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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 2002, p. 2996–3000
0066-4804/02/$04.00ϩ0 DOI: 10.1128/AAC.46.9.2996–3000.2002
Copyright 2002, American Society for Microbiology. All Rights Reserved.
16S rRNA Mutation-Mediated Tetracycline Resistance in
Monique M. Gerrits,1 Marcel R. de Zoete,1 Niek L. A. Arents,2
Ernst J. Kuipers,1 and Johannes G. Kusters1*
Department of Gastroenterology and Hepatology, Erasmus MC—University Medical Center Rotterdam,
1 and Medical Microbiology, Regional Public Health Laboratory
2 The Netherlands
Received 12 February 2002/Returned for modification 18 April 2002/Accepted 29 May 2002
Most Helicobacter pylori strains are susceptible to tetracycline, an antibiotic commonly used for the eradi-
cation of H. pylori. However, an increase in incidence of tetracycline resistance in H. pylori has recently been
reported. Here the mechanism of tetracycline resistance of the first Dutch tetracycline-resistant (Tetr) H. pylori
isolate (strain 181) is investigated. Twelve genes were selected from the genome sequences of H. pylori strains
26695 and J99 as potential candidate genes, based on their homology with tetracycline resistance genes in other
bacteria. With the exception of the two 16S rRNA genes, none of the other putative tetracycline resistance genes
was able to transfer tetracycline resistance. Genetic transformation of the Tets strain 26695 with smaller over-
lapping PCR fragments of the 16S rRNA genes of strain 181, revealed that a 361-bp fragment that spanned
nucleotides 711 to 1071 was sufficient to transfer resistance. Sequence analysis of the 16S rRNA genes of the
Tetr strain 181, the Tets strain 26695, and four Tetr 26695 transformants showed that a single triple-base-pair
3TTC, was present within this 361-bp fragment. This triple-base-pair substitution,
present in both copies of the 16S rRNA gene of all our Tetr H. pylori transformants, resulted in an increased
MIC of tetracycline that was identical to that for the Tetr strain 181.
is a spiral-shaped, gram-negative bacte-
in these genes. The second common mechanism of resistance is
rium that causes chronic infections in the gastric mucosa (6).
mediated through ribosomal protection proteins. These cyto-
This infection will persist for life, unless treated with antibiot-
plasmic proteins confer tetracycline resistance either by a re-
ics. Cure of H. pylori
infection results in ulcer healing and may
duction of the affinity of ribosomes for tetracycline or by re-
reduce the risk of gastric cancer and gastric lymphoma (22, 28).
leasing the bound antibiotic from the ribosome. The ribosomal
The highest cure rates have been obtained with antimicrobial
protection proteins, such as TetM, TetO, and TetS, show ho-
treatments that include two or more antimicrobial drugs, a
mology with the elongation factors EF-G and EF-Tu (Table 1)
bismuth component, and/or a proton pump inhibitor (14, 25).
(4). Beside these two most common tetracycline resistance
For the treatment of H. pylori
mechanisms, two other mechanisms have been described. One
triple or quadruple therapies are often used as a second-line
is based on enzymatic inactivation of tetracycline by the prod-
treatment (7, 9, 17). Until the end of the last century only a few
uct of TetX in the presence of oxygen and NADPH, and the
reports were published on spontaneous tetracycline resistance
other originates from mutations in the 16S rRNA genes that
(18; P. D. Midolo, M. G. Korman, J. D. Turnidge, and J. R.
affect the binding site of tetracycline (4, 21, 24).
Lambert, Letter, Lancet 347:
1194-1195, 1996), and it was gen-
We recently isolated a tetracycline-resistant (Tetr) H. pylori
erally accepted that tetracycline resistance (MIC Ն 4 g/ml) in
isolate (strain 181), from a 72-year-old male dyspeptic patient.
is very rare (5, 12). However, in the last 2 years an
Here we describe the molecular mechanism of tetracycline-
increase in the incidence of tetracycline resistance in H. pylori
resistance in this strain. To achieve this, 12 genes were selected
has been reported (2, 11, 13, 20, 30).
from the published H. pylori
genomes (1, 23) as potential
Tetracycline inhibits the protein synthesis by binding to
candidates, based on their homology with tetracycline resis-
the 30S ribosomal subunit (3, 19). In most bacteria resis-
tance genes in other bacteria (Table 1). These putative tetra-
tance to tetracycline is due to an energy-dependent efflux of
cycline resistance genes were amplified from the genome of the
tetracycline-cation complexes across the cell membrane by
Tetr strain 181 and used for genetic transformation of the
membrane-associated efflux proteins. Export of tetracycline
tetracycline-sensitive (Tets) strain 26695 in order to identify
complexes out of the cell reduces the intracellular drug con-
the changes responsible for tetracycline resistance.
centration and protects the ribosomes from tetracycline (4).
Overexpression of the efflux genes confers tetracycline resis-
tance, while the sensitivity to tetracycline increases by deletions
MATERIALS AND METHODS
Bacterial strains and growth conditions. H. pylori
strains used in this study
were the Tetr strain 181 and the Tets H. pylori
strains 26695 (23), J99 (1), SS1
* Corresponding author. Mailing address: Department of Gastroen-
(15), and ATCC 43504 (American Type Culture Collection). Bacteria were
terology and Hepatology, Rm. L457, Erasmus MC - University Med-
routinely grown on Columbia agar plates (Becton Dickinson, Cockeysville, Md.)
ical Center Rotterdam, Dr. Molewaterplein 40, 3015 GD Rotterdam,
supplemented with 7% lysed horse blood (BioTrading, Mijdrecht, The Nether-
The Netherlands. Phone: 31-10-4632982. Fax: 31-10-4632793. E-mail:
lands) and H. pylori
Dent selective supplement (Oxoid, Basingstoke, United
Kingdom), referred to as Dent plates. Bacteria were inoculated on these plates
TETRACYCLINE RESISTANCE IN HELICOBACTER PYLORI
and incubated for 48 to 72 h at 37°C in a microaerobic atmosphere of 5% O
10% CO2, and 85% N2. Bacterial stocks were prepared by suspending bacteria,
harvested from culture plates with a sterile cotton swab, in brain heart infusion
with 20% glycerol and stored at Ϫ80°C.
Determination of MIC.
The MIC was routinely determined with the E-test
(AB Biodisk, Solna, Sweden) (8). Inocula were prepared from a fresh H. pylori
culture grown routinely for 2 days on Dent plates. Columbia agar plates con-
taining 7% lysed horse blood, but no Dent supplement, were inoculated with
approximately 2 ϫ 108 CFU in 20 l of 0.9% NaCl, the plates were dried for 3
to 4 min, and then the E-test strips were applied to the agar surface. The plates
were incubated at 37°C under microaerobic conditions, and 3 days later the MIC
was determined by the intercept of the zone of inhibition with the graded E-test
strip. By this method the susceptibility was determined for tetracycline, doxycy-
cline, minocycline, amoxicillin, clarithromycin, and metronidazole. The isolates
were considered resistant when the MICs of tetracycline, doxycycline, and mi-
nocycline were Ն4 g/ml and when those of amoxicillin, clarithromycin, and
metronidazole were Ն8, Ն2, and Ն8g/ml, respectively (5, 13).
Natural transformation of H. pylori.
Bacteria were transformed with ϳ1 g of
ϳ250 ng of PCR-amplified gene products from strain 181, as
described previously (27). Tetr transformants were selected on Dent plates con-
taining tetracycline (2 g/ml; Sigma Aldrich Chemie, Zwijndrecht, The Nether-
lands). As controls, bacteria were transformed with either genomic DNA of the
Tetr strain, TE (1 mM Tris-HCl, 0.1 mM EDTA [pH 8.0]), or DNA from the Tets
strains 26695, J99, SS1, and ATCC43504. Individual bacterial colonies present on
tetracycline-containing plates (2 g/ml) were selected, and their MICs of tetra-
Oligonucleotide primers (Isogen, Maarsen, The Netherlands) used for
TCAGAACACTTCAACCCTA TTGCTAGGCACTTCGCCATA CGCTAAAGCCTCTTGCAGTA
CCAAAAGAGCGCCAACAAAC TTTCCATTAGCGTTAGTGTC GCAGTCATTCGCTAATTCAA TCCCTTTTAGCCGCTAGTTC
PCR amplification were based on the genome sequences of H. pylori
26695 and J99 (Table 1; Fig. 1 and 2) (1, 23). PCR was performed in an
automated thermal cycler (I-Cycler; Bio-Rad), in a final volume of 50 l, using
the PCR-core system I (Promega, Madison, Wis.), with approximately 25 pg of
template genomic DNA and 25 pmol of each primer.
Direct sequencing of the obtained PCR products was
performed by Baseclear Inc. (Leiden, The Netherlands). Sequence data were
analyzed with the help of Lasergene (DNAstar, Madison, Wis.), and Sci Ed
Central (Scientific & Educational Software, Durham, N.C.) software.
Nucleotide sequence accession number.
The 16S rRNA gene sequence of Tetr
strain 181 has been deposited into to the GenBank sequence database,
GTTTCCCGCTCCATTTTTA ATGGATGCGGCTAGCGATAA TGATTGGCAAAGGCCGTAGTT
CTTGCGTGTGGTAGTAATGC CTAAAGTTTTGCGCTAAGTG AACGGCTTAGCCTTATACAA ATGACATAGCCCACAAACCC
Determination of the MICs of various antibiotics.
the four antibiotics commonly used in anti-H. pylori
well as those of two antibiotics that belong to the tetracycline
family, were determined by E-test for the Tetr H. pylori
strain181 and the H. pylori
reference strain 26695 (Table 2). The
MIC of tetracycline for strain 181 was 8 g/ml (susceptibility
breakpoint Ն 4 g/ml), while the MIC for strain 26695 was
0.19 g/ml. The MICs of the two other tetracyclines, doxycy-
cline and minocycline, were also significantly higher for strain
181 than for strain 26695. For the three other routinely used
antibiotics (amoxicillin, clarithromycin, and metronidazole)
the MICs varied between Ͻ0.016 and 0.064 g/ml and did not
differ significantly between strains 181 and 26695.
Transfer of tetracycline resistance by natural transforma-
Transformation of H. pylori
strain 26695 (MIC, 0.19 g/
ml) with genomic DNA of strain 181 (MIC, 8
in Tetr colonies with a transformation frequency of 6 ϫ 10Ϫ5.
The MIC of tetracycline for the 10 randomly selected Tetr
transformants (obtained from three independent transforma-
tion experiments), determined by E-test was 8
which is identical to that for the Tetr H. pylori
strain 181. The
Tetr transformants also displayed an increase of MIC of the
tetracycline derivatives, doxycycline and minocycline (Table 2).
Transformation with PCR products of putative tetracycline
FIG. 1. Schematic representation of the 16S rRNA genes of H. pylori
. Alignment of the 16S rRNA genes (rrnA
) from the Tetr strain
181, the Tets strain 26695, and four Tetr 26695 transformants is shown. Sequence analysis of the 16S rRNA genes revealed only a few base pair
substitutions (numbering according to 16S rrnA
of H. pylori
strain 26695) in the Tetr strain 181 that did not occur in the Tets strain 26695. For the
identification of the 16S rRNA region required for tetracycline resistance, the Tets strain 26695 was transformed with smaller overlapping PCR
fragments of the 16S rRNA gene of the Tetr strain 181 (only fragments containing mutations are shown). The transformants were selected on
tetracycline (2 g/ml)-containing Dent plates. Primers used for the amplification of the smaller overlapping PCR fragments started at the outside
of the fragment and each had a length of 20 bp.
Based on their homology with tetracycline
not occur in the Tets strain 26695 (Fig. 1). Three Tetr 26695
resistance genes in other bacteria, 12 genes were selected from
transformants had incorporated the complete 16S rRNA gene
the published genome sequences of H. pylori
strains 26695 (23)
of strain 181, while the fourth transformant contained the first
and J99 (1) (Table 1). The Tets H. pylori
strain 26695 was
part of the 16S rRNA gene of strain 26695 and the second part
transformed with the PCR products of the selected tetracycline
of strain 181. The DNA crossover in this transformant oc-
resistance genes, which were amplified from genomic DNA of
curred after nucleotide 93 and before 127 (numbering accord-
the Tetr strain 181. Only transformation with the PCR product
ing to 16S rrnA
of H. pylori
strain 26695). For each strain or
of the 16S rRNA genes resulted in Tetr transformants, with a
transformant, only one sequence was obtained for the 16S
transformation frequency of 4 ϫ 10Ϫ5. No Tetr transformants
rRNA genes, indicating that these 16S rRNA genes were iden-
were found after transformation with one of the other selected
genes, TE, or DNA from the Tets strain 26695. Similar results
Identification of 16S rRNA mutations involved in tetracy-
were found for the Tets strains J99 (MIC, 0.5 g/ml), SS1
To determine which residues of the 16S rRNA
(MIC, 0.19 g/ml), and ATCC 43504 (MIC 0.125 g/ml). For
genes were responsible for tetracycline resistance in strain 181,
all strains the MIC of tetracycline for 10 randomly selected
the Tets strain 26695 was genetically transformed with smaller
Tetr transformants (obtained from three independent transfor-
overlapping PCR products of the 16S rRNA gene fragments,
mation experiments) determined by E-test was 8 g/ml, which
amplified from genomic DNA of the Tetr strain 181. Tetr
is identical to that for the Tetr donor strain 181.
transformants were only observed after transformation with a
Comparison of the 16S rRNA gene sequences of the Tetr
361-bp DNA fragment that spanned nucleotides 711 to 1071
strain 181, the Tets strain 26695, and four randomly Tetr 26695
(numbering according to 16S rrnA
of H. pylori
transformants (obtained after transformation with genomic
with a transformation frequency of 5 ϫ 10Ϫ6. Transformation
DNA of strain 181) revealed several base pair differences in
with the other DNA fragments did not result in transfer of
the Tetr strain 181 as well as in the Tetr transformants that did
tetracycline resistance. The MIC of tetracycline for 10 ran-
FIG. 2. Both 16S rRNA genes are mutated in H. pylori
tetracycline resistance. rrnA
- and rrnB
-specific sequences were amplified using specific
primers based on sequences which are found outside the two 16S rRNA genes. The rrnA
-specific primers, F1 and F2, are located at position
1207020 and 1207242 (numbers corresponding to the H. pylori
26695 sequence ), respectively, and the rrnB
-specific primers, F3 and F4, are
located at position 1510569 and 1510809, respectively. For amplification, primer R1 (located at position 1208293 and 1511828) was used in
combination with one of the other primers. All primers had a length of 20 bp.
TETRACYCLINE RESISTANCE IN HELICOBACTER PYLORI
TABLE 2. MICs for various H. pylori
for H. pylori
Data shown are the averages of three independent experiments. The isolates
were considered resistant when the MICs of the tetracyclines (tetracycline, doxy-
cycline, and minocycline), amoxicillin, clarithromycin, and metronidazole were
Ն4, Ն8, Ն2, and Ն8 g/ml, respectively (5, 13).
domly selected Tetr transformants determined by E-test was 8
g/ml, which was identical to that for the Tetr donor strain 181.
The only difference found between these Tetr 26695 transfor-
mants and the Tets strain was the triple-base-pair substitution
Both copies of 16S rRNA genes are involved in tetracycline
The primers that were originally used for amplifi-
cation of the 16S rRNA genes did not distinguish between the
two copies present on the H. pylori
chromosome (1, 23). To
FIG. 3. Schematic representation of the primary binding site of
assess the involvement of each copy of the 16S rRNA genes in
tetracycline, based on the 16S rRNA structure of Thermus thermophi-
tetracycline resistance, specific oligonucleotide primers were
proposed by Wimberly et al. (29). The primary binding pocket for
developed (Fig. 2). These specific primers are based on se-
tetracycline is formed by the 16S rRNA residues 1054 to 1056 (box A)
quences which are located approximately 350 to 600 bp outside
and residues 1196 to 1200 (box B) of helix 34 and residues 964 to 967
of helix 31 (box C). The interactions between tetracycline and this
the both 16S rRNA genes, rrnA
. This allowed ampli-
pocket are formed by hydrophobic interactions, hydrogen bonds, and
fication of rrnA
- and rrnB
-specific sequences. rrnA
- and rrnB
salt bridges (3). The triple-base-pair substitution AGA926-9283TTC
containing PCR-fragments were obtained for the Tetr strain
(corresponding to bp 965 to 967 of E. coli
16S rRNA) is located in box
181, the eight 26695 transformants, and Tets strain 26695, and
their DNA sequences were determined. As expected, the rrnA
sequences were identical, where as the sequences
outside the 16S rRNA genes were different. While the Tets
(corresponding to bp 965 to 967 of Escherichia coli
strain-derived fragments contained the AGA sequence in both
present in both copies of the 16S rRNA gene. Tetracycline has
genes, both for strain 181 and the eight 26695 transformants,
one primary and multiple secondary binding sites within the
30S ribosomal subunit (3, 19). In the primary binding site,
in both copies of the 16S rRNA genes.
tetracycline binds exclusively to the 3Ј-major domain of the 16S
rRNA. The primary binding pocket for tetracycline is formed
by the 16S rRNA residues 1054 to 1056 and 1196 to 1200 of
helix 34 and residues 964 to 967 of helix 31 (numbers corre-
Until recently tetracycline resistance in H. pylori
was rare (5,
sponding to E. coli
16S rRNA) (3). The residues 1054 and 1196
12), but in the last 2 years, several Tetr H. pylori
interact primarily with tetracycline through hydrophobic inter-
been isolated (2, 13, 30; Midolo et al., letter). These Tetr
actions, but the majority of the interaction with the drug is made
clinical isolates showed, besides tetracycline resistance, cross-
through hydrogen bonds and salt bridges between tetracycline
resistance to metronidazole (2, 13, 30; Midolo et al., letter).
and the 16S rRNA residues (Fig. 3) (3). In the Tetr H. pylori
The tetracycline resistance present in these strains was always
strain 181, the triple-base-pair substitution AGA926-9283TTC
transferred together with metronidazole resistance to a Tets
is located right in the primary binding site of tetracycline.
strain (13). In these Tetr strains it is not clear whether the
Mutations in this primary binding site are likely to affect the
tetracycline resistance is caused by a known metronidazole
affinity of the drug-ribosome interaction and thus the efficacy
resistance mechanism, a multidrug resistance mechanism, or
of tetracycline as a translational inhibitor.
an unknown tetracycline resistance mechanism (13). In our
In E. coli
, the nucleotides G966 and C967 are located not only
Tetr H. pylori
strain 181, no cross-resistance was found against
in the primary binding site of tetracycline but also in a func-
metronidazole, which indicated that the molecular mechanism
tional region of the ribosome, the P site (16, 26). Mutations in
of tetracycline resistance in strain 181 could be different from
this region may affect protein synthesis (10), either by a change
that of these earlier-described Tetr H. pylori
in binding of tRNA to the P site itself or by blocking the
In H. pylori
strain 181 resistance to tetracycline is mediated
conformational change needed for the tRNA binding to the A
by a single triple-base-pair substitution, AGA926-928 3 TTC
site. In H. pylori
strain 181 and the Tetr transformants of strain
26695, the triple-base-pair substitution AGA
in Escherichia coli
at methyl-modified sites: G966, C967, and G1207. Nucleic
no effect on the growth rate of the bacterium in the presence
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Cibo e Farmaci: un accordo non sempre facile Se la terapia antibiotica si è prolungata più del dovuto o l'antinfiammatorio non ha fatto passare il dolore alla spalla che vi tormenta, se l'antistaminico vi ha fatto addormentare o il diuretico non ha avuto l'effetto sperato forse è colpa di ciò che avete mangiato o bevuto. Sembra strano eppure è proprio così; ci sono alcuni cibi e be
HIGH BLOOD PRESSURE TREATMENT PROTOCOL FOR PATIENTS IN HAITI, INCLUDING WHEN LABORATORY MONITORING IS NOT AVAILABLE There are a number of ways to approach the treatment of the patient with high blood pressure when laboratory testing is not available. The following is one approach. Naturally, the particular circumstances of the individual patient and the experience and clinical judgmen