et al. (20) found AG
transitions at two positions (A2142G and A2143G). Also, an AC
transversion (A2142C) was found to be related to resistance
(15). Recently, Hultén et al. described associated
mutations at two additional positions (G2115A and G2141A)
(10). The conventional method to determine the antibiotic
resistance of H. pylori is based on analysis of cultured
strains by agar diffusion or the E test (12, 13), which is
time-consuming and requires specific expertise. Therefore, DNA-based
diagnostic methods may offer a rapid and reliable alternative approach
for macrolide susceptibility testing. The present study describes a
convenient method, based on PCR and reverse hybridization, for the
detection of the relevant mutations in the 23S rDNA.
Bacterial strains.
A total of 57 H. pylori strains
were tested for 23S rRNA mutations; 29 were obtained from Amsterdam,
The Netherlands, and 28 were obtained from Bordeaux, France. All
strains from Bordeaux and most strains from Amsterdam were freshly
cultured from gastric biopsies. Six strains contained artificial
mutations of the 23S rRNA gene (3). In addition,
Helicobacter reference strains were obtained from the
collection of the Laboratory of Microbiology at the University of Ghent
(LMG): H. pylori (LMG 4654, 5166, 6787, 7162, 6307, 3670, 4046, 6301, and 6222), Helicobacter fennelliae (LMG 13306 and 11759), Helicobacter nemestrinae (LMG 14378),
Helicobacter mustelae (LMG 8776 and 8777),
Helicobacter cinaedi (LMG 9072, 7543, and 8558),
Helicobacter canis (LMG 12640), Helicobacter acinonyx (LMG 12684), and Helicobacter pametensis (LMG
12678, 12637, 12681, and 12682). Also, the following
non-Helicobacter reference strains were used:
Arcobacter butzleri (LMG 11118), Bacillus cereus
(ATCC 11778), Bacillus subtilis (ATCC 6633),
Clostridium perfringens (ATCC 12916), Escherichia
coli (ATCC 11775), Klebsiella pneumoniae (ATCC 13883),
Listeria monocytogenes (NCTC 10527), Proteus
mirabilis (ATCC 29906), Pseudomonas aeruginosa (ATCC
27853), Salmonella typhimurium (ATCC 29946),
Salmonella enteritidis (ATCC 13076), and Wolinella
succinogenes (LMG 7608). Finally, clinical isolates of
Campylobacter jejuni, Campylobacter coli,
Campylobacter lari, Campylobacter upsaliensis,
Enterobacter agglomerans, Enterobacter cloacae,
Haemophilus influenzae, Shigella dysenteriae,
Shigella flexneri, Streptococcus faecalis,
Vibrio cholerae, and Yersinia enterocolitica were tested.
Bacteria were cultured on Trypticase-soy agar plates containing 5%
sheep blood (Becton Dickinson) for 3 to 5 days at 37°C under
microaerobic conditions (5% O2, 10% CO2, and
80% N2). The susceptibility to clarithromycin of each
strain and MICs for each strain were determined with the E test (AB
Biodisk, Solwa, Sweden) (4, 9, 13) or by the standard agar
dilution technique, as described previously (12, 15).
DNA isolation from cultured strains.
The H. pylori
cells were harvested from the plate by suspension in 2 ml of sterile
0.9% NaCl solution and pelleted by centrifugation at 10,000 × g for 2 min. Cells were resuspended in 400 µl of a solution
containing 10 mM Tris-HCl (pH 8.0), 5 mM EDTA, 0.1% sodium dodecyl
sulfate, and 0.1 mg of proteinase K per ml and incubated for 2 to
4 h at 55°C. Proteinase K was inactivated by incubation at
95°C for 10 min. The lysates were diluted 1/100 in sterile water and
directly used for PCR.
Analysis of 23S rRNA mutations by RFLP and/or direct
sequencing.
Mutations A2142G and A2143G were analyzed by
restriction fragment length polymorphism (RFLP) with restriction enzyme
AvaII, BsaI, MboII, or BbsI
as described earlier or by probe hybridization in liquid phase
(15, 16). 23S rRNA sequences were determined by direct
sequencing of PCR products (3, 4).
PCR-LiPA.
A fragment of the 23S rRNA gene was amplified by
PCR. PCRs were performed in a volume of 50 µl, containing 10 mM
Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2, 200 µM
concentrations of deoxynucleoside triphosphates, 0.25 U of AmpliTaq
Gold, and 25 pmol of biotinylated PCR primers. Reaction mixtures were
covered with mineral oil, and PCR was performed in a BioMed-60
thermocycler, under the following conditions: 9 min of preincubation at
94°C, followed by 40 cycles of 30 s at 95°C, 45 s at
62°C, and 45 s at 72°C. A final extension was performed for 5 min at 72°C.
PCR products were analyzed by reverse hybridization in a reverse
hybridization line probe assay (LiPA). This assay is based on
hybridization to a number of oligonucleotide probes, immobilized as
parallel lines on a nitrocellulose strip (Fig.
1). For each strain, 10 µl of the PCR
product was analyzed on the LiPA strip, essentially as described for
the LiPA detection of vacA and cagA genotypes
(18a).
View larger version (36K):
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[in a new window]
|
FIG. 1.
Outline and representative examples of the LiPA strips.
The positions of probes on the strip are shown. Since we did not have
access to strains that contained the A2115G and G2141A mutations,
the specificities of the corresponding probes were determined with
biotin-labelled oligonucleotides, complementary to these probes on the
LiPA. WT, wild type.
|
|
Analysis of 23S rDNA mutations in H. pylori strains by
PCR-LiPA.
PCR primers for the 23S rDNA of H. pylori
were tested on chromosomal DNA from multiple bacterial species. PCR
products of the expected size were obtained only from H. pylori, H. nemestrinae, and H. acinonyx.
Specific probes were developed for reverse hybridization analysis in a
line probe assay, covering the relevant positions of the 23S rDNA. The
PCR fragments from H. nemestrinae and H. acinonyx
showed hybridization only to the wild-type probes.
To evaluate the performance of the PCR-LiPA, a total of 57
H. pylori strains were analyzed. Of these, 51 were clinical isolates,
directly derived from patients, and 6 were laboratory-derived
artificial mutant
H. pylori strains (
3). Of the
57 strains,
52 were resistant, whereas 5 strains were susceptible
(MIC < 2).
Sequences of the 23S rRNA gene also were analyzed by
PCR followed
by a combination of direct sequencing, RFLP, and/or probe
hybridization
as described (
3,
4,
15,
16). All results for
the individual
strains are shown in Table
1 and are summarized in Table
2.
View this table:
[in this window]
[in a new window]
|
TABLE 2.
Comparative results of conventional and PCR-LiPA methods
for the detection of 23S rDNA mutations in natural isolates and
laboratory strains
|
|
In 52 (91.2%) of the 57 isolates studied, PCR-LiPA yielded exactly the
same results as conventional methods (direct sequencing,
probe
hybridization in liquid phase, and/or restriction enzyme
analysis).
These included the natural strains, including the wild
type
(
n = 5) and strains with mutations A2142
G
(
n = 19), A2142
C
(
n = 1), A2143
G
(
n = 19), or a combination (
n = 3),
derived from
patients, as well as five of the six artificial mutants,
with
mutations A2142
G, A2142
C, A2142
T, A2143
G, and
A2143
C, respectively.
No strains were found that contained the
combined A2115
G and
G2141
A mutations (
10), which may
indicate that such mutants
are extremely rare. All strains showing
discrepant results were
retested by the conventional methods as well as
by LiPA. In three
of the clinical strains, multiple mutants were
detected by conventional
methods (strains 1538, LINAN 1567, and PAP),
and PCR-LiPA yielded
exactly the same
results.
The initially seemingly discrepant results observed in five strains can
all be attributed to the high sensitivity of the PCR-LiPA
method.
Strain 140-97, containing the wild-type 23S sequence,
as determined by
conventional methods, yielded a LiPA pattern
indicating the presence of
the wild-type sequence, as well as
A2142
G and A2143
G mutations.
The strain yielded variable MICs,
ranging from less than 2 to more than
256, suggesting the presence
of a mixture of strains. Two isolates
(1264 and 1470) contained
the A2142
G mutation, but PCR-LiPA also
showed the presence of
other mutants. Both strains were macrolide
resistant, and the
MICs for them were higher than 256. Similarly, the
MIC for strain
1075 exceeded 256, and sequence analysis detected only
the A2143
G
mutation, but PCR-LiPA also detected the A2142
G
mutation.
In the clinically macrolide-susceptible strain (ABT2-16), containing
the artificial mutation A2143
T, the wild-type sequence
as well as
the mutant A2143
T sequence was detected by sequence
analysis. The
LiPA does not contain a probe specific for A2143
T,
since it was
shown earlier that the A2143
T mutation was not stable
and rapidly
mutated back to the wild-type sequence during subsequent
passages
(
3). Since we tested the fourth passage of this strain,
the
PCR-LiPA result is consistent with these
data.
Interestingly, strain C3II contained only wild-type sequences, as
determined by all methods. However, this strain was clearly
resistant,
since the MIC for it was higher than 256. This strain
is not a clinical
isolate, but resistance to clarithromycin was
selected in this strain
in the laboratory by exposure to increased
concentrations of
clarithromycin (data not shown). Therefore,
this strain may well have
developed a different mechanism of resistance
(
12).
Compared to the conventional methods, the PCR-LiPA detected additional
mutants and appears to provide more accurate data about
the presence of
bacterial variants in the culture, especially
when multiple strains are
present. In contrast, direct sequence
analysis of PCR products will
often detect only the predominant
sequence. Only if different mutants
are present in approximately
similar concentrations are they detected
by sequence analysis,
whereas the LiPA specifically detects small
amounts of each mutant.
Also, the LiPA format permits the addition of
specific probes
in case additional relevant mutations are found. Thus,
the LiPA
provides accurate information about the presence of different
23S rDNA mutants, even if they represent only a small proportion
of a
bacterial population. Therefore, it could be particularly
suitable to
monitor the development of resistance during antibiotic
therapy. The
method can also be used directly in gastric biopsies,
without the need
for a bacterial culture (data not shown). Sometimes,
the presence of
multiple strains is also indicated by the presence
of apparently
resistant colonies within the growth inhibition
zone on agar plates.
This phenomenon is illustrated by the four
strains in which PCR-LiPA
showed the presence of multiple mutants,
whereas conventional 23 rDNA
analyses detected only a single wild-type
or mutant
sequence.
In conclusion, the PCR-LiPA offers a rapid and easy method for the
detection of clinically relevant mutations in the 23S rRNA
gene of
H. pylori. The high sensitivity of the reverse hybridization
method provides more accurate data, especially when multiple strains
are present. Therefore, this method could facilitate further
epidemiological
and clinical
studies.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Delft Diagnostic
Laboratory, R. de Graafweg 7, 2625 AD Delft, The Netherlands. Phone: 31-15-2604577. Fax: 31-15-2604550. E-mail:
L.J.van.Doorn{at}ddl.nl.
| |
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Antimicrobial Agents and Chemotherapy, July 1999, p. 1779-1782, Vol. 43, No. 7
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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