Mutations in the 23S rRNA gene are associated with clarithromycin resistance in Helicobacter pylori isolates in Brazil.

Abstract

Resistance of Helicobacter pylori to clarithromycin has been associated with A2142G and A2143G point mutations in the 23S rRNA gene. Thus, the purpose of the present study was to determine the prevalence of each mutation in 52 clarithromycin-resistant H. pylori strains and to characterize the influence each type of mutation on the MIC.
The MIC for clarithromycin was determined by the agar dilution method, and the point mutations of H. pylori were detected by PCR followed by restriction fragment length polymorphism.
Clarithromycin MICs ranged from 2 to >256 microgram ml-1 among the 52 strains included in this study. Both the A2142G and the A2143G mutations were present in 94.2% of clarithromycin-resistant H. pylori strains examined. A relationship was observed between the presence of the A2142G mutation and the highest MIC values (p = 0.01).
In an H. pylori-infected population, the A2142G mutation may incur to a greater probability of treatment failure if clarithromycin is used.

Full text

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Annals of Clinical Microbiology and
Antimicrobials
Open Access
Research
Mutations in the 23S rRNA gene are associated with clarithromycin
resistance in Helicobacter pylori isolates in Brazil
Marcelo L Ribeiro, Lea Vitiello, Maira CB Miranda, Yune HB Benvengo,
Anita PO Godoy, Sergio Mendonca and José Pedrazzoli Jr*
Address: Clinical Pharmacology and Gastroenterology Unit, São Francisco University Medical School, Bragança Paulista, SP, Brazil
Email: Marcelo L Ribeiro - marceloribeiro@saofrancsico.edu.br; Lea Vitiello - lea@helicobacter.com.br;
Maira CB Miranda - maira@helicobacter.com.br; Yune HB Benvengo - yune@helicobacter.com.br;
Anita PO Godoy - anita@helicobacter.com.br; Sergio Mendonca - sergiomendonca@saofrancisco.edu.br;
José Pedrazzoli* - pedrazzoli@saofrancisco.edu.br
* Corresponding author
Abstract
Background: Resistance of Helicobacter pylori to clarithromycin has been associated with A2142G
and A2143G point mutations in the 23S rRNA gene. Thus, the purpose of the present study was
to determine the prevalence of each mutation in 52 clarithromycin-resistant H. pylori strains and to
characterize the influence each type of mutation on the MIC.
Methods: The MIC for clarithromycin was determined by the agar dilution method, and the point
mutations of H. pylori were detected by PCR followed by restriction fragment length polymorphism.
Results: Clarithromycin MICs ranged from 2 to >256 microgram ml-1 among the 52 strains
included in this study. Both the A2142G and the A2143G mutations were present in 94.2% of
clarithromycin-resistant H. pylori strains examined. A relationship was observed between the
presence of the A2142G mutation and the highest MIC values (p = 0.01).
Conclusion: In an H. pylori-infected population, the A2142G mutation may incur to a greater
probability of treatment failure if clarithromycin is used.
Background
Helicobacter pylori is a gram-negative bacterium that colo-
nizes the human stomach and is associated with a variety
of digestive diseases, such as chronic gastritis and peptic
ulcer disease [1,2]. Infection with H. pylori can be effec-
tively treated by the combination of a proton pump inhib-
itor with multiple antibiotics. The first-line regimen
consists mainly of a triple therapy, and clarithromycin is
one of the most widely used components. Although the
bacteria can be eradicated in up to 90% of patients, side
effects, poor compliance and resistance to the antibiotics
used are common causes of treatment failure [3,4].
The increasing use of clarithromycin has resulted in the
development of resistance. The prevalence of resistant
strains varies among countries and ranges from 1% in
Norway [5] to 29% in Japan [6]. The mechanism of resist-
ance to clarithromycin in H. pylori seems to be due to a
decrease in binding of macrolides to the ribosome, associ-
ated with point mutations within the peptidyltransferase-
encoding region of 23S rRNA gene [7,8]. Three major
point mutations in two positions have been described in
which an adenine residue is replaced by a guanine or a
cytosine residue at adjacent positions: A2142C, A2142G,
and A2143G [7-10]. Thus, the purpose of the present
Published: 21 November 2003
Annals of Clinical Microbiology and Antimicrobials 2003, 2:11
Received: 06 October 2003
Accepted: 21 November 2003
This article is available from: http://www.ann-clinmicrob.com/content/2/1/11
© 2003 Ribeiro et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all
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study was to determine the prevalence of each mutation in
52 clarithromycin-resistant H. pylori strains and also char-
acterize the influence of the type of mutation upon MIC.
Material and Methods
Bacterial growth conditions and MIC determination
H. pylori strains used in this study were isolated in the
Clinical Pharmacology and Gastroenterology Unit of Sao
Francisco University Medical School, Braganca Paulista,
SP, Brazil. Fifty-two previously isolated clarithromycin-
resistant (claR) strains were inoculated on Brain Heart
Infusion agar plates (Difco, Detriot, MI, USA) supple-
mented with 2.5 g L-1 yeast extract (Difco) and 10% lysed
sheep blood (BBV, Campinas, Brazil), and 6 mg L-1 vanco-
mycin (Sigma Aldrich Chemie, St. Louis, MO, USA), 20
mg L-1 nalidixic acid (Sigma Aldrich Chemie), 2 mg L-1
amphotericin B (Sigma Aldrich Chemie), 40 mg L-1 2,3,5,
triphenyl-tetrazolium chloride (Sigma Aldrich Chemie),
referred to as BHI-YE plates, followed by incubation for 3–
5 days at 37°C under microaerophilic conditions. The
MIC for clarithromycin was determined by the agar dilu-
tion method, using twofold increments (0.125 to 256 µg
ml-1) on Mueller-Hinton agar (Merck, Darmstadt, Ger-
many) supplemented with 10% sheep blood, and incu-
bated at 37°C under microaerophilic conditions [11]. The
isolates were considered resistant when the MIC of clari-
thromycin was > 1 µg ml-1 [12-14].
Detection of mutation associated with clarithromycin
resistance
The point mutations of H. pylori were detected by PCR fol-
lowed by restriction fragment length polymorphism
(RFLP). DNA from 52 claR H. pylori isolates was extracted
by a standard phenol-chloroform method [15]. Part of the
23S rRNA gene was amplified with primers 18 (5'-
AGTCGGGACCTAAGGCGAG-3') and 21 (5'-TTCCCGCT-
TAGATGCTTTCAG-3'), and the amplicon of approxi-
mately 1.4 Kb was digested with MboII (New England
Biolabs, Beverly, MA, USA) or BsaI (New England Biolabs)
to detect the A2142G and the A2143G mutations, respec-
tively [8]. The restriction fragments were analyzed on a
3% agarose gel.
Sequence analysis
When none of the aforementioned mutations were
detected, direct sequencing was used. An internal frag-
ment, that comprises the majority of mutations related to
clarithromycin resistance, was amplified using the follow-
ing primers; 5'-GTAACTATAACGGTCCTAAG-3'
(Cla1995Fw) and 5'-GAAACATCAAGGGTGGTATC-3'
(Cla2274Rev). PCR was performed in a final volume of 50
µl, with approximately 25 pg of template genomic DNA
and 25 pmol of each primer. PCR amplification was car-
ried out through 35 cycles consisting of a denaturation
step of 95° C for 30 sec, a primer-annealing step of 50°C
for 30 sec and an extension step at 72°C for 30 sec, with a
single final extension step of 72°C for 10 min. A 280 bp
fragment was purified with Wizard® SV Gel and PCR
Clean-up System (Promega, Madison, WI) and the direct
sequencing was performed by the Hemocentro/UNI-
CAMP genome center (Campinas, Brazil). Sequence data
were analyzed with Chromas (Technelysium Pty. Ltd.,
Helensvale, Australia).
Nucleotide sequence accession numbers
The nucleotide sequences of the BZ440, BZ447 and
BZ450 regions of the clarithromycin resistant strains were
deposited in GenBank under accession numbers
AY303829 to AY303831, respectively.
Natural transformation
Natural transformation was used to verify if the 23S rRNA
gene is involved in resistance in cases where none of the
mutations related to clarithromycin resistance were
detected. Briefly, the clarithromycin sensitive (claS) refer-
ence strain, 26695, was transformed with ~1 µg of
genomic DNA or ~250 ng of PCR products (1.4 Kb and
280 bp) from the claR strains, as previously described [16].
The resistant transformants were selected on BHI-YE
plates supplemented with clarithromycin at 0.5 and 2 µg
ml-1 (Abbot Laboratories, North Chicago, Ill). As controls,
bacteria were transformed with either DNA from claR
strain BZ586 (MIC >256 µg ml-1), DNA from claS strain
26695 [17] or TE [1 mM Tris-HCl (pH 8.0), 0.1 mM
EDTA].
Statistical analysis
Statistical analyses were performed using either the χ2 test
with Yates continuity correction or Fisher's exact test. A p
value of < 0.05 was considered statistically significant.
Results and Discussion
Clarithromycin is a key component of most current triple-
therapy regimens for treatment of H. pylori infection, how-
ever resistance is a major determinant in the failure of
eradication regimens [3]. Clarithromycin resistance varies
around the world with rates ranging from 1 to 29% [5,6],
in Brazil the resistance rate observed was 16% [18].
Increasing resistance rates have been observed and seem
to be due to the increase in the use of this antibiotic, not
only for H. pylori eradication but also for treatment of res-
piratory tract infections [3].
The molecular mechanism of H. pylori resistance to clari-
thromycin is associated with A-to-G substitution within
the peptidyltransferase-encoding region of the 23S rRNA
gene [7,8]. The clarithromycin MICs ranged from 2 to
>256 µg ml-1 for the 52 strains included in this study.
Either the A2142 mutation or the A2143G mutation were
present in 94.2% of clarithromycin-resistant H. pylori

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strains examined. The majority of isolates (82.7%) con-
tained the A2142G mutation, whereas the A2143G was
present in 11.5% (Table 1). Additionally, when an MIC of
≥ 64 µg ml-1 was used to define a high level of clarithro-
mycin resistance, differences in prevalence of each muta-
tion were observed (Table 1). There was a relationship
between the presence of the A2142G mutation and the
highest MIC values (p = 0.01).
Some authors have shown that the A2143G mutation was
the most frequently detected mutation [19,20], however,
our data and those of others demonstrate that the A2142G
mutation is more prevalent in Brazil [8,21]. Although the
presence of the A2143G mutation was rare in the present
study and the A2142G mutation demonstrated the high-
est frequency observed, the clinical relevance of this dis-
tinction has a limited value, since the presence of either of
these mutations is able to confer clarithromycin resistance
[22]. A high level of resistance to clarithromycin has been
associated with the presence of the A2142G mutation in
H. pylori [13,23]. According to our results, a greater effect
upon MIC was observed among strains possessing the
A2142G mutation. Thus, this mutation may incur a
greater probability of treatment failure in populations
infected by H. pylori [13].
In three isolates, the PCR-RFLP method was unable to
detect these substitutions. The sequence analysis of these
isolates, from the internal fragment that comprises the
majority of point mutations related to clarithromycin
resistance, revealed only a T-to-C transition at position
2245 (Based on GenBank accession no. U27270). To
determine whether the T2245C mutation observed was
responsible for clarithromycin resistance, the 26695 claS
H. pylori strain was transformed with genomic DNA and
specific PCR products. For this purpose, the 23S rRNA was
amplified using primers, 18 and 21 [8], while internal
primers, Cla1995 and Cla2274, created a 280 bp fragment
that only contained the T2245C substitution. The trans-
formants were selected on BHI-YE plates supplemented
with clarithromycin. Although the T2245C substitution
was present in these three resistant strains, clarithromy-
cin-resistant transformants containing the T2245C substi-
tution were only observed after transformation with
genomic DNA, but not after transformation with 23S
rRNA PCR fragments. While transformants were found
after transformation with DNA and PCR products from
the BZ586 claR strain, none of the transformants were
selected after transformation with DNA from the claS
strain 26695 or TE. The MIC values of clarithromycin of
10 randomly selected resistant transformants was deter-
mined by agar dilution and were identical to those of
donor strains.
Although, in the sequence data only a T2245C substitu-
tion was found, natural transformation was unable to
detect an association between the presence of mutations
in the 23S rRNA gene and clarithromycin resistance. Addi-
tionally, analysis of several sequences based on GenBank
indicated that the T2245C substitution was detected in
both resistant and susceptible H. pylori strains
(AB088050-to-AB088065). Thus, the molecular mecha-
nism of clarithromycin resistance remains undetermined
in these three cases.
Conclusions
In conclusion, our results support the hypothesis that the
A2142G and A2143G mutations in the 23s rRNA gene of
H. pylori are linked to clarithromycin resistance, this find-
ing may have a significant impact on patient manage-
ment, providing rapid information for the clinician,
allowing, for example, appropriate antibiotic prescription
and prediction of treatment outcome.
List of abreviation
PCR-RFLP – Polymerase chain reaction – restriction frag-
ment length polymorphism; claR – clarithromycin resist-
ant H. pylori strains; claS – clarithromycin sensitive H.
pylori strains.
Authors' contribition
MLR and MCBM carried out the molecular genetic studies;
APOG, YHBB, LV and SM participated in the antimicro-
bial susceptibility analysis; MLR drafted the manuscript;
MLR and LV performed the statistical analysis; MLR, SM
and JPJ participated in the design of the study and
Table 1: Association between clarithromycin MICs and 23S rRNA mutations
Mutation
No. of strains (%)Total (%) (n = 52)
MIC < 64 µg ml-1
MIC ≥64 µg ml-1
A2142G
A2143G
T2245C
12 (63.2)
5 (26.3)
2 (10.5)
31 (91.1)
1 (4.45)
1 (4.45)
43 (82.7)
6 (11.5)
3 (5.8)

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coordination; All authors read and approved the final
manuscript.
Acknowledgments
This work was supported by the Fundacao de Amparo a Pesquisa do Estado
de Sao Paulo (01/12369-1). YHBB is supported by a fellowship from Funda-
cao de Amparo a Pesquisa do Estado de Sao Paulo (02/08566-9)
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